2022 Host Cell Protein Conference
10th Annual | Virtual | May 16-19, 2022
2022 HCP Speaker Abstracts
Yiling Bi, Sangamo Therapeutics
Title: Challenges and Considerations in the Host Cell Protein Analysis for Gene Therapy Products
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)
Joseph Cohen, Amgen
Title: Case Studies Assessing the Immunogenicity Risk of Host Cell Protein Impurities in Antibody Biotherapeutics
Abstract: (Pending)
Christina de Zafra, Seagen
Title: Risk Assessment: Industry Perspective
Abstract: (Pending)
Erika Friedl, Paul-Ehrlich-Institut
Title: HCP Characterization and Assay Life Cycle Management
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.
Vibha Jawa, Bristol-Myers Squibb Company
Title: Host Cell Proteins: Immunogenicity Risk Assessments
Abstract: (Pending)
Alexey Khrenov, U.S. FDA-CBER
Title: Updated Quality Risk Management Framework and HCP-Related Risks
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.
Stefan Kochanek, Ulm University
Title: Process- and Product-Related Impurities in the ChAdOX1 nCov-19 Vaccine
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?
Barbara Liori, Merck
Title: How HCP ELISA-Specific Antibodies are Born: Generation and Characterization of a Critical Reagent
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.
Guojie Mao, Lonza Biologics plc
Title: Purification Development and Scale-Up of a Sheep Antibody Against CHO Host Cell Proteins
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
Albert Molina Gil, Orchard Therapeutics
Title: Uncovering Protein Profile of Lentiviral Vector Products for Gene Therapy by LC-MS
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.
Ejvind Mørtz, Alphalyse A/S
Title: HCP Analysis in Support of Covid-19 Vaccine Development
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.
Song Nie, Regeneron
Title: Novel Liquid Chromatography Tandem Mass Spectrometry Methods for Deep Profiling of Host Cell Proteins in Therapeutic Antibodies
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.
Niomi Peckham, USP
Title: USP Standards to Support Host Cell Protein Analysis by Mass Spectrometry
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.
Jan Pippel, BioGenes GmbH
Title: 2D Analytics for Accelerated ELISA Development under Time Constraints
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.
Antoine Pissoort, Pharmalex
Title: Development of an HCP ELISA Assay with a New Detection Method. A Statistical Equivalence Study
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.
Martha Stapels, Sanofi
Title: Optimization and Lessons Learned for HCP ELISA Coverage using Affinity Extraction, Mass Spectrometry, and 2D-PAGE
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
Kevin Van Cott, Prolytix
Title: Comparison of Contaminant Profiles for Commercial Preparations of Immune Globulin Intravenous (IGIV)
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.
Fengqiang Wang, Merck; Georgeen Gaza-Bulseco, AbbVie
Host Cell Protein (HCP) Risk Assessment – Where We are with BioPhorum Cross-Company Collaboration and What We Have Learned from an Industry Perspective?
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.
2022 HCP Panel Discussions
Virtual discussion rooms designed as interactive sessions about topics near and dear to the hearts of host cell protein professionals. Two large panel discussions are available after the podium presentations on Day 3 and Day 4. Each session will be facilitated and moderated. These are not typical formal presentations, instead, it is a mix of slides, questions and/or fast presentations to get you talking.
The panel discussions will be hosted in concurrent Zoom breakout rooms. Attendees will be given instructions in advance for logging into Zoom.
Drop in and join the discussion!
NOTE: Zoom Version 5.3.0 or higher is necessary to be able to move freely from one breakout room to another.
MS Standards
Moderators:
Kevin Van Cott, Scientist, Prolytix
Diane McCarthy, Senior Manager, USP
Panelists:
Niomi Peckham, Principal Scientist, USP
Ejvind Mørtz, COO, Alphalyse A/S
Ying Zhang, Principal Scientist, Pfizer
Space:
Topics to be Discussed:
System Suitability
What is the best approach to establishing system suitability for HCP work? How does it compare to approaches used for general proteomics or peptide mapping work?
Is sample prep included in the system suitability or only Lc and MS components of the workflow?
How do you monitor digestion efficiency? Is this needed and what standards are used?
How do you set criteria for system suitability based on the standard you use?
What are the critical performance criteria for HCP detection?
Strategy for Qualification/ Requalification
How does the use of standards differ depending on whether you are focused on quantitation vs identification only?
What is most appropriate for use as a positive control?
How do standards and controls differ depending on whether you are using native or denaturing conditions? For CHO products vs other modalities (AAV, non-mAbs etc.)
When using peptide standards for quantitation, how do select the ones that are most appropriate?
How do you address discrepancies in quantitation – e.g., if different standards provide different quantitative values, how do you decide which to use?
What are the key attributes you would look for in a heavy labeled peptide standard?
What are the key attributes you would look for in a purified protein standard?
Moving LC/MS into a GMP Environment
Moderators:
Martha Stapels, Principal Scientist, Sanofi
Rosie Molden, Senior Scientist II, Just-Evotec Biologics
Panelists:
Thomas Kofoed, CEO, Alphalyse A/S
Veronika Reisinger, Labhead, Novartis
Alexey Khrenov, Biologist, U.S. FDA-CBER
Space:
Topics to be Discussed:
ELISA has been used as the standard method for HCP analysis due to its robustness, reproducibility, and high-throughput nature. LC-MS has so far played a supporting role in HCP characterization. However, LC-MS methods for other critical quality attributes such as PTMs through the MAM method, are starting to be used in QC. Is it also possible to bring LC-MS methods to monitor HCP into QC? This panel will discuss strategies for bringing LC-MS into a GMP environment, with an emphasis on HCP analysis its specific challenges.
Rare Reagents
Moderators:
Oliver Anderka, Associate Director, Novartis Pharma AG
Michael Wiedmann, Roche
Panelists:
Florian Semmelmann, Group Leader, Roche
Jessica Campbell, Pfizer
Catherine Shoemaker-Ramsey, Associate Director, Biogen
Jan Pippel, Senior Scientist, BioGenes GmbH
Stefan Sommerschuh, Group Leader, BioGenes GmbH
Space:
Topics to be Discussed:
Reagent Characterization
What is a good strategy to show suitability of antigen preparation? (2D DIGE of antigen prep and product harvest)
How does one show suitability of antibody response? (antibody coverage)
Are rabbits or goats (or another animal) better for producing HCP antibodies?
Strategy for Qualification/ Requalification
Label/ aliquot working reagents, qualify and trend their response
Keeping Track of Stock
Maintain bulk of reagents in large aliquots in several locations (redundancy)
Lifecycle Management
What do you do if you start to see reagents degrading/ not performing as expected?
Risk Assessment
Moderators:
Denise Krawitz, Principal Consultant, CMC Paradigms LLC
Ying Zhang, Principal Scientist, Pfizer
Panelists:
Christina de Zafra, Director, Toxicology, Seagen
Erika Friedl, Quality Expert, Paul-Ehrlich-Institut
Fengqiang Wang, Principal Scientist, MSD
Joseph Cohen, Senior Scientist, Amgen
Space:
Topics to be Discussed:
This session will cover aspects of risk assessment of HCPs to process development and nonclinical and clinical development. Biologics including monoclonal antibodies, recombinant proteins, and vaccines will be discussed.