who/ draft/ 4 may 2010 english only who/ draft/ 4 may 2010 3 english only 4 5 6 recommendations for...

1 WHO/ DRAFT/ 4 May 2010 2

ENGLISH ONLY 3 4

5

Recommendations for the evaluation of animal cell cultures 6

as substrates for the manufacture of biological medicinal 7

products and for the characterization of cell banks 8

9

Proposed replacement of TRS 878, Annex 1 10

11 NOTE: 12

13

This document has been prepared for the purpose of inviting comments and 14 suggestions on the proposals contained therein, which will then be considered by the 15 Expert Committee on Biological Standardization (ECBS). In particular, suggestions for 16 completing section C will be appreciated. Publication of this early draft is to provide 17 information about the proposed WHO recommendations for evaluation of animal cell 18 cultures as substrates for the manufacture of biological medicinal products and for the 19 characterization of cell banks to a broad audience and to improve transparency of the 20 consultation process. 21

22 The text in its present form does not necessarily represent an agreed formulation 23 of the Expert Committee. Comments proposing modifications to this text MUST be 24 received by 31 May 2010 and should be addressed to the World Health Organization, 25 1211 Geneva 27, Switzerland, attention: Quality Safety and Standards (QSS). 26 Comments may also be submitted electronically to the Responsible Officer: Dr Ivana 27 Knezevic at email: [email protected]. 28 29 The outcome of the deliberations of the Expert Committee will be published in the WHO 30 Technical Report Series. The final agreed formulation of the document will be edited to 31 be in conformity with the "WHO style guide" (WHO/IMD/PUB/04.1). 32 33

© World Health Organization 2010 34

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health 35 Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: 36 [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for 37 noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-38 mail: [email protected]). 39

40

WHO/DRAFT/4 May 2010

The designations employed and the presentation of the material in this publication do not imply the expression of any 41 opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, 42 city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps 43 represent approximate border lines for which there may not yet be full agreement. 44 45 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 46 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. 47 Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 48 49 All reasonable precautions have been taken by the World Health Organization to verify the information contained in 50 this publication. However, the published material is being distributed without warranty of any kind, either expressed 51 or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the 52 World Health Organization be liable for damages arising from its use. 53

54 The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. 55


56

Table of contents 57

58

1. Introduction 59

2. Historical overview 60

3. Scope 61

4. Definitions 62

5. General considerations 63

• Types of animal cell substrates 64

o Primary cell cultures 65

o Diploid cell lines 66

o Continuous cell lines 67

o Stem cell lines 68

• Potential risks and risk mitigation associated with biologicals produced In animal cells 69 o Viruses and other transmissible agents 70

o Cellular DNA 71

o Cellular RNA 72

o Growth promoting proteins 73

74

Part A. General recommendations applicable to all types of cell culture production 75

A.1 Good manufacturing practices 76

A.2 Principles of good cell culture practices 77

A.2.1 Understanding the cells and the culture system 78

A.2.2 Manipulation of cell cultures 79

A.2.3 Training and staff 80

A.2.4 Cell line development and cloning 81

A.2.5 Special considerations for neural cell types 82

A.3 Selection of source materials 83

A.3.1 Introduction 84

A.3.2 Serum and other bovine-derived materials used in cell culture media 85 A.3.3 Trypsin and other porcine-derived materials used for preparing cell culture 86 A.3.4 Media supplements and general cell culture reagents derived from other 87 sources used for preparing cell cultures 88


A.4 Certifications of cells 89

A.4.1 Cell line data 90

A.4.2 Certification of primary cell cultures 91 A.4.3 Certifications of DCLS, CCLs, and SCLs 92

A.5 Cryopreservation and cell banking 93

A.5.1 Cryopreservation 94

A.5.2 Cell banking 95

A.5.3 WHO reference cell banks 96

97

Part B. Recommendations for the characterization of cell banks of animal cell substrates 98

B.1 General considerations 99

B.2 Identity 100

B.3 Stability 101

B.4 Sterility 102

B.5 Viability 103

B.6 Growth characteristics 104

B.7 Homogeneity 105

B.8 Tumorigenicity 106

B.9 Oncogenicity 107

B.10 Cytogenetics 108

B.11 Microbial agents 109

B.11.1 General considerations 110

B.11.2 Viruses 111 B.11.2.1 Tests in animals and eggs 112

B.11.2.1.1 Adult mice 113

B.11.2.1.2 Suckling mice 114

B.11.2.1.3 Guinea pigs 115

B.11.2.1.4 Rabbits 116

B.11.2.1.5 Embryonated chicken eggs 117

B.11.2.1.6 Antibody-production tests 118

B.11.2.2 Tests in cell culture 119

B.11.2.2.1 Applicability 120

B.11.2.2.2 Indicator cells 121


B.11.2.2.3 Additional considerations to the tests in cell culture 122

for insect viruses 123

B.11.2.3 Transmission electron microscopy 124


B.11.2.3.2 General Screening Use 126

B.11.2.3.2.1 Insect Cells 127 B.11.2.4 Tests for retroviruses 128


B.11.2.4.2 Reverse Transcriptase Assay 130

B.11.2.4.3 PCR or other specific in vitro tests 131

B.11.2.4.4 Infectivity test for retroviruses 132

B.11.2.5 Tests for particular viruses 133


B.11.2.5.2 Nucleic Acid Detection Methods 135

B.11.3 Bacteria, fungi, mollicutes and mycoplasmas 136

B.11.3.1 Bacterial and Fungal Sterility 137

B.11.3.2 Mollicutes 138

B.11.3.2.1 Mycoplasma and acholesplasma 139

B.11.3.2.2 Spiroplasma and other mollicutes 140 B.11.3.3 Mycobacteria 141

B.11.4 Transmissible Spongiform Encephalopathies 142

B.11.4.1 Infectivity 143

B.11.4.2 Control measures, sourcing and traceability 144

B.11.4.3 Tests 145

B.12 Summary of tests for the evaluation and characterization of animal cell substrates 146

147

Part C. Risk reduction strategies during the manufacture of biological products derived 148 from animal cell substrates (to be completed) 149

C.1 General considerations 150

C.2 Risks 151

C.2.1 Exogenous agents introduced during manufacturing 152

C.2.1.1 Example: irradiation of bovine sera 153

C.2.1.2 Example: MVM 154


C.2.1.3 Example: equine virus during QC testing using horse serum 155

C.2.2 Endogenous agents 156

C.2.2.1 Example (DNAse) 157

C.2.2.2 Example (viral clearance) 158

C.2.2.3 Example (inactivation): BPL for rabies 159

160

Authors 161

Acknowledgements 162

References 163

Appendix 164

1. Tumorigenicity protocol using athymic nude mice to assess mammalian cells 165

2. Quantitative measurement of residual cell DNA (to be completed) 166

3. List of Abbreviations 167

168

169

170


1. Introduction 171

It is well established that cell substrates themselves and events linked to cell growth can affect 172

the characteristics and safety of the resultant biological products. Therefore, a thorough 173

understanding of the characteristics of the cell substrate is essential in order to identify points of 174

concern and to develop a quality control system that addresses those points. 175

176

Recent advances in the use and quality control of new animal cell substrates, particularly 177

continuous cell lines (CCLs) and insect cells, led to the conclusion that an update of the current 178

WHO Requirements (TRS 878) [ 1] should be prepared. In order to facilitate the resolution of 179

regulatory/ scientific issues related to the use of animal cell cultures, including human, as 180

substrates for the production of biological products. WHO initiated this revision of its 181

Requirements on cell substrates by establishing a Study Group (SG). This document is the result 182

of the SG effort, including wide consultations with individuals and organizations with an interest 183

in this area. After receiving comments from this consultative process, as well as from invited 184

reviewers, further revision of the draft recommendations will be undertaken and presented to the 185

ECBS 2010. 186

187

These recommendations provide guidance to National Regulatory Authorities (NRAs), National 188

Control Laboratories (NCLs) and manufacturers on basic principles and, in some cases, on 189

detailed procedures that are appropriate to consider in the characterization of animal cells that are 190

proposed for use in the manufacture of biological products. 191

192

2. Historical overview 193

Historically, the major concerns regarding the safety of biological medicinal products 194

manufactured in animal cells have been related to the possible presence of microbial 195

contaminants and, in some cases, to the properties and components of the cells themselves such 196

as DNA and proteins. 197

198

For example, in 1954, an experimental adenovirus vaccine was being developed, and human 199

tumour cells (HeLa) were rejected as the cell substrate in favor of ”normal” cells [ 2]. At that 200

time, relatively little was known about the biological mechanism(s) that lead to human cancer, so 201


that the risks to the recipients of a vaccine based on HeLa cells could not be assessed and 202

quantified scientifically. Although ”normal” cells were not defined, that decision led to the use of 203

primary cell cultures (PCCs) from animals such as monkeys, hamsters, and embryonated eggs 204

for vaccine research and development [ 3]. 205

206

The first requirements for cell substrates were published by WHO in 1959 for the production of 207

inactivated poliomyelitis vaccine in PCCs derived from the kidneys of clinically healthy 208

monkeys [ 4]. Those requirements were revised and published in 1966 [ 5]. Subsequently, other 209

PCCs were used for the production of other viral vaccines. 210

211

In the 1960s, human diploid cells (HDCs) were developed and proposed as an alternate to 212

primary monkey kidney cell cultures for polio virus vaccine production as well as for other viral 213

vaccines. The rationale for using HDCs was based on the ability to: a) cryogenically preserve the 214

cells at low population doubling levels (PDLs); b) establish and characterize cryopreserved 215

banks of cells that later could be expanded to provide a standardized source of cells for many 216

decades; c) extensively test recovered cells before use in vaccine production; and d) demonstrate 217

that the cells were free from detectable adventitious agents and that they were unable to form 218

tumors when inoculated into immunosuppressed animals. Thus, HDCs were normal by all of the 219

then existing criteria. It was argued that because HDCs were normal and could be standardized, 220

tested, and used for many years, they were a significant improvement over PCCs. 221

The pathway to acceptance of HDCs was difficult and lengthy, primarily because some members 222

of the scientific community believed that HDCs might contain a latent and unknown human 223

oncogenic agent, and that such a theoretical agent posed a risk to recipients of vaccines produced 224

in HDCs. Numerous conferences and discussions of new data eventually led to the acceptance of 225

HDCs as a substrate for viral vaccine production, and they continue to be used by many 226

manufacturers for various viral vaccines that have a long history of safety and effectiveness. The 227

concept of a master cell bank (MCB) and working cell bank (WCB) system and characterization 228

of the cell substrate were introduced during that period [ 6, 7]. 229

230

Both our understanding of tumor cell biology and the technological tools that were available at 231

that time were much more limited than they are today. As a result, the proponents of using HDCs 232


for vaccine production based their argument that the cells were normal, and therefore safe to use, 233

on four points: a) freedom from detectable adventitious agents; b) the finite life of HDCs; c) the 234

diploid nature of HDCs; and d) the inability of HDCs to form tumors in various in vivo test 235

systems. 236

237

In order to provide a high level of assurance that those four characteristics were stable, the initial 238

lot release tests for each batch of a vaccine derived from HDCs included tests of the cell 239

substrate for adventitious agents, karyology, and tumorigenicity [ 8, 9]. The main question that 240

was being addressed by the routine use of tumorigenicity tests was whether or not the production 241

cell culture had undergone a contamination or transformation event such that it contained a 242

mixture of "normal" and tumorigenic cells. It was eventually agreed that tumorigenicity testing 243

was not sensitive enough to detect a low level of tumorigenic cells, and that it was wasteful of 244

animals and time in repeated testing of a cell line that had been well-characterized and would be 245

used in the context of a cell bank system. Therefore tumorigenicity tests eventually were 246

required only for the characterization of a MCB (using cells at the proposed in vitro cell age for 247

production or beyond) for both HDCs and CCLs [ 10, 11]. 248

249

In the 1970s, there was a clinical research need for more interferon (IFN) than could be produced 250

from primary human lymphocytes. In response to that need, human tumour cells (Namalwa) 251

grown in vitro were proposed as a cell substrate for the production of IFN. The primary concerns 252

about the use of Namalwa cells were that they contained the Epstein-Barr virus (EBV) genome 253

integrated into the cellular DNA, and that either whole virus or DNA containing viral elements 254

could be transmitted to the recipients of the IFN product. Nevertheless, by the end of the 1970s, 255

regulatory agencies had allowed human clinical studies to commence, and the product was 256

eventually approved in several countries. Among the most important factors that contributed to 257

those decisions was the fact that IFN, as opposed to live viral vaccines, was not a replicating 258

agent, and IFN was being used as a therapeutic rather than a prophylactic product, thus 259

representing different risk/benefit considerations. In addition, technology had advanced 260

significantly so that IFN could be highly purified and the purification process could be validated 261

to demonstrate that EBV and cellular DNA were undetectable in the final product, within the 262

limits of the assays then available, which permitted risk mitigation. 263


264

In the 1980s, advances in science and technology led to the development of recombinant DNA 265

(rDNA) proteins and monoclonal antibodies (MAbs). Animal cells with the capacity to grow 266

continuously in vitro (CCLs) were the substrates of choice for those products because of the ease 267

with which they could be transfected and engineered. Also, in contrast to PCCs and HDCs, they 268

grew rapidly to achieve a high density and expressed a variety of products at high concentrations. 269

CHO cell lines became widely used for rDNA products, and hybridomas of various types were 270

required for the production of MAbs. The use of such cells as substrates in the manufacture of a 271

large array of potentially important biological medicinal products raised safety concerns once 272

again. A scientific consensus emerged from numerous conferences that there are three major 273

elements of potential concern related to animal cell substrates: DNA, viruses, and transforming 274

proteins. In 1986, WHO established a SG to examine cell substrate issues in greater depth. 275

276

The SG concluded that there is no reason to exclude CCLs from consideration as substrates for 277

the production of biologicals, and that CCLs are in general acceptable when the manufacturing 278

process is shown to eliminate potential contaminating viruses pathogenic for humans and to 279

reduce DNA to acceptable levels and/or to eliminate its biological activity [ 12]. The SG’s 280

emphasis on infectious agents as the major risk factor was based in large part on actual prior 281

experience in which transmission and disease had occurred through contaminated biological 282

products (e.g., hepatitis B and HIV in Factor VIII). WHO Requirements for Continuous Cell 283

Lines used for Biologicals Production were published in 1987 [ 13]. Based on a review of more 284

recent data, those Requirements were revised in 1998 to raise the acceptable level of rcDNA to 285

10 ng per dose. In addition, it was pointed out that beta-propiolactone, a viral inactivating agent, 286

may also destroy the biological activity of DNA. Use of this agent therefore provides an 287

additional level of confidence even when the amount of DNA per dose may be substantial [ 1]. 288

289

During the 1990s, and on into the 2000s, a variety of CCLs were explored as cell substrates for 290

biological products in development because, like the cell lines already mentioned, they offered 291

significant advantages during production (e.g., rapid growth and high expression). These include 292

the following tumourigenic cell lines: HeLa for adeno-associated virus vectored HIV vaccines; 293

Per.C6 for influenza and HIV vaccines; MDCK for influenza vaccines; and 293ORF6 for HIV 294


vaccines. More recently insect cell lines and stem cell lines (SCLs) have been proposed for the 295

manufacture of biological products, and they introduce a new set of challenges for their 296

evaluation and characterisation . 297

298

The acceptability of a given cell type (primary, diploid, stem, or continuous) as a substrate for 299

the production of a specific biological product depends on a variety of factors including an in 300

depth knowledge of its basic biological characteristics. In that regard, it is important to recognize 301

that the tumorigenic potential of a CCL is but one of many factors to consider such as the extent 302

to which the manufacturing process reduces or eliminates cellular factors that may be of concern. 303

An assessment of the totality of the data available is needed in order to determine whether a 304

product manufactured in a given cell substrate is potentially approvable. 305

306

The following recommendations provide guidance to manufacturers and NRAs/NCLs on the 307

evaluation of animal cell cultures used as substrates for the production of biological medicinal 308

products, and for the characterization of cell banks. 309

310

The main changes from the requirements published in TRS 878, annex 1, include: 311

312

1. general manufacturing recommendations applicable to all types of cell culture production 313

have been updated; 314

2. some considerations for the evaluation of new cell substrates such as insect cells and stem 315

cells have been added; 316

3. definitions have been updated and expanded in number and scope, and moved to an 317

earlier point in the document; 318

4. the structure of the document has been modified to include more background 319

information, and the applicability of various sections to different types of cell substrates 320

is highlighted; 321

5. a section on Good Cell Culture Practice has been added; 322

6. tumorigenicity testing has been updated, and a model protocol for the nude mouse model 323

was added as an appendix; 324

7. oncogenicity testing of tumorigenic cell lysates was added; 325


8. recommendations for microbial agents testing have been updated; 326

9. a summary of testing to be performed at different points in the use of cell substrates based 327

on a cell banking system has been included in a tabulated form; and 328

10. a new section on risk reduction strategies during the manufacture of biological products 329

has been added. 330

331

3. Scope 332

These recommendations supersede previous WHO requirements or recommendations describing 333

procedures for the use of animal cell substrates for the production of biological medicinal 334

products [ 1, 13] . 335

336

It is particularly important that these recommendations be read in conjunction with the 337

requirements or recommendations published by WHO for individual products. In that regard, and 338

recognizing that these recommendations apply to the evaluation and characterization of cell 339

substrates, no recommendations are made in this document on limits of residual cellular DNA 340

(rcDNA) in biological products. Acceptable limits for specific products should be set in 341

consultation with the NRA/NCL taking into consideration the characteristics of the cell substrate, 342

the intended use of the biological product, and most importantly the effect of the manufacturing 343

process on both the size and biological activity of rcDNA fragments. In general, it has been 344

possible to reduce rcDNA in biotherapeutic products to <10 ng per dose, and in many cases <10 345

pg per dose, because they can be highly purified. On the other hand, some products, especially 346

certain live viral vaccines, are difficult to purify without a significant loss in potency, so that the 347

amount of rcDNA in those final products may be significantly higher than 10 ng/dose. Such 348

cases are considered to be exceptional, and should be discussed with the NRA/NCL. 349

350

Some of these recommendations also may be useful in the quality control of specific biological 351

products during the manufacturing process, but it is beyond the scope of this document to 352

recommend quality control release tests. Requirements or recommendations for individual 353

products should be consulted in that regard. 354

355


Cells modified by recombinant DNA technology have been increasingly used in the manufacture 356

of novel medicinal products, and specific considerations for those products are addressed 357

elsewhere [ 1, 10, 14, 15]. Nevertheless, there are a number of generic issues that apply to 358

recombinant as well as other cell substrates. 359

360

These recommendations specifically exclude all products manufactured in embryonated eggs, 361

microbial cells (i.e., bacteria and yeast), and plant cells. Also excluded are whole, viable animal 362

cells such as stem cells when they are used directly for therapy by transplantation into patients or 363

when they are developed into stem cell lines (SCLs) for the purpose of using them as therapeutic 364

agents by transplantation. In those cases, characterization tests should be discussed with the 365

NRA/NCL. However, SCLs used for the production of biological products such as growth 366

factors and vaccines should comply with these recommendations. 367

368

Some of the general recommendations given here (see sections A.1 – A.5) are applicable to all 369

animal cell substrates. More specific guidance for PCCs can be found in the relevant documents 370

published by WHO (e.g., production of poliomyelitis vaccine in primary monkey kidney cells) 371

[ 4, 5]. 372

373

Recommendations published by WHO are intended to be scientific and advisory in nature. The 374

parts of each section printed in normal type have been written in the form of recommendations so 375

that, should a NRA/NCL so desire, they may be adopted as they stand as the basis of national or 376

regional requirements. The parts of each section printed in small type are comments or additional 377

points that might be considered in some cases. 378

379

4. Definitions 380

Adventitious agent: Contaminating microorganisms of the cell culture or source materials 381

including bacteria, fungi, mycoplasmas/spiroplasmas, mycobacteria, rickettsia, protozoa, 382

parasites, TSE agents, and viruses that have been unintentionally introduced into the 383

manufacturing process of a biological product. 384

The source of these contaminants may be from the legacy of the cell 385

line, the raw materials used in the culture medium to propagate the cells 386


(in banking, in production, or in their legacy), the environment, 387

personnel, equipment, or elsewhere. 388

Biological medicinal product: Biological medicinal product is a synonym for biological product 389

or biological described in WHO Technical Report Series. The definition of a medicinal 390

substance, used in treatment, prevention or diagnosis, as a "biological" has been variously based 391

on criteria related to its source, its amenability to characterization by physicochemical means 392

alone, the requirement for biological assays, or on arbitrary systems of classification applied by 393

regulatory authorities. For the purposes of WHO, including the present document, the list of 394

substances considered to be biologicals is derived from their earlier definition as "substances 395

which cannot be fully characterized by physicochemical means alone, and which therefore 396

require the use of some form of bioassay". However, developments in the utility and 397

applicability of physicochemical analytical methods, improved control of biological and 398

biotechnology-based production methods, and an increased applicability of chemical synthesis to 399

larger molecules, have made it effectively impossible to base a definition of a biological on any 400

single criterion related to methods of analysis, source or method of production. 401

Developers of such medicinal products that do not fit the definition of 402

biological medicinal product provided in this document should consult 403

the relevant national regulatory authorities for product classification 404

and licensing application pathway. 405

Biotherapeutic: For the purpose of this document, a biotherapeutic is a biological medicinal 406

product with the indication of treating human diseases. For further information, please refer to 407

Guidelines for similar biotherapeutic products (adopted in 2009). 408

Cell bank: A cell bank is a collection of appropriate containers, whose contents are of uniform 409

composition, stored under defined conditions. Each container represents an aliquot of a single 410

pool of cells (also see illustrations in B.12) 411

The individual containers (e.g., ampoules, vials) should be 412

representative of the pool of cells from which they are taken and should 413

be frozen on the same day, using the same equipment and reagents. 414

Cell culture: The process by which cells are grown in vitro under defined and controlled 415

conditions where the cells are no longer organized into tissues. 416

Cell line: Type of cell population with defined characteristics that originates by serial subculture 417

of a primary cell population, which can be banked. 418


Cloning and sub-coning steps may be used to generate a cell line. The 419

term cell line implies that cultures from it consist of lineages of some of 420

the cells originally present in the primary culture. 421

Cell seed: A quantity of well-characterized cells of human, animal or other origin stored frozen 422

under defined conditions, such as in the vapour or liquid phase of liquid nitrogen in aliquots of 423

uniform composition derived from a single tissue or cell, one or more of which would be used 424

for the production of a master cell bank. 425

Cell substrate: Cells used to manufacture a biological product. 426

The cells may be primary or cell lines, and may be grown in monolayer 427

or suspension culture conditions. Examples of cell substrates include 428

primary monkey kidney, MRC-5, CHO, and Vero. 429

430

Cells used to generate essential components of a final product, such as 431

Vero cells for the generation of reverse genetics virus for use in vaccine 432

production, are considered to be “pre-production” cell substrates. 433

Whereas cells used to manufacture the bulk product (e.g., packaging 434

cell lines for gene therapy vectors; Vero cells for vaccine production; 435

CHO cells for recombinant protein expression) are considered to be 436

“production” cell substrates. 437

Continuous cell line: A cell line having an apparently unlimited capacity for population 438

doublings. Often referred to as "immortal" and previously referred to as "established". 439

Diploid cell line: A cell line having a finite in vitro lifespan in which the chromosomes are 440

paired (euploid) and are structurally identical to those of the species from which they were 441

derived. 442

While this definition is accurate for standard chromosome preparations, 443

a given human diploid cell line may contain genetic variations that will 444

be reflected in a Giemsa-banding pattern that differs from the standard. 445

Gene expression differences also may be found. 446

This definition is based on experience and current understanding of the 447

in vitro behavior of human cells that are not of stem cell origin. 448

449

DNA infectivity: The capacity for exogenous DNA molecules to become integrated into cellular 450

DNA and, as a consequence, may promote the development of proliferating clones of cells 451

containing the exogenous DNA. 452


Endogenous virus: A virus whose genome is present in an integrated form in a cell substrate and 453

that has entered the cell during evolution. Endogenous viral sequences may or may not encode 454

for an intact or infectious virus. 455

End of production cells (EOPC): Cells harvested at or beyond the end of a production (EOP) 456

run, or cells cultured from the MCB or WCB propagated to the proposed in vitro cell age used 457

for production or beyond. 458

In some cases, production cells are expanded under pilot-plant 459

scale or commercial-scale conditions. 460

Functional integrity: The culture sustains the expected performance related to its intended use, 461

under specified conditions e.g. expression of secreted product at a consistent level, production of 462

expected MOI of virus. 463

Indicator cells: Cells of various species used in the in vitro adventitious agent test that are 464

intended to serve as a biological amplification platform to promote the detection of viruses that 465

may be present in cell banks adventitiously. For example, generally, this would include a human 466

diploid cell line, such as MRC-5, a monkey kidney cell line, such as Vero cells, and a cell line of 467

the same species and tissue as the cell bank. The purpose of these cell lines is to indicate a viral 468

infection of the cell bank either through observation of cytopathic effect during and after an 469

appropriate observation period or by hemadsorption and/or hemagglutination at the end of the 470

observation period. Thus, they are referred to as indicator cells. The cell bank may be analyzed 471

on such indicator cells either by co-cultivation or by passage of cell lysates or spent culture 472

supernatants from the cell bank onto the indicator cells. 473

In vitro cell age: Measure of time between thaw of the MCB vial(s) to harvest of the production 474

vessel measured by elapsed chronological time, by population doubling level of the cells, or by 475

passage level of the cells when subcultivated by a defined procedure for dilution of the culture. 476

Latent virus: A virus is considered to be microbiologically latent when the viral genome is 477

present in the cell without evidence of active replication, but has the potential to be reactivated. 478

Master cell bank (MCB): A quantity of well-characterized cells of animal or other origin, 479

derived from a cell seed at a specific PDL or passage level, dispensed into multiple containers, 480

cryopreserved, and stored frozen under defined conditions, such as the vapour or liquid phase of 481

liquid nitrogen in aliquots of uniform composition. The master cell bank is prepared from a 482

single homogeneously mixed pool of cells. In some cases, such as recombinant cells, this may be 483


prepared from a selected cell clone established under defined conditions. It is considered best 484

practice that the master cell bank is used to derive working cell banks (also see illustration in 485

B.12) 486

Oncogenicity: The capacity of an acellular agent such as a chemical, virus, viral nucleic acid, 487

viral gene(s), or a subcellular element(s) to immortalize normal cells and endow them with the 488

ability to form tumours in an animal model. 489

Oncogenicity is distinct from tumorigenicity (see 490

Tumorigencity). 491

Parental cells: Cells that are manipulated to give rise to a cell substrate. For hybridomas, it is 492

typical to also describe the parental cells as the cells to be fused. 493

Manipulation may be as simple as the expansion of a primary cell 494

culture to provide early passage cells, or a more complex activity such 495

as developing a hybridoma or transfected clone, and both processes 496

would provide a cell seed. The parental cells may refer to any stage 497

prior to the preparation of the cell seed. Examples of a parental cell are: 498

WI-38 and MRC-5 at very early passage; Vero at passage 121; and 499

CHO before the introduction of a DNA construct to produce a 500

recombinant cell. In certain situations (e.g. myeloma cells), there may 501

be a lineage of identified stable parental clones, thus, the term "parental 502

cell" would normally refer to the cells used immediately prior to 503

generation of the "seed stock". 504

Passage: Transfer of cells, with or without dilution, from one culture vessel to another in order 505

to propagate them, and which is repeated to provide sufficient cells for the production process. 506

This term is synonymous with “subculture”. Cultures of the same cell 507

line with the same number of passages in different laboratories are not 508

necessarily equivalent because of differences in cell culture media, split 509

ratios, and other variables that may affect the cells. This is a more 510

important consideration for SCLs and CCLs than for DCLs. Population 511

doubling is the preferred method of estimating cell line age, and 512

whenever possible, it should be used instead of “passage”. However, it 513

also may be appropriate to quantitate culture duration of CCLs by the 514

number of subcultivations at a defined seeding density at each passage 515

or time in days. 516

Population doubling: A collection of cells that has doubled in number. 517


Population doubling level (PDL): The total number of population doublings of a cell line or 518

strain since its initiation in vitro. A formula to use for the calculation of "population doublings" 519

in a single passage is: number of population doublings = Log (N/No ) X 3.33 where: N = number 520

of cells in the growth vessel at the end of a period of growth. No = number of cells plated in the 521

growth vessel ( 16). It is best to use the number of viable cells or number of attached cells for this 522

determination. 523

Primary culture: A culture started from cells, tissues or organs taken directly from one or more 524

organisms. A primary culture may be regarded as such until it is successfully subcultured for the 525

first time. It then becomes a "cell line" if it can continue to be subcultured at least several times. 526

Production cell cultures: A collection of cell cultures used for biological production that have 527

been prepared together from one or more containers from the working cell bank or, in the case of 528

primary cell cultures, from the tissues of one or more animals. 529

Specific pathogen free (SPF): Animals known to be free of specific pathogenic microorganisms 530

and reared in an environment that maintains that state. SPF animals usually are raised in 531

biosecure facilities and their health status is monitored on an ongoing basis. The SPF status 532

simply provides an assurance that the stock is not infected with the specified pathogens. SPF 533

animals are not disease free nor are they disease resistant. They may carry pathogens other than 534

those from which they are specified to be free. 535

Transmissible Spongioform Encephalopathy (TSE): The transmissible spongiform 536

encephalopathies (TSEs) are a group of fatal neurodegenerative diseases which include classical 537

and variant Creutzfeldt–Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), 538

fatal insomnia, and Kuru in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic 539

wasting disease (CWD) in mule deer and elk, and scrapie in sheep and goats. 540

Tumorigenicity: The capacity of a cell population inoculated into an animal model to produce a 541

tumor by proliferation at the site of inoculation and/or at a distant site by metastasis. 542

Tumorigenicity is distinct from Oncogenicity (see 543

Oncogenicity). 544

WHO Reference cell bank: A cryopreserved stock of cells prepared from a single, homogeneous 545

pool of cells prepared under defined conditions and subjected to characterization tests. The 546

purpose of such a bank is to serve as a well-characterized cell seed for the preparation of master 547


cell banks that will be extensively characterized by manufacturers, and that has a high 548

probability of meeting these recommendations. 549

Working cell bank (WCB): A quantity of well-characterized cells of animal or other origin, 550

derived from the master cell bank at a specific PDL or passage level, dispensed into multiple 551

containers, cryopreserved, and stored frozen under defined conditions, such as in the vapour or 552

liquid phase of liquid nitrogen in aliquots of uniform composition. The working cell bank is 553

prepared from a single homogeneously mixed pool of cells. One or more of the WCB containers 554

is used for each production culture. 555

556

5. General considerations 557

Types of animal cell substrates 558

Primary Cell Cultures (PCCs) 559

PCCs have played a prominent role in the development of biology as a science, and of virology 560

in particular. Cultures of PCCs from different sources have been in worldwide use for the 561

production of live and inactivated viral vaccines for human use for many decades. For example, 562

PCCs of monkey kidney cells have been used for the production of inactivated and oral 563

poliomyelitis vaccines since the 1950s. 564

565

Major successes in the control of viral diseases, such as poliomyelitis, measles, mumps and 566

rubella, were made possible through the wide use of vaccines prepared in PCCs, including those 567

from chicken embryos and the kidneys of monkeys, dogs, rabbits and hamsters, as well as other 568

tissues. 569

570

PCCs are viable cells of disaggregated tissues that are initiated as in vitro cell cultures usually as 571

adherent cells. Many cell types will be present and a primary culture may be a complex mixture 572

of cells that can be influenced by the process and conditions under which they were harvested, 573

disaggregated and introduced to in vitro culture. Not all cells in a primary culture will have the 574

capacity to replicate. Particular care should be applied to establishing highly reproducible 575

procedures for tissue disaggregation, cell processing and culture initiation and reproducible 576

culture conditions and nutrition. 577

578


PCCs obtained from wild animals usually show a high frequency of viral contamination. For 579

example, monkey-kidney cell cultures can be contaminated with one or more adventitious 580

agents, including simian viruses. 581

582

If PCCs are necessary for the production of a given biological, then the frequency of 583

contaminated cell cultures can be significantly reduced by careful screening of the source 584

animals for the absence of such viruses. Viruses can be detected by in vitro tests such as PCR, 585

and by looking for the presence of circulating antibodies to those viruses in the source animals. 586

The use of animals bred in a carefully controlled colony, especially those that are specific-587

pathogen-free, is strongly recommended. Nevertheless, as suitable alternative cell substrates 588

become available, PCCs are less likely to be used in the future. WHO has promoted the 589

replacement of animals for experimental purposes both from an ethical perspective [ 17] and in 590

the interests of progressive improvement in product safety and quality. 591

592

Advantages: (a) they are comparatively easy to prepare using simple media and bovine serum; 593

and (b) they generally possess a broad sensitivity to various viruses, some of which are 594

cytopathic. 595

596

Disadvantages: (a) contamination by infectious agents is a higher risk than with DCLs and 597

CCLs; (b) the quality and viral sensitivity of cultures obtained from different animals are 598

variable; and (c) although cell cultures derived from nonhuman primates had been in wide use in 599

the past, it has become increasingly difficult to obtain and justify the use of such animals for this 600

purpose, (d) they cannot be tested as extensively as DCLs or CCLs. 601

602

Diploid Cell Lines (DCLs) 603

The practicality of using human DCLs for the production of viral vaccines was demonstrated in 604

the 1960s. The experience gained with oral poliomyelitis and other viral vaccines in successfully 605

immunizing billions of children in many countries has shown clearly that such substrates can be 606

used in the production of safe and effective vaccines [ 3, 18]. 607

608


The essential features of DCLs of human (e.g. WI-38, MRC-5) and monkey (i.e. FRhL-2) origin 609

are: (a) they are cells passaged from primary cultures that have become established as cell lines 610

with apparently stable characteristics over numerous PDLs; (b) they have a finite capacity for 611

serial propagation, which ends in senescence, a state in which the culture ceases to replicate, the 612

cells remain alive and metabolically active, but may show morphological and biochemical 613

changes some of which begin to appear before replication ceases; (c) they are nontumorigenic; 614

and (d) they display diploid cytogenetic characteristics with a low frequency of chromosomal 615

abnormalities of number and structure until they enter senescence. Substantial experience since 616

the 1960s has been accumulated on the cytogenetics of WI-38 and MRC-5, and ranges of 617

expected frequencies of chromosomal abnormalities have been published [ 19, 20]. Similar data 618

are available for FRhL-2 [ 21]. More sophisticated cytogenetic techniques (i.e., high resolution 619

banding) [ 22, 23] have demonstrated the presence of subtle chromosomal abnormalities that were 620

previously undetectable. Recent studies have shown that subpopulations of human DCLs with 621

such abnormalities may appear and disappear over time, and that they are non-tumorigenic and 622

undergo senescence in the same manner as the dominant population. Thus, possessing a stable 623

karyotype might not be such an important characteristic as was previously thought. 624

625

Advantages: (a) they can be well characterized and standardized; (b) production can be based on 626

a cryopreserved cell-bank system that allows for consistency and reproducibility of the 627

reconstituted cell populations. A cell-bank system usually consists of cell-banks of defined 628

population doubling or passage levels that generally include a MCB and a working cell-bank 629

(WCB); and (d) unlike the CCLs and SCLs discussed below, DCLs are not tumorigenic and 630

therefore do not raise the potential safety issues associated CCLs and SCLs. 631

632

Disadvantages: (a) they are not easy to use in large-scale production, but have been cultivated 633

using bioreactor technology employing the microcarrier or multilayer method; (b) in general, 634

they need a more demanding growth medium than other cell substrates; (c) they usually need 635

larger quantities of bovine serum (either fetal or donor calf) for their growth than do PCCs or 636

CCLs; (d) they are difficult to adapt to serum-free growth; (e) they are more difficult than CCLs 637

to transfect and engineer; and (f) they are not permissive for the production of vaccine vectors 638


that require complementation, since they cannot be engineered readily to express complementing 639

proteins without converting the normal cells to tumorigenic cells. 640

641

Continuous cell lines (CCLs) 642

Some CCLs have been used for the production of safe and effective biotherapeutics and vaccines 643

since the 1980s. 644

645

CCLs have the potential for an apparently indefinite in vitro life span, and have been derived by 646

the following methods: (a) serial subcultivation of a PCC of a human or animal tumour (e.g., 647

HeLa cells); (b) transformation of a normal cell having a finite life span with an oncogenic virus 648

or viral sequence (e.g., B lymphocytes transformed by EBV or with viral sequences such as in 649

PER.C6); (c) serial subcultivation of a primary-cell population derived from normal tissue that 650

generates a dominant cell population having an apparently indefinite life span, often described as 651

spontaneous transformation (e.g., Vero, BHK-21, CHO, MDCK, Hi5); (d) fusion between a 652

myeloma cell and an antibody-producing B lymphocyte to produce a hybridoma cell line; or (e) 653

use of ectopically expressed genes involved in the cell cycle such as hTERT telemorase gene to 654

enable indefinite replication of normal human cells. 655

656

CCLs may show chromosome complements with a consistent modal chromosome number (e.g. 657

MDCK, Vero), and although the karyotype of individual cells in a culture at any one time-point 658

may vary, the range of chromosomes per cell will usually show characteristic limits. However, 659

other CCLs, such as highly tumorigenic cells including HeLa may show variation in modal 660

number and a wider drift in the range of the number of chromosomes per cell. 661

662

In the early stage of establishing a cell line, significant diverse karyotypes and changes in 663

karyotype may be observed; but a characteristic typical chromosome component may emerge 664

with continued passage presumably as a clone develops as the dominant cell type. 665

666

Advantages: (a) they can be characterized extensively and standardized; (b) production can be 667

based on a cell-bank system that allows consistency and reproducibility of the reconstituted cell 668

populations for an indefinite period; (c) as a rule, they grow more easily than DCLs using 669


standard media, (d) most can be adapted to grow in serum-free medium; (e) they usually can be 670

grown on microcarriers for large-scale production in bioreactors; and (f) some can be adapted to 671

grow in suspension cultures for large-scale production in bioreactors. 672

673

Disadvantages: (a) CCLs may contain and express endogenous viruses, and some are 674

tumorigenic in immunosuppressed animal models; (b) the potential risk of oncogenicity and 675

infectivity associated with rcDNA that may encode a variety of bioactive elements that might 676

result in neoplasia or infection in the recipient of the product manufactured in such cells; (c) the 677

potential risk of RNA species such as micro RNA (miRNA) that may be able to modulate gene 678

expression; and (d) the potential risk of transforming proteins that may cause normal cells to 679

assume tumor-cell characteristics. The practical significance of these potential risks has not been 680

established. 681

682

Stem Cell Lines (SCLs) 683

SCs differ from other types of cells because they have the capacity to sustain a predominant stem 684

cell population whilst simultaneously producing cell progenitors of differentiated cell types of 685

almost all human tissues (i.e., pluripotent). This property of pluripotency is sustained through 686

numerous cycles of cell division. SCLs may be derived from early stage embryonic, fetal or adult 687

tissues. Typically, specialized media and environmental conditions such as the attachment matrix 688

are required for the growth of SCLs in vitro in order for them to maintain the undifferentiated 689

state. While most SC research and development has been directed towards transplantation of SCs 690

for therapeutic purposes, efforts also have gone into exploring a variety of SCLs as cell 691

substrates for the production of biologicals. In contrast to SCLs that have the capacity to sustain 692

a dominant population of stem cells, DCLs and CCLs also may contain stem cells, but they are 693

not the dominant population and generally do not give rise to differentiated cell types. 694

695

Key considerations for the culture and control of such cell lines have been developed [ 24]. These 696

include the fundamental issues common to the maintenance of all cell lines, but also emphasize 697

the need for appropriate ethical governance regarding donor consent and careful attention to 698

periodic confirmation of phenotype, absence of non-diploid cells and sustained pluripotent 699

capacity. 700


701

Recently it has been shown that conditioned medium from SCLs can have regenerative 702

properties, and such preparations produced from human embryonic SCs have shown regenerative 703

capabilities including repair of myocardial infarction in animal models [ 25]. This raises the 704

possibility of SCs being used as a substrate to produce a variety of biologically active molecules. 705

SCLs can, in some respects, be considered as diploid cells, but they do not appear to have the 706

finite life span characteristic of human diploid fibroblast cultures. In human embryonic SC 707

cultures, clonal variants with chromosomal abnormalities are known to arise. Whilst a diploid 708

and non-transformed nature is to be considered a pre-requisite for cell therapy applications, 709

transformed SCLs might be considered as a form of CCL for the manufacture of biologicals. 710

Since they do not fall easily within any one category of substrate already discussed; SCLs are 711

identified separately in this document. 712

713

Advantages 714

(a) they can be well characterized and standardized; (b) production can be based on a cell-bank 715

system that allows consistency and reproducibility of the reconstituted cell populations for an 716

indefinite period; and (c) some may be adapted to grow in suspension cultures for large-scale 717

production in bioreactors; (d) they may produce unique proteins of potential importance as 718

biotherapeutics; and (e) they have the potential to generate cells and tissue-like structures that 719

may permit the expression of agents currently considered "unculturable" in vitro. 720

721

Disadvantages 722

(a) Subculture techniques commonly used for SCLs are laborious; (b) may produce growth 723

proteins with undefined effects on adult cells/tissues; (c) usually require complex media that may 724

have a TSE risk; (d) rapid development of differentiated cells also means that they are difficult 725

to control in vitro; (e) there is little experience with their use 726

as a cell substrate to manufacture biological products. 727

728

Potential risks and risk mitigation associated with biologicals produced in animal cell 729

cultures 730


The main potential risks associated with the use of biologicals produced in animal cells are 731

directly related to contaminants from the cells, and they fall into three categories: (a) viruses and 732

other transmissible agents; (b) cellular nucleic acids (DNA and RNA); and (c) growth-promoting 733

proteins. In addition, cell-derived inhibiting or toxic substances are theoretically possible. A 734

summary of the risk assessment for each follows. More comprehensive information has been 735

published elsewhere on the risks associated with contaminating DNA and growth-promoting 736

proteins [ 26, 27, 28, 29, 30, 31, 32, 33, 34]. 737

738

Viruses and other transmissible agents 739

There is a long history of concern regarding the potential transmission of viruses and other 740

agents that may be present in cell substrates. This area was reviewed most recently by WHO in 741

1986 by a SG who point out that, as described below, cells differ with respect to their potential 742

for carrying viral agents pathogenic for humans. 743

744

Primary monkey kidney cells have been used to produce billions of doses of poliomyelitis 745

vaccines since they were first developed in the 1950s, and although viruses such as SV40 were 746

discovered in such cells, control measures were introduced to eliminate or reduce as much as 747

possible the risk of viral contamination associated with the manufacture of vaccines in cells 748

containing those viruses. Additional controls may be needed as new viral agents are identified 749

and technologies to detect them are developed. 750

751

Human and nonhuman primate lymphocytes and macrophages may carry latent viruses, such as 752

herpesvirus and retroviruses. CCLs of non-haematogenous cells from human and nonhuman 753

primates may contain viruses or have viral genes integrated into their DNA. In either case, virus 754

expression may occur under in vitro culture conditions. 755

756

Avian tissues and cells may harbour exogenous and endogenous retroviruses, but there is no 757

evidence for transmission of disease to humans from products prepared using these substrates. 758

For example, large quantities of yellow fever vaccines were produced for many years in eggs that 759

contain avian leukosis viruses, but there is no evidence that these products have transmitted 760

disease in their long history of use for human immunization. Nevertheless, the potential for 761


transmitting avian retroviruses should be reduced as much as possible through manufacturing 762

control measures. 763

764

Rodents may harbour exogenous and endogenous retroviruses, lymphocytic choriomeningitis 765

virus, and Hantaviruses. The latter two are exogenous viruses that have caused disease in humans 766

by direct infection from rodents. While contamination with the rodent viruses in the cell harvests 767

of biotherapeutic products derived from CHO cell culture has been reported [ 35, 36, 37] there is 768

no evidence that biological products released for distribution have been contaminated with 769

rodent viruses because they were detected during quality control testing in compliance with 770

GMPs. In addition, it is important to note that there have been no reported cases of transmission 771

of an infectious agent to recipients of recombinant protein products manufactured in animal cells. 772

773

Insect cells recently have been used for vaccine production, and various insect cell lines may be 774

used for the production of biologicals in the future. Insect viruses tend to be ubiquitous in many 775

insect cell lines, and are generally unknown and/or uncharacterized. Many insect cell lines have 776

endogenous transposons and retrovirus-like particles, and some are positive in PERT assays. 777

778

HDCs have been used for vaccine production for many years, and although concern was initially 779

expressed about the possibility of such cells containing a latent pathogenic human virus, no 780

evidence for such an endogenous agent has been reported, and vaccines produced from this class 781

of cell have proved to be free from viral contaminants. 782

783

In light of the differing potential of the various types of cells mentioned above for transmitting 784

viruses that are pathogenic in humans, it is essential that the cells being used to produce 785

biological products be evaluated as well as possible with respect to infectious agents. 786

787

When DCLs, SCLs, or CCLs are used for production, a cell bank system should be used and the 788

cell banks should be characterized as specified in this document. Efforts to identify viruses by 789

testing for viral sequences or other viral markers, especially those not detectable by other means, 790

constitute an important part of the evaluation of cell banks in addition to the standard tests that 791

have been in place for many years. 792


793

When cell lines of rodent or avian origin are examined for the presence of viruses, the major 794

emphasis in risk assessment should be placed on the results of studies in which transmission to 795

target cells or animals is attempted. Risk to human recipients should not be assessed solely on 796

ultrastructural or biochemical/biophysical evidence of the presence of viral or viral-like agents in 797

the cells. 798

799

The overall manufacturing process, including the selection and testing of cells and source 800

materials, any purification procedures used, and tests on intermediate or final products, should 801

ensure the absence of detectable infectious agents in the final product. When appropriate, 802

validation of purification procedures should demonstrate adequate reduction of relevant model 803

viruses with a significant safety factor [ 14]. This is usually required for recombinant protein 804

products. 805

806

There may be as yet undiscovered microbial agents for which there is no current evidence or 807

means of detection. As such agents become identified, it will be important to consider whether to 808

re-examine cell banks for their presence. In general, it is not a practice consistent with GMPs to 809

re-test materials that have already been released, so justification would be necessary before such 810

re-testing is undertaken. Some agents, such as human endogenous retroviruses (HERVs) ( 38) and 811

transfusion-transmitted virus (TTV) ( 39), appear to have no pathological consequences. Positive 812

findings should be discussed with the NRA/NCL. In addition, new testing methods are likely to 813

be developed, and as they become available and validated, they should be considered by 814

manufacturers and NRAs/NCLs for their applicability to the characterization and control of 815

animal cell substrates. 816

817

Cellular DNA 818

PCCs and DCLs have been used successfully for many years for the production of viral vaccines, 819

and the rcDNA deriving from these cells has not been (and is not) considered to pose any 820

significant risk. However, with the use of CCLs that have an apparently indefinite life span, 821

presumably due to the dysregulation of genes that control growth, and with the ongoing 822

development of products from cells that are tumorigenic or were derived from tumors, the DNA 823


from such cells has been considered to have the theoretical potential to confer the capacity for 824

unregulated cell growth, and perhaps tumorigenic activity, upon some cells of a recipient of the 825

biological product. Although the risk of such DNA has been estimated based on certain 826

assumptions and some experimental data, assessing the actual risk of such DNA has not been 827

possible until recently when preliminary data generated from new experimental systems began to 828

quantify those risks [ 40]. 829

830

The potential risks of DNA arise from both of its biological activities: (a) infectivity, and (b) 831

oncogenicity. Infectivity could be due to the presence of an infectious viral genome in the 832

cellular DNA of the cell substrate [ 41, 42]. The viral genome could be that of a DNA virus, 833

whether integrated or extrachromosomal, or that of a proviral genome of a retrovirus. Both types 834

of viral DNA have been shown to be infectious in vitro and in several cases, in vivo [ 40, 41, 42]. 835

The oncogenic activity of DNA could arise through its capacity to induce a normal cell to 836

become transformed and perhaps to become tumorigenic. The major mechanism through which 837

this could occur would be through the introduction of an active dominant oncogene (e.g., myc, 838

activated ras), since such dominant oncogenes could directly transform a normal cell. Other 839

mechanisms require that the rcDNA transform through insertional mutagenesis, and have been 840

considered less likely, since the frequency of integration of DNA in general is low [ 43]. The 841

frequency of integration at an appropriate site, such as inactivating a tumor suppressor gene or 842

activating a proto-oncogene, would be correspondingly lower [ 32]. 843

844

The 1986 WHO SG addressed the risk posed by the oncogenic activity of rcDNA in biological 845

products for human use [ 12]. Risk assessment based on a viral oncogene in an animal model 846

suggested that in vivo exposure to one nanogram (ng) of rcDNA, where 100 copies of an 847

activated oncogene were present in the genome, would give rise to a transformational event once 848

in 109 recipients [ 27]. On the basis of this and other evidence available at that time, the 1986 SG 849

concluded that the risk associated with rcDNA in a product is negligible when the amount of 850

such DNA is 100 picograms (pg) or less per parenteral dose. Based on a review of more recent 851

data, those requirements were revised in 1998 to raise the acceptable level of rcDNA to 10 ng per 852

dose. 853

854


Studies in mice using cloned cellular oncogenes also suggest that the risk of neoplastic 855

transformation by cellular DNA is probably very low [ 34]. However, more recent data have 856

shown that cloned cellular oncogene DNA can induce tumors in selected strains of mice at levels 857

below 1 ng [ 40]. In addition, single oncogenes can also be biologically active [ 44] and initiate 858

the tumour induction process. Because of these data, and the recent description that genes 859

encoding for certain micro-RNA species can be oncogenic in vitro [ 45, 46, 47, 48], thus increasing 860

the number of potential dominant cellular oncogenes, the oncogenic risk of DNA needs to be 861

considered when tumorigenic cells are considered for use in the production of biologicals. This 862

would be especially important for live, attenuated viral vaccines where chemical inactivation of 863

the DNA is not possible, and the only way the biological activity of DNA could be reduced 864

would be by nuclease digestion and the reduction in the quantity of DNA. 865

866

In addition to its oncogenic activity, the infectivity of DNA should be considered. Since a viral 867

genome once introduced could amplify and produce many infectious particles, the infectivity risk 868

is likely greater than the oncogenic risk. The polyoma virus genome is infectious in mice at 869

about 50 pg [ 49], and a recent report demonstrated that 1 pg of a proviral copy of a retrovirus is 870

infectious in vitro [ 50]. Because such low levels of DNA may be biologically active, the amounts 871

of rcDNA should be factored into safety evaluations when tumorigenic cell substrates are used, 872

especially for live viral vaccines. 873

874

Therefore, considerations that need to be taken into account with respect to rcDNA are: (a) any 875

reduction in the amount of the contaminating DNA during the manufacturing process; (b) any 876

size reduction of the contaminating DNA during the manufacturing process; and (c) any 877

chemical inactivation of the biological activity of contaminating DNA during the manufacturing 878

process. A product might be considered by a NRA/NCL to have an acceptable level of risk 879

associated with the DNA of the cell substrate on the basis of (a) and/or (b) and/or (c), when data 880

demonstrate that appropriate levels have been achieved. For example, data have shown that 881

nuclease digestion of DNA or chemical inactivation of DNA with beta-propiolactone, a viral 882

inactivating agent, can destroy the biological activity of DNA [ 50, 51]. Therefore, the use of these 883

procedures may provide an additional level of confidence with respect to DNA risk reduction. 884

885


For products such as monoclonal antibodies manufactured in tumorigenic cell substrates, it is 886

necessary to validate the clearance (removal and/or inactivation) of DNA by the manufacturing 887

process (e.g., through spiking studies). Data should be obtained on the effects of DNA-888

inactivating agents under specific manufacturing conditions so that firm conclusions on their 889

DNA-inactivating potential for a given product can be drawn. 890

891

There may be instances where CCL DNA is considered to pose a higher level of risk because it 892

contains specific elements such as infectious retroviral proviral sequences. Under these 893

circumstances, the steps taken to reduce the risks of rcDNA, such as reducing the size of DNA 894

fragments, should be set in consultation with the NRA/NCL. 895

896

The 1986 WHO SG stated that the risks for rcDNA should be considered negligible for 897

preparations given orally. This conclusion was based on the finding that polyoma virus DNA 898

was not infectious when administered orally [ 49]. For such products, the principal requirement is 899

the elimination of potentially contaminating viruses. Recently, additional data on the uptake of 900

DNA via the oral route have been published [ 52]. These studies demonstrated that the efficiency 901

of uptake of DNA introduced orally was significantly lower than that introduced intra-902

muscularly. Nevertheless, the specifics of the manufacturing process and the formulation of a 903

given product should be considered by the NRA/NCL. 904

905

With respect to the efficiency of DNA uptake via the nasal route, no data have been published 906

comparing the nasal route with parenteral routes. However, data presented publically show that 907

uptake via the intranasal route is less efficient than by the intramuscular route [ 53]. Limits for a 908

specific product should be set in consultation with the NRA/NCL. 909

910

Cellular RNA 911

While protein-coding RNA has not been considered to be a risk factor for biological products 912

due to the unstable nature of RNA and the lack of mechanisms for self-replication the recent 913

description of small non-coding RNA molecules – microRNA (miRNA) – that are more stable 914

and have the capacity to modulate gene expression might necessitate a reassessment. Whether 915

these miRNA molecules are stable in vivo and can be taken up by cells in vivo is unknown. 916


However, as stated above, because certain miRNA genes can be oncogenic [ 45, 46, 47, 48], DNA 917

containing such sequences may need to be considered along with oncogenes when assessing the 918

risk of rcDNA (see B.9 Oncogenicity). However, because this is an evolving area of research, no 919

conclusions can be made regarding the risk of miRNA, and no recommendations are made to 920

control miRNA. 921

922

Growth-promoting proteins 923

Growth factors may be secreted by cells used to produce biologicals, but the risks from these 924

substances are limited, since their growth promoting effects are usually transient and reversible, 925

they do not replicate, and many of them are rapidly inactivated in vivo. In exceptional 926

circumstances, growth factors can contribute to oncogenesis, but even in these cases, the tumours 927

apparently remain dependent upon continued administration of the growth factor. Therefore, the 928

presence of known growth-factor contaminants at ordinary concentrations does not constitute a 929

serious risk in the preparation of biological products manufactured in animal cell cultures. 930

However, some SCLs may secrete higher levels and more potent factors than CCLs. This should 931

be taken into account when designing characterization studies, and the manufacturing process 932

should be designed to address any safety issues that are identified. 933

934


Part A. General recommendations applicable to all types of 935

cell culture production 936

937

A.1 Good manufacturing practices 938

939

The general principles of Good Manufacturing Practices for biologicals should be in place. 940

Requirements or recommendations of the appropriate NRA should be consulted. 941

942

In the preparation of a cell substrate it is considered best practice to establish tiered master and 943

working cell banks (A.5.3) to ensure a reliable and consistent supply of cells which can be fully 944

characterised and safety tested prior to use for production. By definition primary cell cultures 945

cannot be subjected to such banking regimes. However, some manufacturers have utilised pooled 946

and cryopreserved primary cultures which enable completion of lot release testing as in a tiered 947

banking system. The strategy for delivery of primary cells or primary cells recovered from 948

cryopreservation, should be based on the quality and safety that can be assured for the final 949

product according to the overall manufacturing and control processes involved. 950

951

A.2 Principles of Good Cell Culture Practices 952

A.2.1 Understanding the cells and the culture system 953

In all aspects of sourcing, banking and preparing cell cultures, the principles of Good Cell 954

Culture Practices (GCCPs) should be observed (for example, 54). Fundamental features to be 955

considered in the development of cell cultures for production or testing are: 956

1. authenticity including identity, provenance, and genotypic/phenotypic characteristics, 957

2. absence of contamination with another cell line 958

3. absence of microbiological contamination, and 959

4. stability and functional integrity on extended in vitro passage 960

961

An important basic principle for all types of cells is that the donor should be free of transmissible 962

diseases or diseases of uncertain etiology, such as CJD for humans and BSE for cattle. The 963


NRA/NCL may allow specific exceptions concerning donor health (e.g. myeloma, other tumour 964

cells). 965

966

Cells in culture may change their characteristics in response to changes in culture conditions or 967

on extended passage under the same culture conditions. The four cell culture types (PCC, DCL, 968

SCL, CCL) used in manufacture differ in their potential stability and characterisation approaches 969

may need to be adapted in reflection of these differences. 970

971

Cell cultures grow in an in vitro environment that is substantially different from the conditions 972

experienced by cells in vivo and it is not unexpected that they may be susceptible to change or 973

alteration as a result of in vitro culture and processing. It is important to be conscious of the 974

variation that may arise in the cell culture environment as cells may undergo subtle alterations in 975

their cell biology in response to such changes. It is therefore necessary to try to control key 976

known variables that could have significant impact on the cell culture. Medium and specific 977

additives (serum, growth factors, amino acids and other growth promoting compounds) should, 978

where possible, be specified in terms of chemical composition and purity. Where relevant, 979

biological activity should be determined before use. New batches of reagents for cell culture 980

should be supplied with certificates of analysis and origin which enable their suitability to be 981

evaluated against the established specification. The use of serum or other poorly defined reagents 982

is not recommended in the production of new biologicals from cell culture, and wherever 983

possible chemically defined alternatives should be sought. However, given that our current 984

understanding of cell biology is not complete, there is a balance to be made between the benefits 985

that defined media brings in the form of higher reproducibility and reduced risk, and the potential 986

effects of inadequacies of defined culture systems that may not meet the full biological needs of 987

cells. Where complex biological reagents such as FBS remain necessary, they should be 988

carefully controlled, whenever possible, by pre-use selection of batches. Such careful selection 989

also should apply to cell culture surfaces using specified culture vessels or surface coatings 990

where relevant. 991

992

Variation in physical culture parameters such as pH, temperature, humidity, and gas 993

composition can significantly influence the performance and viability of cells and should be 994


specified with established tolerances and the relevant equipment calibrated and monitored. In 995

addition any culture reagents prepared in the laboratory should be documented, quality 996

controlled and released against an established specification. 997

998

A.2.2 Manipulation of cell cultures 999

In vitro processing of cells can introduce additional physical and biochemical stresses that could 1000

have an influence on the quality of the final product. Care should be taken to minimise 1001

manipulations, taking into consideration the specifics of the manufacturing process. In all cases, 1002

a consistent process should be demonstrated. 1003

1004

A.2.2.1 Detachment and subculture 1005

Detachment solutions may adversely affect the cells if exposure is not minimized. Cell 1006

harvesting and passaging procedures should be carried out in a reproducible way ensuring 1007

consistency in the confluency of cells when harvested, incubation times, temperature, 1008

centrifugation speeds and times, and post-passage viable cell seeding densities. 1009

1010

A.2.2.2 Cryopreservation (see A.5.1) 1011

1012

A.2.2.3 Introduction of contamination 1013

As already mentioned, the microbiological status of the donor individual, colony, herd, or flock 1014

is an important consideration in the establishment of PCCs. In order to avoid catastrophic failure 1015

of the production process and to avoid infectious hazards for recipients of products, it is 1016

important to minimise the opportunities for contamination of cell cultures. Therefore, cell 1017

manipulation and open processing steps should be minimised, taking into consideration the 1018

specifics of the manufacturing process. It is critical to adopt rigorous aseptic technique and 1019

provide appropriate environmental controls and air quality for cell culture processing and 1020

preparation of growth media. The presence of any antimicrobial in a biological process or 1021

product is discouraged, although a notable exception is that antibiotic(s) and antifungal(s) may 1022

be required for the first passages of primary cell cultures. Additionally, antibiotics may be used 1023

in some cell line selection systems. Where antibiotics have been used, sterility testing procedures 1024


should account for potential inhibitory effects of the antibiotic on contaminating organisms. 1025

Penicillin or other beta-lactam antibiotics should not be present in production cell cultures. 1026

1027

A.2.3 Training and staff 1028

Training in all cell culture processes is vital to ensure correct procedures are adhered to under 1029

GMPs. Staff should be trained in the underlying principles of cell culture procedures to give 1030

them an understanding of cell culture processes that will enable them to identify events and 1031

changes that could impact on the quality of cells. Key procedures on which such GCCPs training 1032

should focus include passaging of cells, maintenance and use of biological safety cabinets and 1033

cryopreservation. 1034

1035

Cell cultures should be prepared by staff who have not, on the same working day, handled 1036

animals or infectious microorganisms. Furthermore, cell cultures should not be prepared by staff, 1037

who are known to be suffering from an infection. The personnel concerned should undergo a 1038

“return to work” assessment to evaluate any residual risk. 1039

1040

A.2.4 Cell line development and cloning 1041

Wherever a cell culture has passed through a process that may significantly have an influence on 1042

its characteristics such as tumorigenicity, it should be treated as a new (i.e., different) cell line 1043

and should be renamed with a suffix or code to identify this. A MCB should then be prepared 1044

from the post 'treatment' culture. Treatments that may require such rebanking include cell 1045

cloning and genetic manipulation. Any change(s) to the cell culture process should be 1046

demonstrated not to affect product quality. 1047

1048

The details of cloning and selection may vary, depending on the practices of individual 1049

manufacturers, and should be discussed with the NRA/NCL. 1050

1051

For example, in the early stages of cell line development a number of different recombinant 1052

vector systems and cell lines may be used. This will essentially be a research activity but the cell 1053

lines and vectors should originate from well characterised and qualified sources and the cells 1054

from an appropriately qualified seed stock or MCB which will usually be ‘in-house’ host cells 1055


and vectors. The most promising cell/vector combination will then be used to generate a large 1056

number of clones (100s-1000s) after treating the culture with rDNA. Typically these clones will 1057

be screened based on their productivity and a number of the highest productivity (10-50) will be 1058

taken forward for further evaluation. Further testing will then be used to select a small number 1059

(1-5) for establishment as small pre-master cell banks, and a final selection will be made, often 1060

based on stability characteristics and amenability to scale-up, before finally generating a MCB 1061

and WCB. Throughout the process only well characterised and traceable growth media and other 1062

critical reagents will be used (usually the same as for the MCB) and cryopreserved stocks of all 1063

working clones will be made at appropriate stages in the development process (Figure 1). 1064

1065

Figure 1. Simplified Schematic of an Example of the Development of a Genetically 1066

Modified Cell Line 1067

1068

1069

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

In the process of cloning a cell culture, ideally, single cells should be selected for expansion. The 1080

cloning procedure should be carefully documented; including the provenance of the original 1081

Selected for Cell/Vector combination

Cloning procedure

Selection for productivity

Selection for product quality

1-5 clones

Selection on other criteria including

scale-up and stability

Pre-Master Cell Bank(s)


culture, the cloning protocol and reagents used. Cloning by one round of limiting dilution will 1082

not necessarily guarantee derivation from single cells; additional subcloning steps should be 1083

performed. It is important to accurately document the establishment of each clone which should 1084

also have a unique reference. Cryopreserved seed stocks of a significant number of clones should 1085

be established at an early stage. The clones can then be compared in parallel with the parental 1086

culture to establish candidate clones with the best overall characteristics for delivery of the 1087

desired product. The criteria used in the evaluation of the clone selected for production should 1088

include: genomic and phenotypic stability, growth rate, achievable product levels and 1089

integrity/stability of the product. The evaluation of early candidate clones should generate 1090

sufficient information for the manufacturer to make an informed decision on the selection of the 1091

most promising clone(s) for further development. Where recombinant cell clones are under 1092

evaluation, these criteria also should include stability of integrated rDNA. 1093

1094

It is important to bear in mind that even following single cell cloning, epigenetic variation can 1095

result in a cloned culture showing evidence of heterogeneity (i.e., more than one clone). This 1096

should not preclude the use of such a culture for production, unless there are indications of 1097

instability that could affect the quality and/or safety of the final product. 1098

1099

A.2.5 Special considerations for neural cell types 1100

Agents causing transmissible spongiform encephalopathies have been propagated in certain cells. 1101

At the time of writing, the phenomenon has been observed only with very specific pairs of agents 1102

and cells; no cell has been identified that will replicate all agents, although one has been 1103

described that seems to be infectable by many strains of scrapie. The phenomenon is 1104

unpredictable, except that if the line does not express the PrP protein, it may be assumed to be 1105

impossible to infect it, and experience to date suggests that infection is not commonly observed 1106

or easy to maintain. On the other hand the cell types include fibroblastic lines as well as neuronal 1107

cells. The cells have usually been of murine origin because the infecting agents are usually 1108

mouse-adapted scrapie. The fact that certain cells can be infected with certain agents is proof of 1109

principle that cell lines may be infected so that exposure of cells to sources potentially 1110

contaminated with the agents is a concern. The scale of the risk is difficult to judge, and it is 1111

recommended that with respect to safety considerations and TSEs attention focuses on the 1112


selection and documentation of the cell culture reagents and other materials that come into 1113

intimate contact with the cells to provide assurance that they are not contaminated. Strategies to 1114

accomplish this are given in section B.11.4. 1115

1116

A.3 Selection of source materials 1117

A.3.1 Introduction 1118

All materials should be subjected to a risk assessment and testing when necessary, in particular, 1119

raw materials derived from humans and animals that can be a primary source for the introduction 1120

of adventitious agents into the production of biologicals. Therefore careful attention should be 1121

paid to their sourcing, production, handling, testing, and quality control. All cell culture materials 1122

of biological origin that come into intimate contact with the cells during the establishment of cell 1123

cultures, derivation of a new cell line (if any), banking procedures (if any), and production, 1124

should be subjected to appropriate tests, as indicated by risk assessment, for quality and freedom 1125

from contamination by microbial agents to evaluate their acceptability for use in production. It is 1126

important to evaluate the microbiological risks represented by each individual human- and 1127

animal-derived reagent used in a cell culture production process, and it should address: (a) 1128

geographical origin; (b) species of origin; (c) general microbiological potential hazards including 1129

a consideration of the medical history for human-derived reagents; (d) husbandry/screening of 1130

donor animals; (e) testing performed on the product, including Certificates of Analysis (if any); 1131

and (f) the capacity for the preparation, purification, and sterilisation procedures (if any) used to 1132

remove or inactivate contaminants. Other reagents of biological, but of non-animal origin, may 1133

also present risks to product safety and these are discussed further in section A.3.4 1134

1135

Recombinant protein technology now provides many materials formerly derived directly from 1136

animal or human sources. Whilst this eliminates obvious virological risks from donors, the 1137

manufacturing process used for the recombinant proteins should be analysed for any materials of 1138

biological origin and any associated hazards that may need to be addressed, as indicated in items 1139

a-f above. 1140

1141

The NRA/NCL should approve source(s) of animal-derived raw materials, such as serum and 1142

trypsin. These materials should comply with the guidelines on tissue infectivity distribution of 1143


TSEs [ 55]. They should be subjected to appropriate tests for quality and freedom from 1144

contamination by microbial agents to evaluate their acceptability for use in production. Their 1145

origin should be documented to ensure that the sources are from geographical regions with 1146

acceptable levels of microbiological risk (e.g., freedom from Foot and Mouth Disease Virus or 1147

Bovine Spongiform Encephalopathies). In addition, documentation should be gathered on their 1148

manufacturing history, production, quality control, and any final or supplementary processing 1149

that could affect quality and safety, such as blending and aliquoting of serum batches. In 1150

addition, controls should be in place to prevent cross-contamination of one material with another 1151

(e.g., bovine material in a porcine product). 1152

1153

The reduction and elimination from the manufacturing process of raw materials derived from 1154

animals and humans is encouraged, where feasible. 1155

1156

For some human- and animal-derived raw materials used in the cell culture medium, such as 1157

insulin or transferrin, validation of the production process for the elimination of viruses can 1158

substitute for virus detection tests, when justified. 1159

1160

Animal-derived reagents, such as trypsin and serum, which would be substantially damaged or 1161

destroyed in physical sterilisation processes, including heat and irradiation, present the most 1162

likely microbiological hazards to cell culture processes. Batches of reagents, such as trypsin and 1163

bovine serum, have been known to contain Mycoplasma species. and sometimes more than one 1164

viral contaminant. Certain contaminants also have been shown to infect cells in culture. The 1165

processing environment is also a common source of microbiological contamination and should 1166

be controlled to minimize this risk, and prevent growth of contaminants. 1167

1168

A.3.2 Serum and other bovine-derived materials used in cell culture media 1169

The source(s) of serum of bovine origin should be approved by the NRA/NCL. The 1170

responsibility for ensuring the quality of the serum used in the manufacture of cell banks and 1171

biologicals rests with the biologicals' manufacturer. This can be accomplished in more than one 1172

manner. The manufacturer might conduct adventitious agent testing and perform inactivation of 1173

the serum after purchase from the serum manufacturer. Alternatively, the manufacturer might 1174


qualify their serum vendor and only purchase serum from suppliers after conducting thorough 1175

and on-going audits of the serum supplier to ensure that they have properly performed the 1176

manufacture, quality control, and validation necessary to achieve the level of quality of the 1177

serum required for the biological it is being used to produce. In some cases, certificates of 1178

analysis may then be considered sufficient. Some combination of these approaches might be 1179

optimal, and the strategy taken should be considered in evaluating risk. Consultation with the 1180

NRA/NCL also might be advisable. 1181

1182

Serum and other bovine-derived materials should be tested for adventitious agents, such as 1183

bacteria, fungi, mycoplasmas, and viruses, prior to use in the production of MCBs and WCBs 1184

and in the manufacture of biologicals. Particular consideration should be given to those viruses 1185

that could be introduced from bovine-derived materials and that could be zoonotic or oncogenic 1186

(e.g., bovine viral diarrhea virus, bovine circoviruses, rabies, bovine adenoviruses, bovine 1187

parvovirus, bovine respiratory syncytial virus, infectious bovine rhinotracheitis virus, bovine 1188

parainfluenza type 3, reovirus 3, Cache Valley virus, bluetongue virus, and epizootic 1189

hemorrhagic disease virus). In addition, consideration should be given to risk-mitigation 1190

strategies such as inactivation by heat or irradiation to ensure that adventitious agents that were 1191

not detected in the manufacture and quality control of the serum would be inactivated to a degree 1192

acceptable to the NRA/NCL. If irradiation or other inactivation (e.g., heat sterilization) methods 1193

are used in the manufacture of the serum, the tests for adventitious agents should be performed 1194

prior to inactivation to enhance the opportunity for detecting the contamination. If evidence of 1195

viral contamination is found in any of the tests, the serum is acceptable only if the virus is 1196

identified and shown to be present in an amount that has been shown in a validation study to be 1197

effectively inactivated. For serum that is not to be subjected to a virus inactivation/ removal 1198

procedure, if evidence of viral contamination is found in any tests, the serum is not acceptable. 1199

If irradiation is used, it is important to ensure that a reproducible dose is 1200

delivered to all batches and the component units of each batch. The irradiation 1201

dose must be low enough so that the biological properties of the reagents are 1202

retained while being high enough to reduce virological risk. Therefore 1203

irradiation delivered at such a dose may not be a sterilizing dose. 1204

1205


If serum was used in the establishment or passage history of the animal cell substrate prior to 1206

banking by the manufacturer, the cell bank (MCB or WCB) and/or cells at or beyond the level of 1207

production should be tested for adventitious agents of the species of serum used in the 1208

establishment and passage history of the cell substrate. If serum is not used in the production of 1209

the subsequent stages, then this testing would not need to be repeated on those subsequent stages 1210

once the cell bank has been tested and considered free of bovine (or whichever species of serum 1211

that was used) adventitious agents. 1212

1213

Methods used to test for bovine viruses should be approved by the NRA/NCL. The USDA [56, 1214

57] and EMEA [58] have provided guidance on test methods. Infectivity assays are used as the 1215

primary screening method and have resulted in the detection of bovine viral diarrhea virus, 1216

reovirus 3, Cache Valley virus, bluetongue virus, and epizootic hemorrhagic disease virus 1217

amongst others. However, it should be noted that in general, the infectivity screening assay 1218

methods described here do not readily detect some of the viruses (e.g., bovine circoviruses, 1219

bovine polyomaviruses, or bovine anelloviruses) that can be frequent contaminants of serum. 1220

Additional methods may need to be considered, such as the nucleic acid amplification technique 1221

(NAT), although the presence of viral genomic sequences is not necessarily indicative of 1222

infectious virus. In those cases, specific infectivity assays designed to detect the virus of concern 1223

(e.g., bovine polyomavirus) may need to be considered. 1224

1225

A second factor in screening serum is the limited sample volume used compared to the batch 1226

size, which may be on the order of 1000 litres. Consequently, infectious viruses can be missed in 1227

the serum lot testing and consideration should be given to direct screening of the cell bank for 1228

bovine viruses. These assays could include NAT for the presence of bovine viruses that may 1229

infect the cell substrate but undergo abortive and/ or transforming infections. Virus families of 1230

particular concern include polyomaviruses, herpesviruses, circoviruses, anelloviruses, and 1231

adenoviruses. 1232

1233

General screening assays for the detection of infectious viruses in serum or cell substrates 1234

involve the use of at least one indicator cell line, such as bovine turbinate cells, permissive for 1235

the replication of bovine viral diarrhoea virus (BVDV). A second cell line such as Vero also 1236


should be employed to broaden the detection range. Before initiating screening it may be 1237

necessary to evaluate the serum for the presence of antibody, particularly to BVDV, that could 1238

mask the presence of infectious virus. 1239

1240

Typically, indicator cells are cultured in the presence of the serum under test for 21 to 28 days, 1241

passaging the cells as necessary. During this period the cells are regularly examined for the 1242

presence of cytopathic effect (CPE) indicative of virus infection, as well as examined for at the 1243

end of the observation period and at least 7 days since the last sub-culture by cytological staining 1244

to detect CPE. Additional endpoint assays should include haemadsorbtion and haemagglutination 1245

at both 4oC and a higher temperature such as 20 to 25

oC and also immunofluorescence assays 1246

(IFA) for specific viruses. Appropriate controls should be used for each assay, like parainfluenza 1247

virus 3 for heamadsorbtion. IFA is particularly important for BVDV as non-cytopathic BVDV 1248

may be present in the serum. IFA endpoints are also used to detect other viruses that may be 1249

determined by geographical considerations like adenoviruses, bovine parvovirus, bluetongue 1250

virus, bovine syncytial virus, reovirus type 3 and unlikely, but serious, contaminants like rabies 1251

virus. 1252

1253

If serum from another species is used (i.e., other than bovine), the NRA/NCL should be 1254

consulted regarding acceptable testing methods for that species. 1255

1256

A.3.3 Trypsin and other porcine-derived materials used for preparing cell cultures 1257

Trypsin used for preparing cell cultures should be tested for cultivable bacteria, fungi, 1258

mycoplasmas and infectious viruses, including bovine or porcine parvoviruses, as appropriate for 1259

that species and the agents to be sought. The methods used to ensure this should be approved by 1260

the NRA/NCL. 1261

In some countries, irradiation is used to inactivate potential 1262

contaminant viruses. If irradiation is used, it is important to 1263

ensure that a reproducible dose is delivered to all batches and 1264

the component units of each batch. The irradiation dose must 1265

be low enough so that the biological properties of the reagents 1266

are retained while being high enough to reduce virological 1267


risk. Therefore Irradiation cannot be considered a sterilizing 1268

process. 1269

1270

The quality of the trypsin, like serum, is the responsibility of the biologicals’ manufacturer (see 1271

section A.4.2). Recombinant human trypsin is available and should be considered; however it 1272

should not be assumed to be free of risk of contamination and should be subject to the usual 1273

considerations for any reagent of biological origin (see 4.1). 1274

1275

Furthermore, if trypsin was used in the establishment or passage history of the animal cell 1276

substrate prior to banking by the manufacturer, the cell bank (MCB or WCB, and/or EOPC) 1277

should be tested for porcine parvovirus, or appropriate adventitious viruses relevant to the 1278

species of origin of the trypsin used in the establishment and passage history of the cell substrate. 1279

If trypsin is not used in the production of the subsequent stages, then this testing would not need 1280

to be repeated on those subsequent stages once the cell bank has been tested and considered free 1281

of porcine parvovirus (or relevant agents). 1282

1283

A.3.3.1 Trypsin of porcine origin 1284

Trypsin of porcine origin should be tested for porcine parvovirus and preferably, treated to 1285

inactivate any virus potentially present, in a manner accepted by the NRA/NCL. If trypsin from 1286

another species is used, the NRA/NCL should be consulted regarding acceptable testing 1287

methods. 1288

1289

General screening assays for the detection of infectious viruses in trypsin or cell substrates 1290

involve the use of at least one indicator cell line, such as porcine testes cells, permissive for the 1291

replication of porcine parvovirus. Typically, indicator cell cultures would be incubated for 14 1292

days with a sub-culture onto fresh test cells for an additional 14 days. IFA for porcine 1293

parvovirus would be conducted at the end of each culture period, but not less than 4-5 days after 1294

the last sub-culture. 1295

1296

1297


Consideration should be given to screening for other agents such as porcine circoviruses. 1298

Molecular methods, such as PCR, may be used for such purposes. Screening tests such as those 1299

described by USDA [56, 57] and the EMEA [58] also may be considered. 1300

1301

A.3.3.2 Other porcine-derived materials 1302

Testing of other porcine-derived materials might entail testing for more than porcine parvovirus. 1303

For example, testing for porcine adenovirus, transmissible gastroenteritis virus, porcine 1304

haemagglutinating encephalitis virus, bovine diarrhea virus, reoviruses, rabies virus, porcine 1305

anellovirus, hepatitis E virus, and potentially other viruses might be appropriate. Particular 1306

consideration should be given to those viruses that could be introduced from the porcine-derived 1307

material and that could be zoonotic or oncogenic. Additionally, tests for bacterial and fungal 1308

sterility and mycoplasmas depending on the type of porcine-derived material should be 1309

conducted. The NRA/NCL should be consulted in this regard. 1310

1311

A.3.4 Media supplements and general cell culture reagents derived from other 1312

sources used for preparing cell cultures 1313

Media supplements derived from other species should be quality controlled from the perspective 1314

of adventitious agents. Consideration should be given to whether recombinant-derived media 1315

supplements were exposed to animal-derived materials during their manufacture, and if so, 1316

evaluated for the potential to introduce adventitious agents into the manufacture of the cell banks 1317

and biological products. Testing for adventitious agents should assess viruses relevant to the 1318

species from which the supplement was derived. The NRA/NCL should be consulted in this 1319

regard. 1320

1321

Generally, media supplements should not be obtained from human source materials. In 1322

particular, human serum should not be used. However, in special circumstances, and in 1323

agreement with the NRA/NCL, the use of human-derived supplements may be permitted. If 1324

human serum albumin is used at any stage of product manufacture, the NRA/NCL should be 1325

consulted regarding the requirements, as these may differ from country to country. However, as a 1326

minimum, it should meet the revised Requirements for Biological Substances No. 27 1327


(Requirements for the Collection, Processing and Quality Control of Blood, Blood Components 1328

and Plasma Derivatives) [59], as well as the guidelines on tissue infectivity distribution of TSEs. 1329

1330

Recombinant human albumin is commercially available and should be considered; however, it 1331

should not be assumed to be free of risk of contamination and should be subject to the usual 1332

considerations for any reagent of biological origin (see A.3). 1333

1334

As for other cell culture reagents it is important to establish traceability and assess and reduce 1335

microbiological risks as described in section A.3. 1336

1337

A variety of cell culture reagents of biological origin are available, which are derived from non-1338

animal sources; including a range of aquatic organisms, plants and algae. In such cases the exact 1339

hazards involved may be uncertain and unfamiliar. The microbiological risks may be 1340

substantially different to those involved in animal-derived reagents (see section A.3.1-A.3.3) and 1341

other hazards may arise, such as immunogenic, mitogenic and allergenic properties of the 1342

reagent and its components. For example, plant-derived material may carry an increased risk of 1343

mycoplasma and mycobacteria contamination. 1344

1345

A.4 Certification of cells 1346

It is vital that the manufacturer has secured a body of information on the cell substrate that 1347

demonstrates clearly the origin or provenance of the culture, and how the cell banks intended for 1348

production (MCB and WCB) were established, characterised and tested. This should provide all 1349

the information required to demonstrate the suitability of that cell substrate and the established 1350

cell banks for the manufacture of biological products. 1351

1352

A.4.1 Cell line data 1353

Each new cell line should have an associated body of data that will increase as the cell line is 1354

established and developed for manufacturing purposes. This data set is vital to demonstrate the 1355

suitability for use of the cells and should provide information on cell provenance (donor 1356

information and any relevant details on ethical procurement), cell line derivation, culture history, 1357

culture conditions (including reagents), early stage safety evaluation data, banking and cell bank 1358


characterisation and safety testing. This information should be available to the NRA/NCL for 1359

approval of the cells used in manufacture. 1360

1361

A.4.2 Certification of primary cell cultures 1362

Full traceability should be established for PCCs to the animals of origin, husbandry conditions, 1363

veterinary inspection, vaccinations (if any), procedures for administering anesthesia and cell 1364

harvesting, the reagents and procedures used in the preparation of primary cultures and the 1365

environmental conditions under which they were prepared. Extensive testing should be 1366

performed, and this should be documented. 1367

1368

It is important to define the batch or lot of cells used in each individual manufacturing process. 1369

For production purposes a batch or lot is a culture of primary cells derived from single or 1370

multiple animals that has been subjected to a common process of tissue retrieval and 1371

disaggregation, and processing, leading to a single culture preparation of cells. Lots may be 1372

prepared by harvesting and pooling cells in different ways but the cell processing procedures 1373

should be reproducible, and it is especially important to monitor cultures carefully for evidence 1374

of adverse change in the cell culture and microbiological contamination. Prior to any culture 1375

pooling, cells should be examined for acceptability for production. Acceptability criteria should 1376

be established and should include testing for microbiological contamination and the general 1377

condition of the cells (e.g., morphology , number, and viability of the cells). Failure to detect and 1378

eliminate atypical (i.e., potentially virally infected) or grossly contaminated cells will put the 1379

entire production run at risk and could compromise the safety of the product. Cells showing an 1380

unacceptably high proportion of dead or atypical cells should not be used, and ideally 1381

microbiological testing should be completed and passed before the cells are used. 1382

1383

The preparation of cell lots for manufacture should be carefully documented to provide full 1384

traceability from the animal donor(s) to production. Any pooling of cells should be clearly 1385

recorded, as should any deviation from standard operating procedures, as required by GMPs. In 1386

addition, any observations of variation between batches should be recorded; even where such 1387

observations would not necessarily lead to rejection of those batches. Such information may 1388

prove valuable in ongoing optimization and improvement of the production process. 1389


1390

A.4.3 Certification of diploid, continuous, and stem cell lines 1391

All cell lines used for biologicals production should have data available, as indicated in A.4.1. 1392

1393

The original PDL (or passage number, if the PDL is unknown) of the cell seed should be 1394

recorded. 1395

1396

For cell lines of human origin, if possible the medical history of the individual from whom the 1397

cell line was derived should be evaluated in order to better assess potential risks and the 1398

suitability of the cell line. 1399

1400

For SCLs, morphology continues to be an important characteristic, and representative images 1401

and immune-phenotypic profiles of undifferentiated and differentiated cells should be available 1402

for comparison. 1403

1404

A.5 Cryopreservation and Cell Banking 1405

A.5.1 Cryopreservation 1406

When cells are banked, the successful preservation of cells at ultra-low temperatures is critical to 1407

the efficient delivery of good quality cultures (i.e., high viability cultures of the required 1408

characteristics). The need to prepare large stocks of frozen vials of cells for cell banks is 1409

especially challenging, and a number of key principles should be adopted: 1410

• A method that meets current best practice for cell culture preservation should be used (for 1411

example, see ref 60). 1412

• The cooling profile achieved in the cells being frozen should be defined, and the same 1413

cooling process should be used for each separate preservation process (i.e., an SOP 1414

should include documentation of the cooling process in the batch record.) 1415

• Each preservation process should be recorded. 1416

• As a general guide, only cell cultures that are predominantly in the exponential phase of 1417

growth should be used. Cells in such cultures tend to have a low ratio of cytoplasm to 1418

nucleus (v/v) and should be more amenable to successful cryopreservation. It is unwise to 1419


use cells predominantly in the 'lag' phase immediately after passage or in the 'stationary' 1420

phase when the culture has reached its highest density of cells. 1421

• For each bank, cells pooled from a single expanded culture (i.e., not from a range of 1422

cultures established at different times post seeding or different PDLs) should be used and 1423

mixed prior to aliquotting to ensure homogeneity. 1424

• The number of cells per vial should be adequate to recover a representative culture (e.g., 1425

5-10x106 in a 1ml aliquot). 1426

• For new cell banks, antimicrobials should not be used in cell cultures to be banked, 1427

except where this can be justified for early PDL cultures which may carry contamination 1428

from tissue harvesting, and when necessary for the genetic stability of recombinant cell 1429

lines. In any case, if antimicrobials are used, they should not be penicillin or any other 1430

beta-lactam drug. 1431

• When a stock of cells has been frozen, a sample should be recovered to confirm it has 1432

retained viability and the results recorded. It is also important to establish the degree of 1433

homogeneity within the cell bank. Recovery of a sufficient percentage (e.g., 3%) of vials 1434

representative of the beginning, middle and end of the cryopreservation process should be 1435

demonstrated to give confidence in the production process based on the use of that cell 1436

bank. Ultimately, stability (see B.3) and integrity of cryopreserved vials is demonstrated 1437

when the vials are thawed from production and demonstrated to produce the intended 1438

product at scale. 1439

• Cell bank cryostorage vessels should be monitored and maintained to enable 1440

demonstration of a highly stable storage environment for cell banks. Access to such 1441

vessels may cause temperature cycling which in extreme cases can cause loss of viability. 1442

It is therefore prudent to establish a stability testing programme involving recovery of 1443

cells periodically where the frequency of recovery relates to risk of temperature cycling. 1444

New developments in remote monitoring of individual vials may in future eliminate the 1445

need for stability testing. 1446

1447

A.5.2 Cell Banking 1448

When DCLs, SCLs, or CCLs are used for production of a biological, a cell bank system should 1449

be used. Such banks should be approved by and registered with the NRA/NCL. The source of 1450


cells used in cell banking and production is a critical factor in biological product development 1451

and manufacture. It is highly desirable to obtain cells from sources with a documented history 1452

and traceability to the originator of the cell line. 1453

1454

After a sample of the original seed stock is obtained, an early stage pre-master bank of just a few 1455

vials should be established. One or more vials of that pre-master bank is used to establish the 1456

MCB. The WCB is derived by expansion of one or more containers of the MCB. The WCB 1457

should be qualified for yielding cell cultures that are acceptable for use in manufacturing a 1458

biological product. 1459

1460

When using early PDLs from primary cultures for production processes, the preparation of a cell 1461

bank should be considered on a case-by-case basis. This approach has significant benefits as it 1462

gives great flexibility in the timing of the production process, permits quality control and safety 1463

testing to be completed prior to use and reduces the overall burden of testing required in the 1464

production process. 1465

1466

Cell banks should be characterized as specified in Part B of this guidance and according to any 1467

other currently applicable and future guidance published by WHO. The testing performed on a 1468

replacement master cell bank (derived from the same cell clone, or from an existing master or 1469

working cell bank) is the same as for the initial master cell bank, unless a justified exception is 1470

made (also see illustration in B.12). Efforts to detect contaminating viruses and other microbial 1471

agents constitute a key element in the characterization of cell banks. 1472

1473

Having been cryopreserved by qualified methods (see A.5.1), both the MCB and WCB should be 1474

stored frozen under defined conditions, such as in the vapour or liquid phase of liquid nitrogen. 1475

The location, identity and inventory of individual cryovials or ampoules of cells should be 1476

thoroughly documented. It is recommended that the MCB and WCB each be stored in at least 1477

two widely separated areas within the production facility and/or in geographically distinct 1478

locations to assure continued ability to manufacture product in the event of a catastrophe. When 1479

cryopreserved cells are transferred to a remote site, it is important to use qualified shipping 1480

containers and to monitor transfers with probes to detect temperature excursions. All containers 1481


are treated identically and, once removed from storage, usually are not returned to the stock. The 1482

second storage site should operate under an equivalent standard of quality assurance as the 1483

primary site. 1484

1485

A.5.3 WHO reference cell banks (RCBs) 1486

The principle of establishing RCBs under WHO auspices is one that offers potential solutions to 1487

future challenges for the development of vaccines and biotherapeutics in developing regions. 1488

However, WHO does not intend that cells supplied to manufacturers from any RCB be used as a 1489

MCB. The purpose of WHO RCBs is to provide well characterized seed material for the 1490

generation of a MCB by manufacturers with the expectation that such MCBs will comply with 1491

this guidance document and be fully characterized. 1492

1493

The WHO RCBs provide key advantages for vaccine development worldwide that include: 1494

• Traceability to origin of cells and derivation of cell line and materials used in preparation 1495

of seed stock 1496

• Subjected to open international scientific scrutiny and collaborative technical 1497

investigations into the characteristics of the cells and the presence of adventitious agents 1498

• Results of characterisation were peer reviewed and published 1499

• Investigations evaluated under auspices of WHO expert review and qualified as suitable 1500

for use in vaccine production 1501

• Supply of cells free of any adverse Intellectual Property 'reach through' on final products 1502

• Single source of cells with growing and scientifically and technically updated body of 1503

safety testing data and safe history of use; giving increasing confidence for 1504

manufacturers, regulators and public policy makers 1505

1506

The Vero cell line is the most widely used continuous cell line for the production of viral 1507

vaccines over the last two decades. The WHO Vero RCB 10-87 was established in 1987 and was 1508

subjected to a broad range of tests to establish its suitability for vaccine production. This WHO 1509

RCB provides a unique resource for the development of future biological medicines where a cell 1510

substrate with a safe and reliable history of use is desired. A comprehensive review of the 1511


characterization of the WHO Vero 10-87 seed lot was conducted recently, and a detailed 1512

overview is provided on the WHO website at (http://www.who.int/biologicals/). 1513

1514

As concluded by an expert review in 2002, the WHO Vero RCB 10-87 is not considered suitable 1515

for direct use as MCB material. However, the WHO Vero RCB 10-87 is considered suitable for 1516

use as a cell seed for generating a MCB, and its status has changed from "WHO Vero cell bank 1517

10-87" to "WHO Vero reference cell bank 10-87" [61]. 1518

1519

The WHO Vero RCB 10-87 is stored in the European Collection of Animal Cell Cultures 1520

(ECACC, www.hpa.org), Salisbury, Wiltshire, UK, and the American Type Culture Collection 1521

(ATCC, www.atcc.org), in Mannassas, Virginia, USA. These public service culture collections 1522

have distributed ampoules under agreements with the WHO to numerous manufacturers and 1523

other users. The WHO Vero RCB 10-87 is the property of WHO, and there are no constraints 1524

relating to intellectual property rights. The WHO Vero RCB 10-87 is available free of charge on 1525

application to WHO. However, due to the limited number of vials remaining, distribution of 1526

these vials is restricted solely for the production of vaccines and other biologicals. Potential 1527

replacement of the WHO Vero RCB 10-87 is currently under consideration. 1528

1529

WHO also has overseen the establishment of seed stocks of MRC-5 for the production of 1530

vaccines. The WHO MRC-5 RCB was established in 2007 because of stability issues associated 1531

with the original vials of MRC-5 cells that dated to 1966. This RCB was prepared in a GMP 1532

laboratory and subjected to specified quality control testing endorsed by the ECBS. 1533


Part B. Recommendations for the characterization of cell 1534

banks of animal cell substrates 1535

1536

B.1 General considerations 1537

Since the 1986 Study Group report, advances in science and technology have led to an 1538

expanded range of animal cell types that are used for the production of biological 1539

products. In some cases, these new cell types provide significantly higher yields of 1540

product at less cost, while in other cases they provide the only means by which a 1541

commercially viable product can be manufactured. Many products manufactured in CCLs 1542

of various types have been approved, and some examples are listed in Table 1. 1543

1544

Table 1. Examples of Approved Biological Products Derived from CCLs 1545

Product Class Product (disease) Cell Line

Factor VIII

(hemophilia)

CHO

Factor VIIa

(hemophilia)

BHK-21

Therapeutic

Monoclonal antibody

(oncology)

CHO and murine

myeloma (NSO and

SP2)

Poliovirus vaccine Vero

Rotavirus vaccine

Vero

Rabies vaccine Vero

Human papilomavirus vaccine Hi-5

Prophylactic

Influenza vaccine MDCK

1546

Many more products are currently in development, and some use highly tumorigenic cells (e.g., 1547

HeLa; some strains of MDCK), and some involve sources previously unused in production such 1548

as insect cells. Examples are listed in Table 2. 1549

1550


Table 2. Examples of Biological Products in Development Derived from CCLs 1551

Product Class Product (disease) Cell Line(s)

Therapeutic

> 50% of products in development use CHO or murine

myeloma cells as the cell substrate [ 62]. Monoclonal

antibodies are generally produced using CHO, SP2/0, PER.C6,

and NSO cells [ 63].

HIV vaccines CHO, Vero, PER.C6,

293ORF6, HeLa

Herpes Simplex Type 2 vaccine CHO

JE vaccine Vero

Influenza vaccines SF9, Vero, PER.C6

Prophylactic

Rabies vaccine S2

1552 CCLs may have biochemical, biological and genetic characteristics that differ from PCCs or 1553

DCLs that may impose risk for the recipients of biologicals derived from them. In particular, 1554

they may produce transforming proteins, and may contain potentially oncogenic DNA and viral 1555

genes. In some cases, CCLs may cause tumours when inoculated into animals. Nontumorigenic 1556

cells (e.g., PCCs and DCLs) had been thought to be intrinsically safer than tumorigenic cells. 1557

Where tumorigenic cells have been used in the past (e.g., CHO for recombinant proteins), high 1558

degrees of purity have been required with a special emphasis on reduction in size and quantity, 1559

or other approaches to inactivate rcDNA and rcRNA (e.g., BPL for rabies vaccine). 1560

1561

Manufacturers considering the use of CCLs should be aware of the need to develop, evaluate, 1562

and validate efficient methods for purification as an essential element of any product 1563

development programme. However, a minimally purified product such as certain viral vaccines 1564

(e.g., polio) may be acceptable when produced in a CCL such as Vero when data are developed 1565

to support the safety of the product. Such data would include extensive characterization of the 1566

MCB or the WCB and of the product itself. 1567

1568

While tumorigenicity tests have been part of the characterization of CCLs, they comprise only 1569

one element in an array of tests, the results of which must be taken into account when assessing 1570

the safety of a biological produced in a given cell substrate. For example, if a CCL is positive in 1571

a tumorigenicity test, and if the CCL is to be used for the production of a live viral vaccine, an 1572

evaluation of the oncogenic potential of the cells should be undertaken to characterize the 1573


cellular DNA and to detect oncogenic viruses that might be present. Such studies should be 1574

discussed with the NRA/NCL. 1575

1576

Evidence should be provided for any animal cell line proposed for use as a substrate for the 1577

manufacture of a biological product demonstrating that it is free from cultivable bacteria, 1578

mycoplasmas, fungi and infectious viruses, including potentially oncogenic agents to the limits 1579

of the assay’s detection capabilities. Special attention should be given to viruses that commonly 1580

contaminate the animal species from which the cell line is derived, and to cell culture reagents of 1581

biological origin. The cell seed should preferably be free from all microbial agents. However, 1582

certain CCLs may express endogenous retroviruses, or may contain genomic sequences of 1583

adenoviruses, herpesviruses, and papillomaviruses . Tests capable of detecting such agents 1584

should be carried out on cells grown under cell culture conditions that mimic those used during 1585

production and the levels of viral particles should be quantified. Viral contaminants in a MCB 1586

and WCB should be shown to be inactivated and/or removed by the purification procedure used 1587

in production [Part C]. The validation of the purification procedure used is considered essential. 1588

1589

The characterization of any DCL, SCL or CCL to be used for the production of biologicals 1590

should include: a) a history of the cell line (i.e., provenance) and a detailed description of the 1591

production of the cell banks, including methods and reagents used during culture, PDL, storage 1592

conditions, viability after thawing, and growth characteristics; b) the results of tests for infectious 1593

agents; c) distinguishing features of the cells, such as biochemical, immunological, genetic, or 1594

cytogenetic patterns which allow them to be clearly distinguished from other cell lines; and d) 1595

the results of tests for tumorigenicity, including data from the scientific literature. Additional 1596

consideration should be given to products derived from cells that contain known viral sequences 1597

(e.g, Namalwa, HeLa, HEK293, and PER.C6). 1598

1599

The recommendations that follow are intended as guidance for NRAs, NCLs, and manufacturers 1600

as the minimum amount of data on the cell substrate that should be available when considering a 1601

new biological product for approval. The amount of data that may be required at various stages 1602

of clinical development of the product should be discussed and agreed with the NRA/NCL at 1603

each step of the program. 1604


1605

B.2 Identity 1606

Cell Banks should be authenticated by a cell identification method approved by the NRA/NCL. 1607

Wherever practicable, methods for identity testing should be used that give specific identification 1608

of the cell line to confirm that no switching or major cross-contamination of cultures has arisen 1609

during cell banking and production. A number of the commonly used identity testing methods 1610

are compared in Table 3. In the case of human cells, genetic tests such as DNA profiling (e.g., 1611

Short Tandem Repeat analysis, multiple Single Nucleotide Polymorphisms) will give a profile 1612

which is at least specific to the individual from which the cells were isolated. Another test that 1613

might be used for human cells is HLA type. Other tests that may be used but tend to be less 1614

specific include isoenzyme analysis and karyology, which may be particularly useful where there 1615

are characteristic marker chromosomes. In the case of nonhuman cells, these latter techniques 1616

may be the only available identity markers. Therefore in those cases, karyology and isoenzyme 1617

analyses should be performed. However, where more specific genetic markers are available, they 1618

should be considered. For recombinant protein products, cell line identity testing should also 1619

include tests for vector integrity, plasmid copy number, insertions, deletions, number of 1620

integration sites, the percentage of host cells retaining the expression system, verification of 1621

protein coding sequence and protein production levels. 1622

1623

Table 3. Identity Testing for Mammalian Cell Lines 1624

Technique Advantages Disadvantages

Karyology

(especially useful for

DCLs and SCLs)

Gives whole genome

visualisation and analysis that

can identify species of origin for

a very wide range of species

using the same methodology.

Results are generally not specific

to the individual of origin (i.e.,

usually species specificity)

although certain cell lines may

have marker chromosomes that are

readily recognised.

Giemsa banding requires special

expertise and is labour intensive.

Standard analyses of 10-20

metaphase spreads is insensitive

for detecting contaminating cells.

Isoenzyme analysis Determination of species of

origin within a few hours

Analyses for 4-6 isoenzyme

activities will generally identify

species of origin, but is not

specific to the individual of origin


DNA profiling using

variable number of

tandem repeats

(VNTR) analysis

or other PCR method

such as Exon Primed

Intron Crossing-PCR

(EPIC-PCR), or other

techniques such as

restriction fragment

length polymorphism

(RFLP)

Short tandem repeats (STR)

analysis by PCR is rapid and

gives identity specific to the

individual of origin.

Commercial kits are available

which have been validated for a

range of human populations.

EPIC-PCR method is rapid and

gives identity specific to the

individual of origin. It provides

the advantage to cover a broad

spectrum of organisms and cell

lines other than human cells

Some limited, but undefined,

cross-reaction of human STR

primers for primate cells

1625

B.2.1 Applicability 1626

Cell Banks: MCB and each WCB 1627

Cell Types: PCC, DCL, SCL, CCL 1628

1629

B.3 Stability 1630

The stability of cell banks during cryostorage, and the genetic stability of cell lines and 1631

recombinant expression systems are key elements in a successful cell bank program. 1632

1633

B.3.1 Stability during cryostorage 1634

Data should be generated to support the stability or suitability of the cell substrate and any 1635

recombinant expression system or necessary cell phenotype during cultivation to or beyond the 1636

limit of production; and to support the stability of the cryopreserved cell banks during storage. 1637

The latter may be demonstrated by successful manufacture of WCBs or production lots. Periodic 1638

testing for viability is not necessary if continuous monitoring records for storage show no 1639

deviations out of specification, and periodic production runs are successful. If banks are used less 1640

than once every 5 years, then it may be prudent to generate data reconfirming suitability for 1641

manufacturing on a schedule that takes into account the storage condition, once every 5 years. 1642

1643

B.3.1.1 Applicability 1644

Cell Banks: MCB and WCB 1645

Cell Types: DCL, SCL, CCL 1646


1647

B.3.2 Genetic Stability 1648

Additional characterization of the cell biological processes and responses during cultivation (for 1649

instance using global or targeted gene expression, proteomic or metabolic profiles and other 1650

phenotypic markers) might be useful in further developing a broad understanding of the cell 1651

substrate. 1652

1653

Appropriate methods should be applied to assure that cell age is correctly assessed in the event 1654

that cell viability falls dramatically at any given step. Losses in viability are reflected in 1655

increased cultivation times to reach defined levels of growth, and cell age of diploid cells might 1656

also be assessed by measurements of telomere length. 1657

1658

The stability of cell function in terms of productivity within the production process also may 1659

need to be evaluated. Other stability studies may be performed where bioreactor methods are 1660

employed, especially where extended culture periods are involved. 1661

1662

B.3.2.1 Applicability 1663

Cell Banks: MCB taken to EOPC 1664


1666

B.4 Sterility 1667

(see B.11.3.1) 1668

1669

B.5 Viability 1670

High level viability of cryopreserved cells is important for efficient and reliable production 1671

procedures. Typically thawed cells should have viability levels in excess of 80% and a range of 1672

viability tests are available that measure different attributes of cell function (membrane integrity, 1673

metabolic activity, DNA replication). Under certain circumstances, such as pre-apoptotic cells 1674

excluding trypan blue, viability assays may give misleading results and it is important to be fully 1675

aware of the exact information that a particular viability assay provides. 1676


For certain cell cultures such as hybridomas, where a membrane 1677

integrity test is used, additional cell markers such as indicators of 1678

apoptosis should be studied in order to avoid significantly 1679

overestimating viability. 1680

A suitable viability test should be selected and qualified for the cell substrate in question and 1681

typical test values established for cultures considered to be acceptable. It may also be necessary 1682

to select alternative viability assays that are better suited to providing in-process viability data 1683

required during production, e.g., lactate dehydrogenase levels in bioreactor systems. 1684

1685




1689

B.6 Growth Characteristics 1690

For the development of production processes, the growth characteristics of the production cell 1691

line should be well understood to ensure consistency of production. Changes in these 1692

characteristics could indicate any one of a range of events. Accordingly, data on growth 1693

characteristics such as viability, morphology, cell doubling times, cloning and/or plating 1694

efficiency, if applicable, should be developed. For certain cell substrates it may be appropriate to 1695

apply such tests in homogeneity testing (see B 7). Experiments to demonstrate homogeneity and 1696

growth characteristics may be combined although the analysis should be carried out separately. 1697

1698




1702

B.7 Homogeneity Testing 1703

Each cell culture derived from a container of the WCB should perform in the same way (i.e., 1704

within acceptable limits, provide the same number of viable cells of the same growth 1705

characteristics). In order to assure this, it is important to recover a proportion of containers from 1706

each cell bank and check their characteristics as indicated in B.6. The number of containers 1707


tested should be discussed with the NRA/NCL, and broadly in line with those normally sampled 1708

to establish product consistency. Recovery of a sufficient percentage of vials representative of 1709

the beginning, middle and end of the cryopreservation process should be demonstrated to give 1710

confidence in the production process based on the use of that cell bank. Ultimately, stability and 1711

integrity of cryopreserved vials is demonstrated when the vials are thawed from production and 1712

demonstrated to produce the intended product at scale. Instead of testing a portion of container at 1713

different stage of the banking process, an alternative strategy to ensure the homogeneity of the 1714

banks can be used based on the validation of the process method for filling and freezing. 1715

Assessment of growth characteristics (B.6) and homogeneity testing are commonly combined 1716

experimentally, however the analysis and interpretation of each should be distinct. 1717

1718


Cell Banks: WCB 1720


1722

B.8 Tumorigenicity 1723


Several in vitro test systems such as cell growth in soft agar [ 64] and muscle organ culture [ 65] 1725

have been explored as alternatives to in vivo tests for tumorigenicity; however, correlations with 1726

in vivo tests have been imperfect, or the alternate tests have been technically difficult to perform. 1727

Therefore, in vivo tests remain the standard for assessing tumorigenicity. 1728

1729

Although WHO Requirements [ 1] have described acceptable approaches to tumorigenicity 1730

testing, a number of important aspects of such testing were not addressed. Therefore a model 1731

protocol has been developed and is appended to this document. The major points included in the 1732

model protocol are listed below along with comments on each specific item. 1733

1734

Based on long experience with the well characterised DCLs WI-38, MRC-5 and FRhL2, testing 1735

of only new MCBs of these cell lines for tumorigenicity is recommended. 1736

1737


The tumorigenicity tests currently available are in mammalian species whose body temperatures 1738

and other physiologic factors are different than those of avian and insect species. Therefore when 1739

the test is performed on avian or insect cells, the validity of the data is open to question unless a 1740

tumorigenic cell line of the species being tested is included as a positive control. The NRA/NCL 1741

may accept the results of an in vitro test such as growth in soft agar as a substitute for the in vivo 1742

test for avian and insect cell lines. However, as mentioned below, correlations of in vitro tests 1743

with in vivo tests are imperfect. This should be discussed with the NRA/NCL. 1744

1745

A new diploid cell line (i.e. other than WI-38, MRC-5, and FRhL-2) should be tested for 1746

tumorigenicity as part of the characterization of the cell line, but should not be required on a 1747

routine basis. 1748

1749

Many CCLs are classified as tumorigenic because they possess the capacity to form tumors in 1750

immunosuppressed animals such as rodents (e.g., BHK-21, CHO, HeLa). Some CCLs become 1751

tumorigenic at high PDLs (e.g., Vero), even though they do not possess this capacity at lower 1752

PDLs at which vaccine manufacture occurs [ 74, 76]. A critical feature regarding the 1753

pluripotency of embryonic SCLs, even though they display a diploid karyotype, is that 1754

they should form 'teratomas' in immunocompromised mice. 1755

1756

The expression of a tumorigenic phenotype can be quite variable from one CCL to another, and 1757

even within different sub-lines of the same CCL. This range of variability, from nontumorigenic, 1758

to weakly tumorigenic, to highly tumorigenic, has been viewed by some as indicating different 1759

degrees of risk when they are used as substrates for the manufacture of biological products 1760

[ 10, 11]. 1761

1762

If the CCL has already been demonstrated to be tumorigenic (e.g., BHK-21, CHO, HEK293, 1763

Cl27), or if the class of cells to which it belongs is tumorigenic (e.g., hybridomas), it may not be 1764

necessary to perform additional tumorigenicity tests on cells used for the manufacture of 1765

therapeutic products. Such cell lines may be used as cell substrates for the production of 1766

biological products if the NRA/NCL has determined, based on characterization data as well as 1767

manufacturing data, that issues of purity, safety, and consistency have been addressed. A new 1768


cell line (DCL, SCL, or CCL) should be presumed to be tumorigenic unless data demonstrate 1769

that it is not. If a manufacturer proposes to characterize the cell line as non-tumorigenic, the 1770

following tests should be undertaken. 1771

1772

Cells from the MCB or WCB propagated to the proposed in vitro cell age used for production or 1773

beyond should be examined for tumorigenicity in a test approved by the NRA/NCL. The test 1774

should involve a comparison between the cell line and a suitable positive reference preparation 1775

(e.g., HeLa cells), and a standardized procedure for evaluating results. 1776

1777

B.8.2 Type of test animals 1778

A variety of animal systems have been used to assess the tumorigenic potential of cell lines. 1779

Table 4 lists several examples of such tests along with advantages and disadvantages of each. 1780

Because assessing the tumorigenic phenotype of a cell substrate requires the inoculation of 1781

xenogeneic or allogeneic cells, the test animal should be rendered deficient in cytotoxic T-1782

lymphocyte (CTL) activity. This can be accomplished either by the use of rodents that are 1783

genetically immunocompromised (e.g., nude mice, SCID mice) or by inactivating the T-cell 1784

function with anti-thymocyte globulin (ATG), anti-thymocyte serum (ATS), or anti-lymphocyte 1785

serum (ALS). The use of animals with additional defects in NK-cell function, such as, the SCID-1786

NOD mouse, the SCID-NOD-gamma mouse, and the CD3 epsilon mouse, has not yet been 1787

explored for cell-substrate evaluation, but they might offer some advantages. In addition to these 1788

systems, several other in vivo systems, such as the hamster cheek pouch model and ATG-treated 1789

non-human primates (NHPs), have been used in the past, but rarely at present. 1790


Table 4. In vivo tests to assess the tumorigenic potential of inoculated cells 1791

Test & Brief Description Advantage(s) Disadvantage(s) Refs

Adult Athymic mouse (Nu/Nu genotype):

Animals inoculated by the i.m. or s.c. route with cells to be tested

• Animals readily available

• No immunosuppression required

• Higher frequency of spontaneous tumors than in other animal models that are not genetically immunosuppressed

• Low sensitivity for assessing the metastatic potential of the inoculated cells

66

Newborn athymic mouse

Animals inoculated by the s.c. route with cells to be tested


• More sensitive than adults

• Low sensitivity for assessing the metastatic potential of the inoculated cells

• Since litters include heterozygous mice, twice the number of animals must be inoculated in order to be sure that a sufficient number of homozygous mice have been included.

• Cannibalism of newborns by the mother

67

SCID mouse:

Animals receive intra-dermal. or intra-kidney capsule inoculation of test cells


• Potentially increased sensitivity


• Highly susceptible to viral, bacterial, and fungal infections

• Infections can affect the results and reproducibility of studies

• Spontaneous thymic lymphomas may occur

68

Newborn rat:

Animals immunosuppressed with ATG followed by i.m. or s.c. inoculation of cells to be tested


• Sensitive model for detecting metastases

• Very low frequency of spontaneous tumor formation

• Standardized ATG not available as a commercial product

• Careful qualification and characterization of the ATG is required to find the balance between immunosuppressive capacity and toxicity

69

Newborn hamster or mouse:

Animals immunosuppressed with ATS followed by i.m. or s.c. inoculation of cells to be tested


• Cannibalism of newborns by mother

• Standardized ATS not available and difficult to balance toxicity vs. immunosuppressive capacity

70

Newborn hamster or mouse:

Animals immunosuppressed with ALS followed by i.m. or s.c. inoculation of cells to be tested

• Increased sensitivity compared to the HCP test


• Cannibalism of newborns by mother

• Standardized ALS not available and difficult to balance toxicity vs. immunosuppressive capacity

71

Hamster cheek pouch (HCP):

Animals immunosuppressed with cortisone followed by inoculation of the cells to be tested into the cheek pouch


• Lower sensitivity than newer models 72

Nonhuman primates:

Animals immunosuppressed with ATG followed by inoculation of cells to be tested into the muscle of the four limbs

• Species closer to human

• Standardized ATG not available

• Animals not readily available

• Expense and limited availability preclude using large numbers

• Animal welfare principles mandate against use of NHP if same results can be obtained from lower specie

73


Although all of the animal models listed in Table 4 have been used to assess the tumorigenicity 1792

of cells, several sensitivity parameters from studies using positive control cells should be 1793

considered when attempting to compare the various in vivo tumorigenicity models: i) frequency 1794

of tumour formation; ii) time to appearance of tumors; iii) size of tumours; iv) lowest number of 1795

inoculated tumour cells that result in tumour formation; and v) metastatic tumor formation. 1796

Factors i, ii, iii, and v usually depend on the number of cells inoculated (i.e., they are dose-1797

dependent). In addition, the rate of spontaneous tumour formation should be considered. 1798

Although comparisons of two or more assays have been reported in the literature [70,74,75] none 1799

of them take into account all of these factors, nor do they use the same tumorigenic cell lines. 1800

Thus, it is not possible to draw definitive conclusions on the relative sensitivity of the various 1801

tumorigenicity assays. Nevertheless, the following points appear to be generally accepted: i) the 1802

ATS-treated newborn rat and the ATG-treated nonhuman primate systems are the most sensitive 1803

to assess the metastatic potential of inoculated cells; ii) ATS and ATG provide better 1804

immunosuppression than ALS; iii) the nude mouse has a more well-defined level of 1805

immunosuppression than those that depend on ALS, ATS, or ATG, and inter-laboratory 1806

comparisons of nude mouse data is more likely to yield valid conclusions. 1807

1808

The overall experience during the past 30 years, and taking into consideration the points 1809

mentioned above, has led to the conclusion that the athymic nude mouse is an appropriate test 1810

system for determining the tumorigenic potential of cells proposed for use in the production of 1811

biologicals. The major advantages of the athymic nude mouse system are that it is easier to 1812

establish and standardize and is generally available; while the newborn rat system is more 1813

sensitive for assessing the metastatic potential of tumorigenic cells. In some cases, it might be 1814

preferable to use newborn athymic nude mice, as these animals are more sensitive than adults for 1815

the detection of weakly tumorigenic cells [76]. A tumorigenicity testing protocol using athymic 1816

nude mice is provided as Appendix 1. The animal system selected should be approved by the 1817

NRA/NCL. 1818

1819

B.8.3 Point in the life history of the cells at which they should be tested 1820

Investigation of tumorigenicity should form part of the early evaluation of a new cell substrate 1821

for use in production. Cells from the MCB or WCB, propagated to the proposed in vitro cell age 1822


used for production or beyond should be examined for tumorigenicity. The three extra population 1823

doublings ensure that the results of the tumorigenicity test can be used in the assessment of 1824

overall safety of the product even under the assumption of a "worst case" situation, and therefore 1825

provides a safety buffer. 1826

1827

B.8.4 Use of control cells 1828

The tumorigenicity test should include a comparison between the CCL and a positive control 1829

reference preparation such as HeLa cells from the WHO cell bank. This source is preferred in 1830

order to standardize the test among laboratories so that the cumulative experience over time can 1831

be assessed and made available to NRAs/NCLs and manufacturers to assist them in the 1832

interpretation of data. However, other sources for establishing positive-control cells may be 1833

acceptable. The purpose of the positive control is to assure that an individual test is valid by 1834

demonstrating that the animal model has the capacity to develop tumors from inoculated cells 1835

(i.e., a negative result is unlikely to be due to a problem with the in vivo model). If the positive 1836

control cells fail to develop tumours at the expected frequency, then this could be indicative of 1837

problems with the animals or at the testing facility, such as infections, which can reduce the 1838

efficiency of tumour development. 1839

1840

When the cell substrate has been adapted to growth in serum-free medium, which might contain 1841

growth factors and other components that could influence growth as well as detection of a 1842

tumorigenic phenotype, consideration should be given to processing the positive-control cells in 1843

the same medium. Whenever possible, both the test article and the positive-control cells should 1844

be resuspended in the same medium, such as phosphate-buffered saline (PBS) for inoculation. 1845

1846

In designing a tumorigenicity protocol, it is important to recognize that tumors arise 1847

spontaneously in nude mice and that the incidence of such tumors increases with the age of the 1848

mice. Therefore, databases (both published data and the unpublished records/data of the animal 1849

production facility that supplied the test animals) of rates of spontaneous neoplastic diseases in 1850

nude mice should be taken into account during the assessment of the results of a tumorigenicity 1851

test. Generally, negative controls are not recommended because the rates of spontaneous 1852

neoplastic diseases in nude mice are low and small numbers of negative control animals are 1853


unlikely to provide meaningful data. However, if negative control cells such as WI-38, MRC-5, 1854

or FRhl-2 are included, clear justification for including them should be provided. For example, if 1855

serum-free medium is used to grow the cell substrate, it is conceivable that growth factors may 1856

influence the appearance of spontaneous tumors, therefore negative-control cells suspended in 1857

the same medium may be needed to interpret the test results. 1858

1859

B.8.5 Number of test animals 1860

To determine whether the cells being characterized have the capacity to form tumors in animals, 1861

the cells being tested, the reference positive control cells, and if any, the reference negative 1862

control cells should be injected into separate groups of 10 animals each. In a valid test, 1863

progressively growing tumors should be produced in at least 9 of 10 animals injected with the 1864

positive reference cells. 1865

1866

B.8.6 Number of inoculated cells 1867

Each animal should be inoculated intramuscularly or subcutaneously [77] with a minimum of 107 1868

viable cells. If there is no evidence of a progressively growing nodule at the end of the 1869

observation period, the cell line may be considered to be non-tumorigenic. If the cell line is 1870

found to be tumorigenic, the NRA/NCL might request additional studies to be done to determine 1871

the level of tumorigenicity. This can be done with dose-response studies, where doses of 107, 1872

105, 10

3 and 10

1 are inoculated, and the data can be expressed as tumor-producing dose at the 1873

50% endpoint (TPD50 value) [ 11]. 1874

1875

B.8.7 Observation period 1876

Animals are examined weekly by observation and palpation for evidence of nodule formation at 1877

the site of injection. The minimum observation period depends on the test system selected. In the 1878

case of the nude mouse, a minimum of 3 months is recommended. A shorter period is 1879

recommended for the ATS newborn rat because the immunosuppressive effect of the ATS 1880

declines after the final injection at two weeks. 1881

1882

B.8.8 Assessment of the inoculation site over time (progressive or regressive growth) 1883


If nodules appear, they are measured in two perpendicular dimensions, the measurements being 1884

recorded weekly to determine whether the nodule grows progressively, remains stable, or 1885

decreases in size over time. Animals bearing nodules that are progressing should be sacrificed 1886

before the end of the study if the tumor reaches the limit set by authorities for the humane 1887

treatment of animals. Animals bearing nodules that appear to be regressing should not be 1888

sacrificed until the end of the observation period. Cell lines that produce nodules that fail to grow 1889

progressively are not considered to be tumorigenic. If a nodule persists during the observation 1890

period and it retains the histopathological characteristics of a tumor, this should be investigated 1891

further and discussed with the NRA/NCL. 1892

1893

B.8.9 Final Assessment of the inoculation site 1894

At the end of the observation period, all animals, including the reference group(s), are euthanized 1895

and examined for gross and microscopic evidence of the growth of inoculated cells at the site of 1896

injection and in other sites. 1897

1898

B.8.10 Evaluation of animals for metastases 1899

Animals are examined for microscopic evidence of metastatic lesions in sites such as the liver, 1900

heart, lungs, spleen, and regional lymph nodes. 1901

1902

B.8.11 Assessment of metastases (if any) 1903

Any metastatic lesions are examined further to establish their relationship to the primary tumor at 1904

the injection site. If what appears to be a metastatic tumor differs histopathologically from the 1905

primary tumor, it is necessary to consider the possibility that this tumor either developed 1906

spontaneously, or that it was induced by one or more of the components of the cell substrate such 1907

as an oncogenic virus. This may require further testing of the tumor itself or the tumorigenicity 1908

assay may need to be repeated. In such cases, appropriate follow-up studies should be discussed 1909

and agreed with the NRA/NCL. (also see B.9 Oncogenicity) 1910

1911

B.8.12 Interpretation of results 1912

A CCL is considered to be tumorigenic if at least 2 of 10 animals develop tumors at the site of 1913

inoculation within the observation period. However, the reported rate of spontaneous neoplastic 1914


diseases in the test animals should be taken into account during the assessment of the results. In 1915

addition, the histopathology of the tumors must be consistent with the inoculated cells and a 1916

genotypic marker should show that the tumor is not of nude mouse origin. 1917

1918

If only one of 10 animals develops a tumor, further investigation is appropriate to determine, for 1919

example, if the tumor originated from the cell substrate inoculum or the host animal, and whether 1920

there are any viral or inoculated cell DNA sequences present. The NRA/NCL should be 1921

consulted in this regard. 1922

1923

The dose-response of the CCL may be studied in a titration of the inoculum as part of the 1924

characterization of the CCL. The need for such data will depend upon many factors specific to a 1925

given CCL and the product being developed. The NRA/NCL should be consulted in this regard. 1926

1927


Cell Banks: EOPC from the MCB or first WBC 1929


1931

B.9 Oncogenicity 1932

B.9.1 Tests for oncogenicity 1933

While tumorigenicity is the property of cells to form tumors when inoculated into susceptible 1934

animals, oncogenicity is the activity that induces cells of an animal to become tumor cells. As 1935

such, tumors that arise in a tumorigenicity assay contain cells derived from the inoculated cells, 1936

while tumors that arise in an oncogenicity assay are derived from the host. Oncogenic activity 1937

from cell substrates could be due to either the cell substrate DNA (and perhaps other cellular 1938

components), or an oncogenic agent present in the cells. Although there might be a perception 1939

that the cellular DNA from highly tumorigenic cells would have more oncogenic activity than 1940

the DNA of weakly or non-tumorigenic cells, at this time, it is not known if there is a 1941

relationship between the tumorigenicity of a cell and the oncogenicity of its DNA. Nevertheless, 1942

the NRA/NCL might require oncogenicity testing of the cell lysate from a new cell line (i.e., 1943

other than those such as CHO, NSO, Sp2/0, and low passage Vero, for which there is 1944

considerable experience) that is tumorigenic in several animal model systems (see below) 1945


because of the perception that a vaccine manufactured in such a cell line poses a neoplastic risk 1946

to vaccine recipients. 1947

1948

The major complication in assaying cellular DNA in animals arises from the size of the 1949

mammalian genome. Because the mammalian haploid genome is approximately 3 x 109 base 1950

pairs (bp) whereas the size of a typical oncogene could be 3-30 x 103 bp, the concentration of an 1951

oncogene in cellular DNA expression systems would be about 105 to 10

6 fold less concentrated 1952

than a plasmid containing the same oncogene. As a consequence, if 1 µg of an oncogene 1953

expression plasmid induces a tumor in an experimental animal model, the amount of cellular 1954

DNA that would contain a similar amount of the same oncogene is 105 µg to 10

6 µg (i.e., 100 mg 1955

to 1 g). To date, two studies have indicated that ~10 µg of expression plasmids for cellular 1956

oncogenes can be oncogenic in mice. Therefore, more sensitive in vivo assays need to be 1957

developed before the testing of the oncogenic activity of cellular DNA becomes practicable. 1958

Recent results suggest that the sensitivity of the assay can be increased by several orders of 1959

magnitude with the use of certain immune-compromised strains of mice prone to develop 1960

tumours after inoculation with oncogenes [ 40]. Thus, it may be possible to assess the oncogenic 1961

activity of cellular DNA in the future. However, at present there is no standardised in vivo 1962

oncogenicity test for cellular DNA. 1963

1964

Several in vitro systems, such as scoring the neoplastic transformation of NIH 3T3 cells in a 1965

focus-forming assay following transfection of oncogenic DNA [ 78, 79, 80] have been used to 1966

assess oncogenicity, but it is not clear how these assays reflect the oncogenic activity of DNA in 1967

vivo, since they predominantly detect the oncogenic activity of activated ras-family members, 1968

and thus it is unclear how these assays can assist in estimating risk associated with the DNA or 1969

cell lysate from a cell substrate. 1970

1971

Based on experience with DCLs WI-38, MRC-5, and FRhL-2, testing of new MCBs of these cell 1972

lines for oncogenicity is not recommended. Other DCLs for which there is substantial experience 1973

also may not need to be tested. The NRA/NCL should be consulted in this regard. As stated in 1974

Section B.8.1, a new CCL should be presumed to be tumorigenic unless data demonstrate that it 1975

is not. If a manufacturer demonstrates 1976


that a new CCL is non-tumorigenic, oncogenicity testing on cell DNA and cell lysates might not 1977

be required by the NRA/NCL. 1978

1979

When appropriate, and particularly for vaccines, cell DNA and cell lysates should be examined 1980

for oncogenicity in a test approved by the NRA/NCL. 1981

In some countries, the following testing strategy is used. 1982

Type of test animals 1983

Newborn (i.e., <3 days old) nude mice, newborn hamsters, and 1984

newborn rats have been used to assess the oncogenic potential of cell 1985

lines. At this stage, it is not possible to draw definitive conclusions on 1986

the relative sensitivity of the three animal assays for oncogenicity, and 1987

testing is recommended in each of them. 1988

1989

Point in the life history of the cells at which they should be tested 1990

Cells from the MCB or WCB, propagated to the proposed in vitro cell 1991

age for production or beyond should be examined for oncogenicity. The 1992

three extra population doublings ensure that the results of the 1993

oncogenicity test can be used in the assessment of overall safety of the 1994

product even under the assumption of a "worst case" situation and 1995

therefore provides a safety buffer. 1996

1997

Use of controls 1998

The purpose of the positive control is to assure that an individual test is 1999

valid by demonstrating that the animal model has the capacity to 2000

develop tumors from inoculated cell components (i.e., a negative result 2001

is unlikely to be due to a problem with the in vivo test). However, an 2002

appropriate positive control for cell DNA and a cell lysate test is not 2003

clear. As this test is designed primarily to detect oncogenic viruses 2004

rather than oncogenes, the use of a DNA positive control might not be 2005

suitable, both because of the nature of the assay and because DNA 2006

might not be stable in a cellular lysate. 2007

2008

Whether a negative control arm, such as PBS, is included should be 2009

discussed with the NRA/NCL. An advantage of including a negative-2010

control arm is that the frequency of tumor induction with lysates is 2011


expected to be low and may approximate the spontaneous tumour 2012

frequency in the indicator rodent. 2013

2014

Number of test animals 2015

While the number of animals in a tumorigenicity test can be 10 per 2016

group, the number in an oncogenicity test should be larger due to the 2017

lower expected tumor incidence. The number per group should be 2018

discussed with the NRA/NCL. 2019

2020

Inoculation of test material 2021

Cell lysate 2022

A lysate of the cells should be prepared by a method that avoids virus 2023

disruption while allowing maximum virus release (e.g., multiple 2024

freeze/thaw cycles followed by low-speed centrifugation). Each animal 2025

should be inoculated subcutaneously above the scapula with a lysate 2026

obtained from 107 cells. Before inoculation, it should be determined 2027

that no viable cells are present, as the development of tumours from 2028

cells would invalidate the test. If tumours are observed in this assay, the 2029

species of origin will need to be confirmed. The species of tumours that 2030

arise in a tumorigenicity assay is that of the cell substrate, while the 2031

species of tumours that arise in an oncogenicity assay is that of the host 2032

(e.g., rodent). The cell lysate is suspended in PBS and inoculated in a 2033

volume of 50-100 µL into newborn nude mice, newborn hamsters, and 2034

newborn rats [ 81]. If there is no evidence of a progressively growing 2035

nodule at the end of the observation period, the cell line may be 2036

considered not to possess oncogenic activity. 2037

2038

Observation period 2039

Animals are examined weekly by observation and palpation for 2040

evidence of nodule formation at the site of injection. The observation 2041

period should be at least 6 months. 2042

2043

Assessment of the inoculation site over time (progressive or 2044

regressive growth) 2045

If one or more nodules appear, they are measured in two perpendicular 2046

dimensions, the measurements being recorded weekly to determine 2047

whether the nodule grows progressively, 2048


remains stable, or decreases in size over time. Animals bearing nodules 2049

that are progressing should be sacrificed when the nodule reaches a size 2050

of approximately 2 cm in diameter, unless a lower limit has been 2051

established by authorities for the humane treatment of animals. 2052

2053

Final Assessment of the inoculation site 2054

At the end of the observation period, all animals, including the 2055

reference group(s), are sacrificed and examined for gross and 2056

microscopic evidence of tumor formation at the site of injection and in 2057

other sites. Any tumor that is identified is divided into three equal parts: 2058

a) fixed in formalin for histopathology; b) used to establish a cell line, 2059

when possible; and c) frozen for subsequent molecular analysis. 2060

2061

Evaluation of animals for metastases 2062

Animals are examined for microscopic evidence of metastatic lesions in 2063

sites such as the liver, heart, lungs, spleen, and regional lymph nodes. 2064

2065

Assessment of metastases (if any) 2066

Any metastatic lesions are examined further to establish their 2067

relationship to the primary tumor at the site of inoculation. If what 2068

appears to be a metastatic tumor differs histopathologically from the 2069

primary tumor, it is necessary to consider the possibility that this tumor 2070

developed spontaneously. This may require further testing of the tumor 2071

itself. In such cases, appropriate follow-up studies should be discussed 2072

and agreed with the NRA/NCL. 2073

2074

Interpretation of results 2075

If tumours arise in the cell lysate assay, then this could be induced by 2076

an oncogenic virus. Because of the implications for the use of a cell 2077

substrate that contains an oncogenic agent for a biological, the 2078

NRA/NCL should be consulted to consider additional experiments to 2079

identify the oncogenic agent and to determine the suitability of the use 2080

of the CCL. 2081

2082


Cell Banks: MCB or WCB taken to EOP 2084


Cell Types: CCL, SCL (recommended when tumorigenic cells are used in vaccine 2085

production) 2086

2087

B.10 Cytogenetics 2088

B.10.1 Characterization 2089

Chromosomal characterization and monitoring were introduced in the 1960s to support the safety 2090

and acceptability of human DCLs as substrates for vaccine production. Human DCLs differ from 2091

CCLs by retaining the characteristics of normal cells, including the normal human diploid 2092

karyotype. A significant amount of data have been accumulated since then, and this has led to the 2093

conclusion that less extensive cytogenetic characterization is appropriate because of the 2094

demonstrated karyotypic stability of human DCLs used in vaccine production [82]. Thus, the use 2095

of karyology as a quality control test is unnecessary for well-characterized and unmodified 2096

human DCLs (e.g., Wi-38, MRC-5) and FRhL-2. 2097

2098

Cytogenetic data may be useful for the characterization of CCLs especially when marker 2099

chromosome(s) are identified. Such data might be helpful in assessing the genetic stability of the 2100

cell line as it is expanded from the MCB to the WCB, and finally to production cultures (see 2101

B.3). The following recommendations are appropriate for the characterization of DCL and CCL 2102

cell banks. 2103

2104

Cytogenetic recharacterization of DCLs WI-38, MRC-5 and FRhL-2 should not be required, 2105

unless the cells have been genetically modified or the culture conditions have been changed 2106

significantly, since such data are already available ( 19, 20, 21). However, for each WCB 2107

generated, manufacturers should confirm once, that the cells grown in the manner to be used in 2108

production are diploid and have the expected lifespan. 2109

2110

For the determination of the general character of a new or previously uncharacterized DCL, 2111

samples from the MCB should be examined at approximately four equally spaced intervals 2112

during serial cultivation from the MCB through to the proposed in vitro cell age used for 2113

production or beyond. Each sample should consist of a minimum of 100 cells in metaphase and 2114


should be examined for exact counts of chromosomes, and for breaks and other structural 2115

abnormalities. 2116

Giemsa-banded karyotypes of an additional five metaphase cells in each of the four samples may provide additional 2117

useful information. ISCN [ 83] 400 band is the minimum acceptable level of Giemsa-banding analysis for human 2118

cells. 2119

2120

Stained slide preparations of the chromosomal characterization of the cells (i.e., DCL, CCL), or 2121

photographs of these, should be maintained permanently as part of the cell line record. 2122

2123


Cell Banks: MCB 2125

Cell Types: DCL, SCL, CCL (as a test for genetic stability, when appropriate), 2126

2127

B.11 Microbial agents 2128


While many biological production systems require animal cell substrates, such cells are subject 2130

to contamination with and have the capacity to propagate extraneous, inadvertent, or so-called 2131

adventitious organisms such as mycoplasma and viral agents. In addition, some animal cells 2132

contain endogenous agents such as retroviruses that also may be of concern. Testing for both 2133

endogenous (e.g., retroviruses) and adventitious agents (e.g., mycoplasmas) is described in the 2134

succeeding sections. In general, cell substrates contaminated with microbial agents are not 2135

suitable for the production of biological products. However, there are exceptions to this general 2136

rule. For example, the CHO and other rodent cell lines that are used for the production of highly 2137

purified recombinant proteins express endogenous retroviral particles. Risk versus benefit must 2138

be considered when determining the suitability of a cell substrate for the production of a specific 2139

product. Further, risk mitigation strategies during production, including purification (removal) 2140

and inactivation by physical, enzymatic, and/or chemical means, should be implemented 2141

whenever appropriate and feasible. Even though a cell substrate might be unacceptable for some 2142

products such as a live viral vaccine subjected to neither significant purification nor inactivation, 2143

that same cell substrate might be an acceptable choice for a different type of product such as a 2144


highly purified recombinant protein or monoclonal antibody for which risk mitigation has been 2145

achieved by significant and validated viral clearance in the production process. 2146

2147

Testing for various microbial agents can be performed at most stages of manufacturing, 2148

including the MCB, WCB, production harvests and/or EOP cells. Not all tests must be 2149

performed at every stage however. A testing strategy should be developed. One strategy is to 2150

perform exhaustive testing at the MCB level and to perform more limited testing on the WCB 2151

derived from the MCB. This more limited testing would be selected on the basis of those agents 2152

that could potentially be introduced during the production of the WCB from the MCB. Testing 2153

would not need to be replicated for agents that could only have been present prior to the 2154

production of the MCB (e.g., an endogenous retrovirus, or BVDV from serum used for 2155

developing the cell seed or in the legacy of establishing the cell line). 2156

2157

However, if the number of vials of a MCB is limited, an alternative strategy would be to conduct 2158

the more exhaustive testing on each and every WCB made from that MCB, and to limit testing 2159

on the MCB itself. An advantage to the strategy of performing more exhaustive testing on each 2160

WCB is that it provides a greater opportunity for amplification of any agents that may have been 2161

introduced earlier and through to production of the WCBs. There are advantages and 2162

disadvantages to more extensive testing of the MCB or the WCB, and consideration should be 2163

given to what is most appropriate for the particular product(s) to be manufactured using a given 2164

cell bank. Tables 6 and 7 may be consulted to compare and contrast these two strategies. 2165

Consultation with the NRA/NCL should be considered prior to implementation to determine 2166

whether a proposed testing strategy is acceptable. 2167

2168

Additionally, EOPC propagated to or beyond the maximum in vitro age, should be tested once 2169

for each commercial production process. This does not need to be repeated for each WCB. 2170

2171

B.11.2 Viruses 2172

Manifestations of viral infections in cell cultures vary widely among the broad array of virus 2173

families that are potential contaminants; thus, the methods used to detect them vary. Lytic 2174

infections frequently are detected by the CPE they cause. However, in some cases such as non-2175


cytopathic BVDV, no CPE is observed. Viruses also may be present latently (e.g., 2176

herpesviruses) or endogenously (in the germline, e.g., retroviral proviruses). Such inapparent 2177

infections might require specific techniques designed to reveal their presence, such as molecular 2178

and immunological methods, electron microscopy, induction of a lytic infection by exposing the 2179

cells to special conditions (e.g., chemical induction; heat shock), or whole genome sequencing. 2180

2181

The strategy developed to test cell substrates for viruses should take into consideration the 2182

families of viruses and specific viruses that may be present in the cell substrate. Consideration 2183

should be given to the species and tissue source from which the cell substrate originated, and to 2184

the original donor's medical history in the case of human-derived cell substrates or to the 2185

pathogen status of donor animals in the case of animal-derived cell substrates. In addition, 2186

consideration should be given to viruses that could contaminate the cell substrate from the 2187

donors or from animal- or human-derived raw materials used in the establishment and passage 2188

history (legacy) of the cell substrate prior to and during cell banking or production (e.g., serum, 2189

trypsin, animal- or human-derived media components, antibodies used for selection, or animal 2190

species through which the cell substrate may have been propagated), as well as laboratory 2191

contamination from operators or other cell cultures. 2192

2193

Tests should be undertaken to detect, and where possible identify, any endogenous or exogenous 2194

agents that may be present in the cells. Attention should be given to tests for agents known to 2195

cause an inapparent infection in the species from which the cells were derived, making it more 2196

difficult to detect (e.g., simian virus 40 in Rhesus monkeys). 2197

2198

Primary cells are obtained directly from the tissues of healthy animals, and are more likely to 2199

contain adventitious agents than banked, well-characterized cells. This risk with primary cells 2200

can be mitigated by rigorous qualification of source animals and the primary cells themselves. 2201

When feasible, animals from which primary cultures are established should be from genetically 2202

closed flocks, herds, or colonies monitored for freedom from pathogens of specific concern. 2203

Such animals are known as specific-pathogen-free (SPF). The term "closed" refers to the 2204

maintenance of a group (flock, herd, and colony) free from introduction of new animals (new 2205

genetic material that could introduce new retroviral proviruses). Many live viral vaccines are 2206


commonly produced in primary cells and undergo little purification during production. In such 2207

cases, and when feasible, the use of SPF animals is highly recommended. Documentation of the 2208

status of the source animals should be provided to the NRA/NCL. Animals that are not from 2209

closed flocks, herds, or colonies should be quarantined and thoroughly evaluated for a period 2210

sufficient to detect signs of disease or infection (e.g., monkeys are generally quarantined for 6 or 2211

more weeks [ 84]). Such animals also should be screened serologically for appropriate 2212

adventitious agents to determine their suitability as a source for the primary cell substrate. 2213

Animal husbandry practices should be documented. Even so, viral contamination of the cells 2214

may not be excluded from all cultures. For example, contamination of primary monkey kidney 2215

cells with foamy virus or simian cytomegalovirus is common in the absence of specific concerted 2216

efforts to prevent these contaminations. 2217

2218

For primary cell cultures, the principles and procedures outlined in Part C, Requirements for 2219

Poliomyelitis Vaccine (Oral) [ 84], together with those in section A.4 of Requirements for 2220

Measles, Mumps and Rubella Vaccines and Combined Vaccine (Live) [ 85] may be followed. 2221

2222

The production of viral vaccines, such as those against smallpox or rabies, originally required the 2223

use of living animals and the great range of possible viral contaminants only became apparent as 2224

cell culture methods were developed. For example, human enteroviruses were not recognised 2225

until the development of monkey kidney cell cultures in which they could produce cytopathic 2226

effects because the disease produced in humans is either relatively mild or in some cases non-2227

existent. It was also clear that viruses could be detected in some systems but not others; for 2228

instance the polyomavirus SV40 does not produce a cytopathic effect in cultures from rhesus 2229

monkey kidney cells (in which much of the early polio vaccines were produced) but will do so in 2230

cultures from cynomolgous monkey kidney cells. The suspicion was therefore that there were 2231

many viruses in the culture systems of the time and that they were detected only if the assays 2232

were appropriate. This remains an accurate view and has lead to a range of different approaches 2233

to try and catch all contaminants. 2234

2235

Coxsackie viruses are named after the town in New York where they were first identified and 2236

were historically detected by their effects in mice. Coxsackie B viruses produce clinical signs 2237


and death in adult mice while Coxsackie A viruses will affect only suckling mice. For many 2238

years tissue culture methods were a less reliable method of detection of Coxsackie A viruses than 2239

suckling mice and the continued use of these animals in cell bank characterisation reflects this. 2240

2241

In the 1940s, embryonated chicken eggs were a popular substrate for the growth and assay of 2242

viruses such as influenza, measles, mumps, yellow fever and vaccinia. They therefore appear to 2243

have a wide range of susceptibility. Simian viruses such as SV5 or viruses such as Sendai also 2244

grow well in them; as many are paramyxoviruses with haemagglutinating (HA) activity. The 2245

egg-based assays include tests for HA activity as well as death of the embryos. 2246

2247

A range of tissue culture cells is also used, typically including one human, one of the same 2248

species as the production cell and one other. The hope is that the range will catch other viruses 2249

not detected by other means, although in practice it is wise to assume that there is no such thing 2250

as a generic detection method; for example cells from an inappropriate monkey species would 2251

not necessarily detect SV40 whereas other species will (e.g., Vero cells). In certain 2252

circumstances, where a virus is of particular concern specific tests have been applied. For 2253

example, herpes B is a common infection of monkeys in the absence of precautions such as 2254

quarantine and clinical evaluation of the donor animals, and has very serious effects on infected 2255

humans. While herpes B virus was routinely detected by the use of primary rabbit kidney cell 2256

cultures, established rabbit cell lines are now acceptable for this purpose [86]. Another example 2257

is Marburg virus that in the 1970s caused a number of deaths in workers who handled monkeys 2258

that were to be used in a vaccine production facility. The incident might have been avoided had 2259

the animals been adequately quarantined. A specific test in guinea pigs was introduced and 2260

maintained for a number of years to ensure the absence of the agent. 2261

2262

There is a disparate range of tests that have been or are still used with the aim of detecting any 2263

significant contaminant that may be present in cell cultures. Some, such as the rabbit kidney cell 2264

test, are very specific in intent while others may be expected to be more generic. In general 2265

however it is wise to assume that an assay will never be all encompassing whether based on 2266

historical virological approaches or more current methodologies. Thus, the consequences of 2267

deleting tests on the grounds of redundancy must be very carefully evaluated before any action is 2268


taken. On the other hand it is difficult to justify maintenance of a test if it only detects viruses 2269

also detected by other methods of equivalent sensitivity, comparable ease of use, and cost. Each 2270

of these considerations should be given when developing an appropriate testing strategy for the 2271

given cell bank. 2272

2273

B.11.2.1 Tests in animals and eggs 2274

The cells of the MCB and WCB are unsuitable for production if any of the animal or egg tests 2275

shows evidence of the presence of any viral agent attributable to the cell banks. 2276

2277

Generally, MCBs are thoroughly characterized by the methods listed below. WCBs may be 2278

characterized by an abbreviated strategy, when appropriate. However, an alternative strategy to 2279

this general rule may be used, as discussed in section B.11.2 above and as indicated in Table 7. 2280

These tests may be performed directly on cells or supernatant fluids or cell lysates from the bank 2281

itself or on cells or supernatant fluids or cell lysates from passaged cells from the bank that have 2282

been passaged to the proposed in vitro cell age for production or beyond. 2283

2284

B.11.2.1.1 Adult mice 2285

The original purpose of this test was for the detection of lymphocytic choriomeningitis virus 2286

(LCMV). The test in adult mice for pathogenic viruses includes inoculation by the 2287

intraperitoneal route (0.5 mL) with cells from the MCB or WCB, where at least l07 viable cells 2288

or the equivalent cell lysate are divided equally among at least ten adult mice weighing 15-20 g. 2289

2290

In some countries, the adult mice are also inoculated by the 2291

intracerebral route (0.03 mL). 2292

2293

In some countries at least 20 mice are required for each test. 2294

2295

The animals are observed for at least 4 weeks. Any animals that are sick or show any 2296

abnormality are investigated to establish the cause. Animals that do not survive the observation 2297

period should be examined for gross pathology and histopathology in order to determine a cause 2298

of death, and if a viral infection is indicated, efforts should be undertaken to identify the virus. 2299

Viral identification may involve culture and/or molecular methods. The test is not valid if more 2300


than 20% of the animals in either or both of the test and negative control groups (if used) become 2301

sick for non-specific reasons and do not survive the observation period. 2302

2303

In some countries, the adult mice are observed for 21 days. 2304

2305

If the cell substrate is of rodent origin, at least 106 viable cells or the equivalent cell lysate are 2306

injected intracerebrally into each of ten susceptible adult mice to test for the presence of LCMV. 2307

2308

In some countries, after the observation period, the animals are 2309

challenged with live LCMV to reveal the development of immunity 2310

against non-pathogenic LCMV contaminants resulting in otherwise 2311

inapparent infection. 2312

2313

B.11.2.1.1.1 Applicability 2314

Cell Banks: MCB, WCB, or EOPC 2315


2317

B.11.2.1.2 Suckling mice 2318

The original purpose of this test was for the detection of Coxsackie viruses. The test in suckling 2319

mice for pathogenic viruses includes inoculation by the intraperitoneal route (0.1 mL) with cells 2320

from the MCB or WCB; at least l07 viable cells or the equivalent cell lysate are divided equally 2321

between two litters of suckling mice, comprising a total of at least ten animals less than 24-hours 2322

old. 2323

2324

In some countries, the suckling mice are also inoculated by the 2325

intracerebral route (0.01 mL). 2326

2327

In some countries, 20 suckling mice are inoculated. 2328

2329




of death, and if a viral infection is indicated, efforts should be undertaken to identify the virus, 2333


where this is practicable. Viral identification may involve culture and/or molecular methods. In 2334

the case of suckling mice, it is often observed that those that perish are cannibalized by their 2335

mother and this renders determining a cause of death impossible (when they are fully 2336

cannibalized and no remains can be recovered). The test is not valid if more than 20% of the 2337

animals in either or both of the test group and negative control group (if used) do not survive the 2338

observation period. 2339

2340

In some countries, the suckling mice may be observed for a period of 2341

14 days followed by a sub-passage involving a blind passage (via 2342

intraperitoneal and intracerebral inoculation into at least 5 additional 2343

mice) of a single pool of the emulsified tissue (minus skin and viscera) 2344

of all mice surviving the original 14-day test. 2345

2346


Cell Banks: MCB, WCB or EOPC 2348


2350

B.11.2.1.3 Guinea pigs 2351

The original purpose of this test was for the detection of LCMV and Mycobacterium 2352

tuberculosis. When it is necessary to detect Mycobacterium species, a test in guinea pigs is 2353

performed and includes inoculation by the intraperitoneal route (5 mL) with cells from the MCB 2354

or WCB, where at least l07 viable cells are divided equally among the animals. 2355

In some countries, tests in five guinea-pigs weighing 350-450 g are also 2356

inoculated by the intracerebral route (0.1 mL) and observed for 42 days 2357

to reveal Mycobacterium tuberculosis and other species. 2358

2359







than 20% of the animals in either or both of the test and negative control (if used) groups do not 2365

survive the observation period. 2366

2367

The test in guinea-pigs for the presence of Mycobacterium may be replaced by an alternative in 2368

vitro method such as culture or PCR. 2369

2370



Cell Types: PCC, DCL, SCL, CCL (the latter three are dependent on legacy and current use of 2373

media components of animal origin that could result in contamination with Mycobacterial 2374

species) 2375

2376

B.11.2.1.4 Rabbits 2377

The original purpose of this test was for the detection of Herpes B virus. When it is necessary to 2378

detect simian herpes B virus, the test in rabbits for pathogenic viruses is performed and includes 2379

the inoculation by the intradermal (1 mL) and subcutaneous (>2 mL) routes with cells from the 2380

MCB or WCB, where at least l07 viable cells are divided equally among the animals. 2381

2382

In some countries, tests in five rabbits weighing 1.5-2.5 kg are 2383

inoculated by the subcutaneous route with either 2 mL or between 9 2384

and 19 mL. Consultation with the NRA/NCL regarding acceptable 2385

methods should be considered. 2386

2387

The test in rabbits for the presence of herpes B virus in primary simian cultures may be replaced 2388

by a test in rabbit kidney cell cultures ( 86). 2389

2390







than 20% of the animals in either the test or the negative control (if used) groups do not survive 2396

the observation period. 2397

2398




2402

B.11.2.1.5 Embryonated chicken eggs 2403

The original purpose of this test was for the detection of Sendai virus (SV-5). At least 106 viable 2404

cells from the MCB or WCB of avian origin, propagated to the proposed in vitro cell age for 2405

production or beyond are injected into the allantoic cavity of each of at least ten embryonated 2406

hens' eggs, and into the yolk sac of each of at least another ten embryonated hens' eggs. The eggs 2407

are examined after not fewer than 5 days of incubation. The allantoic fluids of the eggs is tested 2408

with red cells from guinea pig and chickens (or other avian species) for the presence of 2409

haemagglutinins. The test is not valid if more than 20% of the embryonated hens' eggs in either 2410

or both of the test group and negative control group (if used) are discarded for non-specific 2411

reasons. 2412

In some countries, the NRA/NCL also requires that other types of red 2413

cells, including cells from humans (blood group IV O), monkeys and 2414

chickens (or other avian species), should be used in addition to guinea-2415

pig cells. In all tests, readings should be taken after incubation for 30 2416

minutes at 0-4°C, and again after a further incubation for 30 minutes at 2417

20-25°C. For the test with monkey red cells, readings also should be 2418

taken after a final incubation for 30 minutes at 34-37°C. 2419

2420

In some countries, inoculation by the amniotic route is used. 2421

2422

Usually, the eggs used for the yolk sac test should be 5-7 days old. The eggs used for the 2423

allantoic cavity test should be 9-11 days old. 2424

2425

Alternative ages for the embryonated chicken eggs and alternative 2426

incubation periods are acceptable if they have been determined to be 2427

equivalent or better at detecting the presence in the test samples of the 2428


adventitious agents this test is capable of detecting when performed as 2429

above. 2430

2431

Embryos that do not survive the observation period should be examined for gross pathology in 2432

order to determine a cause of death, and if a viral infection is indicated, efforts should be 2433

undertaken to identify the virus. Viral identification may involve culture and/or molecular 2434

methods. 2435

2436


Cell Banks: MCB of avian origin, WCB of avian origin or EOPC 2438

Cell Types: avian PCC, DCL, SCL, CCL (also recommended for novel cell substrates) 2439

2440

B.11.2.1.6 Antibody-production tests 2441

Rodent cell lines are tested for species-specific viruses using mouse, rat and hamster antibody 2442

production tests, as appropriate. In vivo testing for lymphocytic choriomeningitis virus, including 2443

a challenge for non-lethal strains, is performed for such cell lines, as described in Section 2444

B.11.2.1.1. 2445

2446



Cell Types: PCC, DCL, SCL, CCL (recommended only for cells of rodent origin) 2449

2450

B.11.2.2 Tests in cell culture 2451

Tests in cell culture are capable of detecting a broad array of viral families. Readouts include 2452

monitoring the cultures periodically for CPE and tests for haemadsorbing and haemagglutinating 2453

viruses, which are conducted at the end of the culture period. In addition to the indicator cells 2454

described below, it may be appropriate to expand the different types of indicator cells used 2455

(beyond 2 or 3) to enable the detection of viruses with differing host requirements. Decisions 2456

about which cell lines to use as indicator cells should be guided by the species and legacy of the 2457

production cell substrate taking into the consideration the types of viruses to which the cell 2458

substrate could potentially have been exposed and thus, the viruses one would like to detect by 2459


this assay method. The cell substrate is unsuitable for production if any of the indicator cell 2460

cultures shows evidence of the presence of any viral agent attributable to the tested cell substrate. 2461

2462




2466

B.11.2.2.2 Indicator cells 2467

Live or disrupted cells and spent culture fluids of the MCB or WCB are inoculated onto 2468

monolayer cultures or cultivated with monolayer cultures of the cell types listed below, as 2469

appropriate. 2470

• Cultures (primary cells or CCL) of the same species and tissue type as that used for 2471

production. 2472

• Cultures of a human DCL. The original purpose of this test, utilizing primary human 2473

cells, was the detection of measles virus. But, where the cell substrate is of human origin, 2474

a simian kidney cell line should be used as the second indicator cell line. The original 2475

purpose of the use of this cell type was the detection of simian viruses. 2476

2477

In some countries, cultures of another (third) cell line from a different 2478

species are required. 2479

2480

In many circumstances, more than two cell lines may be necessary to 2481

cover the range of potential viral contaminants and typically, a third 2482

cell line would be used that is of simian origin, if the cell substrate is 2483

not of simian origin. 2484

2485

For new cell substrates, additional cell lines to detect viruses known to be potentially harmful to 2486

humans could be considered (e.g. for insect cell lines, if the cells selected for the above 2487

mentioned tests are not known to be permissive to insect viruses, an additional detector cell line 2488

should be included in the testing). 2489

2490


The cell bank sample to be tested is diluted as little as possible. Material from at least 107 cells 2491

and spent culture fluids are inoculated onto each of the indicator cell types. The resulting co-2492

cultivated or inoculated cell cultures are observed for evidence of viruses for at least 2 weeks. If 2493

the product is from a cell line known to be capable of supporting the growth of human or simian 2494

cytomegalovirus, HDC cultures are observed for at least 4 weeks. Extended (4-weeks) cell 2495

culture for the purposes of identifying human or simian cytomegalovirus can be replaced by the 2496

use of NAT to detect cytomegalovirus nucleic acid. 2497

2498

In some countries, a passage onto fresh cultures for an additional 2 2499

weeks is recommended for all indicator cultures. In some cases, it may 2500

be difficult to keep the cell cultures healthy for 2 weeks without 2501

subculturing. In those cases, it may be necessary to feed the cultures 2502

with fresh medium or to subculture after two weeks onto fresh cultures 2503

in order to be able to detect viral agents. 2504

2505

At the end of the observation period, samples of each of the co-cultivated or inoculated cell 2506

culture systems are tested for haemadsorbing and/or haemagglutinating viruses, as described in 2507

section B.11.2.1. 5. 2508

2509

B.11.2.2.3 Additional considerations to the tests in cell culture for insect viruses 2510

Many insect cell lines carry persistent viral infections that do not routinely produce a noticeable 2511

CPE. However, the viruses may be induced to replicate by stressing the cells using a variety of 2512

techniques such as increased/reduced culture temperature, heat-shock for a short period, super-2513

infection with other insect viruses, or chemical inducers. Therefore, the probability of detecting 2514

such low-level persistent infections may be increased by stressing the cells prior to analysis. 2515

2516

Intact or disrupted cells from a passage level at or beyond that equivalent to the EOPC is co-2517

cultivated with indicator cells from at least three different species of insect, in addition to the 2518

indicator cells as noted in section B.11.2.2.2. Cell lines should be selected on the following basis: 2519

one of the lines has been demonstrated to be permissive for the growth of human arboviruses, 2520

one has been shown to be permissive for the growth of a range of insect viruses, and the third has 2521

been derived from a species that is closely related to the host from which the MCB is derived (or 2522


another line from the same species). The cells are incubated at 28 ± 1°C, observed for a period of 2523

14 days, and examined for possible morphological changes. The cell-culture fluids from the end 2524

of the test period are tested for haemagglutinating viruses, or the intact cells from the end of the 2525

test period are tested for haemadsorbing viruses. The cells comply with the test if no evidence of 2526

any viral agent is found. 2527

2528

Several mosquito cell lines are available that are permissive for the growth of some human 2529

arboviruses and could be considered for these tests. Alternatively, BHK-21 cells could be 2530

considered for this purpose. The most permissive insect cell lines characterised to date have been 2531

derived from embryonic Drosophila tissues (e.g., Schneider's Line 2 and Line 1). While the 2532

mosquito and Drosophila cell lines may be suitable for some aspects of the testing, it should be 2533

noted that many insect cell lines are persistently infected with insect viruses that usually produce 2534

no obvious CPE. Demonstrating that the indicator cell lines are free from adventitious agents is 2535

an important pre-requisite to their use in the testing outlined above. Consideration should also be 2536

given to risk mitigation strategies as discussed above for highly purified products for which viral 2537

clearance can be achieved and validated. 2538

2539

B.11.2.3 Transmission electron microscopy 2540

At least 200 cells from the MCB, WCB, or EOPC are examined by transmission electron 2541

microscopy (TEM) for evidence of contamination with microbial agents. Methods include 2542

negative staining and thin section. A discussion of these methods is provided by Bierley et al. 2543

[ 87]. It may be appropriate to examine more cells in some cases, such as discussed below for 2544

insect cell lines, and the NRA/NCL should be consulted in this regard. Any unusual or equivocal 2545

observations that might be of microbiological significance should be noted and discussed with 2546

the NRA/NCL. 2547

2548

TEM can detect viral particles in a cell substrate, including certain endogenous retroviruses. 2549

While TEM is fairly insensitive (generally detecting gross contamination, but not necessarily 2550

low-level contamination), it is a generic assay that can detect microbial agents of many types. 2551

TEM also can be used to estimate the concentration of viral particles to support validation of 2552

viral clearance. 2553


2554




2558

B.11.2.3.2 Additional considerations to TEM for insect cells 2559

For MCBs and WCBs derived from insect cells, the general screening test outlined above 2560

applies. In addition, cell lines should be maintained at temperatures above and below that 2561

routinely used for production, and also may be subjected to other treatments such as exposure to 2562

chemical stressors prior to examination by transmission electron microscopy. Further, increasing 2563

the number of cells examined may also improve the probability of detecting an agent. The 2564

maintenance temperatures and treatments used should be agreed with the NRA/NCL along with 2565

the number of sectioned cells to be examined. 2566

2567

B.11.2.4 Tests for retroviruses 2568

All vertebrate and insect cells that have been analysed possess endogenous, genetically acquired 2569

retroviral sequences integrated into chromosomal DNA in the form of proviruses. These 2570

sequences may be expressed, or be induced, as mRNA. In some cases, the mRNA is translated 2571

into viral protein and virus particles (virions) are produced. In many cases, these virions are 2572

defective for replication (e.g., avian endogenous retrovirus EAV, Chinese Hamster Ovary cell 2573

line gamma-retrovirus [88]) whereas in others (e.g., X-MuLV) the retroviruses may be capable 2574

of infecting cells of other species including human cells. 2575

2576

Consideration should also be given to the possibility that cell banks may be infected with non-2577

genetically acquired retroviruses (exogenous retroviruses) either because the donor animal was 2578

infected or through laboratory contamination. 2579

2580

It should be noted that infection by retroviruses is not necessarily associated with any cytopathic 2581

effect on the cells and therefore it may require screening assays, like the Product-Enhanced RT 2582

(PERT) assay for reverse transcriptase or TEM to reveal their presence. 2583

2584


The cells of the MCB or WCB are unsuitable for production if the tests for infectious 2585

retroviruses, if required, show evidence of the presence of any viral agent attributable to the 2586

substrate that cannot be demonstrated to be cleared during processing. Generally, the 2587

downstream manufacturing process for products (e.g., monoclonal antibodies) made in cell 2588

substrates that produce retroviral particles (e.g., CHO cells) or infectious endogenous retrovius 2589

(i.e., NS0, SP2/0 cells) is validated to provide adequate viral clearance [ 14]. The margin of viral 2590

clearance required should be agreed with the NRA/NCL. 2591

2592

Chick embryo fibroblasts (CEF) contain defective retroviral elements that frequently produce 2593

defective particles with reverse transcriptase activity. This has been the subject of many studies 2594

and WHO consultations because they are used for live viral vaccine production [ 89, 90, 91, 92]. If 2595

evidence is presented that the donor flock is free of infectious retroviruses and there is no 2596

evidence that the cultures are contaminated with infectious retroviruses, then the cultures can be 2597

considered acceptable with respect to retrovirus tests. 2598

2599

Rodent cell lines express endogenous retroviruses, and thus infectivity tests should be performed 2600

to determine whether these endogenous retroviral particles are infectious or whether the presence 2601

of non-infectious endogenous retroviral particles is masking an infectious retrovirus 2602

contamination. 2603

2604

Cell lines such as CHO, BHK, NS/0, and Sp2/0 have frequently been used as substrates for drug 2605

production with no reported safety problems related to virus contamination of the products and 2606

may be classified as "well-characterized" because the endogenous retrovirus particles have been 2607

studied extensively. Furthermore, the total number of retrovirus-like particles present in the 2608

harvest is evaluated quantitatively (TEM or QPCR) on a representative number of lots and 2609

retrovirus clearance is demonstrated with significant safety factors. Thus, in these situations 2610

testing for infectious retrovirus may be reduced; e.g. test one lot then discontinue testing, but 2611

repeat when there is a significant change in the cell culture process such as a change in scale. In 2612

all cases, samples derived from unprocessed bulk are the appropriate test article and it usually is 2613

not necessary to test purified bulk since titers of any virus potentially present would be highest in 2614

unprocessed samples. Sponsors are encouraged to consult with the NRA. 2615


2616


MCBs and cells that have been propagated to the proposed in vitro cell age for production or 2618

beyond. Alternatively, this testing could be performed on WCBs. In such cases, each new WCB 2619

is tested. 2620



2623

B.11.2.4.2 Reverse Transcriptase (RT) Assay 2624

Test samples from the MCBs or WCBs propagated to the proposed in vitro cell age for 2625

production or beyond are examined for the presence of retroviruses: 2626

2627

Culture supernatants are tested by a highly sensitive, quantitative, polymerase chain reaction 2628

(PCR)-based RT assay or PERT assay [ 93, 94, 95, 96]. 2629

2630

RTase activity is not specific to retroviruses and may derive from other sources, such as 2631

retrovirus-like elements that do not encode a complete or infectious genome 2632

[92, 97, 98, 99, 100, 101] or cellular DNA-dependent DNA polymerases [ 102, 103]. Attempts to 2633

reduce the PERT activity associated with cellular DNA-dependent DNA polymerases have been 2634

reported [ 103, 104, 105], although no treatment can eliminate all activity, and thus the results of 2635

such highly sensitive assays need to be interpreted with caution. Use of appropriate controls in 2636

the assay might assist in this regard. Since RT activity can be associated with the presence of 2637

defective retrovirus-like particles and since polymerases other than reverse transcriptase can 2638

result in apparent RT activity, a positive result in an RT assay is not conclusive evidence of the 2639

presence of infective retrovirus. Positive results may require further investigation, i.e., carrying 2640

out infectivity assays (see Section B.9.1.4.4). Also, it may be useful to utilize the conventional 2641

RT assay in this investigation to determine whether the RT activity is Mg++ or Mn++ dependent. 2642

Such testing should be agreed in advance with the NRA/NCL. 2643

2644

CEFs and other cells of avian origin are known to express retroviral elements. With such cells, 2645

the appropriateness of this test should be discussed with the NRA/NCL. For example, it may be 2646


appropriate to direct testing strategies at the detection of infectious avian retroviruses, such as 2647

avian leukosis viruses and reticuloendotheliosis virus. Additionally, it is known that insect cells 2648

have retroviral elements that are detected by a PERT assay, and so they too can test positive by 2649

this assay. 2650

2651

B.11.2.4.3 PCR or other specific in vitro tests for retroviruses 2652

When the PERT test gives unclear results, or when it is unavailable, it may be appropriate to 2653

screen the cell substrate for species-specific retroviruses by molecular methods, such as the PCR, 2654

immunofluorescence, ELISA, or other virus-specific detection methods. Molecular methods, 2655

such as PCR, also may be used for quantification of retrovirus like particles in the production 2656

harvests provided that the method is validated accordingly. Consultation with the NRA/NCL 2657

regarding the acceptability of this approach is recommended. 2658

2659

B.11.2.4.4 Infectivity test for retroviruses 2660

When the test sample is found to have RT activity, it might be necessary to carry out infectivity 2661

assays to assess whether the activity is associated with replicating virus. 2662

2663

Because rodent cells generally express endogenous retroviruses, the infectivity of such 2664

retroviruses or contaminating exogenous retroviruses should be assessed. Test samples from the 2665

MCB or WCB, propagated to the proposed in vitro cell age for production or beyond should be 2666

examined for the presence of retroviruses by infectivity assays. Cells to be used for these assays 2667

should be able to support the replication of a broad range of viruses; this might require using 2668

cells of various species and cell types. The testing strategy should be agreed with the NRA/NCL. 2669

2670

It is often possible to increase the sensitivity of assays by first inoculating the test material onto 2671

cell cultures that can support retroviral growth in order to amplify any retrovirus contaminant 2672

that may be present at low concentrations. For non-murine retroviruses, test cell lines should be 2673

selected for their capacity to support the growth of a broad range of retroviruses, including 2674

viruses of human and non-human primate origin [ 106, 107]. 2675

2676


For murine retroviruses, it is important to assess whether the cells release infectious retroviruses 2677

and, if so, to determine the host range of those viruses. The testing for murine retroviruses can be 2678

complex, and the NRA/NCL should be consulted for guidance. Murine and other rodent cell 2679

lines (CHO, NS0, SP2/0) or hybrid cell lines containing a rodent component should be assumed 2680

to be inherently capable of producing infectious retroviruses or non-infectious retrovirus-like 2681

particles. In such cases, the clearance (removal and/or inactivation) of such retroviruses during 2682

the manufacturing process should be quantified and provide a level of clearance acceptable to the 2683

NRA/NCL. 2684

2685

Any testing proposed by the manufacturer should be agreed with the NRA/NCL. 2686

2687

B.11.2.5 Tests for particular viruses not readily detected by the tests described in 2688

sections B.11.2.1, B.11.2.2, B.11.2.3, and B.11.2.4 and their sub-sections 2689

Some viruses, such as Hepatitis B or C or Human Papillomaviruses, cannot be detected readily 2690

by any of the methods described above because they are not known to grow in cell culture or are 2691

restricted to human host range. In such circumstances, it may be necessary to include testing for 2692

selected particular viruses that cannot be detected by more routine means. While broad general 2693

tests are preferable for detecting unknown contaminants, some selected viruses may be screened 2694

by using specific assays, such as molecular techniques (e.g., nucleic acid amplification). 2695

Antibody-based techniques might also be employed, such as immunofluorescence assays. 2696

2697

Generally, once the MCB, WCB, or EOPC has been demonstrated to be free of selected viruses, 2698

it might not be necessary to test the cells at later stages (i.e., at the production level) if such 2699

viruses could not be introduced readily during culture. 2700

2701

Human cell lines should be screened using appropriate in vitro techniques for specific viruses 2702

that are the cause of significant morbidity and for those viruses that might establish latent or 2703

persistent infections, and that may be difficult or impossible to detect by the techniques 2704

described in sections B.11.2.1, B.11.2.2, B.11.2.3, and B.11.2.4 and their sub-sections. Selection 2705

of the viruses to be screened should take into account the tissue source and medical history of the 2706

donor, if available, from whom the cell line was derived. 2707


2708

Under circumstances in which the cell origin or medical history of the donor, if available, would 2709

suggest their presence, it may be appropriate to perform specific testing for the presence of 2710

human herpesviruses, human retroviruses, human papillomaviruses, human hepatitis viruses, 2711

human polyomaviruses, or difficult-to-culture human adenoviruses. 2712

2713

Consideration should be given to screening insect cell lines for specific viruses that have been 2714

reported to contaminate that particular cell line (e.g., nodaviruses) or viruses that may be present 2715

persistently in insect cell lines and that are known to be infectious for humans. 2716

2717


The NRA/NCL should be consulted in regard to the specific pathogens or selected viruses that 2719

should be included in the testing strategy as these will be directed on a case-by-case basis 2720

depending on the species and origin of the cell and the medical history of the donor, if available. 2721


Cell Types: PCC (as needed), DCL, SCL, CCL 2723

2724

B.11.2.5.2 Nucleic acid detection methods 2725

Tests for selected viruses usually are performed using nucleic acid amplification and detection 2726

methods. PCR can be performed directly on DNA extracted from the cells or on cell lysates or 2727

supernatant fluids by DNA amplification, or on RNA by reverse transcription followed by DNA 2728

amplification (RT-PCR). In this manner, both DNA and RNA viruses can be detected, as can the 2729

proviral DNA of retroviruses. PCR primers can be directed against variable regions of viral 2730

nucleic acids in order to ensure detection of a specific virus or viral strain, or against conserved 2731

regions of viral sequences shared amongst strains or within a family in order to increase the 2732

opportunity of detecting multiple related viruses. Standard PCR analysis can be coupled with 2733

hybridization methods to increase its versatility, sensitivity, and specificity. For example, the use 2734

of probes to various regions of the amplicon might be useful in identifying the virus strain or 2735

family. However, PCR methods have the limitation that viral genes might not be sufficiently 2736

conserved among all members of any particular viral family to be detected even when conserved 2737

regions are selected. 2738


2739

New, sensitive, molecular methods, with broad detection capabilities are being developed. These 2740

are not yet in routine use, but as they become widely available and validated, they will play an 2741

increasing role in the evaluation of cell substrates. One of the advantages of some of these new 2742

methods is that they have the potential to discover new viruses. These methods include 2743

degenerate PCR for whole virus families with analysis of the amplicons by hybridisation, 2744

sequencing or mass spectrometry; and random PCR amplification followed by analysis of the 2745

amplicons on large oligonucleotide arrays of conserved viral sequences and digital subtraction of 2746

expressed sequences. The latter method, which can be applied to integrated transforming viruses, 2747

uses high throughput sequencing to analyse the 3' ends of expressed sequences. Those sequences 2748

present within genomic and expression databases of cells of the appropriate species are 2749

eliminated, leaving a small subset that can be analysed for their relationship to viral sequences. 2750

Some of these methods (e.g., PCR followed by mass spectrometry) also can be used for 2751

identification purposes [ 108, 109] 2752

2753

B.11.3 Bacteria, fungi, mollicutes, and mycobacteria 2754

The most common contaminants of cell culture are non-viral. These can be introduced easily 2755

from the environment, materials, personnel, etc. Furthermore, many such organisms multiply 2756

rapidly and can be pathogenic for humans. 2757

2758

Biological starting materials should be characterized to ensure that they are free from extraneous 2759

infectious organisms such as bacteria, fungi, cultivable and non-cultivable mycoplasmas, 2760

spiroplasma (in the case of insect cells or cells exposed to plant-derived materials), and 2761

mycobacteria. For a substance to be considered free of such contaminants, the assays should 2762

demonstrate, at a predefined level of sensitivity, that a certain quantity of the substance does not 2763

contain detectable levels of that contaminant. Testing should be conducted in an aseptic 2764

environment under appropriate clean room conditions to avoid false-positive results. Testing 2765

plans should include a policy to account for the need for repeat testing to deal with potentially 2766

false-positive results, and a prequalification plan for reagents used in the tests. 2767

2768


Mycobacterial testing might be applied to cell bank characterization if the cells are susceptible to 2769

infection with Mycobacterium tuberculosis or other species. Such testing also should be 2770

performed on primary cell cultures. It may be necessary to lyse the host cells in some cases in 2771

order to detect Mycobacteria because some strains may be primarily intracellular. 2772

2773

Detection of mycoplasma/spiroplasma may require different growth conditions from methods 2774

used for mammalian cells - although at least one, Spiroplasma, can be cultivated at 30ºC. 2775

Positive controls for these tests (particularly for Spiroplasmas) are an issue that needs to be 2776

resolved. Spiroplasma have been reported as infectious agents in a number of insect species and 2777

insect cell lines, and in addition have been reported to cause pathogenic effects in mammals. 2778

2779

B.11.3.1 Bacterial and Fungal Sterility 2780

Tests are performed as specified in Part A, section 5.2 [ 110] of the Requirements for Biological 2781

Substances No. 6 (General Requirements for the Sterility of Biological Substances), by a method 2782

approved by the NRA/NCL. Additional information can be found in national pharmacopoeias 2783

and ICH documents [111,112,113,128]. For the MCB and WCB, the test is carried out using for 2784

each medium 10 mL of supernatant fluid from cell cultures. In addition, the test is carried out on 2785

at least 1% of the filled containers (i.e., cyropreservation vials) with a minimum of two 2786

containers. For supernatant fluid, the recommended method is the membrane filtration method. 2787

For cell bank vial testing, it may be necessary to use the direct inoculation method. 2788

2789


Cell Banks: MCB, WCB 2791


2793

B.11.3.2 Mollicutes 2794

This class of bacteria is distinguished by an absence of a cell wall. They are parasites of various 2795

animals and plants, living on or in the host's cells. They are also a frequent contaminant of cell 2796

cultures. In addition to their potential pathogenicity, mycoplasmas compete for nutrients, induce 2797

chromosomal abnormalities, interrupt metabolism and inhibit cell fusion of host cells. M. 2798

pneumoniae is pathogenic for humans, although there are no reported cases of human infections 2799


with this organism arising from exposure to cell cultures or cell-derived products. Nonetheless, 2800

cell banks should be demonstrated to be free of such contamination in order to be suitable for the 2801

production of biologicals. 2802

2803

B.11.3.2.1 Mycoplasma and acholesplasma 2804

Tests for mycoplasmas are performed as specified in Part A, sections 5.2 and 5.3 of the 2805

Requirements for Biological Substances No. 6 (General Requirements for the Sterility of 2806

Biological Substances) [ 114], or by a method approved by the NRA/NCL. Both the culture 2807

method and the indicator cell-culture method should be used. NAT alone, in combination with 2808

cell culture, or with an appropriate detection method, might be used as an alternative to one or 2809

both of the other methods after suitable validation and discussion with the NRA/NCL. In this 2810

case, a comparability study should be carried out. The comparability study should include a 2811

comparison of the respective detection limits of the alternative method and official methods. 2812

However, specificity (mycoplasma panel detected, potential false-positive results due to cross-2813

reaction to other bacteria) should also be considered. More details are available in the European 2814

Pharmacopoeia chapter 2.6.7 [ 115]. One or more containers of the MCB and each WCB are 2815

used for the test. 2816

2817


Cell Banks: MCB and each WCB 2819


2821

B.11.3.2.2 Spiroplasma and other mollicutes 2822

2823

2824


Cell Banks: MCB, WCB (recommended for insect cells) 2826

Cell Types: DCL, SCL, CCL (recommended for insect cell substrates and when raw materials 2827

of plant origin are used during the cell bank preparation or production process). 2828

2829

B.11.3.3 Mycobacteria 2830


The test for mycobacteria is performed as described below or by a method approved by the 2831

NRA/NCL. 2832

2833

Inoculate 0.2 mL of the sample in triplicate onto each of 2 suitable solid media (such as 2834

Löwenstein-Jensen medium and Middlebrook 7H10 medium). Inoculate 0.5 mL in triplicate into 2835

a suitable liquid medium at 37°C for 56 days. 2836

2837

In some countries, the incubation period is 42 days. 2838

2839

An appropriate positive control test should be conducted simultaneously with the sample under 2840

evaluation, and the test should be shown to be capable of detecting the growth of small amounts 2841

of mycobacteria. In addition, the fertility of the medium in the presence of the preparation to be 2842

examined should be established by a spiking inoculation of a suitable strain of a Mycobacterium 2843

sp., such as BCG. If at the end of the incubation time no growth of mycobacteria occurs in any of 2844

the test media and the positive control and spiked control show appropriate growth, the 2845

preparation complies with the test. 2846

2847

Nucleic acid amplification techniques might be used as an alternative to this culture method, 2848

provided that such an assay is shown to be comparable to the compendial culture method. An 2849

appropriate comparability study should be carried out that includes a comparison of the 2850

respective detection limits of the alternative method and culture method. However, specificity 2851

(mycobacteria panel detected, potential false-positive results due to cross-reaction to other 2852

bacteria) should also be considered. An in vivo method, as described in the test in guinea pigs, 2853

also may be used (B.11.2.1.3). 2854

2855


Cell Banks: MCB or WCB 2857


2859

B.11.4 Transmissible Spongiform Encephalopathies 2860


TSEs are a group of slowly developing fatal neurological diseases affecting the brain of animals 2861

and humans. The accepted view at present is that they are caused by non-conventional infectious 2862

agents known as prions (PrPtse

), which are made up of a normal host protein (PrP) in an 2863

abnormal conformation. TSEs include BSE of cattle, scrapie of sheep, CJD and its variant form 2864

(vCJD) and kuru in humans, CWD in elk and deer, and transmissible mink encephalopathy 2865

(TME) [ 116, 117]. Normal PrP (PrPc) protein may be expressed on cell surfaces, but in vivo this 2866

protein can mis-fold and become the abnormal disease-causing type PrPtse

, which is able to 2867

catalyse the conversion of PrPc protein into the abnormal conformation. PrPtse

is relatively 2868

resistant to common proteolytic enzymes, such as proteinase K, compared to PrPc. 2869

2870

BSE in cattle has been transmitted to humans in the form of vCJD. Approximately 200 2871

individuals have been affected either directly through exposure to BSE-infected material or 2872

through secondary transmission by non-leukocyte-depleted red blood cells. Classical CJD has 2873

also been transmitted by medical procedures including administration of cadaveric growth 2874

hormone [ 118], corneal transplant [ 119], and the use of dura mater [ 120, 121, 122], and vCJD 2875

may be transmissible by the same routes. Cattle-derived proteins, including serum, have often 2876

been used in the growth of cells in culture and the production of biological products, including 2877

vaccines and recombinant products. Therefore, it is important to ensure that any ruminant-2878

derived material used in biopharmaceutical manufacture is free of the agents that cause TSE. 2879

Moreover, as there is a possible but unquantifiable risk that cells can become infected by the 2880

agents of TSE, it is important that possibly contaminated ruminant material has been excluded 2881

from the start of the development of any cell line used. When there is insufficient traceability in 2882

the legacy of a cell line, a risk assessment should be undertaken to aid in decision-making about 2883

the suitability of the cell line for the intended use. There is currently no practical validated test 2884

that can be used for biological products or cell line testing for the agents of TSE, other than 2885

infection of susceptible species where the experiments are very difficult because of the length of 2886

the incubation. More usable tests such as protein misfolding cyclic amplification (PMCA) which 2887

is analogous to PCR for nucleic acids, and epitope protection assays [ 123] are under 2888

investigation, but their performance characteristics when used to detect TSE agents in biological 2889

products or cell lines have not been defined. Strategies for minimising risk have therefore 2890


focused so far on sourcing materials from countries believed to be at very low risk of infection 2891

and on substituting non-animal-derived materials. 2892

2893

B.11.4.1 Infectivity categories of tissues 2894

Ruminant tissues are categorized by the World Health Organization and other scientific bodies 2895

such as the European Medicines Evaluation Agency into three Categories (Category A - High 2896

infectivity, Category B - Lower infectivity, and Category C - no detectable infectivity) [124]. 2897

Category A includes brain and Category C includes materials such as testes and bile. Assays of 2898

improved sensitivity have shown infectivity in tissues such as muscle, previously thought to be 2899

free of infectious agents, and the implication is that while certain tissues contain large amounts 2900

of infectivity, many other tissues may contain low levels that are difficult to detect (124,125, 2901

126, 127). 2902

2903

B.11.4.2 Control measures, sourcing and traceability 2904

Where effective alternatives to ruminant-derived material are available, they should be used in 2905

cell culture/manufacturing procedures. Examples include: 1) cell culture medium free of animal 2906

material, 2) polysorbate and magnesium stearate of plant origin, 3) enzymes, such as rennet, of 2907

microbial origin (used in lactose production), and 4) recombinant insulin and synthetic amino 2908

acids. However, it is not always possible to use ingredients free of animal materials, and raw 2909

materials of non-ruminant origin. For example, foetal bovine serum might have to be used in the 2910

development of cell lines or for fermentation. Under these circumstances, the raw materials 2911

should be sourced from countries classified by the World Organisation for Animal Health (OIE) 2912

as negligible BSE risk or GBR I, as classified by the European Food Safety Authority (EFSA). 2913

Raw materials of Category C may be sourced from countries that are classified as controlled risk 2914

provided there is assurance that no cross-contamination with materials of Category A or B could 2915

have occurred during collection and processing, with the caveat that while they have 2916

undetectable levels of infectivity, it could conceivably be present. Manufacturers should 2917

maintain records, so that the finished product from any batch is traceable to the origin of any 2918

ruminant ingredient used in its manufacture that might pose a risk of exposure to a TSE agent, 2919

and each ruminant ingredient is traceable to the finished product. This includes ingredients used 2920

to develop and produce the MCB and WCB, and as far as is possible, to the derivation of the cell 2921


line itself. This traceability in both directions is important for appropriate regulatory action if 2922

new scientific research indicates that there is a TSE infectivity risk in the materials used, or if 2923

there is an association of the products’ use with vCJD. Category A and B ruminant materials, 2924

originating from BSE-enzootic countries, should not be used in vaccine production under any 2925

circumstances. Because new BSE cases continue to occur despite feed bans, because suitable 2926

tests for TSE agents in raw-materials are not available, and because developments in scientific 2927

research indicate the presence of pathological prions in Category C materials, the best approach 2928

to TSE safety is not to use animal-derived protein. The next best approach is to source raw 2929

materials from countries classified as free of BSE bearing in mind that cases may be detected in 2930

future. 2931

2932

B.11.4.3 Tests 2933

Currently, there are no suitable screening tests available for TSE agents in raw materials of 2934

human/ruminant origin similar to serological or PCR assays for the screening for viral agents. 2935

Newer tests are being developed to screen for the presence of TSE agents in blood (such as 2936

PMCA, epitope-protection assay, etc.). Such tests, once validated, could eventually become 2937

suitable for the screening of raw materials. 2938

2939

B.12 Summary of tests for the evaluation and characterization of 2940

animal cell substrates 2941

The following sections provide an overview of tests that are recommended for the evaluation and 2942

characterization of animal cell substrates proposed for use in the production of biological 2943

products. Not all of the tests are appropriate for all animal cell substrates, but each of them 2944

should be considered and a determination made as to its applicability for a given cell substrate in 2945

the context of its use to manufacture a specific product. In addition, the point(s) at which a test 2946

should be applied needs to be rationalized. The overall testing strategy should provide assurance 2947

that risks have been mitigated to reasonable levels for the product and its intended use. The 2948

testing strategy should be agreed with the NRA/NCL. 2949

2950

B.12.1 Cell Seed 2951


The cell seed generally is derived from a cell or tissue source of interest because of its potential 2952

utility in the development of a biological product. In some cases, the cell may be expected to 2953

serve as the substrate for the production of multiple products. The cell seed is usually of limited 2954

quantity so that extensive testing is not feasible. Some of the seed is therefore used to produce a 2955

supply of cells in a quantity that allows more extensive testing as well as providing a long-term 2956

source of cells for use in manufacturing. This secondary cell source is usually termed the master 2957

cell bank (MCB). However, the cell seed also may be used to produce additional low passage 2958

material that can be banked (pre-MCB) and used to generate MCBs that then are characterized as 2959

described in this document. 2960

2961

Tests on the cell seed can be done at any point before the establishment of the MCB. Usually 2962

those tests are limited to information essential to make the decision to commit resources to the 2963

preparation of a MCB. Such tests typically include: viability, morphology, identity (e.g., 2964

karyotype, isoenzymes), and sterility (e.g., bacterial, fungal, mycoplasma). These data serve as 2965

important background information, but they cannot substitute for the full characterization of the 2966

MCB. 2967

2968

B.12.2 Master Cell Bank (MCB) and Working Cell Bank (WCB) 2969

The MCB generally will be developed to generate a sufficient quantity of cells to supply enough 2970

vials of cells to produce many WCBs over an extended period of time (usually years). MCBs 2971

typically contain at least 200 vials, and often 1000 or more. There also should be a sufficient 2972

number of vials in the WCB to provide material for the characterization of the cell line. 2973

2974

Some tests on the WCB are conducted on cells recovered directly from the bank itself; but other 2975

tests will be conducted on cells that have been propagated to a passage at or beyond the level that 2976

will be used for production (EOPC). In addition, some tests may be appropriate to use as in-2977

process control (IPC) tests. In such cases, they should be identified and described in the 2978

recommendations applicable to specific products. 2979

2980

Figure 2 presents an overview of the relationships among the parental cell, cell seed, pre-2981 MCB, MCB, WCB, and EOPC. 2982


2983 2984 Table 5. Characterisation of primary cell cultures 2985 Test Primary

Identity -

Sterility1 +

Viability -

Microbial agents2

+ 2986 1. Bacteria, fungi, and mycoplasma; and spiroplasma for insect cell lines 2987 2. Endogenous and exogenous agents using in vivo, in vitro, and physical methods as appropriate for the cell 2988

system and culture conditions. 2989 2990 2991


Table 6. Testing of Cell Substrates when the strategy is taken to focus extensive 2992 characterization on the Master Cell Bank and limit characterization of Working Cell 2993 Banks 2994 2995

Test Cell

Seed MCB WCB ECB

1

1. IDENTITY AND PURITY

Morphology + + + +

Identification e.g., isoenzymes,

immunological and cytogenetic markers,

DNA fingerprinting + + + +

Karyotype + + - +

Life span (diploid cell lines) - - - +

Viability - + + +

Genetic stability (engineered cell lines) - + - +

2. EXTRANEOUS AGENTS

Sterility2

+ + + +/-

Electron microscopy - + - +

Tests in cell cultures - + + +

Retroviruses3

- - - +

Tests in animals and eggs - + + +

Selected viruses by molecular methods - - - +

Antibody-production tests (rodent cell

lines4)

- - - +

Bovine and/or porcine viruses - - - +

3. BIOLOGICAL CHARACTERISTICS

Growth Characteristics - + + +

Tumorigenicity5

- - - +

Oncogenicity6

- - - + 2996 1 The characterization of the ECB means to characterize the MCB by using cells at or beyond the maximum population doubling 2997 level used for production passaged from the MCB. So, while the material in the test derives from the ECB, the results constitute 2998 extensive characterization of the MCB. 2999 2 Bacteria, fungi, and mycoplasma; and spiroplasma for insect cell lines; mycobacteria if appropriate 3000 3 May be limited to PERT or for PERT-positive or rodent cell lines, may include infectivity assays 3001 3 Testing is performed only on rodent cell lines in a species-specific manner. 3002 4 The MRC-5, WI-38, and FRhL-2 cell lines are recognised as being non-tumorigenic and they need not be tested. Tests are also 3003 not carried out on cell lines that are known or assumed to be tumorigenic, such as rodent cell lines. 3004 5 Testing would only be performed in cases in which the cell line is tumorigenic and proposed for use in producing prophylactic 3005 viral vaccines. Testing would be performed on cell lysates from cells passaged from the MCB using cells at or beyond the 3006 maximum population doubling level used for production. 3007 3008

3009

3010

3011

3012


Table 7. Testing of Cell Substrates when the strategy is taken to limit characterization of 3013 the Master Cell Bank and focus extensive characterization on Working Cell Banks 3014 3015

Test Cell

Seed MCB WCB ECB

1

1. IDENTITY AND PURITY

Morphology + + + +

Identification e.g., isoenzymes,

immunological and cytogenetic markers,

DNA fingerprinting + + + +

Karyotype + - + +

Life span (diploid cell lines) - - - +

Viability - + + +

Genetic stability (engineered cell lines) - - + +

2. EXTRANEOUS AGENTS

Sterility2

+ + + +/-

Electron microscopy - - + +

Tests in cell cultures - + + +

Retroviruses3

- - - +

Tests in animals and eggs - + + +

Selected viruses by molecular methods - - - +

Antibody-production tests (rodent cell

lines4)

- - - +

Bovine and/or porcine viruses - - - +

3. BIOLOGICAL CHARACTERISTICS

Growth Characteristics - + + +

Tumorigenicity5

- - - +

Oncogenicity6

- - - + 3016 1 The characterization of the ECB means to characterize the WCBs by using cells at or beyond the maximum population doubling 3017 level used for production passaged from the WCB. So, while the material in the test derives from the ECB, the results constitute 3018 extensive characterization of the WCB. 3019 2 Bacteria, fungi, and mycoplasma; and spiroplasma for insect cell lines; mycobacteria if appropriate 3020 3 May be limited to PERT or for PERT-positive or rodent cell lines, may include infectivity assays 3021 3 Testing is performed only on rodent cell lines in a species-specific manner. 3022 4 The MRC-5, WI-38 and FRhL-2 cell lines are recognised as being non-tumorigenic and they need not be tested. Tests are also 3023 not carried out on cell lines that are known or assumed to be tumorigenic, such as rodent cell lines. 3024 5 Testing would only be performed in cases in which the cell line is tumorigenic and proposed for use in producing prophylactic 3025 viral vaccines. Testing would be performed on cell lysates from cells passaged from the MCB using cells at or beyond the 3026 maximum population doubling level used for production. 3027 3028


Part C. Risk reduction strategies during the manufacture of 3029

biological products derived from animal cell substrates 3030

3031

C.1. General considerations 3032

The manufacturing process for the production of biologicals in CCL substrates should take these 3033

factors into account in order to ensure the safety of the product. Generally, purification 3034

procedures will result in the extensive removal of cellular DNA, other cellular components and 3035

potential adventitious agents, as well as allergens. Procedures that extensively degrade or 3036

denature DNA might be appropriate for some products (e.g. rabies vaccine). When CCLs are 3037

being contemplated for use in the development of live viral vaccines, careful consideration must 3038

be given to potentially oncogenic and infectious cellular DNA in the final product [see B.8 and 3039

B.9]. 3040

3041

C.2 Risks 3042

C.2.1 Exogenous agents introduced during manufacturing 3043

C.2.1.1 Example (irradiation of serum) 3044

C.2.1.2 Example (MVM) 3045

C.2.1.3 Example (equine virus during QC testing of horse serum) 3046

C.2.2 Endogenous agents 3047

C.2.2.1 Example (DNAse) 3048

C.2.2.2 Example (viral clearance) 3049

C.2.2.3 Example (inactivation) (BPL for rabies vaccine) 3050

3051

3052

3053


Authors 3054

3055

Acknowledgements 3056

3057

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3375


Appendix 1 3376 3377

Tumorigenicity Protocol Using Athymic Nude Mice to Assess Mammalian 3378

Cells 3379 3380 During the characterization of a MCB (or WCB), the cells should be examined for 3381 tumorigenicity in a test approved by the national regulatory authority (NRA) or the national 3382 control authority (NCA). 3383 3384 The following model protocol is provided to assist manufacturers and NRAs/NCLs in 3385 standardizing the tumorigenicity testing procedure so that the interpretation and comparability of 3386 data among various laboratories and regulatory authorities can be facilitated. 3387 3388

1. Test Animals 3389 The test article cell line and the control cells are each injected into separate groups of 10 3390 athymic mice (Nu/Nu genotype) 4-7 weeks old. 3391

Because male athymic mice often display aggressive traits 3392 against each other when housed together, loss of some mice 3393 during the observation period occurs often. Therefore, the use 3394 of only female mice should be considered. 3395 3396

2. Test article cells 3397 Cells from the MCB or MWCB that have been propagated to at least 3 population 3398 doublings beyond the limit for production are examined for tumorigenicity. 3399 3400

3. Control cells 3401 a. Positive control cells 3402

HeLa cells from the WHO cell bank are recommended as the positive control 3403 reference preparation. Portions of that bank are stored at the ATCC (USA) and 3404 NIBSC (UK). 3405

Other cells may be acceptable to the NRA/NCL if HeLa cells 3406 from the WHO cell bank are not available. 3407 3408

b. Negative control cells 3409 Negative control cells are not required. Databases (both published data and the 3410 unpublished records/data of the animal production facility that supplied the test 3411 animals) of rates of spontaneous neoplastic diseases in nude mice may be taken 3412 into account during the assessment of the results of a tumorigenicity test. 3413 3414 If negative control cells are included, clear justification must be provided. In 3415 particular, the number of animals used must provide meaningful data, and the 3416 rationale for generating additional data must be persuasive to the NRA/NCL in 3417 the context of animal welfare regulations. 3418 3419

4. Validity 3420 In a valid test, progressively growing tumors should be produced in at least 9 of 10 3421


animals injected with the positive control reference cells. At least 90% of the inoculated 3422 control and cells and test cells must be viable for the test to be valid. 3423

3424 5. Inoculum 3425

The inoculum for each animal is 107 viable cells (except as described in 11.b), suspended 3426

in a volume of 0.1 mL of PBS. 3427 Cell culture media without serum has been used in the past to 3428 suspend the cell inoculum. However, many current media are 3429 serum-free and contain one or more growth factors that may 3430 affect the result of the tumorigenicity assay. Therefore careful 3431 consideration should be given to the choice of the liquid into 3432 which the cells are suspended. 3433 3434

6. Injection route and site 3435 The injection of cells may be by either the intramuscular (IM) or subcutaneous (SC) 3436 route. If the IM route is selected, the cells should be injected into the thigh of one leg. If 3437 the SC route is selected, the cells should be injected into the supracavicular region of the 3438 trunk. 3439

Based on published studies, the intracerebral route may be 3440 more appropriate in some cases. For example, lymphoblastoid 3441 cells have been shown to proliferate best when inoculated by 3442 the intracerebral route. 3443

3444 7. Observation period 3445

All animals are examined weekly by observation and palpation for a minimum of 12 3446 weeks (i.e., 3 months) for evidence of nodule formation at the site of injection when the 3447 route of inoculation is IM or SC. Examinations need not be more frequent than twice a 3448 week for the first 3 to 6 weeks, and once a week thereafter. 3449

In some countries, the observation period is 4 to 7 months, 3450 depending on the level of concern associated with the specific 3451 cell substrate in the context of the product being developed. 3452 Whether a longer observation period is needed should be 3453 agreed with the NRA/NCL. Also see 8 below. 3454 3455

8. Assessment of the inoculation site over time 3456 If a nodule appears, it is measured in two perpendicular dimensions, the measurements 3457 being recorded weekly to determine whether the nodule grows progressively, remains 3458 stable, or decreases in size over time. Animals bearing nodules that appear to be 3459 regressing should not be sacrificed until the end of the observation period. Cell lines that 3460 produce nodules that fail to grow progressively are not considered to be tumorigenic. 3461

If a nodule that fails to grow progressively, but persists during the 3462 observation period and it retains the histopathological morphology of a 3463 neoplasm, this should be discussed with the NRA/NCL to determine 3464 whether additional testing will be required. Such testing could include 3465 extending the observation period or switching to a newborn nude 3466 mouse, ATS-treated newborn rat, or other in vivo model to assess the 3467 tumorigenicity of the cell substrate. 3468 3469 If the cells that are injected fail to form tumors or to persist during the 3 3470 month observation period, it might be necessary to extend the 3471 observation period or switch to a newborn nude mouse, ATS-treated 3472


newborn rat, or other in vivo model to assess the tumorigenicity of the 3473 cell substrate. This will depend on the level of concern associated with 3474 the specific cell substrate in the context of the product being developed. 3475 Whether such additional testing is needed should be agreed with the 3476 NRA/NCL. 3477

3478 9. Final Assessment of the inoculation site and other sites 3479

At the end of the observation period, or at an earlier time if required due to the death of 3480 an animal or other justifiable circumstances, all animals, including the reference group(s), 3481 are sacrificed and examined for gross and microscopic evidence of the proliferation of 3482 inoculated cells at the site of injection and in other sites such as the heart, lungs, liver, 3483 spleen, kidneys, brain, and regional lymph nodes since some CCLs may give rise to 3484 tumors at distant sites without evidence of tumor at the injection site. The tissues are 3485 fixed in 10% formol saline and sections are stained with hematoxylin and eosin for 3486 histological examination to determine if there is evidence of tumor formation and 3487 metastases by the inoculated cells. 3488 3489

10. Assessment of metastases (if any) 3490 Any metastatic lesions are examined further to establish their relationship to the primary 3491 tumor. If what appears to be a metastasis to a distant site differs histopathologically from 3492 the primary tumor, consideration should be given to the possibility that the tumor either 3493 developed spontaneously, or that it was induced by one or more of the components of the 3494 cell substrate such as an oncogenic virus. 3495

If the histopathology or genotype of any tumors that develop 3496 are inconsistent with the inoculated cell type, or are of a 3497 histopathological type that has not been recognized as 3498 occurring spontaneously in the test species, additional tests 3499 should be undertaken to determine whether such tumors are 3500 actually spontaneous or are induced by elements within the 3501 cell substrate itself such as oncogenic viruses or oncogenic 3502 DNA sequences. In such cases, appropriate follow-up studies 3503 should be discussed and agreed with the NRA/NCL. 3504

3505 11. Interpretation of results 3506

a. The test in nude mice is considered positive if at least 2 of 10 animals inoculated 3507 with the test article cells develop tumors that meet all of the following two 3508 criteria: 3509

3510 i. Tumors appear at the site of inoculation or at a metastatic site 3511

ii. Histologic or genotypic examination reveals that the nature of the cells 3512 constituting the tumors is consistent with that of the inoculated cells 3513

In the past, chromosomal markers have been useful to demonstrate that 3514 the tumor cells are of the same species as that from which the 3515 inoculated cells were derived. However, the use of cytogenetics for this 3516 purpose has largely been replaced by genetic and antigenic markers. 3517 3518

b. If only one of 10 animals develops a tumor that meets the three criteria in 11.a, 3519 the cell line should be considered possibly tumorigenic and examined further. 3520 Such testing could include one or more of the following: a) repeating the test in an 3521 additional 10 nude mice, b) extending the observation period, c) increasing the 3522


size of the inoculum, or d) switching to the newborn nude mouse model, the ATS-3523 treated newborn rat model, or other in vivo model. In such cases, appropriate 3524 follow-up investigations should be discussed and agreed with the NRA/NCL. For 3525 example, it may be appropriate to determine if the tumor is of nude mouse origin 3526 and whether there are any viral or inoculated cell DNA sequences present. 3527

Assessment of dose-response may provide additional 3528 information on the characteristics of the CCL. If such studies 3529 are undertaken, the design should be based on the in vivo 3530 titration of the inoculum in groups of 10 animals per dose 3531 level. For example, if 10 of 10 animals develop tumors with an 3532 inoculum of 10

7 cells, the titration could be done with 10

5, 10

3, 3533

and 101 cells in groups of 10 animals each. 3534

3535


Appendix 2 3536

3537

Quantitative measurement of residual cell DNA 3538


Appendix 3 3539

3540

List of Abbreviations 3541 3542 3543 ALS anti-lymphocyte serum 3544 ATG anti-thymocyte globulin 3545 ATS anti-thymocyte serum 3546 BSE bovine spongiform encephalopathy 3547 BVDV bovine viral diarrhoea virus 3548 CCLs continuous cell lines 3549 CJD Creutzfeldt–Jakob

disease 3550

CPE cytopathic effect 3551 CTL cytotoxic T-lymphocyte 3552 CWD chronic wasting disease 3553 DCLs diploid cell lines 3554 EBV Epstein-Barr virus 3555 EFSA European Food Safety Authority 3556 EOP End of production 3557 EOPC End of production cells 3558 EPIC-PCR Exon Primed Intron Crossing-PCR 3559 GMPs good manufacturing practices 3560 GSS Gerstmann-Sträussler-Scheinker syndrome 3561 HA haemagglutinating 3562 HCP hamster cheek pouch 3563 HDCs human diploid cells 3564 HERVs endogenous retroviruses 3565 ICH International Conference on Harmonization 3566 IFA immunofluorescence assays 3567 IFN interferon 3568 IM intramuscular 3569 IPC in process control 3570 LCMV lymphocytic choriomeningitis virus 3571 MAbs monoclonal antibodies 3572 MCB master cell bank 3573 miRNA microRNA 3574 NAT nucleic acid amplification techniques 3575 NCLs National Control Laboratories 3576 NRAs National Regulatory Authorities 3577 OIE World Organisation for Animal Health 3578 PCCs primary cell cultures 3579 PCR polymerase chain reaction 3580 PDL population doubling level 3581 PERT product enhanced reverse transcriptase 3582 PMCA protein misfolding cyclic amplification 3583 RCBs Reference Cell Banks 3584


rcDNA residual cellular DNA 3585 rDNA recombinant DNA 3586 RFLP restriction fragment length polymorphism 3587 RT reverse transcriptase 3588 SC subcutaneous 3589 SCs stem cells 3590 SCLs stem cell lines 3591 SG Study Group 3592 SIVB Society for in Vitro Biology 3593 SOP standard operating procedure 3594 SPF specific-pathogen-free 3595 STR short tandem repeats 3596 SV40 simian virus 40 3597 TEM transmission electron microscopy 3598 TME transmissible mink encephalopathy 3599 TPD50 tumor-producing dose at the 50% endpoint 3600 TSEs transmissible spongiform encephalopathies 3601 TTV transfusion-transmitted virus 3602 VNTR variable number of tandem repeats 3603 WCB working cell bank 3604 3605

3606

who/ draft/ 4 may 2010 english only who/ draft/ 4 may 2010 3 english only 4 5 6 recommendations for...

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