Host Cell Protein Analysis in Biologic Drug Development

Scientific White Paper Host Cell Protein Analysis in Biologic Drug Development Jennifer S. Chadwick1,‡, Robert Garnick2, Chen Li1 and Shiaw-Lin Wu1 1BioAnalytix, Inc., Cambridge, MA 02139 2Lone Mountain Biotechnology and Medical Devices, Inc., Las Vegas, NV 89129 ‡To whom correspondence should be addressed: [email protected] Host cell proteins (HCPs) are common impurities in biologic drug products and are an obligatory critical quality attribute (CQA). The individual protein contaminants that make up the HCP profile, however, may vary significantly among individual biologic products and expression systems. As such, the HCP profile must be analyzed to identify even low levels of potentially concerning species in a biologic drug to ensure safety and efficacy. This whitepaper reviews fundamental aspects of HCPs pertaining to biologic drug development and current and emerging approaches to HCP analysis. Host Cell Proteins remain in a biologic product following purification. Once identified, HCPs often are monitored, and Biologic drugs are produced using living biological the levels controlled to consistently yield a safe systems, which have highly complex milieus and efficacious product.1,2,3 While ELISA methods containing several thousand to tens of thousands have historically been the main approach to of unique macromolecular species. The majority of detecting HCPs, LC-MS based HCP detection biotherapeutics today are recombinant proteins approaches have increasingly become an expressed in cultured cells, while others are expected orthogonal standard in successful cellular products. Because biologics are derived biologic development. The specific advantages from cellular systems, host cell proteins (HCPs) and application of ELISA and LC-MS methods for that are co-produced with the biotherapeutic are HCP profiling are discussed below (Table 1). abundant at harvest and represent the major class of impurities in the final product. As such, HCPs are The specific components and amounts of HCPs in an obligatory critical quality attribute (CQA) of a drug product depend on numerous factors, biologic drug products because these residual including the type of host cell used for production impurities can affect product quality, efficacy and (bacteria, yeast, insect, mammalian, plant), safety as well as induce or enhance culture media conditions and purification immunogenicity. To ensure consistency of product approaches.2,4-6 As such, residual HCP profiles can manufacture and shelf-life stability and to vary widely, particularly with different hosts, and minimize potential adverse reactions, significant impurities from individual hosts can result in attention must be paid to identifying HCPs that unique risks in clinical practice. Production in E. 790 Memorial Drive, Cambridge, MA 02139 • 857-829-3200 • www.bioanalytixinc.com 1 ISSUE 1 | SEPTEMBER 2018 coli or yeast (e.g., S. cerevisiae, P. pastoris) leads systems. The glycan composition profile can affect to a smaller pool of potential HCPs due to the pharmacokinetics, pharmacodynamics and relative simplicity of these organisms, which have biodistribution of a protein,16,17 for example, by approximately 4300 and 5300 protein-encoding affecting clearance rate, but more concerning is genes, respectively, compared to mammalian cells that small differences can alter immunogenic (e.g., CHO) with approximately 30,000 genes and potential. Some host cells may generate foreign plants, such as tobacco, which can have over PTMs that potentially promote undesirable 60,000 protein-encoding genes. The type of immunological reactions, for example unique proteins expressed in microbial or plant cells are glycan structures on HCPs may be present that are more distantly related to human proteins and not observed in humans and may be recognized as have potential to be recognized as foreign by a foreign.11,16,18 In such cases, detection of the recipient’s immune system, and could lead to relevant impurity would require not only suspension of a clinical trial due to immune detection of the protein but recognition of the reactions to the foreign HCPs.10,11,12 On the other specifically modified form by the HCP assay. hand, problems can also arise from HCP impurities Modifications often appear at low-levels, and that are closely related to the biologic drug. The consequently, both specificity and sensitive presence of a homologous protein from the host detection are needed to identify and monitor cell in a drug product can diminish potency and modified species. efficacy, stemming from competitive binding, and has led to serious reactions such as induction of Within a specific host cell type (e.g., CHO cells) neutralizing anti-drug antibodies (ADA) to the there is more consistency, and studies have recombinant therapeutic product.11,13,14 In a few reported significant overlap among HCP species specific cases, further complications have detected in recombinant protein products, occurred due to ADA cross-reactivity with the providing some general awareness about effects patient’s endogenous protein.11,13 This type of HCP of a few defined process-related impurities.4,7,19 is a common concern, particularly for enzyme For example, mAbs are typically purified using replacement therapies (ERT), where homologous Protein A resin, which can leach into the product or closely related host cell proteins to the during purification and cause toxicity as well as intended therapeutic product also are expressed result in retention of similar HCPs.7 However, it in the host cell. Because the homologous host also has been observed that most HCPs found in protein is so similar, separation is difficult and co- purified products accompany the active molecule purification with the recombinant therapeutic despite extensive purification because of protein often occurs.10,12,15 The ability to appreciable binding to it. Consequently, distinguish such HCPs from the intended protein differences in HCP content among even very product, therefore, is a key aspect of developing closely related biotherapeutic molecules (e.g., two an effective HCP assay. An additional IgG1 mAbs) can be significant and meaningfully consideration is that post-translational alter clinical response.8 Substantive differences in modifications vary among different host cells. HCPs also have been reported for the same Glycan modifications, for example, are molecule following altered process changes, heterogeneous inherently and differences in which often occur during the normal progression occupancy and relative amounts of individual of drug development, scale up and post-market.2,6 species can result from different expression 2 ISSUE 1 | SEPTEMBER 2018 Method Advantages Limitations High sensitivity Lengthy development time (LOQ <0.1 ppm) (months) HCP-specific Requires generation and validation of Immunoassays High throughput immunoassay custom immunoreagents Finite shelf-life of immunoreagents; QC compatible assay requires periodic redevelopment Multiple reaction Sensitive detection Isotopically labeled peptides or protein monitoring (MRM); (single digit ppm) required Targeted absolute Excellent accuracy Analysis takes weeks quantitation and precision Select top peptides Fast estimate of Significant margin of error Mass for quantitation concentration (within ~2-fold of actual concentration) spectrometry Rapid assessment Spectral counting of relative amounts Lower precision than other MS for relative Effective approaches quantitation comparison among samples One important consideration in HCP assessment is Other enzymes have been reported to degrade that not all HCPs are equal with respect to stabilizing surfactants.27 This finding is broadly pharmaceutical stability and clinical impact.2 applicable because the majority of protein Specific HCPs have been shown to diminish the therapeutics are monoclonal antibodies (mAbs), integrity of biologic products in distinct ways and, and the majority of mAb products are formulated recently, have been responsible for adverse with polysorbate 20 (PS20) or 80 (PS80). Two reactions leading to the termination of two clinical lipases, lipoprotein lipase (LPL) and phospholipase trials.9,10 Not only do individual HCP components B-like 2 (PLBL2), have been shown to hydrolyze need to be identified but the level at which each is PS20/80, cleaving them into fatty acid and alcohol present should be determined and, whenever components.27 Polysorbates are added to protect possible, correlated with risk. Residual enzymes biologics from surface-induced aggregation and present at very low levels in drug products have their degradation can result in increased been reported to cause product degradation.6,20 aggregate formation as well as phase separation Several studies have identified proteases, such as and/or increased interaction of the degradants Cathepsin D, as the source of fragmentation of with the biologic molecule.28-30 Depending on the active protein therapeutics.21-24 Protein disulfide extent of breakdown and the specific product, this isomerase and other proteins with reactive may reduce shelf-life, diminish potency and/or cysteine residues can promote thiol/disulfide promote an immune reaction. Clinically, specific exchange, and these residual contaminants have HCPs may pose a greater risk than others. In some been found to cause aggregation of proteins.25,26 cases, this may manifest as reduced efficacy due 3 ISSUE 1 | SEPTEMBER 2018 to chemical degradation or loss of active to chromatography coupled to mass spectrometry

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