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10.5731/pdajpst.2016.006502Access the most recent version at doi: 300-31170, 2016 PDA J Pharm Sci and Tech

Jeff Blake, David Estapé, Ken Green, et al. BPOG Response to Annex 2

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COMMENTARY

BPOG Response to Annex 2JEFF BLAKE (NOVAVAX), DAVID ESTAPE (M�W GROUP), KEN GREEN (SHIRE),KAVITA RAMALINGAM IYER (MERCK), JEFF JOHNSON (MERCK), PHIL MCDUFF (BIOGEN),MARC PELLETIER (CRB), ALAN POWELL (DME ENGINEERING),SCOTT PROBST (BAYER TECHNOLOGY SERVICES),PAUL SMOCK (MERIDIAN BIOGROUP LLC AND WAS EMPLOYED BY ASTRAZENECA AT THE TIMETHE WORK WAS COMPLETED), ROBIN PAYNE (BPOG FACILITATOR), TIM CORBIDGE* (BPOG FACILITATOR)

Introduction

This paper is an interpretive response to Annex 2 ofEudralex—Volume 4; the European Union (EU)guidelines for good manufacturing practice (GMP) ofmedicinal products for human and veterinary use (1).It was written collaboratively, by a team of experts inclosed system processing, from 26 companies in thebiopharmaceutical industry facilitated by BioPhorumOperations Group (BPOG), who have a history ofassessing the potential for new technologies to benefitthe industry (2, 3). The authors listed on the title pagewere lead contributors to the content of this document,writing sections, editing, and liaising with colleaguesto ensure that the messages it contains are represen-tative of current thinking across the biopharmaceuticalindustry. This paper is a consensus view of a responseto Annex 2, but it does not represent fully the internalpolicies, views, or opinions of the authors’ respectivecompanies.

The purpose of this paper is to share an interpretationof key areas of Annex 2, providing enhanced clarityfor the industry. This paper supports a scientific andrisk-based approach that identifies the biological ac-tive substance manufacturing requirements, and thetypes of control that meet those requirements.

Volume 4 of the EU GMP guidelines was originallydeveloped to complement EU Commission Directive91/356/EEC (subsequently amended by 91/412/EECand 2003/94/EC). It contains two annexes (Annex 1

and Annex 2) that are highly influential and provideguidance on the design, building, and regulatory in-spections of biopharmaceutical facilities. Their titlesare:

Annex 1: Manufacture of Sterile Medicinal Products.

Annex 2: Manufacture of Biological Active Substancesand Medicinal Products for Human Use.

Annex 1, pertaining to sterile manufacturing opera-tions and sterile products, will be revised through2015 and into 2016 (4).

Annex 2 focuses on the manufacture of biologicalactive substances and biological medicinal productsfor human use.

Annex 2 states the importance of quality risk manage-ment (QRM) principles in the development of controlstrategy [such as those principles described in ICH Q9(5)]. However, Annex 2 references Annex 1 in anumber of places, leading to potential misinterpreta-tion, that is, that the environmental classification cas-cades and associated controls described in Annex 1(sterile products) are required for facilities and man-ufacturing systems in scope for Annex 2 as well. Onthe other hand, QRM enables selection of controls thatare proportionate to the risk identified, as opposed tonon-risk-based conventional approaches. The authorsof this paper value the QRM approach, and it is theiropinion that QRM should drive the design of processand control strategies for the mitigation of contami-nation and cross-contamination risks.

A goal for QRM, in terms of facility design, is to applyrisk-based approaches to the management of contam-ination and cross-contamination that add the highestvalue regarding product quality and patient safety.It is not always necessary to employ conventionalmitigation methods like environmental controls. For

* Corresponding Author: Tim Corbidge, BiophorumOperations Group, 5 Westbrook Court, Sharrow ValeRoad, Sheffield, S11 8YZ, United Kingdom; E-mail:[email protected]; Telephone: � 44 (0)7970340073.

doi: 10.5731/pdajpst.2016.006502

DISCLAIMER: The following article is a special editorial contribution from the BioPhorum Operations Group (BPOG).Please note that it did not go through the PDA Journal of Pharmaceutical Science and Technology regular peer reviewprocess.

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example, if one uses a completely closed system ofoperation, QRM evaluation may determine that envi-ronmental controls are not required for the closedprocess steps. In this paper, the authors focus on theapplication of QRM to the following topics:

1. References to Annex 1 in Annex 2, which could beinterpreted to imply nonsterile facilities, shouldapply sterile facility controls.

The authors recognize Annex 1 will be revised (4) andsee this as an opportunity to clarify guidance in linewith some of the reasons for commenting in this paper.

2. Potential alternatives to specific text in Annex 2,pertaining to “Premises and Equipment”, “Startingand Raw Material” and “Operating Principles”.These are considered to be open to a range ofinterpretations and are frequently the subject oflengthy debate when biopharmaceutical facilitiesare designed and built.

1. References To Annex 1 In Annex 2

Annex 2 applies to the manufacture of biologicalactive substances and medicinal products for humanuse. The two instances in which Annex 2 refers toAnnex 1, which applies to the manufacture of sterilemedicinal products, sometimes lead the reader to theinterpretation that Annex 1 also applies verbatim tothe manufacture of biological active substances. Thatinterpretation is contrary to the QRM principles es-poused in Annex 2.

Annex 1 is referenced only to provide principles andguidance for establishing classified environments tomitigate contamination risks as deemed necessary.Annex 2 is clear that the environmental controlsshould be conceived and established through the exe-cution of a QRM process. The following sections showthe instances where Annex 2 references Annex 1 andhow these references instead could be applied to bio-pharmaceutical processes using the QRM framework.

1.1. Annex 2, Paragraph 6

Paragraph 6, in the “Premises and Equipment” section,states:

“6. Manufacturing and storage facilities, processes andenvironmental classifications should be designed toprevent the extraneous contamination of products. Pre-

vention of contamination is more appropriate thandetection and removal, although contamination islikely to become evident during processes such asfermentation and cell culture. Where processes are notclosed and there is therefore exposure of the product tothe immediate room environment (e.g., during addi-tions of supplements, media, buffers, gasses, manipu-lations during the manufacture of ATMPs) controlmeasures should be put in place, including engineeringand environmental controls on the basis of QRM prin-ciples. These QRM principles should take into accountthe principles and guidance from the appropriate sec-tions of Annex 118 to EudraLex, Volume 4, whenselecting environmental classification cascades and as-sociated controls.”

Footnote 18 provides clarification regarding the appli-cation of Annex 1 for active pharmaceutical ingredient(API) manufacturing:

“18 Although the title of Annex 1 refers to the manu-facture of sterile medicinal products it is not the in-tention to force the manufacture of sterile product at astage when a low bioburden is appropriate and autho-rized. Its use is because it is the only EU GMP sourceof guidance on all of the classified manufacturingareas including the lower grades D and C.”

Reason for Commenting

The application of QRM principles to the manufactureof biological products often indicates the use of de-signs that differ from the environmental control mea-sures dictated by Annex 1. The statement that controlmeasures should be based on QRM principles, fol-lowed by the statement that these should take intoaccount guidance from relevant sections in Annex 1,could lead to an interpretation that Annex 1 should beapplied directly to the manufacture of biological activesubstances. The statement in footnote 18, that it is notthe intention of Annex 1 to force sterile manufacturingwhen “. . . a low bioburden is appropriate and autho-rized”, provides some clarification but is open to num-ber of different interpretations.

Interpretation

It is proposed that decisions to assign area classifica-tion and implement other controls to prevent contam-ination should consider the nature of the operation,stage of the manufacturing process, and QRM out-come.

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Clearly, the ingress of contaminants into process fluidsor materials should be minimized to prevent adverseimpact on the product. The risk-based approach re-quires development of justified control measures topotential sources of environmental contamination.This requires justification beyond simply assumingthat filtration or other downstream purification stepsremove environmental contaminants that enter duringprocessing, or that contamination will be detected.

The following case illustrates the tenet that controlmeasures should be commensurate with the risks (5)and the consequences of simple adherence to Annex 1for biopharmaceutical processes:

One case where the risk profile of biological activesubstances manufacturing differs from sterile productmanufacturing is an open aseptic process for inoculumpreparation for cell culture. The operations often in-volve open manipulations of cell culture using asepticprocessing techniques. Across the industry, Grade Aconditions are achieved in the unidirectional air flowhood often used for such operations. The objective isto avoid microbial or viral contamination of the cul-ture during these operations.

In the above example, adherence to all Annex 1 re-quirements for Grade A environments is not necessar-ily required to avoid contamination of the culture.However, since Annex 2 refers the reader to Annex 1for principles and requirements of classified areas, onemight conclude that an open operation, like inoculumpreparation, where microbial contamination will resultin failure of the batch, requires adherence to all Annex1 background and in-process monitoring requirementsfor a Grade A environment.

Specifically, paragraph 33 from Annex 1 in the section“Aseptic Preparation” states:

“33. Handling and filling of aseptically prepared prod-ucts should be done in a grade A environment with agrade B background.”

Hence, adherence to Annex 1 aseptic requirementswould require a Grade B background environment forthe Grade A environment for aseptic operations inbiological active substances manufacturing. However,this interpretation may result in conditions that impedereliable manufacture of cell culture products. Appli-cation of Annex 1 requirements should be challengedand the design justified using QRM, to ensure thepatient is being protected in the most appropriate way.

In the case of open operations that fill sterile medicinalproducts, Annex 1 applies and requires a Grade Aenvironment with a Grade B background. This makessense, as there are no additional steps to remove orinactivate microbial contaminants and limited oppor-tunity to detect contamination before the product isgiven to the patient. In such a context, the Annex 1requirement provides appropriate controls.

However, the Annex 1 requirements are less appropri-ate for inoculum preparation. Additional layers ofprotection are provided in the process and controls andmitigate the impacts of potential contamination:

● A microbial load or viral contamination wouldlikely be detected through cell culture testing. An-nex 2 acknowledges this point in paragraph 6where it states “. . . contamination is likely tobecome evident during processes such as fermen-tation and cell culture.”

● Inoculum preparation is at the very beginning ofmany API manufacturing processes, with addi-tional detection points and downstream processsteps that remove contaminants.

Therefore, the risk profile of inoculum preparationdiffers from that of filling of sterile medicinal prod-ucts. QRM evaluation will often indicate differentrequirements for this process step.

In current practice, industry has significant experienceperforming cell culture expansions in a Grade A hoodwith Grade C or Grade D background environmentswith very good success and exceedingly low contam-ination rates.

Regulatory authorities have supported this practice.For example, the Irish Medicines Board, Reference 6states: “Typically, Grade C background with Grade Asupply BSC is considered acceptable”. An Aide Me-moire (071210BN) issued by the German Health Au-thority (ZLG) indicates that a Grade A unidirectionalflow hood in a Grade C background environment isappropriate for establishing a cell bank (7).

1.2. Annex 2, Paragraph 33

Paragraph 33 of Annex 2 in the “Starting and RawMaterials” section also refers to Annex 1:

33. Given that the risks from the introduction of con-tamination and the consequences to the finished prod-




uct is the same irrespective of the stage of manufac-ture, establishment of a control strategy to protect theproduct and the preparation of solutions, buffers andother additions should be based on the principles andguidance contained in the appropriate sections of An-nex 1. The controls required for the quality of startingand raw materials and on the aseptic manufacturingprocess, particularly for cell-based products, wherefinal sterilization is generally not possible and theability to remove microbial by-products is limited,assume greater importance. Where an MA [MarketingAuthorization] or CTA [Clinical Trial Authorization]provides for an allowable type and level of bioburden,for example at active substance stage, the controlstrategy should address the means by which this ismaintained within the specified limits.”


The statement that the consequences of contaminationare “. . . the same irrespective of the stage of manu-facture”, and that a control strategy should be based on“. . . appropriate sections of Annex 1”, though “appro-priate sections” are not specified, indicates that verystrong contamination controls should be in placethroughout every process. However, a QRM approachwould indicate that the risk of contamination is lowerupstream in the process than downstream, as down-stream filtration and purification steps remove andprovide opportunities to detect contamination. Stron-ger controls may be required further downstream inthe process, where there are fewer subsequent purifi-cation steps and opportunities for detection.

While the initial statement of the paragraph is not inaccordance with QRM, the statement at the end of theparagraph that “the Marketing Authorization (MA) orthe Clinical Trial Authorization (CTA) may providefor an allowable bioburden level and that controlsneed to be in place to ensure those specified bioburdenlevels” are consistent with the risk-based approachthat is prescribed throughout Annex 2. Apparentlycontradictory statements in paragraph 33 may lead toconfusion and misinterpretation for an end user.

Interpretation

As stated in the previous section, paragraph 6 statesthat prevention of contamination is more appropriatethan detection and removal. The authors agree withthis approach so long as it is interpreted within a QRMframework. Some operations, such as solution prepa-

ration, are typically open operations, and microbialcontamination is successfully removed by filtration.However, applying Grade C conditions to solutionpreparation in biological active substance manufactur-ing operations (as specified under paragraph 17 ofAnnex 1) may not be an appropriate control for arisk-based approach where many factors are consid-ered.

In the following example for solution preparation, it isobvious that raw material is the primary contaminationsource as opposed to influences from the environment.Hence, controls developed to address bioburden fromraw materials will also address bioburden from theenvironment.

Consider a solution preparation operation in which1000 L of solution is prepared with a total solidsconcentration in the solution of 50 g/L. The solidsused in the solution are manufactured under con-trolled, nonclassified conditions (at best). None of thesolids are tested for viruses, but all are tested forbioburden and have a maximum bioburden specifica-tion of 100 CFU/g. This means that the final solutioncould contain 5,000,000 CFU of bioburden from theraw materials alone. This does not include microbialpropagation in the solution during preparation.

The solids are added through an open port on thesolution preparation vessel in an open manner. Envi-ronmental conditions should be set so that the poten-tial additional contamination is negligible in compar-ison to the inherent microbial load in the startingmaterials. Even if the air contained 1000 CFU/m3, fivetimes higher than the recommended values for GradeD specification in operation, 50 m3 of air (50,000 L)would have to enter the tank during the addition pro-cess in order to introduce 50,000 CFUs of bioburden,which is 1% of the maximum allowable amount ofbioburden based on the raw material specifications.For a briefly exposed operation, exchange of 50 m3 ofair between the vessel and surroundings is not credi-ble, because both must be at the same pressure for theopen operation. No driving force exists for rapid airexchange between the tank and the room environment.In this context, QRM should drive the appropriatemanufacturing controls.

However, the tables in paragraph 17 and paragraph 32of Annex 1 both mandate a Grade C environment forthe preparation of solutions that are subsequently fil-tered when those solutions are used in a process in

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which the product is not terminally sterilized. Thiscould lead to the interpretation that solution prepara-tion for biological active substances, when the productis not terminally sterilized, should also be conductedin a Grade C environment. This should not be the case.Due to the inherently greater capability to detect andremove contaminants, the risk profile of biologicalactive substance manufacturing is very different frommanufacturing sterile medicinal products. Therefore, arisk-based approach should be taken when setting en-vironmental conditions for such operations. See Ap-pendix A for an example of the application of riskanalysis to solution preparation operations. As amodel, this example is intended to help when deter-mining appropriate area classifications for such oper-ations.

In summary, the two instances in which Annex 2refers to Annex 1 may lead the reader to believe thatAnnex 1 also applies to the manufacture of biologicalactive substance. Annex 1 is only referenced becauseit provides considerations and guidance for establish-ing classified environments that may be used to miti-gate contamination risks if deemed necessary. In sce-narios where Annex 2 is applicable, appropriateenvironmental controls should be implemented whenthe requirement is determined through the executionof a QRM process.

2. Potential Alternatives To Specific Text InAnnex 2

The second part of this paper describes interpretationsof statements in Annex 2 that the authors consideroverly prescriptive and/or that may be interpreted inways that are inconsistent with a risk-based approach.

2.1. Paragraph 5

“As part of the control strategy, the degree of envi-ronmental control of particulate and microbial con-tamination of the production premises should beadapted to the active substance, intermediate or fin-ished product and the production step, bearing in mindthe potential level of contamination of the startingmaterials and the risks to the product. The environ-mental monitoring program should be supplementedby the inclusion of methods to detect the presence ofspecific microorganisms (i.e., host organism, yeast,molds, anaerobes, etc.) where indicated by the QRMprocess.”


Paragraph 5 highlights the need for an environmentalmonitoring program and supplemental requirements asindicated by the QRM process. This might be inter-preted to mandate a strong and conservative environ-mental monitoring program of a type that might berequired only in some instances for open processing.

Also, at the end of the paragraph, very specific rec-ommendations for an environmental monitoring pro-gram are given. However, the QRM process for aclosed system likely would show minimal risk ofparticulate or microbial contamination when located ina controlled nonclassified (CNC) space where require-ments for environmental monitoring are minimal ornonexistent. Usage of the abbreviation “i.e.” also in-dicates an expectation that tests for the listed organ-isms will be included. Therefore, the team believesthat the recommendations in this paragraph relating toan environmental monitoring program are overly pre-scriptive.

Interpretation

The degree of environmental control for contamina-tion should be risk based and not driven by conven-tional practices or preset expectation. Elements to beconsidered when determining the risk of contamina-tion from the environment are:

● Properties of the process materials, for example,growth promoting characteristics could affect holdtime.

● The degree of closure of equipment.

For closed and functionally closed systems, the QRMprocess may determine that the risk of particulate ormicrobial contamination is minimal and that a CNCenvironment is appropriate, in which case the environ-mental monitoring program can be minimized or elim-inated as required and appropriate for CNC environ-ments. In the example below, the risk from roomenvironment during cell expansion steps is renderednegligible by an approach to closing the operations.

A typical cell culture seed train process progressesthrough multiple cell expansion steps and increasesbatch volume in a process that requires multiple steriletransfers. In each transfer, connections are requiredthat increase the process contamination risk by poten-




tially exposing process contact surfaces to the outsideenvironment. To minimize this risk it is critical tokeep process vessel connections closed until ready touse and then to join them together while minimizingexposure to the outside environment.

The availability and successful use of sterile closedsystems like presterilized disposable media bags andbioreactor bags that can be fitted with transfer, addi-tion, and sample lines significantly reduce or eliminatethe process contamination risks that otherwise can bea concern. The closed systems can be integratedthrough the use of techniques such as tube weldingand/or disposable aseptic connectors, therefore ensur-ing that the process remains closed even when theseconnections are made. Because the entire seed traincan now be performed as a closed system, it is notexposed to the room environment and the operationhas been successfully performed without contamina-tion in CNC areas.

The nature of the process step being executed can, ofcourse, have an impact on how best to protect the drugsubstance, upstream versus downstream of viral clear-ance. The following example illustrates this for man-ufacture of a drug substance intermediate:

● Operations like fermentation are a classic exampleof an upstream drug substance manufacturing pro-cess. Fermentation processes are conducted in aclosed system to prevent contamination (i.e., main-tain pure culture) and QRM evaluation would sup-port conducting such operations in CNC space.

● Downstream processes like purification of a drugsubstance have typically been located in classifiedareas. The higher room classification would oftenhave been selected following a QRM evaluation,because purification operations typically havefewer layers of protection and often include openoperations.

Implementation of QRM presents the opportunity toperform downstream operations in CNC or lower clas-sification areas. Modern downstream processes oftenachieve a high degree of closure, such as utilizingfunctionally closed systems and single-use technolo-gies (e.g., tube welding and sterile connections). Witheffectively closed systems coupled with other layers ofprotection, QRM evaluation may support locating theprocess in a CNC space, with minimal or no environ-

mental monitoring requirements as determined byQRM evaluation.

For most systems, an effective way to minimize therisk of contamination is not to control the environmentbut to close the system so that the risk associated withthe environment is minimized or eliminated.

Recommendation

Currently paragraph 5 describes environmental moni-toring requirements that might be applicable for opensystems. It will be beneficial if Annex 2 is revised toindicate that QRM evaluation should determine if EMis required and to what extent for a process step.

2.2. Paragraph 50

“A control strategy for the entry of articles and mate-rials into production areas should be based on QRMprinciples. For aseptic processes, heat stable articlesand materials entering a clean area or clean/con-tained area should preferably do so through a dou-bled-ended autoclave or oven. Heat labile articlesand materials should enter through an air lock withinterlocked doors where they are subject to effectivesurface sanitization procedures. Sterilization of ar-ticles and materials elsewhere is acceptable pro-vided that they are multiple wrappings, as appropri-ate to the number of stages of entry to the cleanarea, and enter through an airlock with the appro-priate surface sanitization precautions.”


While paragraph 50 supports the implementation of aQRM approach and the authors fully agree with thefirst sentence, the remainder of the text describes insome detail the expectations for aseptic processingareas. While this direction may be appropriate forareas where open aseptic processing is conducted, thedetailed attention to aseptic processes might lead areader to conclude that similar controls are expectedfor a broader spectrum of processes, that is, typicalbulk biopharmaceutical manufacturing processes thatare not open aseptic processes. The amount of textdevoted to aseptic processes and the failure to distin-guish closed processes from open processes may in-fluence the end-user to implement designs that do notalign with QRM principles, providing an overburdenof controls.

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Interpretation

Paragraph 50 focuses on the requirements appropriatefor aseptic processing with no mention of the typicalbulk biopharmaceutical nonaseptic requirements. TheQRM process for entry into nonaseptic areas willindicate lower risk than for aseptic areas and minimalrisk for entry into nonaseptic areas with functionallyclosed systems with no need for multiple wrappingsand/or multiple stages of entry to the production area.Most of the discussion in paragraph 50 applies for thehigh-risk aseptic processes, but there is no discussionof lower risk options. Paragraph 50 could be inter-preted as requiring multiple wrapping and/or stages ofentry and surface sanitization requirements even forlow contamination risk situations, such as for nona-septic operations in closed systems. It is importantto have the understanding that QRM evaluation andoutcome should determine and inform the controlstrategy instead of incorporating process controlsbased on the nature of the process whether it isaseptic or nonaseptic.

Recommendation

Suggested wording: “A control strategy for the entryof articles and materials into production areas shouldbe determined based on QRM principles as applicableto the stage and nature of the process.”

2.3. Paragraph 54

“Centrifugation and blending of products can lead toaerosol formation and containment of such activitiesto minimize cross-contamination is necessary.”


Centrifuges are often used in manufacturing, often forupstream cell and cellular debris separations. The for-mation of aerosols may transfer live microorganismsor other contaminants into the environment.

The requirement for “containment of such activities”in paragraph 54 is interpreted as an unconditional needto isolate centrifuges without a rational assessment ofthe likelihood that aerosols will contaminate the envi-ronment and present a significant risk for cross-con-tamination. As a result, there is a clear expectation thata centrifuge must be located in a separate room, inde-pendent of the type, scale, operation, operating time,etc.

Most of the centrifuges now used in biopharmaceuticaloperations operate as functionally closed systems thatdo not release aerosols into the environment. Forexample, while windage and mechanical action mayresult in considerable aerosol formation inside a discstack centrifuge, the design of the casing and connec-tions for ventilation, solid and liquid discharges renderthe system closed and effectively prevent release ofaerosols. While other requirements may dictate theisolation of centrifuges, for example, noise abatement,vibration control, proper maintenance, and othersafety requirements, QRM may show that a centrifugemay be located and operated in the same room withother unit operations in the same space without a riskof cross-contamination due to aerosols.

Paragraph 54 also refers to blending of products as apotential source of aerosols that present a risk ofcross-contamination. Similarly, a perceived risk ofblending operations like buffer and media preparationor additions to product vessels is the spread contami-nants. When these operations are open and may re-lease particles or aerosols, then QRM may well dictateuse of dedicated areas or rooms for such operations.However, QRM also considers the process and othercontrols, for example, the nature of the blending op-eration, material properties (e.g., chemical or raw ma-terial type), levels of protection (e.g., closed systems,temporal separation, air flow direction), time andlength of the operation, etc., to determine whetherseparate rooms are required.

In summary, dedicated rooms are often built to containoperations like centrifugation or solution preparationeven though other methods to prevent aerosol contam-ination may be more appropriate and may already beintrinsic to the design.

Isolation of other operations that can lead to potentialaerosol formation, like homogenization, is often notconsidered because these operations are not explicitlymentioned in Annex 2.

Interpretation

It is essential to understand the probability or level ofaerosol formation and spread and to evaluate the ac-tual risk and consequences for cross-contaminationposed by aerosols. Proportional containment measure-ments should be adopted according to the risk that hasbeen determined. This may not require segregation orisolation of such operations when more robust solu-tions (like closed processing) are used.




This QRM approach is not limited to centrifuges andblending operations, the two examples mentioned inAnnex 2. It should include any unit operation that canlead to aerosol formation.

2.4. Paragraph 57

“57. In cases where a virus inactivation or removalprocess is performed during manufacture, measuresshould be taken to avoid the risk of recontamination oftreated products by non-treated products.”


Although methods to prevent recontamination are notprescribed in Annex 2, a frequent subject of discussionwithin industry and regulatory agencies is where andhow to segregate pre- and post-viral product processstreams to comply with paragraph 57. These discus-sions and expectations result in inconsistant levels ofimplementation and strategies.

If a firm decides not to segregate, it is likely that atsome point the firm will engage in difficult discussionswith regulatory agencies. This is perceived as a busi-ness risk rather than a product quality issue. As aresult, separate rooms are frequently used for pre- andpost-virus removal processing even if a QRM ap-proach indicates that alternative techniques are appro-priate.

Interpretation

Achieving viral safety of biological active substancesrequires more attention than simply undertaking pre-and post-virus reduction steps into separate rooms.Based on a sound QRM process and appropriate jus-tification, use of physical barriers between pre- andpost-virus operations (e.g., separate rooms or closedprocessing) is just one component of a complete riskmitigation strategy for virus contamination control.Other components include: barriers to virus entry(e.g., characterization of cell banks), raw materialssourcing, robust virus clearance steps, and testingduring production to detect any contaminating virus(8).

Robust control strategies will only be achievedthrough a sound QRM process and not by solelyfollowing the current common practice. Where a virusinactivation or removal process is performed duringmanufacturing, the QRM approach should provide

measures to identify and mitigate the risk of recon-tamination of treated products by nontreated products,components, or equipment.

Conclusions

While Annex 2 of the EU GMP regulatory guidanceclearly embraces QRM, it refers to Annex 1 in waysthat could be misinterpreted to imply controls used forsterile product processes should be applied to low-bioburden processes. Recognizing that the ultimategoal of regulatory guidance documents is protection ofpatients, thus prior to final product being renderedsterile, the bioprocess manufacturing scheme mustprovide layers of protection or barriers to protect theprocess from environmental contamination. However,our interpretation of the guidance is that controls forsterile products (i.e., those referred to in Annex 1) arenot typically required for biological active substancesprior to them being rendered sterile.

Use of a QRM approach ensures the selection ofcontrol measures that are commensurate with the po-tential risks to product and ultimately to the patient. AQRM approach should define the control strategy inline with Annex 2. Protecting the drug substance andintermediates by closing the process is a consistent,reproducible, and robust approach—more so than at-tempting to control the environment around the man-ufacturing process. Closing the process yields a lowerrisk to the manufactured drug substance, and ulti-mately to the patient.

The authors are aware that there will be a revision ofAnnex 1 and believe this represents an opportunity toclarify guidance as suggested in this paper (4).

Acknowledgments

BPOG and the authors wish to acknowledge the workof the following people, who acted as reviewers of thispaper and who support and endorse the content andproposals made:

Jose Caraballo (Bayer), Liz Dooley (Janssen), PaulDriesprong (Janssen), Marcella Goodnight (AstraZeneca Biologics), Richard Gummer (Baxalta), LarsHovmand-Lyster (Novo Nordisk), Jorgen Magnus(Bayer), Benjamin Montano (Baxalta), Russ Moser(Janssen), Iv Neov (Novavax), Lawrence Pranzo(Merck), Stephanie Ramsey (Baxalta), and GeraldUitz (Baxalta).

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Conflict of Interest Declaration

The author(s) declare that they have no competinginterests.

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6. Moody, P. Irish Medicines Board GMP Informa-tion Seminar, September 27, 2012.

7. Aide memoire Bio- und Gentechnologie071210BN, Zentralstelle der Länder fur Gesund-heitsschutz bei Arzneimitteln und Medizinproduk-ten, June 16, 2004.

8. Aranha, H. Virus Safety of Biopharmaceuticals.Contract Pharma 2011, 13.

9. ISPE Baseline® Guide. Risk-Based Manufacture ofPharmaceutical Products (Risk-MaPP), 1st ed.;2010; Volume 7.

Appendix: A

As previously stated in the discussion of Annex 2,paragraph 33, Grade C background for solution prep-aration activities is often accepted as a defacto regu-latory requirement.

The following risk assessment example demonstratesthat environmental classification controls are only oneconsideration, and probably not the most significantconsideration, when evaluating risks associated withthe preparation and storage of buffers used in drugsubstance purification. In this example, the risk ofmicrobial endotoxins entering the purification processwith buffers is evaluated. Application of the scoringcriteria indicate that the operation is low risk overallwith all the controls in place and that environmentalsources of bioburden-related endotoxin are bothhighly unlikely and highly detectable, rendering themthe lowest risk of all.

The example buffer preparation process shown inFigure 1 is conducted in a CNC environment. TableI shows a number of measures that are in place toavoid introduction and propagation of microorgan-isms that could produce endotoxins. The preparationvessel remains closed, and when solution compo-nents are added, they are added in a protected man-ner that minimizes exposure of the internal surfacesof the tank to the external environment. After prep-aration, the buffer is held for a short maximum holdtime (12 h) then filtered into a sterile storage bag toreduce bioburden that may be present in raw mate-rials. The buffer is stored under conditions in whichthere is no, or very low, bioburden and thereforelimited opportunity for endotoxins to form duringthe storage period.

Table I provides a risk assessment for the base casescenario shown in Figure 1. Severity, probability ofoccurrence, and detection point are all evaluated withrespect to the failure mode (microbial endotoxins enterprocess equipment from storage bag). Table I alsoprovides a risk assessment for a select set of alterna-tives to the base case (in red and blue text). Values forthese alternative cases are also assigned with respectto the failure mode. The analysis of the alternatives



http://ec.europa.eu/health/documents/eudralex/vol-4/index_en.htm

http://ec.europa.eu/health/documents/eudralex/vol-4/index_en.htm

http://ec.europa.eu/health/files/eudralex/vol-4/2008_11_25_gmp-an1_en.pdf

http://ec.europa.eu/health/files/eudralex/vol-4/2008_11_25_gmp-an1_en.pdf

http://ec.europa.eu/health/files/eudralex/vol-4/vol4-an2__2012-06_en.pdf

http://ec.europa.eu/health/files/eudralex/vol-4/vol4-an2__2012-06_en.pdf

http://www.biopharminternational.com/challenging-cleanroom-paradigm-biopharmaceutical-manufacturing-bulk-drug-substances



http://www.biopharminternational.com/new-challenges-cleanroom-paradigm-multi-product-facilities



http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2015/02/WC500181863.pdf



http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q9/Step4/Q9_Guideline.pdf




shows that increasing the classification of the back-ground environment does not reduce the risk associ-ated with the microbial load or endotoxins in thesolution; nonenvironmental factors have a greater im-pact. The potential contribution of microbial load tothe solution from the environment is already negligi-ble in the base case, with controls, in comparison toother potential sources of microbial load. However,other factors can influence the risk. For example,doubling the allowable preparation and filtration timeprovides more opportunity for microbial contaminantsto grow. Reduced preparation temperature lowers therate at which microbes will proliferate (with only asmall risk-reduction impact). Finally, if the addition

of solids to the tank is not as protected as describedin the base case, there is more opportunity formicrobial load to enter from the background envi-ronment or personnel.

Table II shows the scoring guidelines for severity,probability of occurrence, and detection point thathave been used in this example. This guidance hasbeen taken directly from the ISPE Baseline® Guide:Risk-Based Manufacture of Pharmaceutical Products(Risk-MaPP) (9).

An addendum is added to provide a context forinterpreting the resultant Risk Process Number[RPN].

Figure 1

Process for preparing and storing a purification buffer in a CNC environment. Solids are added so thatexposure of the interior of the vessel to the external environment is minimized. After preparation, thebuffer is 0.2 micron filtered into a gamma-irradiated storage bag. The solution remains in the storage baguntil it is ready for use. The buffer is dispensed to the target equipment when needed through gamma-irradiated tubing.

309Vol. 70, No. 3, May–June 2016



Tab

leI.

Ass

essm

ent

ofth

eR

isk

Tha

tM

icro

bial

End

otox

ins

Cou

ldE

nter

the

Pur

ifica

tion

Pro

cess

Stre

amG

iven

the

Pre

para

tion

Equ

ipm

ent

and

Pro

cedu

res

Illu

stra

ted

inF

igur

e1.

Bas

eC

ase

Ana

lysi

sA

naly

sis

ofA

lter

nati

veC

ases

Fai

lure

Mod

eP

oten

tial

Eff

ects

ofF

ailu

reM

ode

Seve

rity

Pot

enti

alC

ause

sC

urre

ntC

ontr

ols

Occ

urre

nce

Det

ecti

onR

PN

Per

mut

atio

nSe

veri

tyO

ccur

renc

eD

etec

tion

RP

NC

omm

ent/

Exp

lana

tion

Mic

robi

alen

doto

xins

ente

rpu

rifi

cati

oneq

uipm

ent

from

buff

er.

End

otox

ins

may

ente

rth

edr

ugsu

bsta

nce

proc

ess

stre

aman

dm

ayno

tbe

rem

oved

.H

igh

endo

toxi

nco

ncen

trat

ions

inth

edr

ugpr

oduc

tw

ill

resu

ltin

adve

rse

pati

ent

reac

tion

s.

10[A

]B

iobu

rden

(tha

tca

nfo

rmen

doto

xins

*)is

pres

ent

insa

lts.

[B]

Bio

burd

enpr

esen

tin

CW

FI.

[C]

Bio

burd

enen

ters

from

the

envi

ronm

ent

(CN

Cba

ckgr

ound

)[D

]B

iobu

rden

tran

sfer

red

toin

teri

orof

tank

byop

erat

ors.

[E]

Bio

burd

enpr

esen

ton

exte

rior

surf

aces

ofra

wm

ater

ial

pack

agin

gan

dis

tran

sfer

red

toin

teri

orof

prep

arat

ion

vess

el.

[F]

Pre

para

tion

vess

elan

d/or

tran

sfer

line

isin

adeq

uate

lycl

eane

dan

dsa

niti

zed

befo

reus

e.T

here

isbi

obur

den

pres

ent

inth

eve

ssel

ortr

ansf

erli

nepr

ior

toth

est

art

ofth

eba

tch.

[G]

Sol

utio

nsi

tsin

vess

ela

long

tim

eat

room

tem

pera

ture

befo

refi

ltra

tion

into

stor

age

bag

and

biob

urde

ngr

ows

wit

hin

the

tank

.[H

]In

itia

lle

vel

ofbi

obur

den

wit

hin

the

solu

tion

isto

ohi

ghan

dfil

ter

isun

able

tore

tain

the

biob

urde

n.[I

]0.

2m

icro

nfi

lter

isno

tin

tegr

al.

Bio

burd

enca

npa

ssth

roug

hdu

ring

filt

rati

on.

[J]

Sin

gle-

use

tubi

ngor

stor

age

bag

isno

tin

tegr

al.

Bio

burd

enca

nen

ter

thro

ugh

inte

grit

ybr

each

.[K

]S

ingl

e-us

etu

bing

orst

orag

eba

gin

adeq

uate

lysa

niti

zed

byth

eve

ndor

and

cont

ains

biob

urde

n.[L

]B

iobu

rden

accu

mul

ates

wit

hin

the

bag

duri

ngst

orag

e.O

rgan

ism

sdi

ean

dre

leas

een

doto

xins

into

the

form

ulat

ion

buff

er.

[A]

Bio

burd

ensp

ecifi

cati

onli

mit

sfo

rsa

lts.

[B]

Man

yde

sign

and

engi

neer

ing

cont

rols

inpl

ace

toav

oid

biob

urde

nin

CW

FI.

[B]

Per

iodi

csa

niti

zati

onof

CW

FI

dist

ribu

tion

syst

em.

[B]

Reg

ular

envi

ronm

enta

lm

onit

orin

gof

CW

FI.

[C,

D,

E]

Soli

dsad

ded

soth

atex

posu

reof

the

inte

rior

toth

eve

ssel

toth

een

viro

nmen

tis

min

imiz

ed.

[F]

Tan

kan

dtr

ansf

erli

near

ede

sign

edfo

rpr

oper

clea

ning

and

drai

ning

.A

vali

date

dcl

eani

ngan

dsa

niti

zati

onse

quen

ceis

exec

uted

for

the

vess

elan

dth

etr

ansf

erli

nepr

ior

toea

chlo

t.P

erio

dic

envi

ronm

enta

lm

onit

orin

g(i

.e.,

rins

esa

mpl

ing)

tove

rify

cont

inue

def

fect

iven

ess

ofcl

eani

ngpr

oced

ure.

[G,

H]

Val

idat

edti

me

esta

blis

hed

from

begi

nnin

gof

batc

hpr

epar

atio

nto

end

offi

ltra

tion

.T

ime

stam

psar

ech

ecke

din

the

batc

hre

cord

toen

sure

batc

hw

aspr

epar

edan

dfi

lter

edw

ithi

nth

eal

low

edti

me.

[I]

Fil

ter

vend

orqu

alit

ysy

stem

.[I

]P

ost-

use

inte

grit

yte

stpe

rfor

med

onea

chfi

lter

.[J

,L

]S

ingl

e-us

eve

ndor

qual

ity

prog

ram

.[J

]O

pera

tor

trai

ning

for

insp

ecti

ngsi

ngle

-use

elem

ents

prio

rto

use

and

toav

oid

dam

agin

gsi

ngle

-use

elem

ents

duri

ngha

ndli

ng.

[J]

Inte

grit

ybr

each

may

beco

me

evid

ent

duri

ngfi

ltra

tion

and

tran

sfer

.[K

]R

adia

tion

indi

cato

rson

gam

ma

ster

iliz

edel

emen

tsth

atar

ere

ceiv

ed.

[L]

Sol

utio

nis

0.2

mic

ron

filt

ered

prio

rto

stor

age.

Mea

sure

sJ

and

Kar

ein

plac

eto

prev

ent

the

buff

erfr

ombe

ing

cont

amin

ated

duri

ngth

est

orag

epe

riod

afte

rfi

ltra

tion

.N

OT

E:

Dru

gsu

bsta

nce

iste

sted

for

mic

robi

alen

doto

xins

.T

hem

ater

ial

isno

tre

leas

edfo

rfi

nal

drug

prod

uct

man

ufac

turi

ngif

the

spec

ified

lim

itis

exce

eded

.

31

30

[C’1

]So

luti

onpr

epar

atio

nan

dst

orag

eis

perf

orm

edw

ithi

na

Gra

deA

back

grou

nden

viro

nmen

tra

ther

than

ina

CN

Cen

viro

nmen

t.[D

’1,

E’1

)]So

lid

com

pone

nts

are

adde

dth

roug

han

open

man

way

rath

erth

anvi

aa

prot

ecte

dco

nnec

tion

.[G

’1,

H’1

]P

repa

rati

onan

dfi

ltra

tion

tim

eis

incr

ease

dfr

om12

to24

h.[G

’2]

Dec

reas

epr

epar

atio

nte

mpe

ratu

refr

om20

°Cto

5°C

.

10 10 10 10

3 5 7 3

1 1 1 1

30 50 70 30

[C’1

]P

roba

bili

tyof

occu

rren

cedo

esno

tch

ange

ifop

erat

ion

ispe

rfor

med

ina

Gra

deA

back

grou

nden

viro

nmen

t.T

hein

gres

sof

mic

robi

allo

adfr

omth

een

viro

nmen

tis

alre

ady

negl

igib

lein

the

base

case

inco

mpa

riso

nto

othe

rpo

tent

ial

sour

ces

(e.g

.,fr

omra

wm

ater

ials

).[D

’1,E

’1]

The

open

man

way

prov

ides

agr

eate

rop

port

unit

yfo

rbi

obur

den

from

the

envi

ronm

ent

orth

eop

erat

orto

ente

rth

eve

ssel

duri

ngpr

epar

atio

n.[G

’1,H

’1]

Incr

easi

ngth

eal

low

able

prep

arat

ion

and

filt

rati

onti

me

coul

din

crea

seth

epo

tent

ial

mic

roor

gani

sms

pres

ent

inth

era

wm

ater

ials

anop

port

unit

yto

grow

and

prod

uce

endo

toxi

ns.

[G’2

]D

ecre

asin

gth

epr

epar

atio

nte

mpe

ratu

rede

crea

ses

the

grow

thra

teof

pote

ntia

lm

icro

bial

cont

amin

ants

pres

ent

inra

wm

ater

ials

.

*T

his

isas

sum

edw

hene

ver

biob

urde

nis

men

tion

edw

ithi

nth

isex

ampl

e.




Table II.Failure Mode and Effects Analysis Scoring Guidelines Used in the Example Presented in Appendix A.These guidelines have been taken from the ISPE Baseline® Guide: Risk-Based Manufacture ofPharmaceutical Products (Risk-MaPP) (9).

Value Severity Occurrence Detection

10 Injury to a patient oremployee

More than once per batch Not detectable bycurrent methods

7 Cause extreme customerdissatisfaction

Once per batch All manuallyinspected

5 Something likely toresult in a complaint

Once per six months Statisticalsampling

3 Minor nuisanceresulting in no loss

Once every one to threeyears

100% inspection

1 Be unnoticed and notaffect performance

One occurrence in greaterthan five years

Obvious ormonitored oralarmed

Interpretation of RPN Values:RPN �350, High Risk (Minimum -10 S X 7O X5D)–Additional controls must be identified and implementedRPN 105–350, Medium Risk (Minimum 5S X 7O X 3D)–Additional controls should be considered for implemen-tationRPN �105, Low Risk–No additional controls are required

311Vol. 70, No. 3, May–June 2016



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