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Recommendations for Cell Banks Used in GXP Assays Preparation, Characterization, and Storage Ana T. Menendez, Nadine Ritter, Jonathan Zmuda, Darshana Jani, and Jaya Goyal

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ells and cell-derived reagents form the basis of an operationally challenging class of test methods used in execution of product potency testing (stability and lot release), assessments of pharmacokinetic/ pharmacodynamic (PK/PD) profiles, detection of antidrug antibodies (ADAs) or neutralizing antibodies (NAB), and characterization and comparability testing of biopharmaceutical products. Frequently, cell-based assays provide the only measurement of the tertiary/quaternary structure of each batch of product at the time of lot release and during stability testing to assist in determining product shelf-life. Cultured cells themselves are often used to generate monoclonal antibodies (MAbs), enzymes, or substrates for use as critical reagents in other types of tests, including ligand binding and enzymatic assays. In all these applications, the cells serve as highly critical, highly complex “reagents” that require distinct characterization and control measures to ensure operational consistency over time. Cells are sensitive to innumerable chemical and physical elements that can alter expression of their cellular proteome and change growth characteristics or responsiveness to ligands/biopharmaceuticals. Establishment and characterization of homogeneous, stable cell banks are necessary to ensure that starting cellular material for each assay is as consistent as possible. Appropriately established and stored master and working cell banks (MCBs and WCBs) provide a continuous supply of viable cells to generate accurate, reliable results within specified test methods or provide the cell-derived reagents used in those methods. Although the strategy for preparing cell banks is clearly defined by regulators for cells used to produce biotechnology products, it is less clear what strategies should be applied to

cells that are used solely as part of analytical or bioanalytical test methods. An early FDA points-to-consider (PTC) guidance on characterization of cell lines for producing biologicals (1) outlined many MCB and WCB characterization requirements that were later adopted into ICH Q5D for production cell lines (2). But it is still common to find the phrase PTC testing used in relation to the list of tests applied to nonproduction cell banks even though that guidance was not intended to apply to such banks. In the absence of clarifying information about the significant differences in intended use between production cell lines and those used for analytical/bioanalytical methods, some laboratories choose to apply the entirety of the PTC guidance to both. Conversely, others fail to establish cell banks at all for analytical use, severely jeopardizing the desired state of assay control. The US Pharmacopeia recently published a suite of chapters — Development (3), Validation (4), and Assay Analysis (5) — providing guidance on good manufacturing practice (GMP) potency bioassays. Several paragraphs in USP provide a general outline for MCB and WCB preparation; however, specific details are not provided to assist laboratories with the less obvious but still critical aspects of creating, characterizing, and storing MCBs and WCBs. Recommendations presented herein support and significantly elaborate on principles noted in USP for establishment and characterization of mammalian and bacterial cell banks used to support analytical/bioanalytical testing. These approaches may also be extrapolated (when applicable) to include cells used for reagent production, for growing viruses used in test methods, and so on. These

B i o P r o c e s s special report strategies represent our combined opinions on best practices in establishment, characterization, and maintenance of controlled and consistent cell sources using a risk-based and productphase–appropriate approach. Each sponsor should determine which recommendations to adopt and when each will be performed based on the level of risk acceptable in developing and validating methods that use cells or cell-derived reagents.

Laboratory Best Practices for Cell Banking

Before generating MCB/WCBs either for use in cell-based assays or for generating cell culture-derived reagents, consider the expected practices under which such laboratory work is done. As a part of quality-by-design (QbD) strategies being adopted for analytical test methods, reviewers and auditors are increasingly expecting to see MCB/WCB activities reported in (or linked to) method qualification and/or validation reports (6). Banking and characterizing cells is critical for designing quality principles into associated test methods. To support the objective of analytical QbD, it is thus recommended to include phase-appropriate MCB/WCB details in assay development, qualification, or validation reports that form the method’s life-cycle documentation. Such documents can be critical should a method change in performance through operational drift, technology transfer, or from changes to other critical assay components (7). The 1993 FDA PTC guidance on characterizing cell banks for biological production (1) indicates that tests performed to establish production MCBs/WCBs should be conducted under 21 CFR Part 58 good laboratory practices. Contaminated or unstable cell lines can have a major impact on patient safety through changes to expressed proteins, so it is reasonable to consider establishment of production cell banks to be covered by GLP regulations as are the viral inactivation/clearance studies performed in support of product safety (1). Because nonproduction cell lines used for bioassays never contact the product manufacturing stream, patient safety is not directly at risk from cell bank contamination. So this guidance does not directly apply to cell lines used only for analytical/ bioanalytical test methods. It is important to note here the clear distinction between work performed under GXP regulations and activities performed to achieve GXP compliance. For example, executing quality control (QC) release or stability testing of a product using a validated cell-based potency assay included in a product’s master specifications must be under GMP regulations as a part of laboratory controls (8). Similarly, using a validated cell-based assay for NAB 28 BioProcess International

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testing as part of a GLP and/or good clinical laboratory practice (GCLP) protocol is done under GLP (9) or GCLP (10). By contrast, activities (such as cell banking) performed to generate the test methods that will be used as part of subsequent GXP testing are not subject to GXP regulation. So it is our intent to describe non-GXP best practices that can ensure that cell banking activities are performed and documented in a way that’s consistent with the future use of cells in GXP analytical and bioanalytical test methods. Furthermore, for our purposes, the term GXP cell bank defines the intended use of MCBs and WCBs in methods used for GXP studies or tests. It does not imply that cell banking activities to support assay development were performed under GXP regulations; however, these best practices cover documenting the developmental history of key activities performed to generate the cell banks for such methods. The laboratory work conducted to establish them in support of GXP method development and validation thus may be performed by an R&D group following sound scientific principles and good documentation practices. Without formal regulatory requirements on R&D laboratory quality practices, some laboratory groups choose to conduct MCB/WCB activities under internally defined GXP practices as a part of their overall quality management system. That is not a requirement, however. Regardless of which (or whether) quality systems are implemented in R&D, we have successfully used the specific practices noted here for capturing details pertaining to the generation of cell banks intended for use as part of regulated studies or tests. We assume here that laboratory personnel involved in cell banking activities are skilled in standard aseptic cell culture techniques, that instruments used for measurement and delivery (e.g., pipettes, balances, pH meters, and spectrophotometers) are calibrated, and that all reagents and materials have been stored appropriately and have not expired. Procedures dictating establishment and use of the cell banks are assumed to be captured in controlled documents to be followed by trained laboratory personnel. GXP compliance is not a legal requirement to generate nonproduction cell banks; but use of proper documentation and best practices provides a high level of confidence in the continued performance of such cells over time. Our recommendations illustrate details of the technical, regulatory, and quality principles and practices that have been implemented successfully in major biotechnology organizations for establishing and characterizing MCBs and WCBs used in cell-based methods or production of cell-derived reagents. Sponsors should determine the best practices that minimize risks to their own programs and adopt quality systems that align with specific guidance provided to them by regulatory reviewers or inspectors for their products and laboratories.

The Role of Cell Banks in Bioassay Development

Cell-based assay development is an ongoing process divided into separate stages. This section is not meant to be a guide to bioassay development or validation — other references focus on the subject (1, 3, 4, 11). Instead we highlight the role of cell banks in development of bioassays or production of critical reagents.

Risk-Based Approaches for Initial Cell Characterization Before Banking: The extent of initial cell line characterization

before cell banking should be risk-based depending on multiple factors, including the cell source, history, and documentation available. We recommend that lower-risk cells — obtained from reputable sources with available history and documentation — be minimally shown to be mycoplasmafree (if not already demonstrated by the vendor) using a fast technique (e.g., polymerase chain reaction, PCR), sterile in the absence of antibiotics, and responsive in the bioassay for which they will be used before banking. For higher-risk cells without history or documentation — or those with identity, species, activity, or sterility in question — it may be desirable to perform additional testing before cell banking. This should be considered case by case: e.g., species identification by isoenzyme analysis, verification of cell line identity by short-tandem repeat (STR) profiling, screening for adventitious agents, and PTC mycoplasma/sterility testing. Such methods may be considered en masse or individually to address specific risks and provide confidence before efforts are expended in generating a cell bank. In addition to cell-specific issues, other variables such as the intended use of a bioassay should be considered when determining the extent of initial cellular characterization before banking. For example, a more thorough knowledge may be desired for cells that will be used as part of lotrelease assays during initial/early clinical development than for cells that will be used only as part of qualitative or characterization assays. This example highlights potential benefits of early characterization for a high-risk cell line with a risk-based approach: Cells were received in a regulated testing laboratory from an academic lab with the understanding that they were human cells transfected with a human receptor. They were certified sterile and mycoplasma-free and showed desired bioassay activity. A seed vial was propagated and banked, with successful method development and validation during phase 3 clinical trials. Isoenzyme analysis was not performed until just before commercialization, and results demonstrated that this cell line was of rodent origin because it expressed the transfected rodent receptor. Fortunately, adequate homology existed between human and rodent receptors, so that discovery had no negative effects on the bioassay’s intended, and the rodent cell line was accepted as the target cell. Had it been determined that these cells were a mixture of human and rodent cells, that outcome would have created a setback in product development because the situation would have been unacceptable. A heterogeneous cell population is in danger of causing bioassay drift and can lead to inaccurate results.

Figure 1:  Example flow chart of cell banking process Obtain seed vials of cells from source and initiate culture.

Subculture cells at various densities and monitor growth.

Identify preliminary log-phase growth conditions.

Verify activity of cells in the bioassay.

Expand cells based on identified log-phase growth conditions.

Freeze master cell bank.

Initiate seed vial testing.

Thaw master cell bank for creation of working cell bank.

Expand cells based on identified log-phase growth conditions.

Freeze working cell bank.

Initiate master cell bank testing.

Thaw working cell bank.

Initiate working cell bank testing and characterization.

Develop and qualify assay.

Validate assay.

Feasibility Assessment: The feasibility stage consists of initial experiments that examine cells and conditions (concentrations, timing, endpoints, technology platforms, and so on) that will be used in a given method. This provides enough information to commit to one particular cell line for future method development and thus cell-bank generation. Bacterial bioassay cells also should undergo preliminary testing to demonstrate their purity, lacking phages and other contaminants that can cloud data interpretation. Examples of ideal performance expectations for a cell line used in a potency assay are • A signal–background ratio >5:1 (lower ratios may be acceptable if results are reproducible) • Appropriate dose response and linearity ≥0.95 across the intended range of the assay • Slope of the dose-response curve ≥1.0 • Intraassay replicate precision 5; slope > 1.0; R2 > 0.98

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Cells were thawed on 12 November 2009; assay executed at passage 4; all specs passed (signal:noise = 8, slope = 1.2, and R2 = 0.99)

quality general laboratory operations are all critical to minimizing cell-based assay failures. None of those activities, however, can ensure method accuracy and precision if the starting cell bank is contaminated, unstable, or inconsistent. Lack of control over the cultures that form the foundation of an assay (or that provide critical reagents) will inevitably lead to performance problems in the assay itself. Ultimately, individual sponsors are responsible for developing their own strategies regarding the level of characterization and the rigor of cell bank testing performed based on risk, pertinent internal policies, and regulatory input. The strategies presented here have proven successful for supporting development and validation of methods used in product testing and laboratory studies to support a number of GXP applications. So they should provide useful general guidance for generating and maintaining these types of cell banks.

Acknowledgments The authors thank the following individuals for their critical review and input for this manuscript: Kendall Carey, Medimmune; Paul Duncan, Merck; Paula Hochman, Wei Zhang, Helena Madden, and Svetlana Bergelson of Biogen Idec; Helen Loughrey of Theratechnologies (Canada); Natalie Cohen of NC Biologics Consulting (Israel), formerly of Teva); Jane Robinson of NIBSC (United Kingdom); and several FDA CDER reviewers from the Office of Biotechnology Products’ Divisions of Therapeutic Proteins and Monoclonal Antibodies.

References

1 CBER. Points to Consider in the Characterization of Cell Lines Used to Produce Biological Products. US Food and Drug Administration: Rockville, MD, 1993; www.fda.gov/downloads/biologicsbloodvaccines/ safetyavailability/ucm162863.pdf. 2 ICH Q5D: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products. US. Fed. Reg. 63(182) 1998: 50244–50249. 3 USP Development and Design of Biological Assays. Pharmacop. Forum 36(4) 2010: 956. 4 USP Validation of Biological Assays. Pharmacop. Forum 36(4) 2010: 986. 5 USP Analysis of Biological Assays. Pharmacop. Forum 36(4) 2010: 1005. 40 BioProcess International

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6 ICH Q8(R2): Pharmaceutical Development.US Fed. Reg. 71(98) 2008: www.ich.org/fileadmin/Public_Web_Site/ICH_Products/ Guidelines/Quality/Q8_R1/Step4/Q8_R2_Guideline.pdf. 7 Ritter NM, Wiebe M. Validating Critical Reagents Used in CGMP Analytical Testing: Ensuring Method Integrity and Reliable Assay Performance. BioPharm May 2001: 12–21. 8 Stability Testing: Testing and Release for Distribution. US Code of Federal Regulations, Title 21, Part 211.165. US Food and Drug Administration: Rockville, MD. 9 Good Laboratory Practices. US Code of Federal Regulations, Title 21, Part 58. US Food and Drug Administration: Rockville, MD. 10 Good Clinical Laboratory Practice. World Health Organization: Geneva, Switzerland, 2009; http://apps.who.int/tdr/publications/tdrresearch-publications/gclp-web/pdf/gclp-web.pdf. 11 Rieder N, et al. The Roles of Bioactivity Assays in Lot Release and Stability Testing. BioProcess Int. 8(6) 2010: 33–43. 12 Meza RA, et al. Study of the Stability in Real Time of Cryopreserved Strain Banks. Universitas Scientiarum, Pontificia Universidada Javeriana 9(2) 2004: 35-42. 13 Gilliland SE, Speck ML. Relationship of Cellular Components to the Stability of Concentrated Lactic Streptococcus Cultures at –17 C. App. Micro. 27, 1974: 793–796, 14 Technical Report 47: Preparation of Virus Spikes Used for Virus Clearance Studies. Parenteral Drug Association: Bethesda, MD, 2010. 15 Menendez AT. Accelerating Bioassay Transfer in a GMP Environment. BioProcess Int. 7(11), 2009 : 26–33. 16 Duguid J. Top Ten Validation Considerations When Implementing a Rapid Mycoplasma Test. Am. Pharmaceut. Rev. 2010: 26–32. 17 Nims RW, Herbstritt CJ. Cell Line Authentication Using Isoenzyme Analysis. BioPharm Int. June 2005. 18 ATTC Standards Development Organization Workgroup ASN0002. Nat. Rev. Cancer 10, 2010: 441–448. •

Ana T. Menendez, PhD, is president of Menendez Bio-Consulting LLC. Corresponding author Nadine Ritter, PhD, is senior CMC consultant for Biologics Consulting Group, 400 North Washington Street, #100, Alexandria, VA 22312; 1-240-372-4898; nritter@bcg-usa. com, www.bcg-usa.com. Jonathan Zmuda, PhD, is associate director of R&D for Life Technologies. Darshana Jani is translational medicine manager, and Jaya Goyal, PhD, is principal investigator in translational medicine, both at Biogen Idec.