Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Host Cell Protein Immunoassay Comparability and Best Practices for Reagent Changes or Bridging to a New Assay

• Speaker: Eric Bishop, Cygnus Technologies
• Abstract: A well-developed, broadly reactive, and qualified HCP ELISA remains a gold standard method effectively used during the purification process to ensure removal of HCP and to demonstrate process consistency and final drug substance purity. In this presentation we will discuss the best practices in HCP ELISA bridging necessitated by certain critical reagent changes that sometime might occur due to one or more critical reagent depletions. A case study using a specific HCP ELISA Assay and advanced orthogonal Antibody Affinity Extraction and MS methods for antibody and assay qualification will be presented.
• (Day 3: HCP ELISA Development and Lifecycle Management Session)

Applicability of a Host Cell Protein Platform Assay for CHO-Expressed Products

• Speaker: Michael Chaplet, GSK
• Abstract: During expression of recombinant protein-based vaccine, complex mixtures of additional proteins are also produced by normal expression machinery of the host cell. These Host Cell Proteins (HCP) can have a potential impact on patient safety and product efficacy, and therefore, must be well characterized. Also, the ability of the process to clear these proteins must be demonstrated. Due to the complexity of HCP, the method(s) used for their monitoring must be demonstrated able to provide sufficient information about relevant proteins. The most commonly used analytical method for monitoring HCP is an enzyme-linked immunosorbent assay (ELISA). To ensure development of a suitable HCP ELISA, careful selection of critical reagents such as anti-HCP antibodies and HCP reference standard, is crucial.In the context of the development of 3 new potential vaccine candidates produced in CHO cell line, we evaluated the suitability of an existing HCP ELISA used for the commercial release of a licenced product, for the monitoring of the HCP population in these new products. The goal was to define if this registered HCP ELISA could become a platform assay for these new CHO-based products.
  The comparison between each CHO null cell culture (representing HCP antigen of each new product) and the potential platform HCP standard, analysed by 2D-DiGE and LC-MS/MS, showed high similarity in HCP populations with a number of common expressed proteins around 90%. The potential platform anti-HCP antibodies demonstrated adequate coverage by detecting a wide variety of proteins across different molecular weights and pIs. When assayed using the potential platform HCP sandwich immunoassay, all CHO null cell cultures and the HCP standard, loaded at the same nominal concentration, showed similar dilution curves in shape and amplitude, and calculated HCP concentration recoveries were between 98 and 106%. Surprisingly, lower recoveries (around 40%) were observed when using anti-HCP antibodies specifically produced from HCP antigen of one of the new products (process specific antibodies) in the assay. Also, differences in HCP levels were detected when these anti-HCP antibodies (both platform or specific) were used for residual HCP quantification in in-process and drug substances samples.This work demonstrated that although the characterization data obtained on HCP antigen and HCP immune-tools are relevant and satisfactory (representativeness of standard material, antibodies coverage), these reagents will not necessarily be appropriate to be used as platform for the quantification by ELISA. The suitability of the HCP platform assay was demonstrated for two out of the three new candidates while a process specific assay will be required for the quantification of HCP in the third.
• Contributing Authors: Vincenzo Vinci, Stéphanie Gilis, Isabelle Antoine, Amaury Brasseur, Varnika Roy and Michael Chaplet
• (Day 3: HCP ELISA Development and Lifecycle Management Session)

Host Cell Proteins – Passing Regulatory Hurdles

• Speaker: Erika Friedl, Paul-Ehrlich-Institut
• Abstract: A big challenge in bioprocessing includes the control of process-related impurities such as host cell proteins (HCPs). Host cell proteins are considered Critical Quality Attributes, since their presence in biopharmaceuticals may affect the quality including activity and stability of the therapeutic protein. In addition, HCPs could cause immunogenicity in patients. Therefore, removal and tight control of these impurities are required and should be in line with the guidance available in the European Pharmacopoeia and the US Pharmacopoeia. Practical approaches for removal, characterization, and control of HCPs throughout the product life cycle are briefly outlined in these guidance documents. However, developing a potent control strategy is often challenging. The use of appropriate validated assays as well as potent HCP characterization strategies will be discussed in this presentation. Paving the way to licensing, the flexibilities around regulatory decisions will be supported by case studies.
• (Day 1: 2021 Regulatory Trends for HCPs Session)

Coverage Analysis Without a Mock Cell Line

• Speaker: Joe Hirano, Cytiva
• Abstract: The most widely used method to monitor Host Cell Protein (HCP) levels in biologics is Enzyme-Linked Immunosorbent Assay (ELISA). It uses specially designed polyclonal antibodies to detect as many HCPs as possible. The proportion of HCPs recognized by the polyclonal antibody is evaluated by a coverage analysis on mock or null cell lines. Coverage analysis becomes more challenging when one only has access to a production cell line. This is due to the relatively high concentration of drug substance in these samples, which obscures a significant population on HCPs in the analysis. In this study, we share our recent experiences of coverage assay development on a drug substance expressing sample. We demonstrate removal of a monoclonal antibody drug substance from a production sample, which enables the visualization of previously masked HCPs.
• (Day 3: HCP ELISA Development and Lifecycle Management Session)

Orthogonal Methods for HCP Analysis in Gene Therapy and Vaccine Development

• Speaker: Jared Isaac, Cygnus Technologies
• Abstract: CHO cells are widely used to produce biologicals and mAbs. Regulatory authorities are used to seeing the IND and BLA submissions including HCP ELISA qualification data including antibody coverage assessment. The rise of gene and cell therapies has brought about the usage of cell lines such as HEK 293, Sf9, Vero and PG13. Clearance of HCPs from these bioprocesses is less well defined. Guidelines used in monoclonal antibody and recombinant protein development such as ELISA monitoring of host cell proteins during purification can still be used and assessing whether your ELISA can effectively quantify the HCPs in the bioprocess is still needed. A key step in downstream purification of gene therapies is the selection of a platform ELISA kit or developing a custom ELISA assay. One part of determining that the ELISA antibodies are fit for purpose is performing antibody coverage analysis. The selection of the proper antibody reaction technique and detection method can be used to determine antibody coverage analysis for gene therapy drug substances. We will present data showing how detection method determines ELISA antibody coverage, how AAE calculates antibody coverage through CHO mAb and HEK gene therapy bioprocesses, and when to develop a custom ELISA.
• (Day 1: Rapid Fire Talks)

Moving LC-MS HCP Analysis into an GMP Environment

• Speaker: Thomas Kofoed, Alphalyse A/S
• Abstract: For new biologics under development validated HCP impurity assays are needed for GMP release test to show product purity. Often, the available generic HCP-ELISAs does not have sufficient HCP coverage for the new manufacturing process, and in some cases, it is not possible to develop a product-specific ELISA. Mass spectrometry (MS) has been used as orthogonal method to ELISA to identity and quantify individual and potentially problematic HCPs. However, it has been very difficult to move HCP analysis by LC-MS into QC laboratories due to the complexity of the sample preparation and data analysis, the sophistication of the high-end mass spectrometers, and lack of reproducibility of the complete workflow.At Alphalyse, we have developed a robust and automated workflow based on data independent and data dependent mass spectrometry and use of intact proteins as internal standards for robust and reproducible HCP analysis. Using data from more than 100 different projects, we have investigated the robustness and reproducibility of the method, over a period of 3 years and on a variety of different sample matrices. The parameters evaluated include different expression systems (e.g., CHO, E. coli, HEK, Adenovirus), types of drug (e.g., mAbs, recombinant proteins, vaccines, and gene therapy products) and complexity of the samples (e.g., from early process sample to final purified drug substance).All projects were run with the same set of 7 standard proteins as internal standards, making it possible to evaluate different parameters of importance for method validation and to transfer the method to GMP level including:
  – Variation in peptide identification and quantification (retention time, MS, and MS/MS signal intensity)
  – Variation at protein level (identification, quantification)
  – Important method parameters – cleaning of MS instrument, new column,
  – Automation of sample preparation)
• (Day 2: MS Improvements That Lead to Reliable HCP Profiling Session)

The PICRH Genome and HCP Analysis of a Model CHO System

• Speaker: Kelvin Lee, NIIMBL / Univ Delaware
• Abstract: The recent update to the Chinese hamster reference genome known as PICRH, relies on chromosome conformation capture. With this latest generation of the reference genome for the community, >97% of the genome is placed on chromosome-length scaffold sequences. Such a complete, high quality genome can serve as a useful basis for LC-MS searches of host cell proteins. We will also discuss observations from a recent study of changes in HCP as a function of cell age from an industrially-relevant model CHO system.
• (Day 1: 2021 Regulatory Trends for HCPs Session)

Identification of Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling

• Speaker: Shawn Li, Merck
• Abstract: Polysorbate is widely used to maintain stability of biotherapeutic proteins in formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.
• Contributing Authors: Shawn Li, Divya Chandra, Simon Letarte, Gregory C. Adam, Jonathan Welch, Rong-Sheng Yang, Smaranda Bodea, Alex Dow, An Chi, Christopher A. Strulson, and Douglas D. Richardson
  Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
• (Day 2: Management of Individual HCP Impurities Session)

Control Strategy for Difficult to Remove HCPs in Biopharmaceutical Development

• Speaker: Suli Liu, Biogen
• Abstract: As an orthogonal method to ELISA, LC-MS based analytical approach offers unique features of identification and quantitation of individual HCPs. A high throughput, high sensitivity and robust LC-MS based platform has been developed to support HCP clearance monitoring during process development. In this presentation, two residual HCPs produced from a CHO cell line were identified as ‘difficult to remove’ during downstream purification process. Risk assessment was performed to understand the impact of the HCPs and to guide process development. An end-to-end approach from upstream to downstream was utilized to understand HCP removal capability at each step to establish a robust process for biopharmaceutical manufacturing.
• (Day 1: High-Throughput HCP Analysis Session)

Development and Qualification of an HCP Immunoassay for a Drug Substance Hitchhiker

• Speaker: Cullen Mason, Biogen
• Abstract: A specific HCP was found to be hitchhiking along with a product DS. This case study discusses the development of an immunoassay for the detection of this specific HCP, and the comparison of results from this assay to LC-MS.
• (Day 2: Management of Individual HCP Impurities Session)

The Paradox of Host Cell Protein Coverage by Two Different Generations of Commercial Vero HCP ELISA Kits in Vero Cell Produced COVID-19 Live Virus Vaccines

• Speaker: Shruti Patel, Merck & Co.
• Abstract: The Vero cell line is one of the most commonly acceptable continuous cell lines for virus propagation and has been used as a cell culture platform for a range of new viral vaccines including those developed to prevent COVID-19 spread at Merck & Co., Inc., Kenilworth, NJ, USA. HCPs from Vero cells constitute a major part of process-related impurities during the manufacturing of those viral vaccines. Their removal from downstream purification and levels in final bulk drug substance (BDS) need to be monitored to demonstrate process and batch consistency. For this purpose, a commercial Vero HCP ELISA kit (Cygnus Technologies F500) was used in early phase of development and then replaced with a 2nd generation kit (Cygnus Technologies F975) due to that the F500 kits are running out of supply. To bridge HCP testing results between F500 kit and F975 kit, a set of process intermediates and three lots of BDS from one of our investigational viral vaccine produced from Vero cell culture were used to compare the HCP testing results obtained by the F500 and F975 kits side-by-side. Our data show that the 2nd G F975 kit was able to detect 2-8 times more HCP than the F500 kit in the three BDS lots and more purified process intermediates but ~2-3 times less HCP than the F500 kit in the viral harvest (VH) and clarified harvest (CH). Additionally, in another investigational viral vaccine BDS lot, F975 kit also detects much less HCP than the old F500 kit. An investigation on the coverage of Vero HCPs from different process intermediates by the respective antibody reagents used in the two kits was performed using 1D SDS-PAGE and 1D western blots. Our data indicate that the F975 kit has better coverage to the HCPs enriched in the ultrafiltered final retentate (UFFR) while the F500 kit preferentially detects slightly more proteins bands in the VH and CH, which explains the paradoxical HCP detection difference in different process intermediates and BDS observed when comparing the two Vero HCP ELISA kits.
• Contributing Authors: Shruti Patel, Fengqiang Wang, Richard Rustandi, Christopher Tubbs, Dennis Driscoll, Rico Gunawan
  Author’s affiliation: Analytical Research & Development, Merck & Co., Inc., Kenilworth, NJ, USA
• (Day 1: Rapid Fire Talks)

High-Throughput Platform and Automated Sample Preparation for Host Cell Protein Analysis to Support Downstream Process Development

• Speaker: Suzana Petrovic, Sanofi
• Abstract: Host cell Proteins (HCPs) are process related impurities typically monitored using immunological techniques such as enzyme‐linked‐immunosorbent assay (ELISA). HCPs may result in adverse events in patients or impact product potency and stability, depending on their identity and quantities. As the final residual amount needs to be assessed for the product quality and aims to reach a minimal level, effective HCPs clearance is of the main objective for process development. In this context, analytical demands during process development are steadily growing triggering more HCPs quantity determinations. A high throughput platform using the Gyrolab xP workstation and the CHO‐HCP E3G kit using the proprietary CHO HCP 3G ELISA antibody from Cygnus Technologies has been successfully developed and implemented at Sanofi to be able to support accelerated bioprocess development. Moreover, sample preparation is still a time‐consuming step. Therefore, automated sample dilution step was developed using the Bravo platform (Agilent) to increase throughput, increase results confidence, reduce dilution error, hands‐on time and eventually reduce the time to introduce new molecules in clinical studies.
• Contributing Authors: Suzana.Petrovic@sanofi.com; Yann.Fromentin@sanofi.com
  Affiliation: Sanofi R&D, Biologics development, Bioanalytics Vitry
• (Day 1: High-Throughput HCP Analysis Session)

Identification of Hamster MCP1 Host Cell Protein Impurity as the Potential Culprit for Clinical Adverse Events

• Speaker: Karen Price, Bristol-Myers Squibb Company
• Abstract: Identification of Hamster MCP1 Host Cell Protein Impurity as the Potential Culprit for Clinical Adverse Events Karen D Price, MS, DABT, Scientific Director, Nonclinical Safety – Bristol -Myers Squibb Abatacept, a CTLA4Ig fusion protein, is a selective costimulatory inhibitor marketed for the treatment of rheumatoid arthritis. During early clinical development (circa 1997), the program was put on clinical hold due to a series of adverse events in a Phase 2a psoriasis trial. The clinical adverse events could generally be separated into two categories: those that were histamine-like characterized by lacrimation, flushing, rhinitis, edema face/tongue, throat tightness, blurred vision, urticaria, or an anaphylactoid reaction, and those that were constitutional symptoms including fever, chills, tremors, arthralgia, and myalgia. In response to these findings, an integrated comprehensive investigation as to the possible causative factor(s) (drug, excipient, impurity) was immediately initiated. As part of that investigation, the ability of abatacept drug product to induce basophil histamine release in vitro was assessed using blood from five subjects in the IM101-005 study. These data showed that an impurity present in abatacept drug product could induce in vitro histamine release in whole blood from one subject that experienced a histamine-like reaction. It further demonstrated that this impurity was not present in the new-process material, at least at a concentration that could induce histamine release in the blood of that particular subject. This histamine-releasing activity appeared to reside predominantly in peak 2 following C3 reverse-phase chromatography, and the impurity in peak 2 had significant sequence homology with monocyte chemoattractant protein-1 (MCP-1), a potent inducer of histamine release.
• (Day 2: Management of Individual HCP Impurities Session)

Analysis of Process-Related Impurities in Cell and Gene Therapy Products by Mass Spectrometry

• Speaker: Rikke Raaen Lund, Alphalyse A/S
• Abstract: Analysis of process-related impurities in cell and gene therapy products is not straightforward: The manufacturing processes and the purified products are complex and contain process-related impurities from multiple sources. Commercial HCP-ELISA kits are often not available for the cell line or are not suitable for the specific process due to a low Host Cell Protein (HCP) coverage. Further, the short development time for some products does not allow for the development of a process-specific ELISA. Impurity analysis, by combining data dependent and data independent mass spectrometry, is overcoming several of these challenges and has multiple advantages; A) it enables quantification of the total HCP amount in ng/ml drug substance, B) it identifies and quantifies each individual HCP, C) it provides quantification of other process-related impurities, such as benzonase, and bovine serum albumin, and D) it enables identification and quantification of each individual viral protein. Here, we present case examples from LC-MS analysis of viral products, including AAV and lentivirus, and immunotherapy products based on oncolytic viruses in human cell lines.
• (Day 1: Rapid Fire Talks)

Processing of HCPs and Impacts on Biopharmaceutical Products

• Speaker: Martha Stapels, Sanofi
• Abstract: Host cell proteins (HCPs) are process-related impurities that are monitored to ensure product purity, stability, efficacy, and safety. Mass spectrometry (MS) is a highly sensitive technique used to identify HCPs, as it enables detection of individual HCPs down to 1 ppm. Using untargeted MS on samples from throughout the purification processes for multiple products, high sequence coverage has been obtained for many HCPs. This allows an increased depth in understanding for many HCPs, including problematic ones such as proteases or lipases. Certain HCPs are processed into active forms due to upstream condition changes. Other HCPs are processed into separate chains during cell culture and while one chain can be purified away, the other chain is observed as a hitchhiker. This knowledge has been used to optimize purification processes as well as generate targeted MS methods for the more problematic HCP isoforms. The processing of a variety of HCPs will be discussed, along with the potential impacts these proteins might have on product quality.
• Contributing Authors: Martha Stapels, Helena Awad, Michelle Busch, Fateme Tousi, and Maral Adeli-Koudehi
  Biologics Development, Sanofi Corporation
• (Day 2: MS Improvements That Lead to Reliable HCP Profiling Session)

Trust, but Verify – Mass Spectrometry Support for Generic ELISAs Used in AAV Gene Therapy

• Speaker: Olaf Stamm, Charles River
• Abstract: Residual Host Cell Proteins (HCP) are inevitable impurities in any biopharmaceutical product. In contrast to traditional recombinant proteins like monoclonal antibodies manufactured in CHO cells, AAV-based gene therapy products present additional HCP control challenges from a strategic process development perspective. AAV are typically manufactured using human cell lines like HEK293 which present a potentially complex proteome background. Contrasting with recombinant proteins purified from the cell culture supernatant, AAV harvest typically relies on cell lysis, meaning the entire proteome may contribute to the HCP load presented to the downstream purification process. Additionally, instead of the chemically defined media used in CHO production processes, virus production relies on protein growth factors and protein-containing additives including serum originated from other non-host cell species. Finally, the purification steps used for viral vectors are limited in number, impacting their overall capacity to separate the AAV product from other proteinaceous impurities. Adding to the technical challenges, gene therapy programs are often intended for severe indications and are therefore more likely to be placed on an accelerated development pathway. This time pressure conflicts with the timeline required to develop a product-specific immunoassay, so commercially available HCP detection ELISA kit use is common practice from early stage through late stage AAV product development. This approach poses significant risk because the generic HCP antigen used to generate antibodies for a generic ELISA kit may not provide accurate HCP quantitation since the actual HCP antigen in an AAV product will de-facto be different. As shown here, after comparing HCP content in drug substance samples of AAV vectors measured using a generic kit-based ELISA, the actual content determined by quantitative LC-MS showed consistently higher HCP values across many lots than were observed using generic ELISA, indicating commercial HCP kits may in some cases significantly under-estimate HCP content by a wide margin. The impact of this is especially relevant to high-dose AAV programs. The root cause of these findings can be explained as insufficient coverage by the antibodies used in the ELISA, as demonstrated by 2D SDS-PAGE and Western Blot.The HCP content is a critical quality control attribute highly relevant to the safety of a gene therapy product. Therefore, the analytical methods used in the process development and quality control should be suitable for their intended use by providing accurate results. In cases where the development timelines are forcing use of generic reagents, those reagents should be checked for specificity and coverage (accuracy) and the results obtained be confirmed by an unbiased approach such as Mass Spectrometry. Quantification by LC-MS is more accurate because known protein/peptide standards can be spiked into the test articles providing a reliable quantitative readout of actual HCP content. Furthermore, LC-MS provides identification of the individual proteins detected, guiding refinement of AAV purification strategies on a faster timescale than product-specific ELISA development.
• (Day 3: HCP ELISA Development and Lifecycle Management Session)

Monitoring HCPs in an Orphan Drug Gene Therapy Program

• Speaker: Jessica Thrall, Novartis
• Abstract: (Pending)
• (Day 1: 2021 Regulatory Trends for HCPs Session)

An Enzymatic Assay for Lipase Detection in Support of Process Characterization and Improvement

• Speaker: Todd Wright, Seagen
• Abstract: Persistence of specific classes of host-cell proteins (HCPs) pose both product and patient risks in antibody-based biologics. Lipases, hydrolases, and esterases capable of degrading common formulation excipients polysorbate 20 (PS20) and polysorbate 80 (PS80) have been pinpointed as a root cause of particle formation. Such observation motivated development of techniques to characterize and control these phenomena. Current methods to measure enzymatic activity are available but suffer from ease of implementation and time to perform. Here, we outline a simple plate-based technique using commercially available materials to quantify enzymatic activity. This method benefits from improved sensitivity and reduced run time due to optimization of its critical reagents. Method qualification has shown it can detect multiple recombinant lipases, including recombinant lipoprotein lipase (rLPL) below 10 ng/mL on-plate. This enzymatic assay was further used to identify purification process improvements. These changes led to a marked reduction in lipase activity. Long-term, we aim to standardize this technique as a risk mitigation tool to increase clinical success of both early and late phase programs.
• (Day 1: Rapid Fire Talks)