Defining Your Product Profile and Maintaining Control Over It, Part 2 Challenges of Monitoring Host Cell Protein Impurities

B IO P ROCES S TECHNICAL

Kathleen Champion, Helena Madden, John Dougherty, and Emily Shacter

n this second session of the CMC monitored in biopharmaceutical Strategy Forum (19–20 July 2004), preparations. Because host cell derived representatives from the proteins are highly complex mixtures Ibiopharmaceutical industry and of polypeptides, measuring them poses the FDA discussed the challenges of some challenging analytical dilemmas. monitoring host cell protein (HCP) Nonetheless, HCP assays are important impurities during the development tools for demonstrating product purity and commercialization of recombinant and consistency of manufacture. protein drugs. Our discussion followed a session about process-related impurities MEASUREMENT BY IMMUNOASSAYS in general. The sidebar provides more Host cell protein populations are information about this meeting from typically measured using multianalyte Part 1 of this four-part article. enzyme-linked immunosorbent assays Recombinant protein (ELISAs) with polyclonal antibodies. pharmaceuticals are purified to a high The HCP values are expressed in units degree from the host cells that produce of ng/mL or parts per million (ng HCP them. Whether a recombinant protein per mg product). These values reflect is secreted from Chinese hamster ovary the degree of specific immunoreactivity ANDREI TCHERNOV (WWW.ISTOCKPHOTO.COM) (CHO) cells, for example, or remains because they are a measure of the within a bacterial host (such as epitope population in a sample. That method offers high resolution Escherichia coli), it is necessary for host Nevertheless, the value obtained in because it separates proteins by both cell derived proteins to be separated an ELISA is a reasonable measure isoelectric point and molecular mass. from the recombinant protein and for of HCP mass, provided that the Application of 2D-PAGE may be residual host cell proteins to be antibody reagent and immunoassay useful in certain cases, but SDS- are characterized and can be shown to PAGE is generally sufficient. When PRODUCT FOCUS: ALL RECOMBINANT accomplish the intended purpose. bands are detected in polyacrylamide PROTEINS It is important to recognize both gels, Western blotting with antibodies the limitations and strengths of using PROCESS FOCUS: PROCESS to product and HCPs can be used in immunoassays to measure host cell DEVELOPMENT (PRODUCT an attempt to distinguish product- proteins. The key limitation is that CHARACTERIZATION) related bands from HCP bands, nonimmunoreactive or weakly respectively. The conditions used in immunoreactive proteins are not WHO SHOULD READ: MANUFACTURING Western blotting can be adjusted to detected by these assays. To detect AND PROCESS DEVELOPMENT, PROJECT allow binding by low affinity anti- such proteins, an independent method MANAGERS, AND ANALYTICAL PERSONNEL HCP antibodies that do not bind in is used, usually SDS-PAGE with a the ELISA format. KEYWORDS: HOST-CELL PROTEINS, sensitive protein staining method One strength of immunoassays is MICROBIAL AND ANIMAL CELLS, PROCESS- (e.g., silver or SyproRuby). Some that they can detect host cell proteins RELATED IMPURITIES, IMMUNOASSAYS sponsors also use two-dimensional at levels undetectable in gels. This is polyacrylamide gel electrophoresis LEVEL: BASIC because an ELISA measures the (2-D PAGE) to assess product purity. collective sum of immunoreactive 52 BioProcess International SEPTEMBER 2005 PROCEEDINGS OF THE WCBP CMC STRATEGY FORUM, 19–20 JULY 2004 Well-characterized biopharmaceutical products are facilitate efficient and cost-effective development, defined during development by the identification and production, and availability of safe and beneficial new quantification, when at all possible, of both process- drug products. related and product-related impurities. However, The CMC Strategy Forum on Defining and Controlling expectations for the permissible levels of residual Product Profile: The sixth Well-Characterized processing components or product variants throughout Biotechnology Pharmaceutical (WCBP) Chemistry, the product development life cycle are unestablished. Manufacturing, and Controls (CMC) Strategy Forum was Several factors have contributed to the difficulty in held on 19–20 July 2004 at the Lister Hill Auditorium on standardizing those requirements, including safety of the the NIH Campus in Bethesda, Maryland. Sponsorship residual components and process capability to reduce of the event was provided by the California Separation unwanted components. These, and other variables, Science Society (CaSSS; www.casss.org) as part of a have resulted in the “case-by-case” model for regulatory series of discussions between industry and regulatory assessment of impurities to be specified and acceptable participants exploring current practices in analytical limits for control. and bioprocess technologies for development and communication of consensus concepts. The absence of clear guidance contributes to uncertainty in the fitness of product development plans, potential The purpose of this two-day forum was to survey which misalignment of priorities with the true safety risk posed methods are most useful in identifying and measuring by each impurity, delay in regulatory review of filings as process-related and product-related impurities — and these factors are evaluated anew with each dossier, and identify strategies and specifications to ensure a inconsistent standards from product-to-product or consistent product profile. The first day concentrated on sponsor-to-sponsor. Establishment of appropriate process-related impurities (Parts 1 and 2 of this article), standards for acceptable levels of process-related and whereas on day two the focus was shifted to product- product-related impurities, along with strategies for related impurities (Parts 3 and 4). removal and requirements for specification would proteins. In other words, a signal is Comparing Product-Specific and The polyclonal antibodies used in obtained from a number of different Multiproduct Immunoassays: multiproduct immunoassays are raised proteins present at low levels. Biopharmaceutical companies usually in moderate-sized mammals (typically Therefore, even when a recombinant develop HCP immunoassays for their rabbits or goats) against a complex product is sufficiently pure that there own use, maintaining proprietary mixture of host cell proteins, such is not enough of any particular host assays and antibody reagents. as from a whole cell extract. cell protein species to be detected in a Multiproduct ELISAs can be Multiproduct assays differ from polyacrylamide gel, an HCP ELISA developed for all products derived process-specific assays, which can provide a measurement of residual from a particular cell type or generally use antibodies against an host cell proteins. Overall, ELISAs expression sytem. For example, a in-process pool taken at the first are more sensitive than methods sponsor may develop one assay for downstream step in a purification for monitoring individual HCPs measuring CHO cell proteins and use process. Such pools are thought to except that they do not measure it for all of that sponsor’s CHO cell- represent the most likely impurities nonimmunogenic proteins. derived products. Such multiproduct for a product rather than all the Given the above considerations, assays are acceptable, provided that immunogenic host cell proteins that a complete assessment of product both the antibody reagent and could be present. In some cases, such purity with regard to host cell proteins immunoassay are characterized and process-specific assays may be more should include information from more shown to be suitable for assessessing sensitive or more specific than than one type of analysis. It should HCPs with each product. A broad multiproduct assays. include the results of a meaningful spectrum of host cell proteins should Product-specific assays also have ELISA as well as SDS-PAGE and be recognized by the antibodies. An several shortcomings. For example, possibly immunoblot analysis. Some antibody preparation that recognizes the applicability of an antibody sponsors have seen significant only a few proteins is insufficient for preparation generated against partially quantities of a particular host cell- monitoring HCP populations and fractionated material depends on the derived protein present in their removal during product reproducibility of the manufacturing recombinant protein preparations purification. HCP populations step(s) used to generate the HCP despite the lack of detection by differing widely from one product immunogen. Using an assay that is ELISA. Detection of such impurities to the next would suggest that the selective for host cell proteins that has been accomplished using PAGE multiproduct ELISA is not widely typically copurify with the product or MALDI-TOF mass spectrometry. applicable, whereas similar HCP does not permit detection of those populations would suggest that it is. that may be present when a deviation

SEPTEMBER 2005 BioProcess International 53 or change in the process has occurred. well as the complexity of these the levels of residual host cell proteins Similarly, if an HCP assay is impurity populations. Most in their products. developed early in product biotechnology products reviewed by If a particular host cell protein development, a new one will be the FDA contain ELISA-based host copurifies with a therapeutic protein required when the culture and recovery cell protein levels of 1–100 ppm. and can be detected by SDS-PAGE processes are finalized, and yet another To date, only limited clinical adverse analysis, it should be identified and its may be required if postapproval events have been attributed to HCP level in the final product estimated and process changes are implemented. impurities. Nevertheless, there are general controlled. Identification of protein Introduction of a new assay makes it concerns about residual host cell proteins bands in gels is readily accomplished difficult to compare HCP levels across in biopharmaceuticals. Lingering safety using peptide mass mapping or Edman different processes and therefore limits concerns stem from the potential for degradation sequence analysis. The information concerning the adjuvant effects or allergic reactions to level of the HCP impurities should manufacturing history of a product. host cell proteins. In this regard, be estimated by conducting titrations When using a multiproduct measuring HCP impurities using an with SDS-PAGE and/or immunoblot immunoassay, it is unnecessary to ELISA is appropriate. An ELISA analysis, even though such analyses are wait until the process is locked down measures the HCP immunoreactivity only semiquantitative. before developing the residual HCP in a sample and thereby may provide Similar levels of sensitivity are assay. Furthermore, using the same insight into potential immunoreactivity achieved by silver or SyproRuby immunoassay for multiple products in humans. A Western blot immunoassay detection (1). Whereas SyproRuby permits development of method can be very useful (and sometimes staining offers a much wider dynamic expertise and consistent data necessary) to determining whether an range than silver staining, that range interpretation. From a practical immunogenic response in patients is is generally too narrow to provide perspective, less training of personnel against a residual HCP impurity or the relative levels of residual HCPs is required when one method is used product itself. present. Although the nature of each rather than several methods. Reagent Several factors influence particular HCP species is a factor to generation and maintenance are determination of an acceptable level of consider when setting an acceptable simplified and less expensive. Overall, residual host cell proteins. For example, level, it is generally better to both the process-specific and the species from which HCPs are reengineer a purification process to multiproduct approaches to HCP derived is important because some remove that impurity to levels assay development are acceptable. cell types (e.g., yeasts) yield potentially undetectable in gels. This is The advantages and disadvantages more allergenic proteins than others. particularly true if the presence of each should be considered before Additional factors include the of host cell proteins hinders reliable determining which one to select. manufacturing capability and history characterization of the active of a sponsor, the sponsor’s ability to pharmaceutical ingredient. ACCEPTABLE LEVELS characterize the product, and the OF RESIDUAL HCPS product safety profile. The drug dose COMPARING MULTIPRODUCT Because host cell protein ELISAs are must be considered because higher WITH GENERIC IMMUNOASSAYS developed independently, and because doses result in higher HCP loads to When a sponsor develops an HCP the sensitivity and specificity of each patients. The route (e.g., intravenous or immunoassay that can be used for all assay depends on the host cell used and subcutaneous) and schedule (e.g., acute products derived from a given cell type the way in which the assay was or chronic) of administration for a drug (e.g., E. coli or CHO cells), that assay developed, ppm (parts per million) product should also be considered when is considered a “multiproduct” assay. It values obtained at one company are not setting an acceptance level because they is typically maintained as a proprietary necessarily equivalent to those obtained both significantly influence assay with a proprietary antibody at a different company. The value immunogenicity. For example, concerns reagent. Such assays should be provided by an ELISA indicates the about residual HCPs may be greater in distinguished from generic assays. collective sum of all immunoreactive a drug product administered in high, We use the term generic to refer to HCPs present. It is not generally known repeated doses subcutanously for a those assays developed by independent whether that value is derived from the number of years than in one given in a companies for use by a variety of signals of 10 or 100 different proteins. So single intravenous dose. Even though biopharmaceutical sponsors. immunoblot analysis should be used as no major safety issues have arisen from Theoretically, commercially available part of the development and assessment residual host cell protein impurities, it generic assays should be widely of all HCP ELISAs. is considered good practice to minimize applicable to products made using a In contrast with their position on HCP levels and thus limit the potential given cell type at different companies. DNA, regulatory authorities have not for unexpected adverse events such as To date, however, several sponsors set a global limit on host cell protein molecular mimicry, anaphylaxoid have found insufficient sensitivity levels. Setting an acceptable level for reactions, and adjuvant effects. For with such assays and a lack of residual host cell protein impurities is these and other reasons, the FDA immunoreactivity with certain host complicated by the different assays as recommends that sponsors minimize cell proteins.

54 BioProcess International SEPTEMBER 2005 Any immunoassay used to measure that an antibody reagent continues proteins by peptide mass mapping HCPs in biopharmaceutical to recognize a broad spectrum of straightforward. preparations should be evaluated and potential contaminants, its suitability To assess potential differences proven capable of measuring numerous for HCP detection should be between E. coli host strains and host cell proteins of various sizes that reestablished following major fermentation conditions used in various could potentially copurify with a manufacturing changes. production processes, four products in product. Characterization of the Process validation can be sufficient development or on the market were antibody reagent should include SDS- to replace a bulk drug substance selected for study (3). These host cell PAGE immunoblots showing specification for host cell proteins. proteins were analyzed without reactivity with numerous HCP bands. ICH guideline Q6B on process- interference from product-related Assessment of a generic HCP assay related impurities states, “For certain proteins by performing control (blank) should involve comparing a Western impurities, testing of either the drug fermentations with host strains carrying blot of the sponsor’s own control substance or the drug product may plasmid vectors without the product sample with a standard from the not be necessary and may not need genes. All host strains were derived commercial supplier (typically the to be included in the specifications from a common strain (W3110) and immunogen used to produce the if efficient control or removal to differed from one another by a few antibody reagents). Overall, generic acceptable levels is demonstrated by genetic markers (for details, refer to (commercial) assays are considered suitable studies” (2). Process validation Reference 3). Cells were cultured under acceptable by the FDA provided that and monitoring of host cell proteins is conditions that had been optimized for they are properly qualified. production of different recombinant proteins.Fermentations differed in HOW IMMUNOASSAYS ARE APPLIED duration, dissolved oxygen level, HCP immunoassays are primarily temperature, and other parameters. used in the biopharmaceutical industry Upon completion of each fermentation to demonstrate consistency of process, cells were harvested and whole manufacturing processes and to cell lysates analyzed by 2D-PAGE. The characterize product purity. They can be resulting proteomic profiles reflected used to monitor host cell protein levels the physiological state of the cells and at several stages in biopharmaceutical BIOGEN IDEC (WWW.BIOGENIDEC.COM) the protein mixture that would development and commercialization. potentially enter a purification process Early in drug development, (3). Comparisons of these protein a widely used approach in which HCP immunoassays can aid in development profiles revealed a high degree of immunoassays are used to validate the and optimization of purification similarity and revealed no dramatic capabilities of a manufacturing process processes. These assays provide differences in the protein and and demonstrate lot-to-lot consistency information about the ability of a new corresponding epitope populations of a final product. However, until purification process step to remove from one E. coli host strain and culture sufficient information on process HCP impurities. In fact, by using an condition to the next. This suggested capabilities and product appropriate HCP immunoassay early that a multiproduct ECP ELISA characterization is available, a release in drug development, a sponsor was feasible. test for HCPs in a drug substance is increases the utility of its To further investigate the usually required. If HCP testing is not manufacturing history. applicability of multiproduct ECP used for drug substance lot release, As drug development progresses, immunoassays, E. coli cells grown with then HCP clearance should be HCP immunoassays are used during or without recombinant protein over- reassessed following major recovery process characterization to expression were compared. Control manufacturing changes. evaluate and identify process and production fermentations for parameters that affect the ability of USE OF PROTEOMIC STUDIES human growth hormone (hGH) purification steps to remove HCP. manufacturing were compared using A series of proteomic studies was Finally, HCP ELISAs are used to silver-stained 2-D PAGE. The undertaken at Genentech to increase monitor HCP levels during recovery potential effect of fermentation scale understanding of HCP populations process validation, and they are often on the host cell protein profile was that potentially enter purification used as a release test for bulk drug also evaluated by comparing 10-L processes and to assess the feasibility substance. When used in process and 1000-L fermentations for human of multiproduct HCP ELISAs. These validation, they should be qualified growth hormone production. As studies were initiated using E. coli host to demonstrate their appropriateness. expected, the most striking distinction cells rather than CHO cells because When used for drug substance testing, between the control and production the E. coli protein (ECP) population is they should be validated. (Distinctions fermentations was the large amount theoretically simpler than that of a between and details of assay of hGH present in the production mammalian cell line. Furthermore, the qualification and validation were not fermentations. An elevated level of E. coli genome has been completely addressed in this session.) To ensure a stress protein in the control sequenced, which makes identifying 56 BioProcess International SEPTEMBER 2005 fermentation turned out to be due to 5 Champion, et al. Manuscript in DISCLAIMER preparation. an experimental artifact — namely, a  The content of this manuscript truncated vector-encoded gene (4). reflects discussions that occurred Overall, neither the scale of Corresponding author Kathleen during the CMC Forum workshop in fermentation nor overexpression of Champion is a senior scientist in the addition to the personal viewpoints department of analytical development at recombinant human growth hormone and experiences of the authors. This Genentech, Inc., MS#62, 1 DNA Way, South had a large impact on the host cell San Francisco, CA 94080; [email protected]. document does not represent protein profile. The results of these Helena Madden is a senior scientist at officially sanctioned FDA policy or proteomic studies suggest that Biogen Idec Inc. John Dougherty is a opinions and should not be used in multiproduct HCP ELISAs are not regulatory scientist at Eli Lilly and lieu of published FDA guidance only feasible, but they can be readily Company. Emily Shacter is chief of the documents, points-to-consider applied across products generated by biochemistry laboratory in the office documents, or direct discussions different strains of biotechnology products division of with the agency. and fermentation processes at different therapeutic proteins at the US FDA’s Center scales. for Drug Evaluation and Research. Finally, experiments involving 2D- PAGE Western blots and immunoaffinity fractionation were performed to evaluate immunoreactive ECPs. Results showed numerous immunoreactive ECPs that covered a broad range of molecular weights and isoelectric points. The results also suggested that there were high-affinity antibodies to certain E. coli proteins and low- or moderate-affinity antibodies to others. This was confirmed in a multiproduct ECP ELISA, and the details will be described elsewhere (5).

ACKNOWLEDGMENTS We would like to thank Kim Michener for helpful discussions. We would also like to thank the conference organizers, Sid Advant et al., for an excellent setting for open discussions about matters important to industry and regulators alike.

REFERENCES 1 Nishihara JC, Champion KM. Quantitative Evaluation of Proteins in One- and Two-Dimensional Polyacrylamide Gels Using a Fluorescent Stain. Electrophoresis 23(14) 2002: 2203–2215. 2 International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products Q6B, recommended for adoption at Step 4 of the ICH Process 10 March 1999; www.ich.org. 3 Champion KM, et al. Similarity of the Escherichia Coli Proteome upon Completion of Different Biopharmaceutical Fermentation Processes. Proteomics 1(9) 2001: 1133–1148. 4 Champion KM, et al. Comparison of the Escherichia coli Proteomes for Recombinant Human Growth Hormone Producing and Non-Producing Fermentations. Proteomics 3(7) 2003: 1365–1373.