USP Standards for BiologicsTina S. Morris, Ph.D., Vice President, Biologics & Biotechnology USP-NF

Fouad Atouf, Ph.D., Director, Biologics & Biotechnology, USP-NF

User ForumJanuary 17th, 2013Istanbul, Turkey

USP Standards—Biologicals

Major B&B Themes and Initiatives for 2010-2015

Modernization of MonographsOutreach to Industry for New MonographsProcedural Chapters and Characterization/Measurement of

Ancillary and Process MaterialsGrowing the portfolio to support all biologicsModernization of analytical approaches

Biological Potency Tests:Implementation of new USP ChaptersAssay transitions: animal to cell, cell to binding, activity to amount of

substance – questions of equivalence and units

Product Class Chapters:Monoclonal antibodiesGlycoconjugate vaccines

Vertical Standards – Monographs

Role and Use: Clearly define identity, strength and purity, as well as other

important quality attributes at the product level Allow independent testing and verification based on a public

standard

Considerations for Standard Development: Complexity of specifications and system suitability criteria Biological potency assignments and unit maintenance

– Across manufacturers– Internationally

Product- specific vs. common product class requirements For well-characterized and legacy products: inclusion and

bridging to new analytical technology

Official USP Biologics Monographs by Product Class

Product Class Number of monographs

peptide 47

enzyme 12

complex extract 11

carbohydrate 11

glycosaminoglycan 9

other 5

Tissue product 6

IgG/serum 9

Blood component/protein 5

Vaccine 3

39%

10%9%

9%

8%

4%

5%

8%

4%3%

1%

B&B Overall Monograph Distribution by Product Class

peptide enzyme complex extract carbohydrate

glycosaminoglycan other Tissue product IgG/serum

Blood component/protein Vaccine Other

Current Biologics in the US Market

From Kozlowski et al., NEJM 265;5, 2011

Biologics with no official USP Monograph

Betaseron

Novoseven

Epogen/Procrit

Lucentis

Rebif

Cerezyme

Herceptin

Erbitux

Kogenate FS

Humira

Aranesp

Rituxan

Neulasta

PEGASYS

Tysabri

Synagis

Remicade

Recombinate

Avastin

Avonex

Enbrel

2008

2010

2013

2013

2013

2013

2014

2014

2014

2014

2014

2015

2015

2015

2015

2015

2018

2019

2019

2026

2028

Patent Expiry Horizon

Current Key New Monograph Development Projects

BB1:– Insulin Glargine– Fondaparinux– Dalteparin Sodium– Epoetin– Octreotide

Soon in ballot:– Filgrastim

BB2:– Factor IX, plasma

derived

Soon in ballot:• Sipuleucel T

Current Key Monograph Modernization Projects

BB1:– Heparin Sodium, stage 3– Menotropins– Pancreatin/pancrelipase– Insulins

Horizontal Standards - Procedural

Benefits: Access to validated procedures and procedure performance

criteria early in development

Solid anchor point for product characterization

Facilitate comparability during all development stages

Directly apply ICH guidance, including:– Q5E– Q6B

Establish consistency in analytical expectations

From General to Specific - Procedures

<1084>Glycoprotein and

Glycan Analysis – Introduction and Choice of Analysis Methods

<212>Oligosaccharide Analysis

<210>Monosaccharide Analysis

Monosaccharide Mixes 1–4

Oligosaccharide mixturesfrom - Human α acid

Glycoprotein- Fetuin- RNAse B- Human IgG

Single Monosaccharides

Guidance & Information

Procedures & Performance

Criteria

Procedural (Horizontal)

Standards for System Suitability

& Validation

<1084> - Glycoprotein Analysis Strategies

<1084> Glycoprotein Analysis Strategies

Test Chapters – Current Major Initiatives

Impurities

• <30> Residual DNA

Physicochemical Tests• <212> Oligosaccharide Analysis

• <210> Monosaccharide Analysis

• <121.1> Insulin Physicochemical Analysis

• <209> Low Molecular Weight Heparin Molecular Weight Determinations

• <XXX> Collagen

Potency Assays and Content

Measurement• <57> Protein Determination Procedures

• <123> Glucagon Bioidentity Tests

• <124> Epoetin Bioassays

• <126> Somatropin Bioassays

• <208> Anti-Factor IIa and Xa Assays for Unfractionated and Low Molecular Weight Heparins

Vaccine Chapters

<1235> Vaccines for Human Use (completed)

<1238> Bacterial Vaccines (completed)

<1239> Viral Vaccines (in progress)

<XXXX> Toxins/Toxoids

Sub-<1000> Analytical Chapters for Key Quality Attributes and RS

Possible PC Chapters

<1234> Polysaccharide and Glycoconjugate

<XXXX> Live Attenuated

<XXXX> Other Subunit

<XXXX> Live Attenuated

<XXXX> Killed Viruses

<XXXX> Subunit Vaccines

<129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies

Will contain a collection of validated compendial procedures with established system suitability criteria for therapeutic MAbs

Will be accompanied by USP MAb System Suitability RS

Will not contain product or class specific acceptance criteria

Will be supported by a suite of >1000 Information Chapters that discuss quality attributes, manufacturing and quality control aspects for MAbs

Benefits: Quality specifications and standards for complex ancillary and

process materials – Give access to process ingredients of consistent quality– Control process variability– Facilitate testing of process intermediates and bulks

Challenges in Standard Development: Fast evolution of process materials:

– Serum-free, proprietary custom media– Engineered 2nd and 3rd generation materials, e.g. Protein A

Defining key quality attributes for complex materials, e.g. Fetal Bovine Serum (FBS)

Horizontal Standards– Ancillary & Process Materials

Raw/Ancillary Materials

Raw materials may or may not remain in the final therapeutic product as active substances or excipients

– Ancillary materials are a subset of raw materials

Ancillary products may exert an effect on a therapeutic substance – (e.g. a cytokine may activate a population of cells), but they are not intended to

be in final formulation

Concerns related to raw materials qualification have been amplified

by recent materials supply issues: – Glycerin, Heparin, Melamine– Animal derived material (serum) used in manufacturing of some biologics

Some components are more critical than others! Risk assessment strategies are required to ensure quality – A critical material will come in contact with cells with a potential to alter either the

growth characteristics of the cells or the ability of the cell culture to meet lot release specifications.

Ancillary Materials Standards: USP Approach

<1043> Ancillary Materials for Cell-, Gene-, and Tissue-Engineered

Products

Specific AM Chapters

<90> FBS Quality Attributes<92> Cytokines and Growth Factors Quality Attributes

Reference Standards:- FBS- Interleukin-4- FGF-2- Transferrin- G-CSF

General Information

Chapter (Guidance)

Ancillary Material Requirements for

Specific AMs

Ancillary Material Reference Standards

Tier 1 – Low-Risk, Highly Qualified Materials with Intended Use as Therapeutic Drug or Biologic, Medical Device, or Implantable Material

Tier 2 – Low-Risk, Well Characterized Materials with Intended Use as AMs, Produced in Compliance with GMPs

Tier 3 – Moderate-Risk Materials Not Intended for Use as AMs (frequently produced for in vitro diagnostic use or reagent grade materials)

Tier 4 – High-Risk Materials, Materials not Produced in Compliance with cGMPs and materials not intended to be used in cell manufacturing

Risk-based Classification of Ancillary Materials

Risk-based Qualification – USP <1043>

Elements of Qualification and/or Risk ReductionActivities Tier 1 Tier 2 Tier 3 Tier 4

Master File cross reference X X X X

Certificate of analysis X X X X

lot-to-lot effect on process performance X X X X

Removal from finished product X X X X

Stability as stored and used in specific process X X X X

Confirm Certificate Analysis Test X X X

Vendor Audit X X X

Upgrade Manufacturing to GMP level X X

Develop internal specifications X X

Lot to lot comparability may be needed X X

Testing for adventitious agents may be needed X X

Traceability to country of origin, safety from animal diseases X

Adventitious agent testing for animal source-relevant viruses X

Recombinant Human Interleukin 4 -USP

Interleukin-4 Standard Requirement– Specific Activity: Not less than 0.5 x 107 USP Units /mg of total

protein• Labeled potency of RS will be based on bioactivity using TF-1 cell line

– Purity: 98% (SDS-PAGE and silver stain)– Identity: N-term protein sequencing (10 residues) and Western Blot

Associated Reference Standard – Lyophilized powder– Calibrated against International Standard (WHO)

How is the standard used?– Preparation of IL-4 for which the activity was determined by

calibration against the USP standard, will provide consistency in the manufacturing of the cell therapy product, by way of using the right amount of material.

Fetal Bovine Serum – Quality Attributes and Functionality Tests

Official uses of the USP FBS Reference Standard – Identification – Radial Immunodiffusion

– FBS Functionality Tests – Growth Promotion Curve• Five cell lines are recommended for use• Functionality tests are performed on 3 cell lines

○ Two from the recommended cell line list

○ Third is cell line relevant to the user’s application

USP General Chapter <90>

Fetal Bovine Serum (FBS)- USP

FBS Standard Requirements– Osmolality: 280-360 mOsm/Kg

– Total Protein: 30-45 mg/mL

– pH: 7.00 - 8.00

– Endotoxin: Not more than 10 units/mL

– Hemoglobin level: Not more than 30 mg/dL

– Identification: Radial Immunodiffusion (RID): species ID, IgG levels

– Functionality Assays (Growth Curve and Clonal Assay)

Associated Reference Standard (RS), under development– Liquid frozen, 10 mL– Collaborative study to include several laboratories to test:

• Identification (FBS sample positive for bovine IgG and content is < 500 mg/L)• Growth curve (doubling time in test sample is not less than 90% compared to

RS)

How the FBS Standard is Used: Growth Curve

Challenges: Cell Line, Cell Density, Cell Counting, Days in Culture

Three cell densities, determine viable cell counts on days 0,1,2,3,4, and 7. Select the

cell density that exhibit a growth curve with 3 phases: Lag, Log, Stationary; and linearover 3 time points or more

Use the selected cell density to assess thetest FBS side by side with the referencestandard FBS

Doubling time is estimated using a growth curve that is linear over three or more time points.

Acceptance Criteria:R2≥ 0.98Doubling time of test sample should be notless than 90% of doubling time of RS

Growth Curve for prostate cell line, WPE1-NB26-64

1.00E+03

1.00E+04

1.00E+05

1.00E+06

0 1 2 3 4 5 6 7Days in Culture

A

B

C

Growth Factors and Cytokines – additions to <92>. – Next revision adds FGF

Standards for enzymes as ancillary materials– Trypsin, others?

Second and third generation identification tests for complex, naturally derived materials:– Application of modern immunology and proteomics

approaches?

Future Directions for USP Ancillary Materials Standards

Thank you!