Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Challenges and Considerations in the Host Cell Protein Analysis for Gene Therapy Products

• Speaker: Yiling Bi, Sangamo Therapeutics
• Abstract: Host Cell Proteins (HCPs) are process-related impurities in a drug product that need to be measured and continuously monitored due to their potential safety concerns. In comparison to traditional biologics, the quantitation and control of HCPs in gene therapy products face significant challenges as manufacturing processes of these products are highly complex with widely different upstream and downstream processes. The HCPs have traditionally been measured by commercially available ELISA assays which may or may not represent the specific process. In addition, HCPs contain both low and high molecular weight species and it is important to get sufficient coverage to detect majority if not all HCPs generated during the process. The HCP coverage can be measured by various methods including Antibody affinity extraction (AAE), 2D-Western analysis and LC-MS based approaches. In this presentation, we will discuss a case study on roadmap for HCP analysis for an AAV-based gene therapy product. In addition, some considerations for choosing (a) specific approach(es) during different clinical development stages will also be discussed.
• Contributing Authors: Santoshkumar Khatwani (Sangamo Therapeutics)
• (Day 2: Product Specific HCP Profiling Session)

Case Studies Assessing the Immunogenicity Risk of Host Cell Protein Impurities in Antibody Biotherapeutics

• Speaker: Joseph Cohen, Amgen
• Abstract: (Pending)
• (Day 1: Risk Assessment Session)

Risk Assessment: Industry Perspective

• Speaker: Christina de Zafra, Seagen
• Abstract: (Pending)
• (Day 1: Risk Assessment Session)

HCP Characterization and Assay Life Cycle Management

• Speaker: Erika Friedl, Paul-Ehrlich-Institut
• Abstract: In order to guarantee high quality medicines, a tight impurity control strategy in the manufacturing process for recombinant medicines is required. In that respect, HCPs are especially in the focus due to their potential impact on the safety and efficacy of medicinal products. Regulatory requirements regarding HCPs are specified in the European Pharmacopoeia and the US Pharmacopoeia. The development of a potent control strategy is challenging. Therefore, appropriate HCP characterization tools including the use of validated assays as well as a convincing life cycle management of the HCP assay are necessary. In regulatory submissions, new ideas regarding assay choices and reagent changes are presented and reviewed. Lately, the assay choices as well as the change of critical assay reagents and the pitfalls connected came into focus and will be discussed in this presentation. Regulatory decisions will be supported by selected case studies.
• (Day 2: Product Specific HCP Profiling Session)

Host Cell Proteins: Immunogenicity Risk Assessments

• Speaker: Vibha Jawa, Bristol-Myers Squibb Company
• Abstract: (Pending)
• (Day 1: Risk Assessment Session)

Updated Quality Risk Management Framework and HCP-Related Risks

• Speaker: Alexey Khrenov, U.S. FDA-CBER
• Abstract: The ICH Q9 guidance offers a systematic approach to, and provides principles and examples of tools for Quality Risk Management (QRM) which is a systematic process for the assessment, control, communication, and review of risks to the quality of the drug product across the product lifecycle. The primary principles of QRM are that evaluation of the risk to product quality should be based on scientific knowledge and ultimately link to the protection of the patient; and that the level of effort, formality, and documentation of the QRM process should be commensurate with the level of risk. Based on the experience in the implementation of the original ICH Q9 guidance published in 2005, revisions are currently underway to address several deficiencies, including high levels of subjectivity in risk assessments and QRM outputs; failure to adequately manage the risks related to product supply and availability; lack of understanding as to what constitutes formality in QRM work; and lack of clarity on the risk-based decisionmaking process.Host Cell Protein (HCP) impurity is a critical quality attribute for biological products because it can affect product quality, efficacy, and safety. At the same time, diversities within the HCP population along with their associated analytical challenges, and limited information regarding the effects of specific HCP impurities on the product pose multiple challenges with regard to risk assessment and management. The use of systematic QRM frameworksmay be beneficial in addressing these challenges.This presentation will discuss the proposed revisions to the ICH Q9 guidance, and considerations for the application of QRM principles to manage HCP-related risks, focusing on the areas covered by the revisions.• My comments are an informal communication and represent my own best judgement. These comments do not bind or obligate FDA.
• (Day 1: Risk Assessment Session)

Process- and Product-Related Impurities in the ChAdOX1 nCov-19 Vaccine

• Speaker: Stefan Kochanek, Ulm University
• Abstract: Stimulated by rare severe side effects mainly observed following vaccination with ChAdOx1 nCov-19, involving thrombosis of the cerebral venous sinuses and low platelet counts, we looked at adenovirus-based SARS-CoV-2 vaccines from different angles: content, activity and expression. Unexpectedly, several lots of the ChAdOx1 nCov-19 vaccine contained higher than expected levels of host cell proteins (HCPs) and of free viral proteins, due to incomplete purification of the vaccine product, in fact significantly exceeding the specification limit. Possible implications, needs and future research directions are outlined.
  We also faced a dilemma: how to communicate with the public, in the middle of a pandemic, when experimental data indicate quality issues of an approved, effective and widely used vaccine?
• (Day 2: Product Specific HCP Profiling Session)

How HCP ELISA-Specific Antibodies are Born: Generation and Characterization of a Critical Reagent

• Speaker: Barbara Liori, Merck
• Abstract: Host Cell Proteins (HCPs) are process-related impurities and critical process parameters in the manufacturing of protein-based pharmaceuticals. The fundamental issue is how to adequately measure and control the HCPs such a large number of protein species (potentially thousands of components) to ensure safe and efficacious products.¹A noble solution is to use an immunoassay (enzyme‐linked immunosorbent assay [ELISA]) based on polyclonal antibodies raised in the host cell (biological system) used to synthesize a particular therapeutic product.² Although generic ELISA kits are commercially available to quantify HCP from different recombinant system, a specific assay for biologicals is required before registration.For this reason, the development of a mock cell line to mimic HCP from the expression cell line and the immunization strategy to generate anti-HCP antibodies will be discussed.The HCP measurement is entirely dependent on both the antibody serum used and the characterization approach which dictates the sensitivity of the assay and the degree of coverage of the HCP spectrum.In this presentation will be presented the strategy and the challenges observed during all the steps of development and characterization of specific reagents generation, with a special focus on the sera purification steps.
• References:
  ¹ USP 39 Published General Chapter Residual Host Cell Protein Measurement in Biopharmaceuticals.
  ² Biotechnology Bioengineering volume 112(9):1727-37.
• (Day 4: ELISA Method Development Session)

Purification Development and Scale-Up of a Sheep Antibody Against CHO Host Cell Proteins

• Speaker: Guojie Mao, Lonza Biologics plc
• Abstract: Host cell proteins (HCP) are host cell-derived impurities during the manufacturing process. HCP can pose risks to patient safety and product efficacy. Removal of HCP is a critical step and one of the biggest challenges in biomanufacturing. Therefore, residual HCP should be measured and monitored in final biopharmaceutical products. Regulatory authorities recommend immunoassays to monitor residual HCP in drug substance or product before it is released.Lonza monitors HCP impurity with a platform HCP ELISA during the manufacturing process. HCP reagents management is essential for supporting release testing. The quality of polyclonal antibodies against HCP is critical for a reliable HCP immunoassay and includes antigen source and antibody immunocoverage.We developed a two-step method to purify antibodies from sheep antisera raised against HCP from a mock-transfected null GS Xceed® host cell line. In the first step, total immunoglobulins (IgG) were purified from sheep sera with a Protein A based capture purification. Despite the known weak interaction of sheep IgG to Protein A, our capture step successfully purified total sheep IgG. In a second step, we purified HCP-specific antibodies from total IgG by affinity purification based on immobilised HCP from Lonza´s GS Xceed® host cell line. In the HCP affinity purification step, the majority of sheep IgG (about 99%) showed no immune-response to HCP and were removed from final HCP antibody.This two-step method was scaled up to process over 90L of sheep antisera. Multiple HCP sources including supernatant, cell lysate and a low molecular weight HCP fraction purified from cell lysate were used as HCP antigen. A pilot scale purification of sheep HCP antibodies confirmed consistent performance and robustness of the two-step purification method.With this two-step method, Lonza HCP antibodies showed improved immunocoverage against HCP over 83% by spots-matching 2D western blot. Subsequently, we developed a HCP ELISA based on the purified HCP antibodies, resulting in a limit of quantitation (LOQ) below 10 ng/ml in a validation study. Both purification and HCP antigens design ensured the future-proof quality of HCP antibody.After a successful validation, the GS Xceed® HCP ELISA was implemented as a platform assay to monitor HCP impurities in process intermediates and final bulk drug substance
• Contributing Authors: Guojie Mao, Hanna Shore, Jean Aucamp, Rita Tommasi, Antonietta Corrado and James Graham
  Lonza Biologics plc, 228 Bath Road, Slough, UK, SL1 4DX
• (Day 4: ELISA Method Development Session)

Uncovering Protein Profile of Lentiviral Vector Products for Gene Therapy by LC-MS

• Speaker: Albert Molina Gil, Orchard Therapeutics
• Abstract: Lentiviral vectors used in the production of cell and gene therapy products present multiple sources of impurities derived from sources such as viral, starting materials, process and host cell derived constituents. While residual protein characterization for these products has typically been performed via conventional enzyme-linked immunosorbent assays (ELISAs), the robust identification and quantification of individual residual proteins from multiple sources requires an appropriately matched analytical approach to ensure a solid and thorough characterization.In this work, we have analyzed and compared residual protein levels of different lentiviral vector products across the different steps of the manufacturing process using liquid chromatography – mass spectrometry analysis. More than 200 media- and host cell-related proteins were identified by 2 or more peptides and >100 were quantified above lower limit of quantification for intermediate samples across different products. Interestingly, therapeutic and viral proteins were also quantifiable with the relative levels of the latter correlating with the expected stoichiometry. The analysis and respective data will be discussed in the context of Identification/quantification of the different vector components and process characterization and assessed as a tool to evaluate the consistency of lentiviral vector manufacturing platforms regarding protein profile.
• (Day 2: Product Specific HCP Profiling Session)

HCP Analysis in Support of Covid-19 Vaccine Development

• Speaker: Ejvind Mørtz, Alphalyse A/S
• Abstract: Preparedness against pandemic diseases demands rapid-response vaccine technology and ready-to-use analytical methods to support CMC activities. Manufacturing process consistency and comparability between GMP batches are of key importance when the process is scaled up and the production is performed at several manufacturing facilities, to provide a pure and safe product to vaccinate billions of healthy individuals.
  Process-related impurity assays for pandemic vaccines, should therefore ideally be a) very fast without long assay development time, b) generic for common manufacturing processes and vaccine types, and c) provide reproducible impurity measurements over time and in-between laboratories.
  Here, we present results and learnings from LC-MS analysis of several viral-vector and protein-based Covid-19 vaccines on the market and under development. The results include identity and quantity (ppm and ng/ml) of HCPs in marketed Covid-19 vaccines and comparison of observed HCP profiles. We have found that the product purity and HCP profiles varies substantially between marketed Covid-19 vaccines: Some vaccines having almost 1:1 ratio between viral proteins and HCPs, other vaccines having low ppm level impurities.
  The presentation includes evaluation of assay performance regarding reproducibility and intermediate precession to support CMC manufacturing consistency and batch comparability.
• (Day 2: Product Specific HCP Profiling Session)

Novel Liquid Chromatography Tandem Mass Spectrometry Methods for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies

• Speaker: Song Nie, Regeneron
• Abstract: Liquid chromatography coupled to mass spectrometry (LC-MS) is a powerful tool for the analysis of host cell proteins (HCP) during antibody drug process development due to its sensitivity, selectivity, and adaptability. However, the enormous dynamic range between the therapeutic antibody and accompanying HCPs poses a significant challenge for LC-MS based detection of these low abundance impurities. To address this challenge, enrichment of HCPs via immunoaffinity, protein A, 2D-LC, or other strategies is typically performed. However, these enrichments are time-consuming and sometimes require a large quantity of sample. Here, we report a simple and sensitive strategy to analyze HCPs in therapeutic antibody samples without cumbersome enrichment by combining an ultra-low trypsin concentration during digestion under non-denaturing conditions, a long chromatographic gradient, and BoxCar acquisition (ULTLB) on a quadrupole-Orbitrap mass spectrometer. Application of this strategy to the NIST monoclonal antibody standard (NISTmAb) resulted in the identification of 453 mouse HCPs, which is a significant increase in the number of identified HCPs without enrichment compared to previous reports. Known amounts of HCPs were spiked into the purified antibody drug substance, demonstrating that the method sensitivity is as low as 0.1 ppm. Thus, the ULTLB method represents a sensitive and simple platform for deep profiling of HCPs in antibodies.
• (Day 3: Uses of LCMS for HCP Profiling Session)

USP Standards to Support Host Cell Protein Analysis by Mass Spectrometry

• Speaker: Niomi Peckham, USP
• Abstract: Residual host cell proteins (HCPs) in biotherapeutic products can pose a risk to patients and the quality of the product. Therefore, the HCPs must be carefully monitored and controlled. Mass spectrometry (MS) has become an increasingly common approach for the identification and quantitation of HCPs in biotherapeutic products. Based on feedback from industry stakeholders, USP has been developing both documentary and physical standards to support MS analysis of HCP. The USP Host Cell Protein Expert Panel is working on a new general chapter on best practices for identification and quantitation of HCPs by MS. This presentation will focus on the proposed contents of the new USP general chapter and approaches to HCP analysis by MS. USP is also developing physical standards to support the identification and quantitation of HCPs of particular concern. We will discuss USP’s strategy for development and characterization of physical standards to support HCP analysis by MS.
• (Day 3: Uses of LCMS for HCP Profiling Session)

2D Analytics for Accelerated ELISA Development under Time Constraints

• Speaker: Jan Pippel, BioGenes GmbH
• Abstract: The current pandemic has shown the need for a drastic time-to-market reduction in the development of novel drugs and vaccines. As part of a customer project, a tight schedule demanded the rapid implementation of functional HCP ELISA monitoring to measure these important process-related impurities in a novel API production process. To reduce the development time of a process-specific ELISA, a combination of pre-existing reagents developed for the HCP monitoring of two closely related manufacturing processes (A and B) was investigated.In this case study, the compatibility of the antigen standard material from process A and the specific antibody panel for process B were evaluated for the development of a combined ELISA. State of the art 2D technology for similarity assessment and coverage analyses demonstrated excellent suitability. An HCP coverage value of 84% was determined by immunoaffinity chromatography (IAC) with 2D DIGE, which exceeded the performance of a previously tested generic HCP antibody. This allowed for the successful development of a process-specific ELISA with high sensitivity (working range of ~1.5 – 100 ng/mL), which is now being used for HCP detection in the novel drug manufacturing process.This example demonstrates how, when the urgency of a project requires it, careful reagent characterization by means of elaborate 2D technology and sophisticated suitability testing enables much more rapid and cost-efficient ELISA development.
• (Day 4: ELISA Method Development Session)

Development of an HCP ELISA Assay with a New Detection Method. A Statistical Equivalence Study

• Speaker: Antoine Pissoort, Pharmalex
• Abstract: The goal is to show that a new method is equivalent to the old method for this Host Cell Protein (HCP) assay. Different scenarios have been explored in terms of modelling and of transformation of the response. The use of four parameter-logistic model with log10 transformation, with or without an offset are shown to have luminescence results that are equivalent to that of the old method. Equivalence was shown using two approaches, namely:
  1. Bivariate least square regression method on the back-calculated concentrations of both methods.
  2. The total error approach on the back-calculated concentrations that compares both methods by incorporating all sources of error.
• (Day 4: ELISA Method Development Session)

Optimization and Lessons Learned for HCP ELISA Coverage using Affinity Extraction, Mass Spectrometry, and 2D-PAGE

• Speaker: Martha Stapels, Sanofi
• Abstract: Host cell proteins (HCPs) are process-related impurities that are monitored to ensure product purity, stability, efficacy, and safety of biopharmaceutical products. Mass spectrometry (MS) is a highly sensitive technique used to identify HCPs, as it enables detection of individual HCPs down to 1 ppm. HCP ELISAs are the standard method for quantifying HCPs in QC for product release and process development. For each platform-specific ELISA, it is beneficial to show that the ELISA antibodies recognize the most abundant HCPs in the purified protein drug as well as in upstream samples. The combination of affinity enrichment along with 2D PAGE and mass spectrometry is useful to demonstrate good coverage as well as understanding of the individual abundant HCPs in a process. In order to perform affinity enrichment of in-process samples, different matrices were tested, including harvest material as well as other in-process eluates. Due to matrix effects in the column eluates, the harvest material was shown to yield the best recovery after antibody enrichment. Interestingly, affinity enrichment using different ELISA antibodies led to the enrichment of Cathepsin B, even though it was not particularly abundant. Coverage from the ELISA will be calculated by the standard 2D PAGE approach as well as by mass spectrometry and the results will be compared. Practical tips and lessons learned in the development of these methods will also be discussed.
• Contributing Authors: Martha Stapels, Joanne Cotton, Neelambari Joshi, and Michelle Busch
  BioAnalytics, Sanofi Corporation
• (Day 4: ELISA Method Development Session)

Assessing Immunogenicity Risk of Host Cell Proteins in Vaccines and Biologics

• Speaker: Frances Terry, EpiVax
• Abstract Purpose: We have developed a tool that evaluates HCPs that copurify with therapeutic protein, peptide, or vaccine products for immunogenicity. Residual host cell proteins (HCPs), known as “hitchhiker” proteins, can induce detrimental immune responses in patients and compromise the safety and efficacy of a biologic. Indeed, the presence of Chinese hamster ovary (CHO)-derived HCPs contributed to the suspension of two clinical trials in 2012. Our immunogenicity risk assessment tool for therapeutic proteins, the Interactive Screening and Protein Re-engineering Interface, has been in use by industry clients and collaborators since 2003. We have recently developed a web-based tool to evaluate the immunogenic potential of HCPs based on T cell epitopes and their similarity to the human proteome, and to evaluate sets reported HCP impurities.Methods: We analyzed a set of HCP identified by a consortium of monoclonal antibody producers [Jones et al.], as well as host cell and viral vector proteins identified in a lot of ChAdOx1 nCov-19 vaccine [Krutzke et al.]. Immunogenicity of biologics is driven by activated CD4 + T cells, and anti-drug antibody (ADA) responses may develop in reaction to components of the drug product or contaminants from production host cell lines. The web accessible ISPRI_HCP tool assesses the immunogenicity risk posed by HCPs by evaluating both the potential presence of an HCP of interest and its potential immunogenicity. Each input sequence is parsed into overlapping 9-mer frames and evaluated for potential to bind HLA using the extensively validated EpiMatrix immunoinformatic algorithm, and then evaluated for humanness using the JanusMatrix algorithm. Briefly, JanusMatrix searches for potentially cross-reactive sequences based on the preservation of the TCR-facing residues and established HLA binding preferences of given 9-mer ligands. Predicted ligands from an HCP that share the same HLA restriction and TCR-facing contour as epitopes derived from self (human) are presumed to be tolerated.Results: Our results indicate that the immunogenicity of HCPs can vary dramatically. A careful analysis of identified HCPs within product preparations can help to determine the degree of immunogenic risk of the HCP. Furthermore, visualization of ISPRI HCP results in context with relative abundance in individual product lots can help drug producers to quickly identify the impurities with the highest likelihood of recognition by a patient immune system. Results are depicted on a plot where the Y axis indicates density of predicted T cell epitopes (EpiMatrix Immunogenicity Score), the X axis indicates average humanness of those epitopes (JanusMatrix Score), and the size of each marker indicates the relative abundance (e.g. ppm) measured in the lot. As shown for the Jones dataset of monoclonal antibody HCPs, Phospholipase B-Like 2 Protein is an example of a CHO protein found with high frequency in monoclonal antibody lots, and its position in the upper left quadrant (high epitope density, low cross-conservation with human) supports the observation of specific immune response to this impurity [Fischer et al.]. In the case of the ChAdOx1 nCov-19 vaccine, several viral vector proteins have elevated immunogenicity scores and low human cross-conservation, indicating considerable risk for impurity-driven immune response. Additionally, multiple human host cell proteins persist in the clinical material, with widely ranging immunogenicity scores and human cross-conservation. In a best-case scenario, highly cross-conserved human epitopes are well tolerated in this material. In a worst-case scenario, however, it is possible that immune responses against the copurifying human host cell proteins or other human epitopes with which they are cross-conserved, might be capable of stimulating auto-immunity, particularly if response against residual viral vector proteins provides an adjuvating effect.Conclusion: Further development of the ISPRI_HCP tool, in collaboration with biologics industry partners, will enable exploration of immune responses in vitro, enhancing the prediction of immunogenic and tolerated T cell epitopes from HCPs, as well as evaluate the correlations with resultant ADA when patient data are available.References:
  1. Bailey-Kellogg C, Gutierrez AH, Moise L, Terry F, Martin WD, De Groot AS, et al. CHOPPI: a web tool for the analysis of immunogenicity risk from host cell proteins in CHO-based protein production. Biotechnology and Bioengineering. United States; 2014 Nov 2; :2170–82. PMID: 24888712
  2. Koren E, De Groot a. S, Jawa V, Beck KD, Boone T, Rivera D, et al. Clinical validation of the “in silico” prediction of immunogenicity of a human recombinant therapeutic protein. Clin Immunol. 2007; 124:26–32. PMID: 17490912
  3. De Groot AS, Martin W. Reducing risk, improving outcomes: Bioengineering less immunogenic protein therapeutics. Clin Immunol. United States: Elsevier Inc.; 2009 May; 131(2):189–201. PMID: 19269256
  4. Moise L, Gutierrez AH, Bailey-Kellogg C, Terry F, Leng Q, Abdel Hady KM, et al. The two-faced T cell epitope: Examining the host-microbe interface with JanusMatrix. Human Vaccines and Immunotherapeutics. 2013. p. 1577–86. PMID: 235842515. Fischer SK, Cheu M, Peng K, Lowe J, Araujo J, Murray E, McClintock D, Matthews J, Siguenza P, Song A. Specific Immune Response to Phospholipase B-Like 2 Protein, a Host Cell Impurity in Lebrikizumab Clinical Material. AAPS J. 2017 Jan;19(1):254-263. doi: 10.1208/s12248-016-9998-7. Epub 2016 Oct 13. PMID: 27739010.
  5. Jones M, Palackal N, Wang F, Gaza-Bulseco G, Hurkmans K, Zhao Y, Chitikila C, Clavier S, Liu S, Menesale E, Schonenbach NS, Sharma S, Valax P, Waerner T, Zhang L, Connolly T. “High-risk” host cell proteins (HCPs): A multi-company collaborative view. Biotechnol Bioeng. 2021 Aug;118(8):2870-2885. doi: 10.1002/bit.27808. Epub 2021 May 31. PMID: 33930190.
  6. Krutzke, L., Rösler, R., Wiese, S., & Kochanek, S. Research Square 2021. Process-related impurities in the ChAdOx1 nCov-19 vaccine.
  7. Fischer SK, Cheu M, Peng K, Lowe J, Araujo J, Murray E, McClintock D, Matthews J, Siguenza P, Song A. Specific Immune Response to Phospholipase B-Like 2 Protein, a Host Cell Impurity in Lebrikizumab Clinical Material. AAPS J. 2017 Jan;19(1):254-263. doi: 10.1208/s12248-016-9998-7. Epub 2016 Oct 13. PMID: 27739010.
• (Day 2: Product Specific HCP Profiling Session)

Comparison of Contaminant Profiles for Commercial Preparations of Immune Globulin Intravenous (IGIV)

• Speaker: Kevin Van Cott, Prolytix
• Abstract: Immune globulin intravenous (IGIV) has grown steadily in the volume of use and numbers of clinical indications since the first modern commercial preparation was approved by FDA in 1981. All US-marketed IGIV products are approved by FDA to treat primary immune deficiency and immune thrombocytopenia purpura. Currently, most IGIV infusions are for treating autoimmune diseases. Dosing levels of IGIV for autoimmune diseases can be in the range of 2 g/kg over a 3-5 day period, and repeated monthly. IGIV has historically been isolated from pools of human plasma (at least 1,000 donors, but typically ranging from 10,000 donations in the US up to 60,000 donations in some EU nations) using Cohn-Oncley cold ethanol fractionation. Most firms now supplement this process with chromatographic steps to remove Factor XIa, other promoters of complement activation, and other Ig isotypes. Despite high purity, a range of adverse symptoms, varying widely by product, is associated with IGIV infusions. To date, a possible correlation between adverse event profiles and contaminant profiles for IGIV products has not been studied and remains poorly understood. FDA requires donor blood tests for absence of hepatitis, HIV and other viral diseases and final container Ig products must show potency against measles, diphtheria and polio virus and an absence of prekallikrein activator. However, no further characterization of contaminant proteins is currently required.We undertook comparative impurity analyses of nine different IGIV products approved or in development for the US market. Impurity analysis was performed using high resolution mass spectrometry (HRMS) and immunoassays (single protein ELISA and multiplexed immunoassay). IGIV is polyclonal, and thus orders of magnitude more complex than monoclonal antibodies or other recombinant proteins – the number of unique protein sequences in an IGIV product is currently unknowable and likely at least in the hundreds of thousands. Therefore, impurity analysis by mass spectrometry has significant challenges compared to HCP analysis of recombinant proteins. We developed methods that use both a standard denaturing digestion and the native digestion developed by Huang et al. (Analytical Chemistry, 2017). As expected, the standard digestion method was less sensitive, due to the complexity of the product; we obtained detection limits in the 40-50 ng/mg range. The native digestion method developed for mAbs was adapted for the polyclonal IgG products; we obtained detection limits of 1-20 ng/mg. Both digest methods, combined with 1D-UPLC and DDA MS/MS detection, had good precision among replicates. The ELISAs were designed to detect primarily biomarkers related to cytokine regulation, IgA, and IgM. There was some overlap between impurities detected by HRMS and those determined by ELISA, and the HRMS and ELISA results were complementary, but quantitative results between the methods differed by a significant amount in some cases. Contaminants detected by MS included IgA, IgM, IgD, β2-glycoprotein-1 (ApoH), α2-macroglobulin, albumin, transferrin, coagulation cascade proteins, catalase, hemopexin, properdin, multiple apolipoproteins, and multiple complement cascade proteins. IL6, IL-8 and IL-10 were detected in a few products by immunoassay, but in no case more than 3-fold above the quantitation limit. The tumor markers CEA, αFP, CA-125, CA 19-9 and TNF-α were not found in any product.
• (Day 3: Uses of LCMS for HCP Profiling Session)

Host Cell Protein (HCP) Risk Assessment – Where We are with BioPhorum Cross-Company Collaboration and What We Have Learned from an Industry Perspective?

• Co-Speakers: Fengqiang Wang, Merck; Georgeen Gaza-Bulseco, AbbVie
• Abstract: Despite greater understanding of individual residual HCP composition in drug substance by increased use of LC-MS based analytical technologies, HCP risk assessment remains an industry-wide challenge due to limited knowledge available on both the intrinsic properties of individual HCPs and the extrinsic settings that make those HCPs problematic. BioPhorum Development Group (BPDG) HCP Workstream addresses this challenge by pooling knowledge from the industry leaders and subject matter experts within its 34 member companies. Since its inception, it has published white papers and best practices including the methodologies used for HCP process risk assessment and a list of “high-risk” HCPs frequently detected in CHO-produced biologics to guide HCP risk assessment. In this presentation, we will share an update on the latest development from BPDG HCP Workstream on this topic and some key learnings from our cross-company collaboration and discussions.
• (Day 1: Risk Assessment Session)