0484-0491 [1043] Ancillary Materials for Cell, Gene, And Tissue-Engineered Products

8/15/2019 0484-0491 [1043] Ancillary Materials for Cell, Gene, And Tissue-Engineered Products

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484 1041 Biologics / General Information USP 35

Vehicles and Added Substances— Vehicles and added cell, gene, and tissue-engineered products. These materialssubstances suitable for biologics are those named in the include plasma- or serum-derived products, biological ex-Food and Drug Regulations. tracts, antibiotics, cytokines, culture media, antibodies, poly-

meric matrices, separation devices, density gradient media,Containers for Injections— Containers for biologics in-toxins, conditioned media supplied by “feeder cell layers,”tended to be administered by injection meet the require-

fine chemicals, enzymes, and processing buffers. Many of ments for Containers for Injections under Injections 1 .these items are used to ensure the survival and promote theVolume in Container— The volumes in containers of bio- growth of certain cell populations, although their mecha-

logics intended to be administered by injection meet the nism of action may not be entirely understood. Examplesrequirements for Volume in Container under Injections 1 . include fetal bovine serum (FBS) and various media supple-Labeling— Biologics intended to be administered by in- ments. Other items, such as highly purified cholera toxin,

jection comply with the requirements for Labeling under In- are introduced into the processing stream during manufac- jections 1 . In addition, the label on the final container for turing to exert a specific biochemical effect and are immedi-each biologic states the following: the title or proper name ately washed out in subsequent processing steps to avoid(the name under which the product is licensed under the unwanted toxicity at a later point. The finished biologicalPublic Health Service Act); the name, address, and license products produced in such processes are often complexnumber of the manufacturer; the lot number; the expiration mixtures that, in some cases, cannot be completely charac-date; and the recommended individual dose for multiple- terized. Careful scrutiny of the materials used in manufactur-dose containers. The package label includes all of the above, ing is necessary to prevent the introduction of adventitiouswith the addition of the following: the preservative used agents or toxic impurities, as well as to ensure the ultimateand its amount; the number of containers, if more than safety, effectiveness, and consistency of the final product.one; the amount of product in the container; the recom- In cell, gene, and tissue-engineered product manufactur-mended storage temperature; a statement, if necessary, that ing, these reagents and materials are collectively called an-

freezing is to be avoided; and such other information as the cillary materials (AMs). AMs have also been referred to asFood and Drug regulations may require. ancillary products, ancillary reagents, processing aids, and

Packaging and Storage— The labeling gives the recom- process reagents. AMs were first discussed under the syno-mended storage temperature (see General Notices ). Precau- nym ancillary products in the U.S. Food and Drug Adminis-tions should be taken where products labeled to be stored tration Notice, “Application of Current Statutory Authoritiesat a temperature between 2 ° and 8 ° are stored in a refriger- to Human Somatic Cell Therapy Products and Gene Therapyator, in order to assure that they will not be frozen. Diluents Products” (Federal Register 58(197), October 14, 1993, pp.packaged with biologics should not be frozen. Some prod- 53248–53251). This document established the FDA’s au-ucts (as defined in Section 600.15) are to be maintained thority to regulate human somatic cell therapy products andduring shipment at specified temperatures. gene therapy products. AMs are also synonymous with

“processing materials” that were defined in 21 CFR PartExpiration Date— For compendial articles the expiration1271, “Current Good Tissue Practice for Manufacturers of date identifies the time during which the article may beHuman Cellular and Tissue-Based Products; Inspection andexpected to meet the requirements of the PharmacopeialEnforcement; Proposed Rule” (Federal Register 66(5), Janu-monograph, provided it is kept under the prescribed storageary 8, 2001, pp. 1508–1559). AMs can be analogous toconditions. This date limits the time during which the prod-“components,” and in some cases, “containers” as de-uct may be dispensed or used (see General Notices , page 1).scribed in the current good manufacturing practice (cGMP)However, for biological products, the stated date on eachregulations for finished pharmaceuticals as outlined in 21lot determines the dating period, which begins on the dateCFR 211.80 through 211.94 and 211.101(b) and (c).of manufacture (Section 610.50) and beyond which the

The defining property of AMs is that they are not in-product cannot be expected beyond reasonable doubt totended to be present in the final product. They are materials yield its specific results and to retain the required safety,used as processing and purification aids or agents that exertpurity, and potency (Section 300.3 (1) and (m)). Such atheir effect on the therapeutic substance. Materials or com-dating period may comprise an in-house storage period dur-ponents that are intended to be in the final product dosageing which it is permitted to be held under prescribed condi-

form (e.g., genetic materials, biopolymeric supports, physio-tions in the manufacturer’s storage, followed by a periodlogical buffers) are not AMs. Cell banks and virus banks areafter issue therefrom. The individual monographs usually in-also not considered AMs; there are a number of guidancesdicate both the latter period and (in parentheses) the per-that describe requirements for their certification. However missible in-house storage period. If the product is held in“helper” viruses and “helper” plasmids may be consideredthe manufacturer’s storage for a longer period than that in-

AMs when they are not intended to be part of the finaldicated (in parentheses), the expiration date is set so as toproduct.reduce the dating period after issue from the manufacturer’s

The quality of an AM can affect the stability, safety, po-storage by a corresponding amount.tency, and purity of a cell, gene, or tissue-engineered prod-uct. For example, the mechanism by which an AM exerts itseffect may not be known, and the impact of normal varia-tion of the AM on the quality and safety of the therapeuticproduct may not be understood. Alternatively, AMs of human or animal origin may present an infectious diseasetransmission risk. Other AMs, if administered to humans,1043 ANCILLARY MATERIALS may cause an immune reaction. Finally, an AM with toxicproperties that is introduced into a manufacturing processFOR CELL, GENE, AND TISSUE- and is not adequately removed in subsequent processingsteps will expose the patient to a toxic substance and mayENGINEERED PRODUCTS impair the effectiveness of the therapeutic entity. These risksto the quality and safety of the therapeutic product areoften heightened with cell, gene, and tissue-engineeredproducts, due to the limited ability to conduct extensive in-process and release tests. For example, lack of in-processINTRODUCTION holding steps or limited shelf life may create the need toadminister the cell, gene, or tissue-engineered products

A wide variety of reagents and materials, many of which before in-process or final-release testing results are available.are unique or complex, are required for the manufacture of In other cases, the scarcity of suitable donor tissue or the

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USP 35 General Information / 1043 Ancillary Materials 485

complex logistics in the transport of biological materials Selection and Suitability for Usemay limit the amount of material available for testing. Tominimize these risks, whenever possible, it is necessary to Developers of cell, gene, and tissue-engineered productsimplement rigorous material qualification and prudent appli- should establish and document selection criteria for AMscation of manufacturing process controls. and qualification criteria for each vendor early in the design

Frequently, these novel therapeutic products are created phase of product development. Selection criteria should in-using complicated biological processes. The AMs employed clude assessments of microbiological and chemical purity,in these procedures may be selected primarily for their identity, and biological activity pertinent to the specificunique functional contributions or biological effects. When- manufacturing process. It is important to address these is-ever possible, it is preferable to source AMs that are ap- sues early in product development because certain AMs thatproved or licensed therapeutic products because they are are initially considered necessary may be impossible or pro-well characterized, have an established toxicological profile, hibitively expensive to qualify, thereby justifying the investi-and are manufactured according to controlled and docu- gation of alternatives or replacements. Examples includemented procedures. Conversely, the AM may be intended some animal- or human-derived materials that in some cases“for research use” and may, therefore, lack the level of qual- have alternate (i.e., plant or chemically synthesized) sources.ification necessary for use in the production of a therapeutic AMs of animal or human origin should be selected cau-product. In either case, the manufacturer of the cell, gene, tiously due to the potential infectious or zoonotic diseaseor tissue-engineered product should develop comprehensive risks associated with these materials. Vendors should be se-and scientifically sound qualification plans to ensure the lected that can supply documentation regarding the countrytraceability, consistency, suitability, purity, and safety of the of origin for animal-derived AMs to address concerns re-

AM. In cases where AMs are products approved for use for garding transmissible spongiform encephalopathies andtherapeutic purposes, the level of qualification will probably other diseases of agricultural concern, like tuberculosis andbe less extensive than that for a material intended for re- brucellosis. In many cases, the chain of custody for animal-search purposes. However, their suitability in the manufac- derived AMs (i.e., abattoir → intermediate processing center turing process will still need to be established when the AM → final processing center) will need to be documented.is being used beyond the scope of its intended use or label- Vendors of human-derived AMs should be able to supplying. The purpose of this chapter is to provide guidance in documentation regarding material traceability. For instance,developing appropriate qualification programs for AMs em- human plasma-derived AMs should be sourced from li-ployed in cell, gene, and tissue-engineered product censed facilities that control the donor pool and appropri-manufacturing. ately screen the individual donors for relevant human infec-

tious diseases. In some cases, vendors of animal- andhuman-derived AMs supply different grades of materials,QUALIFICATION OF ANCILLARY MATERIALS some of which will be more suitable for use in cell, gene,and tissue-engineered product manufacturing than other Qualification is the process of acquiring and evaluating grades. For example, FBS can be obtained that has beendata to establish the source, identity, purity, biological processed to reduce the risk of bovine viral contaminationsafety, and overall suitability of a specific AM. The responsi- by subjecting it to validated irradiation and nanofiltrationbility for AM qualification resides with the developer or processes. Also, many animal and human plasma-derivedmanufacturer of the cell, gene, or tissue-engineered prod- components are subjected to chemical (detergent or solventuct. This section outlines the basis by which a manufacturer treatment) or physical (heat exposure for extended periodscan establish rational and scientifically sound programs for of time) treatments that have been shown through valida-qualifying AMs, although the broad nature of the cell, gene, tion studies to significantly reduce the risk of adventitious

and tissue-engineered products and of the AM used to pro- microbial or viral contamination associated with startingduce these products make it difficult to recommend specific AMs. Such AMs are preferred for use in cell, gene, and tis-tests or protocols for a qualification program. Thorough sue-engineered product manufacturing processes becausedocumentation is the cornerstone of any qualification they significantly reduce the risks associated with the origi-program. nal material. A well-designed qualification program becomes more The complexity of risk assessment can be reduced by em-comprehensive as product development progresses. In the ploying one of a number of quantitative or semiquantitativeearly stages of product development, safety is the primary approaches, such as failure mode effects analysis (FMEA), focus. In the later stages, AM production and qualification quality function deployment (QFD), or hazard analysis andactivities should be comprehensively developed to support critical control point (HACCP). These programs typically as-eventual licensure of the cell, gene, and tissue-engineered sign a point value to each risk parameter for an AM thatproduct. On some occasions, complex or unique substances results in cumulative scores that make it easier to prioritizethat have been shown to be essential for process control or effort and resources for decreasing the risks associated withproduction may not be available from suppliers that pro- AMs. For example, an AM that has a strong safety profileduce them in compliance with cGMP. In these situations, and is used in minimal amounts in upstream steps of thethe manufacturer will have to develop a scientifically sound manufacturing process and is thoroughly washed from thestrategy for qualification. A qualification program for AMs system would accumulate a low point score. Conversely, anused in cell, gene, and tissue-engineered product manufac- AM that is known to be toxic and is employed in down-turing should address each of the following areas: (1) identi- stream processing would, therefore, possess a higher poten- fication, (2) selection and suitability for use in manufactur- tial for appearing as a residual in the final product anding, (3) characterization, (4) vendor qualification, and (5) would be assigned a higher point value. One can also assignquality assurance and control. points based on the risk classification (see Risk Classification).

Identification CharacterizationThe first step in any qualification program is the listing of Specific quality control characterization tests need to beall of the AMs used in a given product manufacturing and developed or adopted and implemented for each AM. Thewhere in the manufacturing process they are to be em- set of tests for each AM should assess a variety of qualityployed. The source and intended use for each material attributes, including identity, purity, functionality, and free-should be established, and the necessary quantity or con- dom from microbial or viral contamination. The appropriatecentration of each material should be determined. Also, al- level of testing for each AM is derived from its risk assess-ternate sources for each material should be identified. ment profile and the knowledge gained during develop-




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486 1043 Ancillary Materials / General Information USP 35

ment. Test specifications should be developed for each AM therapeutic products are preferable because they are well-to ensure consistency and performance of the manufactur- characterized with an established toxicological profile anding process. Acceptance criteria should be established and are manufactured according to controlled and documentedjustified on the basis of the data obtained from lots used in procedures. Licensed biologics, approved drugs, and ap-preclinical and early clinical studies, lots used for demonstra- proved or cleared medical devices or implantable materialstion of manufacturing consistency, and relevant develop- that have been incorporated into cell, gene, or tissue-engi-ment data, such as those arising from analytical procedure neered product manufacturing processes present a knowndevelopment and stability studies. or more favorable safety profile for the patient than nonap-

Some AMs that are biological in nature may be difficult to proved or nonlicensed versions. Qualification programs for fully characterize. Because these materials exert their effects these AMs should reflect the extensive scrutiny that thesethrough complex biological activities, and biochemical test- items were subjected to in their development and manufac-ing may not be predictive of the AM’s process performance, ture. Consequently, greater emphasis should be placed on

functional or performance testing may be needed. Perfor- the investigation of the impact of inherent variability of mance variability of such materials may have a detrimental these AMs on final product function. For instance, a manu-impact on the potency and consistency of the final thera- facturer may utilize human serum albumin, intended for peutic product. Examples of complex functionality testing human administration, as a supplement to a cell cultivation

for AMs include growth promotion testing of individual lots medium for a cell-based product. Because the cell-basedof FBS on the cell line used in manufacturing, performance product is marketed as a licensed biological, one need nottesting of digestive enzyme preparations, and in vitro tissue repeat all the testing already performed by the supplier asculture cytotoxicity assays. (see aspects of Performance part of material qualification. In contrast, the impact of lot-Testing ). to-lot variability on cell growth rate or maintenance of an

important differentiated cellular property may be a prudentarea of investigation. Alternatively, the stability of this mate-Vendor Qualification rial at the concentration employed in processing or its po-tential for interaction with other processing components

Vendors supplying AMs should be qualified at the earliest may also be areas worthy of investigation. Such approachesopportunity. An early audit of the vendor’s manufacturing to AM qualification therefore focus on the AM as a potential facility, including their GMP and AM testing program, are source of variability that may influence final product po-basic elements of a vendor qualification program. A review tency and safety. Qualification programs for these AMsof the vendor’s processing procedures and documentation should be comprehensive to minimize consumer risk andprogram is essential in establishing confidence in the vendor ensure that unacceptable lots or adulteration will beas a reliable supplier. Additionally, vendors that have been detected.certified through an ISO inspection program or audited by The qualification program must also take into account theother governmental agencies tend to have robust quality quantity of the AM employed in manufacturing as well as itssystems in place. Reports of past audits of U.S. suppliers point of introduction in the manufacturing process. A rele-obtained through the Freedom of Information (FOI) Act may vant example is the use of FBS as a supplement to a tissueaugment the qualification process. culture medium used to expand a stem cell population from

It is important to develop a good working relationship a specific tissue for eventual administration to a patient (seewith a vendor. In some cases, the vendor may provide Manufacturing Overview under Cell and Gene Therapy Prod-higher manufacturing standards, custom formulation ser- ucts 1046 ). A qualification program for such an AM wouldvices, or replacement of substandard components upon re- include (a) assurance that the serum was sourced from aquest, with or without additional costs. A good rapport is country or region known to be free of bovine spongiformessential if further investigation into AM suppliers is war- encephalopathy (BSE); (b) assurance that the source herdsranted. It is also critical to ensure that the vendor takes ap- are monitored and test negative for specific diseases relevantpropriate steps to prevent cross contamination between its in agricultural settings (e.g., tuberculosis, brucellosis, footproducts during manufacture. Vendors should be familiar and mouth disease); (c) testing of the serum for sterility,with the principles of validation, especially cleaning valida- mycoplasma, endotoxin content, and adventitious bovine vi-tion, as well as viral inactivation and sterilization validation. ruses known to be associated with the material; 1 (d) theFinally, systems should be established where vendors supply review and archiving of the supplier’s certificate of analysis;written certification of processing or sourcing changes to (e) lot-to-lot assessment of the ability of the serum to con-customers, well in advance of the implementation of the sistently expand a representative cell population using achanges so that customers can evaluate the potential impact standardized cell culture quality control assay; and (f) on-siteof such changes. audit of the supplier to ensure that the material is sourced

and processed in a manner deemed acceptable by a respon-sible QA unit.Quality Control and Quality Assurance To aid manufacturers and developers in the design of their qualification programs for a variety of AMs, tiers of Because the components of the qualification program are sample risk categories are presented in Tables 1–4 and aremultifaceted and need to be in compliance with cGMP, they provided as a guide. Risk is also dependent on the amount

should be monitored by a quality assurance/quality control and the stage at which the AM is used in the manufacturingunit (QAU). Typical QAU activities include the following sys- process. Tables 1–4 do not address the impact of quantity or tems or programs: (1) incoming receipt, segregation, in- stage of use.spection, and release of materials prior to use in manufac-Tier 1— These AMs are low-risk, highly qualified materialsturing, (2) vendor auditing and certification, (3) certificate

that are well-suited for use in manufacturing. The AM isof analysis verification testing, (4) formal procedures andeither a licensed biologic, an approved drug, an approvedpolicies for out-of-specification materials, (5) stability testing,or cleared medical device, or it is intended for use as anand (6) archival sample storage.implantable biomaterial. Generally these components or materials are obtained as a sterile packaging system or dos-age form intended for their label use, but are instead uti-RISK CLASSIFICATION1 Most suppliers test for adventitious agents according to 9 CFR 113, which

A scientifically sound and rational qualification program was developed by the Center for Veterinary Biologics, Animal and PlantHealth Inspection Service, United States Department of Agriculture. Theseshould be designed for each AM and should take into ac-tests may differ from those used to test products developed for human usecount the source and processes employed in its manufac- (e.g., mycoplasma).ture. Whenever available, AMs that are approved or licensed




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Table 1. AM Risk Tier 1 Low-Risk, Highly Qualified Materials with Intended Use as Therapeutic Drug or Biologic,

Medical Device, or Implantable Material

Typical Use in Cell, Gene, or Tissue-Engineered Product Qualification or Risk Reduction

Example Manufacturing Activities

DMF cross reference (when possible or Recombinant insulin for injection Cell culture medium additive practical)Organ preservation fluid Process biological fluid employed in tissue Certificate of analysistransport or processing

Assess lot-to-lot ef fect on processHuman serum albumin for injection Cell culture medium performance 1

Sterile fluids for injection Process biological fluid employed in tissue Assess removal from final producttransport, cell processing, purificationImplantable biomaterials (formed collagen, Scaffolds, matrices for immobilized cellular Stability assessment on AM as stored for silicone, polyurethane constructs intended for cultivation use in manufacturing 2surgical implantation)Recombinant deoxyribonuclease for inhalation or Process enzyme employed in viral vector

injection manufacturing, stem cell processing Antibiotics for injection 3 Cell culture medium and biopsy transport fluid

additive to reduce risk of bacterialcontamination

Injectable monoclonal antibodies Immunologically targeting specific cellpopulations for selection or removal

Injectable cytokines Cell culture medium Vitamins for in jection; defined nutrients, Cell culture medium additive employed in cell

chemicals, or excipients intended for injection expansion, controlled cellular differentiation/activation step, or manufacture of a viral vector

IV bags, transfer sets and tubing, Storage vessels or container closure systems,cryopreservation bags, syringes, needles closed aseptic transfer systems

1 See Performance Testing .2 Often AMs are aliquoted or stored at different concentrations, in different buffers, or under conditions that are different from those stated on thelabel or previously validated. Data should be generated that demonstrate the stability and preservation of activity of the AM under the conditionsthat are specific to the manufacturing application.3 Beta lactam antibiotics should not be used as AMs due to the risk of patient hypersensitivity.

lized “off label” in the manufacturing process for the cell, assess consistency of removal of a known toxic substance or gene, or tissue-engineered product. lot-release testing to demonstrate reduction levels consid-

ered to be safe; or (e) validation of the manufacturing pro-Tier 2— These AMs are low-risk, well-characterized mate-cess of the cell, gene, or tissue-engineered product to assessrial that are well-suited for use in manufacturing. Their in-consistency of removal or inactivation of adventitioustended use is for drug, biologic, or medical device manufac-agents, disease-causing substances, or specific contaminantsture, including cell, gene, and tissue-engineered products asassociated with the material. Developers in the early stages AMs, and they are produced under relevant cGMPs. Mostof development should evaluate the necessity of theseanimal-derived materials are excluded from this category.materials and explore alternative substances or sources.Tier 3— These AMs are a moderate risk material that will

require a higher level of qualification than previous tier materials. Frequently, these materials are produced for in PERFORMANCE TESTINGvitro diagnostic use and are not intended for use in theproduction of cell, gene, or tissue-engineered products. In In cases where AMs are chosen for their ability to providesome cases, upgrade of AM manufacturing processes may a particular biological function in producing the therapeuticbe necessary in order to employ the AM in manufacturing product, performance testing becomes an essential compo-of these products (e.g., modification of the production pro- nent of their overall qualification. This is especially truecess for a diagnostic grade monoclonal antibody to include when the AM plays a critical role in modulating a complexrobust viral removal steps in purification). biochemical effect and has a large impact on product man-

Tier 4— This is the highest risk level for AMs. Extensive ufacturing yield, purity, or final product potency. These AMsqualification is necessary prior to use in manufacturing. The tend to be complex substances or mixtures, are frequentlymaterial is not produced in compliance with cGMPs. AMs biologically sourced, and can exhibit significant lot-to-lot va-are not intended for use in the production of cell, gene, or riability. As a result, these AMs usually have no simple iden-tissue-engineered products. This risk level includes highly tity test, nor can they be easily characterized by physical or toxic substances with known biological mechanisms of ac- chemical tests. The development of well-defined perfor-tion, and also includes most complex, animal-derived fluid mance assays for complex AMs will not only ensure processmaterials not subjected to adventitious viral removal or inac- reproducibility and final product quality, but in many casestivation procedures. These materials may require (a) an up- will satisfy the identity testing criteria in accordance with 21grade of AM manufacturing processes; (b) treatment of AMs CFR 211.84(d).to inactivate or remove adventitious agents, disease-causing In some cases, the initial qualification of an AM for use insubstances, or specific contaminants (e.g., animal viruses, manufacturing should be the investigation of the effect of prions); (c) testing of each lot of material to ensure that it is the amount of the AM on the desired response (increased

free of adventitious agents, disease-causing substances, or yield, purity, or potency of the therapeutic product). Thespecific contaminants; (d) validation of the manufacturing amount of the AM used in manufacturing should be chosenprocess of the cell, gene, or tissue-engineered product to to consistently yield the desired effect while minimizing is-




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

Typical Use in Cell, Gene, or Tissue- Qualification or Risk ReductionExample Engineered Product Manufacturing Activities

DMF cross reference (when possible or Recombinant growth factors, cytokines 1 Cell culture medium additive practical)Immunomagnetic beads Immunomagnetic separation of cells Certificate of analysis

Assess lot-to-lot effect on processHuman AB serum Cell culture medium additive performance 2

Progesterone, estrogen, vitamins, purified Cell culture medium additives, induction agents, Assess removal from final productchemicals (USP-grade) buffer componentsSterile process buffers Process biological fluid employed in tissue Stability assessment on AM as stored for

transport, cell processing, purification use in manufacturing 3

When relevant, confirm certificate of Biocompatible polymers, scaffolds, hydrogels Scaffolds, matrices for immobilized cellular analysis test results critical to productcultivation (could include functional assay)Proteolytic enzymes Process enzyme Vendor auditTissue culture media Cell culture medium additiveMonoclonal antibodies Immunologically targeting specific cell

populations for selection or removalDensity gradient media Cell separation via centrifugation1

These AMs should be produced from nonmammalian, recombinant sources (i.e., microbially grown in the absence of animal-derived growthmedium components).2 See Performance Testing.3 Often AMs are aliquoted or stored at different concentrations, in different buffers, or under conditions that are different from those stated on thelabel or previously validated. Data should be generated that demonstrates the stability and preservation or activity of the AMs under the conditionsthat are specific to the manufacturing application.

Table 3. AM Risk Tier 3 Moderate-Risk Materials Not Intended for Use as AMs

(frequently produced for in vitro diagnostic use or reagent grade materials)

Typical Use in Cell, Gene, or Tissue- Qualification or Risk ReductionExample Engineered Product Manufacturing Activities

DMF cross reference (when possible or Recombinant growth factors, cytokines Cell culture medium additivepractical)

Certificate of analysis Assess lot-to-lot effect on process

performance 1

Assess removal from final productStability assessment on AM as stored for use in manufacturing 2

When relevant, confirm certificate of analysis test results critical to product(could include functional assay)

Tissue culture media Cell culture medium additive Vendor auditMonoclonal antibodies (diagnost ic-grade Immunologically targeting specific cell Upgrade manufacturing process for

produced in cell culture) populations for selection or removal material to GMPProcess buffers Process biological fluid employed in tissue Develop stringent internal specificationstransport, cell processing, purificationNovel polymers, scaffolds, hydrogels Scaffolds, matrices for immobilized cellular Determine if lot-to-lot biocompatibility,

cultivation cytotoxicity, or adventitious agenttesting are needed

Proteolytic enzymes Process enzymePurified chemicals (reagent-grade) Culture medium additives, induction agents,

buffer components1 See Performance Testing.2 Often AMs are aliquoted or stored at different concentrations, in different buffers, or under conditions that are different from those stated on thelabel or previously validated. Data should be generated that demonstrate the stability and preservation or activity of the AM under the conditionsthat are specific to the manufacturing application.

sues by removing the AM in subsequent processing steps. each lot of AMs produces the expected rate andSuch testing frequently assesses the important functional at- amount of cellular proliferation or the expected level of tribute expected of the AM in a scaled-down or simulated secreted therapeutic agent.manufacturing process. Some examples follow: • If a monoclonal antibody is used to purify a particular

• If an AM is added to the culture media because it pro- cell type, the new lot of monoclonal antibody could bemotes cellular proliferation or the secretion of a critical shown to purify the cell population with the expectedtherapeutic agent, the assay could demonstrate that recovery and purity for the desired cell type.




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Table 4. AM Risk Tier 4High-Risk Materials

Typical Use in Cell, Gene, or Tissue- Qualification or Risk ReductionExample Engineered Product Activities

FBS Cell culture medium additiveSame as in Table 3, plus

Animal-derived (including human) extracts Cell culture medium additive Animal-derived polymers, scaffolds, hydrogels Scaffolds, matrices for immobilized cellular

cultivation Verify traceability to country of originPurified enzymes Process enzyme

Ascites-derived antibodies or proteins Immunologically targeting specific cell Assure country of origin is qualified aspopulations for selection or removal

safe with respect to source-relevant Animal or human cells used as feeder layers Cell culture substratum or source of medium animal diseases, including TSE

componentsChemical entities with known toxicities (i.e. Selection agents used in cell culture to improve

methotrexate, cholera toxin, Staphylococcus or maintain transgene expression, enhance Adventitious agent testing for animalaureus pore-forming hemolysin, Staphylococcus cellular proliferation, improve cell survival upon source-relevant virusesenterotoxins A and B, toxic shock syndrome cryopreservation, superantigens for thetoxin) activation of T cells

• If a deoxyribonuclease is used to degrade cellular DNA, in the recipient or have a detrimental effect on product po-new lots could be tested for the ability of the deoxyri- tency. Undesired effects in humans include direct toxicity of bonuclease to degrade DNA. the AM or an unwanted immunogenic response. Some ex-

• If a particular type of density gradient material is used amples include the following:to purify a vector or cell, new lots of the material used • In the generation of a tumor vaccine using a patient’sto make the gradient could be shown to purify the vec- tumor biopsy as the starting material, a chemical entitytor or cell to an acceptable level. is introduced to denature the cell surface proteins and

• If a plasmid or viral vector is used in the production of tumor antigens to enhance their antigenicity. Thea gene therapy vector (e.g., helper function), new lots chemical entity is known to be highly toxic.of the helper vector could be shown to produce the • Antibiotics may be added to a transport solution for expected amounts of the gene therapy vector. human cells to address microbial contamination issues

• If a cell therapy is produced in a hollow-fiber bi- associated with the procurement procedure. Residualoreactor, new lots of the bioreactor could be shown to levels of the antibiotic may affect the proliferative ca-produce the anticipated amount of cell product. pacity of the final engineered cellular product. Residual

The actual assay used may well evolve as the manufactur- antibiotics could also cause an anaphylactic response ining process is developed further and the critical relationships some individuals.of the AM and the final product are better understood. • FBS, employed in the cultivation of an engineered

Because most performance testing yields relative results, it human skin graft, may cause the development of a hu-is often helpful to assay a new lot of AMs side by side with moral antibody response directed against bovinean approved lot of AMs or an official reference standard, if proteins.available. This simultaneous comparison helps to reduce the • Aggregated mouse immunoglobulin, a trace impurity invariability due to different lots of cells or vectors and will a purified preparation of mouse monoclonal antibodyhelp discern variability associated with the different lots of used to target a cell population for immunoselection,

AMs. If performance testing involves assays to demonstrate may be immunogenic.that the new lot of AMs does not affect the impurity profile • A cytokine, employed as an immunomodulator in theof the final therapeutic product, either by generating new generation of a gene-modified autologous tumor vac-impurities or by increasing the level of existing impurities, it cine product, may elicit a severe reaction in theis helpful to assay both for the total level of impurities, as recipient.well as look for the presence of new impurities. An immuno- • Cholera toxin, employed as part of a cell culture me-logically-based binding assay can typically assess only the dium for a cell therapy product intended for intrave-total level of impurities. For example, a Western blot of the nous administration, will be highly toxic to the recipientgene therapy product that is probed both with antibodies if it is not removed during processing.to the product and antibodies to host cell proteins is useful These risks can be mitigated through the design of

for detecting new protein species and significant increases in processes to include steps to adequately remove the AMthe levels of host cell impurities. This initial qualification is through dilution, separation, or inactivation, as well as theenhanced by a performance assay that has a quantitative development of analytical detection assays to assess the AMreadout with a clear change in the signal when a significant levels during processing and in the final therapeutic prod-change in the amount of AMs is introduced into the assay uct. Assessment and removal strategies for residual AMs(e.g., dose response). A threshold-type response (i.e., there should be considered in the early phases of process develop-are two levels of response to the AM and neither large ment. There are two different approaches for assessing re-changes in an AM below a certain dose nor above a certain sidual AM levels in the final therapeutic product: (1) Valida-dose change the response) can make it more difficult to tion studies can demonstrate that the process is capable of select a concentration of AM that consistently results in the removing more of the AM than would be present in adesired effect and minimizes the residual levels of the AM in worst-case scenario. (2) The residual levels of an AM can bethe final therapeutic product. measured for each lot at an appropriate step in the manu-

facturing process. Validation of an AM removal is often best performed by

ANCILLARY MATERIALS RESIDUAL LEVEL spiking the impure product with “worst case” or higher lev-ASSESSMENT AND REMOVAL els of the AM and showing the purification process is capa-

ble of removing the AM to “undetectable levels.” Clearance AMs are not intended to be present in the final dosage factors can then be generated for each purification step in a

form in cell, gene, and tissue-engineered products. Their manner analogous to that done in viral clearance studies.presence in the final product could lead to undesired effects When designing the validation studies, the following three




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considerations should be included: (1) The assay should be final product and endanger the health of patients. Thus, im-able to accurately quantitate the AM in each sample matrix. plementing an AM qualification program that addresses the(2) If the validation is conducted at a scale smaller than that risks associated with the AM, the stage of manufacture atused for routine lot production, the comparability of this which it is used, and the amount of the AM used duringsmaller scale process to the full scale process needs to be manufacture will ensure the safety and effectiveness of thedemonstrated. This usually means that the smaller scale pro- final product.cess is operated using the same critical parameters as the

full scale process with the product generated at each step APPENDIXhaving a similar purity and yield. (3) As with any spikingstudy, one has to demonstrate that the additional, higher

AMs used in cell, gene, and tissue-engineered productslevel of AM has not affected the purification process. If thewill be regulated in the context of the manufacturing pro-second approach of measuring residual levels of the AM incess of the cell, gene, and tissue-engineered products. Cer-each lot is used, the specification for the maximum amounttain AMs may already be approved for uses other than for of AM in the final therapeutic product is based on thecell, gene, and tissue-engineered product manufacture. It isamount of the AM in the lots used in toxicological or preferable to source AMs that are approved therapeuticclinical studies or known toxicological data.products when they are available because they are well-The development of sensitive and reproducible analyticalcharacterized with an established toxicological profile andassays for AMs is another important component of a riskare manufactured according to controlled and documentedreduction approach. Two types of assays are useful in assess-procedures. The following list of documents should provideing the levels of residual AM impurity: a limit test and arelevant regulatory guidance and a description of best prac-quantitative test. Either test should be accurate, precise, ro-tices in product and process development, manufacturing,bust, and have a low limit of detection. Assays for residualquality control, and quality assurance: AMs may be performed on the product before it is formu-

• Biological Reactivity Tests, In Vitro 87lated (e.g., on the drug substance) to avoid any interference• Biological Reactivity Tests, In Vivo 88of the components used in the formulation with the assay• Biotechnology-Derived Articles 1045 for residual AMs or in the final drug product. Spike-recovery • Cell and Gene Therapy Products 1046controls are often included in such assays to demonstrate• Biotechnology-Derived Articles—Amino Acid Analysis that the sample matrix does not inhibit the detection of the

1052 AM. Preferably, assays should be designed to detect all• Biotechnology-Derived Articles—Capillary Electrophoresis forms of AMs including aggregates, fragments, or conju-

1053gates. Aggregated protein has been shown to be particularly• Biotechnology-Derived Articles—Isoelectric Focusing 1054immunogenic.• Biotechnology-Derived Articles—Peptide Mapping 1055Immunoassays such as ELISA are most commonly used to• Biotechnology-Derived Articles—Polyacrylamide Gel Electro-assess residual levels of AMs. An ELISA for bovine serum al-

phoresis 1056bumin (BSA) has been used to assess residual levels of FBS.• Biotechnology-Derived Articles—Total Protein Assay 1057Polymerase chain reaction (PCR) technology has been em-• 21 CFR 211 Subpart E, 211.80 through 211.94 andployed to assess residual levels of host cell DNA. Labeling

211.101cells with 3 H thymidine or performing PCR for a feeder cell-• 21 CFR 312specific gene sequence are two ways to assess for residual• 21 CFR 314levels of feeder cells. If “wash out” of the AM is achieved by• 21 CFR 801.109 (b) (1)exhaustive dilution associated with further processing activi-• 21 CFR 807.81 through 21 CFR 807.97ties, it may be useful to calculate the dilution factor for the• 21 CFR 812 AM during this processing. In some cases, this is sufficient to • 21 CFR 814ensure that the AM has been reduced to safe levels for early• FDA Center for Biologics Evaluation (CBER) “Draft Guid-clinical development. Data should be obtained later in

ance for Monoclonal Antibodies Used as Reagents inclinical development to confirm the wash out of the AM atDrug Manufacturing” (1999)the expected step(s). This approach is particularly useful

• FDA Center for Biologics Evaluation (CBER) “Points towhen there is pre-existing knowledge of the therapeutic lev-Consider in the Characterization of Cell Lines Used toels and toxicity of the AM. In other cases, information re-Produce Biologicals” (1993)garding the safety and tolerability of the AM should be col-

• FDA Center for Devices and Radiological Health (CDRH)lected (in preclinical toxicology studies or later with human“Class II Special Controls Guidance Document: Tissueclinical studies) in order to determine the safe or nontoxicCulture Media for Human ex vivo Tissue and Cell Cul-levels that must be achieved. These data may be neededture Processing Applications; Final Guidance for Industryeven for an AM that is approved for use for therapeuticand FDA Reviewers” (May 16, 2001)purposes if it is being used in a manner inconsistent with its

• CDRH Blue Book Memorandum G95-1intended use or labeling or if the route of administration or • ISO 10993-1: 1997 “Biological Evaluation of Medicaldosage level of the AM may present risks not previously

Devices—Part 1: Evaluation and Testing”encountered or considered.• International Conference on Harmonization (ICH) Q5A

“Guidance for Viral Safety Evaluation of BiotechnologyCONCLUSION Products Derived from Cell Lines of Human and AnimalOrigin”

While many types of AMs are used during the manufac- • International Conference on Harmonization (ICH) Q5Dture of cell, gene, and tissue-engineered products, they have “Guidance on Quality of Biotechnological/Biologicalreceived less emphasis than the final products. However, the Products: Derivation and Characterisation of Cell Sub-importance of AM quality to the quality of the final product strates Used for Production of Biotechnological/Biologi-cannot be overstated. Good quality AMs should perform as cal Products”intended in a consistent manner, batch-to-batch, if they are • Public Health Service Guideline on Infectious Diseasescarefully selected and appropriately used. AMs of insufficient Issues in Xenotransplantation (October 18, 2000)quality will affect the quality and the effectiveness of the




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USP 35 General Information / 1045 Biotechnology-Derived Articles 491

though it is theoretically possible to develop public stan-1045 BIOTECHNOLOGY- dards for a macromolecular article, it is not possible todevelop specific standards that incorporate all prospectiveDERIVED ARTICLES methods of production. The compendial perspective is todevelop public standards that can be applied to a finalproduct without comprehensive knowledge of productiondetails but which can ensure maintenance of safety, identity,Macromolecular substances can be obtained by a number strength, quality, and purity.

of methods including extraction from natural sources, modi- Testing for identity, purity, and activity generally requires fication of naturally occurring protein, mammalian cell cul- the use of USP Reference Standards. It will be necessary toture in vitro, mammalian cell culture in vivo, production by consider what USP Reference Standards might be requiredmicroorganisms, and chemical syntheses. From a com- and how relevant they might be to the method of produc-pendial perspective, macromolecular articles derived from tion as it relates to a final product’s characteristics. Suchbiotechnology processes—or more specifically from recombi- decisions will be made on a product-by-product basis.nant-DNA (rDNA) technology, hybridoma technology, and Favorable consideration will be given to the use of USP Ref-transformed continuous cell lines—are those articles for erence Standards that are representative of the specificwhich official names have been established. These articles products that have undergone clinical testing and are fullyhave official public standards for identity, strength (po- characterized.tency), quality, and purity. Advances in genetics and the Although early adoption in USP of general methods of applications of genetic engineering have made the produc- analysis of macromolecular drugs could be conducive totion of new and existing macromolecular articles technologi- early standardization of methods, the technology and ana-cally and economically feasible. lytical procedures are evolving very rapidly. Analytical proce-The technologies involved in producing a protein by bi- dures—chemical, physical, microbiological, and immunologi-otechnological processes have been widely documented and cal—will be included in the specific product monographs.general guidelines have been established by the federal gov-

ernment. The products of biotechnology may be regulatedas drugs, biologics, or diagnostics, depending on their SCOPE OF BIOTECHNOLOGY IN THEsource, composition, and intended use. The novel ap- DEVELOPMENT OF PHARMACOPEIALproaches permitted by biotechnology can make it difficult

ARTICLESto apply classic definitions of these categories and FDA hasadvised manufacturers to seek clarification in the earlystages of development for how a product will be regulatedwhen classification is not obvious. 1 The overall regulatory Definition of Biotechnology—Historicalscheme for biotechnology-derived products is the same as

Perspective for products in the same category produced by traditionalmanufacturing methods, with the addition of specific re-

In its broadest definition, biotechnology refers to the usequirements suited to the biotechnology-derived product.of living organisms, including isolated mammalian cells, inThe general requirements are described primarily in the ap-the production of products having beneficial use. This defi-plicable parts of the Code of Federal Regulations, Title 21.nition would place alcohol, antibiotic production, and dairyNIH has published a guideline for rDNA research that isprocessing, for example, within the scope of biotechnology.mandatory for both public and private NIH-supported re-However, the current interest in biotechnology is primarily asearch. This guideline has wide acceptance and voluntaryresult of two major advances. The first advance was the de-

compliance is common by institutions and corporations not velopment of rDNA technology, which allowed the genes of specifically governed by it. 2 Laboratory safety practices, par-one species to be transplanted into another species. Thus,ticularly protection from potentially infectious materials, aregene coding for the expression of a desired protein (usuallya concern. 3 Producing macromolecular articles by bi-human) could be inserted into a host prokaryotic or eukary-otechnological processes involves initially the cloning of aotic cell in such a manner that the host cell would thenspecific gene in the laboratory, or the construction of a syn-express usable quantities of the desired protein. The secondthetic gene, with subsequent insertion into a host cell andmajor advance was the development of techniques for pro-subcloning in a microorganism or cell culture; then a pro-ducing large quantities of monoclonal antibodies (i.e., anti-cess development on a pilot scale to optimize yield andbodies arising from a single lymphocyte).quality; and finally large-scale fermentation or cell culture

Biotechnology within the pharmaceutical industry gener-processes. The next step, which is the most relevant to theally refers either to the production of protein products usingdevelopment of compendial monographs, is the purificationrDNA techniques or to the production of monoclonal anti-of the macromolecular proteins. This is followed by animalbodies. Other technologies, such as transgenic animals andtesting, clinical testing, regulatory approval, and marketing.plants, gene therapy, and antisense DNA, may have poten-Development of relevant public standards for thesetial implications for the pharmaceutical industry in the fu-macromolecular articles is generally closely linked to theture but are not within the scope of this chapter.processing technology used and the physicochemical and

biological characteristics of a specific drug. Characterizationsof these articles to ensure safety, purity, and activity should rDNA Technologyincorporate classical techniques as well as methods specificto the technology. There is always the possibility that these The major steps in the application of rDNA technology for articles may cause some untoward effects in patients using production of a desired protein are outlined in this section.them due to immunological sensitization as a result of a The critical first step is identification of the protein that is tosingle (or multiple) molecular modification. Such a possibil- be produced, followed by the isolation of the gene of inter-ity requires precise characterization of these substances. Al- est (i.e., the DNA sequence coding for the desired protein).1 A series of documents entitled Points to Consider are available from the Di- Once this gene is isolated and fully characterized, it is in-rector, FDA Center for Biologics Evaluation and Research, HFB-1, 8800 Rock- serted into a suitable vector such as a plasmid, which is anville Pike, Bethesda, MD 20892. extrachromosomal segment of DNA usually found in certain2 This guideline was originally published in the Federal Register, Guidelines for Research Involving Recombinant DNA Molecules 1986; 51 (88): 16957-16985. bacteria. The plasmid is then inserted into the host cell.Copies may be obtained from the Office of Recombinant DNA Activities, Clones of the transformed host cell line are isolated, and12441 Parklawn Drive, Suite 58, Rockville, MD 20852. those that produce the protein of interest in the desired3 A comprehensive guideline, Biosafety in Microbiological and Biomedical Labo-

quantities are preserved under suitable conditions as a cellratories , is available from the Superintendent of Documents, U.S. GovernmentPrinting Office, Washington, DC 20402, stock #107-040-000508-3. bank. As manufacturing needs arise, the cloned cells can be

ffici from y 1 2012
