2023 Hybrid HCP Conference
11th Annual | Dubrovnik, Croatia | March 22-24, 2023
2023 HCP Speaker Abstracts
Sookyen E, Regeneron Pharmaceuticals
Title: Identification and Absolute Quantification of a Problematic HCP with Lipase Activity to Support Therapeutic Protein Development
Abstract: Monitoring of residual host cell proteins (HCPs) in therapeutic protein is essential to ensure product quality, safety and efficacy. The LC-MS/MS-based proteomics method has evolved as an important tool to identify and quantify HCPs. Despite the development of advanced mass spectrometry techniques and optimized workflows, identifying and quantifying problematic HCPs present at low levels remain challenging. Here, we established a practical, effective strategy for the identification and quantification of low abundance HCPs. A case study of using this strategy to investigate a high-risk lipase HCP, liver carboxylesterase (CES), will be discussed. This newly developed workflow has been applied to investigate the cause of PS80 degradation, facilitate downstream process development and effectively remove high-risk HCP. Compared to previous studies, the current LC-MS/MS-MRM method improves the sensitivity of HCP quantification by over 10 folds, thus is able to meet the challenges for the accurate quantification of certain problematic HCPs at sub-ppm to ppb levels during drug development.
Erika Friedl, Paul-Ehrlich Institute
Title: HCP Impurity Control Covering Various Product Classes
Abstract: High quality medicines and patient safety are dependent on the efficient control of process-related impurities such as HCPs. The control of HCPs is an important issue for many product classes such as blood products, monoclonal antibodies, gene and cell therapy products as well as vaccines. Similar aspects apply to reach the goal of tight HCP control. Importantly, these aspects comprise the choice of suitable HCP assays during product development, the assay validation and also covering the life cycle management of the critical assay reagents. Removal of HCPs to the lowest possible level is generally recommended by a variety of guidance documents. Combined with the HCP identification by established and new technologies a suitable HCP control strategy should be implemented throughout the product life cycle. Case studies will be discussed to highlight the decision points also covering various product classes to facilitate licensing applications for the European and the US market.
Elisa Giordano, Merck
Title: In-House CHO HCP ELISA Platform Feasibility Study
Abstract: Host cell proteins (HCPs) are a major class of process-related impurities derived from the host organism, such as Chinese Hamster Ovary (CHO) cells used to produce recombinant therapeutic products. Even if in residual amounts, they have the potential to affect product quality, safety, and efficacy. [1]The workhorse of HCPs monitoring and quantitation during bioprocessing manufacturing is sandwich enzyme-linked immunosorbent assay (ELISA), which employs polyclonal anti-HCP antibodies for both capture and detection. This assay format offers a combination of high sensitivity, specificity, throughput, automation potential, rapid turnaround, quantitative results, and low cost per assay. [2]Depending on the stage of clinical development, different types of HCP-ELISAs are often applied. Commercial ELISA kits developed from CHO are widely applied in early drug development stages (preclinical, phase I, and phase II). For phase III and beyond, the upstream process-specific method is preferred and recommended by the Health Authorities [3]In this presentation will be showed a feasibility study divided into two main parts:
• The first part was dedicated to the selection of the best antigen-antibody couple among materials from four different CHO molecules.
• The second part was dedicated to the development of an in-house CHO HCP ELISA platform using the antigen-antibody couple selected and comparing its performance with the current commercial ELISA kit.The goal of this study is to demonstrate the greater reproducibility and robustness of the proprietary inhouse ELISA method in recognizing the HCP populations compared to the commercial one during the drug product development cycle until phase III.
References:
[1] Xinrong Liu, Ying Chen, Yiwei Zhao, Virginia Liu-Compton, Wesley Chen, Gillian Payne, Alexandru C. Lazar, Identification and characterization of co-purifying CHO host cell proteins in monoclonal antibody purification process, Journal of Pharmaceutical and Biomedical Analysis, Volume 174, 2019, Pages 500-508.
[2] USP 39 Published General Chapter Residual Host Cell Protein Measurement in Biopharmaceuticals.
[3] Pilely, K., Johansen, M.R., Lund, R.R. et al. Monitoring process-related impurities in biologics–host cell protein analysis. Anal Bioanal Chem 414, 747–758 (2022).
Robert Hooper, Rockland Immunochemicals, Inc.
Title: Qualification of a Generic CHO HCP Assay by Orthogonal Methods and Recommendations for Management of Critical Reagent Supply for the Validated HCP-ELISA
Abstract: Use of a generic off-the-shelf HCP ELISA has several challenges both before and after it is validated for use in bioprocess monitoring. First, it is difficult demonstrate that an HCP ELISA is fit-for-purpose to use in bioprocess control strategy and second consistent long-term reagent supply is an established risk for HCP ELISAs.
To validate HCP reagents, ELISA and 2D coverage are common orthogonal techniques that together demonstrate that the anti-HCP ELISA is fit for purpose. Mass spectrometry provides another way to assess HCP impurities, and unlike ELISA provides identification of the individual HCP proteins. As such mass spectrometry is now commonly used as both a primary and an orthogonal method for HCP assessment along with 2D “DIGE/DIBE immunoblots and ELISA.
Here we describe the characterization of a generic CHO HCP assay using standard methods of 2D, ELISA, coupled with an orthogonal LC-MS analysis of the HCP standard and the evaluation of potential problematic HCPs as part of the qualification. We also propose recommendations for demonstrating fit-for-purpose to the specific bioprocess and for securing long term HCP reagent supply to reduce the risk of premature critical reagent depletion.
Yunli Hu, Regeneron Pharmaceuticals
Title: Probing Residual Host Cell Proteins in Monoclonal Antibody and its High Molecular Weight Species: A Different Way to Study Difficult-to-Remove HCPs
Abstract: Host cell proteins (HCPs) are process-related impurities introduced during therapeutic protein production. The presence of problematic HCPs in the purified drug substance (DS) poses challenges in drug development as it may induce adverse events in patients and affect product quality and shelf life. The problematic HCPs retained in the final DS are mainly due to specific and non-specific interactions with column resin/ligands or mAbs. To support process development and mitigate risks, effective methods with high sensitivity and rapid turnaround times are required to identify and quantify these difficult-to-remove HCPs. However, host cell protein (HCP) analysis has seldom been conducted to characterize the impurities in aggregates. In this work, HCP analysis of enriched HMW species and drug substance (DS) from five different monoclonal antibodies (mAbs) was performed. More HCPs were identified in the enriched HMW than in the DS across all five mAbs. Some frequently identified and problematic HCPs were present at higher levels in the enriched HMW fractions than DS, thus indicating a potential interaction between HCPs and HMW. Especially, the most abundant HCP from mAb1, C-C motif chemokine, was 46 times more abundant in enriched HMW than DS. The enriched HMW was further fractionated into enriched dimers and enriched very HMW (vHMW) fractions. The C-C motif chemokine was found to interact mainly with mAb1 dimer species rather than vHMW fraction. Removing the HMW species from mAb1 significantly decreased the C-C motif chemokine level in the final mAb1 DS. The current study of HCP profiling of HMW expands knowledge regarding the HCPs present in mAb preparations and their interaction mechanisms, thus aiding in understanding of HMW, immunogenicity, HCP identification, and removal for overall development of therapeutic protein drugs.
Jared Isaac, Cygnus Technologies
Title: Case Studies Using LC-MS to Mitigate Problematic HCPs
Abstract: Mass Spectrometry (MS) is an orthogonal technique to ELISA. ELISA is a well-established method for lot release testing, and MS supports ELISA during early-stage drug substance (DS) purification, DS batch testing, and when transferring biomanufacturing process between CDMOs. During downstream process development (DSP) and manufacturing scale up problematic HCPs may be enriched to above specification levels. MS can be used to perform antibody coverage analysis to ensure the QC ELISA is not missing HCPs and identify potentially problematic HCPs. We will present several case studies of problematic HCPs that were not cleared during the DSP. AAE-MS was applied to show that the ELISA was appropriately monitoring the DSP and used to identify problematic HCPs.
Kelvin Lee, University of Delaware
Title: Analyzing CHO Host Cell Proteins: Impact of Cell Age and of Digest Conditions
Abstract: In this presentation, we will explore a number of issues related to HCP analysis including how HCP profiles change as a function of cell age during the course of 30, 60, and 90 doublings as well as the impact of sample processing conditions prior to LC-MSMS based HCP analysis. We will discuss how a small subset of HCPs, which may be difficult to remove, demonstrate age-dependent expression and over the time-scale in question most show decreased expression. In addition, we explore how comparisons of standard and native digest conditions might impact the assessment of HCPs in a sample.
Suli Liu, Biogen
Title: Comprehensive Characterization of In-House ELISA Host Cell Protein Reagents by IAC and LCMS
Abstract: Host cell proteins (HCPs) derived from host expression systems are critical process-related impurities of recombinant protein biopharmaceuticals, which may impact drug product safety and efficacy. HCPs are traditionally monitored using a multi-analyte enzyme-linked immunosorbent assay (ELISA). The coverage of the critical reagent of an ELISA has been traditionally assessed by 2D gel electrophoresis methods. In this study, we present a workflow to assess ELISA reagent coverage by combining immunoaffinity chromatography (IAC) and liquid chromatography mass spectrometry (LCMS). Null cell antigens of multiple cell lines were loaded and eluted on immunoaffinity columns coated with anti-HCP reagent antibodies, followed by in-depth LCMS proteomics characterization. The results provided protein-specific coverage analysis of different ELISA reagents. This new workflow will assist in selection of the appropriate reagent at early phase of biopharmaceutical development and enable strategy for new reagent generation. Also, database of reagent coverage and prior HCP information can guide downstream development to select the purification parameters for effective and targeted HCP removal.
Ejvind Mørtz, Alphalyse
Title: ELISA Reagent Characterization Using Advanced LC-MS Methods
Abstract: When using a commercial ELISA kit or a process-specific ELISA for Host Cell Protein (HCP) impurity measurements it is required by regulatory reviewers that the ELISA reagents have been characterized for suitability to the specific product. The ELISA characterization typically includes HCP Coverage analysis using 2D PAGE for comparison of spot patterns, and dilutional linearity experiments. As a CRO with experience in HCP characterization from many different projects, it is our observation that frequent challenges in ELISAs are: A) Low HCP coverage, B) ELISA not measuring abundant HCPs, C) lack of dilutional linearity, D) different HCP values obtained when using new ELISA reagents. These challenges can however not be answered by 2D PAGE analysis and require higher resolution analytical methods to provide more detailed information about the HCP coverage.
Here, we present results and learnings from ELISA reagent characterization using advanced LC-MS techniques on a variety of different biologics and expression systems. The analyses include:
-HCP Coverage analysis of early process sample using ELISA immunocapture combined with mass spectrometry (ELISA-MS)
-Coverage of individual HCPs of potential concern
-Coverage of individual HCPs in the purified drug substance
-Comparison of the HCP standard with the early process sample
-Evaluation of high-responding HCPs and non-specific binding likely leading to lack of dilutional linearity
-Evaluation of cross-reactivity to the drug substance
This more comprehensive ELISA reagent characterization enables more detailed understanding of the HCP-ELISA reagents, and documentation of its suitability to the specific manufacturing process and purified drug substance. The LC-MS analyses also enables bridging between ELISA reagents and explains differences in HCP measurements between old and new reagents.
Jeremy Osko, Merck
Title: LUMINEX – An Innovative Method for Host Cell Protein Quantification with Multiplexing Capabilities
Abstract: Enzyme-Linked Immunosorbent Assay (ELISA) is a hallmark technique that is routinely used for the quantification of Host Cell Proteins (HCPs) during biologics development and manufacturing. Despite its high sensitivity and throughput, total HCP ELISA is highly dependent on the polyclonal antibody reagents used in the assay and often leaves proteins with insufficient antibody coverage undetected or under detected. To address this problem, orthogonal characterization of HCP by proteomic LC-MS/MS often supplements ELISA and enables the identification of problematic HCPs. To accurately tracking the clearance of problematic HCPs, new technologies such as LUMINEX have multiplexing capability to allow the monitoring a panel of potential problematic HCPs in one assay, and thus has much higher throughput and is considered more cost-effective than the use of multiple individual HCP ELISAs. In this presentation, we will introduce LUMINEX as a new multiplex assay platform and present case studies comparing its performance with individual HCP ELISAs monitoring problematic HCPs such as lipases.
Sherin Panikulam, University of Applied Sciences and Arts Northwestern Switzerland
Title: Analysis of Host Cell Proteins in Protein A Eluates of mAb-based Therapeutic Proteins
Abstract: Protein A chromatography is the first step in purifying therapeutic antibodies and remov ing various impurities. One of the major process-related impurities of therapeutic protein production based on cellular systems are host cell proteins (HCPs). Analyzing Protein A eluates of 23 therapeutic proteins using LC-MS/MS revealed the propensity of specific HCPs to co-elute. This co-elution appears to be due to functional protein networks these HCPs may form.
Contributing Authors: Sherin Panikulam1,2, Frieder Kroener2, Oliver Anderka2, Georgios Imanidis1, Thomas Villiger1, and Nicolas Lebesgue2
1 University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland;
2 Novartis Pharma AG, Basel, Switzerland
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 to the quality of the product. Mass spectrometry (MS) has become an increasingly common approach for characterization of HCP impurities due to its ability to identify and quantitate individual HCPs in biotherapeutic products, which can support risk assessment and process optimization as well as characterization and quality control. This presentation will provide an overview of USP efforts to support the quality and consistency of mass spectrometry-based HCP analysis through development of both documentary and physical standards to support MS analysis of HCP. Best practices for HCP identification and quantitation will be shared from a new general chapter, <1132.1> Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry, which has been completed by the USP Expert Panel and will be published in Pharmacopeial Forum for public comment. USP is also developing both intact protein and peptide standards to support identification and quantitation of high risk and high abundance HCPs. We will present USP’s strategy for development and characterization of physical standards to support HCP analysis by MS and an update on standards currently in development.
Suzana Petrovic, Sanofi
Title: High Throughput Platform for Multiplex Analysis of Total and Specific Host Cell Proteins (HCPs) Content to Support mAb Process Development
Abstract: Host cell Proteins (HCPs) are process related impurities typically monitored using immunological techniques such as enzyme-linked-immunosorbent assay (ELISA). HCPs are a major class of process related impurities as they 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 total HCPs quantity determinations. A high throughput platform using the Gyrolab xP workstation and Gyrolab CHO-HCP E3G kit has been successfully developed and implemented at Sanofi to support accelerated bioprocess development for total HCP determination. Moreover, some specific HCPs are identified as “High Risk” HCPs for mAb process development due to their immunogenicity and/or impact on product quality. Therefore, a multiplex approach was evaluated to be able to perform a specific HCP assay in the same Gyrolab run as the total HCP assay in order to enhance process development support with reduced time, cost and modification of the initial workflow.
Corresponding authors: Suzana.Petrovic@sanofi.com
Affiliation: Sanofi R&D, Global CMC Development, BioAnalytics Vitry
Katie Porter, EpiVax, Inc.
Title: Comprehensive Assessment of Immunogenicity Risk of Host Cell Proteins in Biologics Using in silico and in vitro Methods
Abstract:
Purpose
Host cell protein (HCP) impurities are a critical quality attribute (CQA) because they may trigger undesired immune responses with the potential to compromise the safety and efficacy of biologics. The presence of immune responses to Chinese hamster ovary (CHO)-derived HCPs has contributed to the suspension of clinical trials in the past. EpiVax has developed a web-based tool to evaluate the immunogenicity of HCP impurities. This tool, the Interactive Screening and Protein Re-engineering Interface for Host Cell Proteins (ISPRI-HCP) has been in use by industry clients and collaborators since 2003. ISPRI-HCP evaluates the immunogenic potential of proteins based on T cell epitope content and their similarity to the human proteome. We used ISPRI-HCP, to perform an in-silico immunogenicity risk assessment of 140 HCPs identified by a consortium of monoclonal antibody producers. EpiVax will further evaluate and validate the ISPRI-HCP platform by performing in-vitro T cell assays for this set of commonly found HCP impurities.
Methods
In-silico immunogenicity risk assessment of host cell protein impurities was performed using, a web-based platform, ISPRI-HCP. Each input sequence is parsed into overlapping 9-mer frames and evaluated for the potential to bind HLA using the extensively validated EpiMatrix immunoinformatic algorithm. “
Positive hits are considered likely to be T cell epitopes, which can drive anti-HCP immune responses and antibody formation. The density of T cell epitope per unit protein is calculated for each HCP, and then the T cell epitopes in the CHO protein are compared to similar epitopes in the human genome, using JanusMatrix. Each of the 9-mers in the HCP protein are evaluated for humanness by searching for potentially cross-reactive sequences based on the preservation of the TCR-facing residues of HLA 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. The potential immunogenicity risk was then determined using established thresholds for potential immunogenicity and tolerogenicity. This analysis can identify which HCP have high or low potential for immunogenicity, enabling drug sponsors to focus efforts to remove HCP that have the highest immunogenicity risk.>br>
Results
While detailed reports for each protein will be obtained for the 140 HCP, usually ISPRI-HCP results are depicted on a plot where the Y axis indicates the density of predicted T cell epitopes (EpiMatrix Immunogenicity Score), the X axis indicates the average humanness of those epitopes (JanusMatrix Score), and the size of each marker indicates the relative abundance (e.g. ppm). For this set of proteins, it is clear that HCP immunogenicity varies quite dramatically. For example, for the Jones et al. dataset of monoclonal antibody HCPs, we observe that the results for Phospholipase B-Like 2 Protein show that the protein has a very high T cell epitope density and low cross-conservation with human supports the observation of specific immune response to this impurity. Results for additional HCP will be provided in the presentation. We will be selecting at least 6 HCP for further investigation in vitro, to determine whether the in silico results correlate with human immuine response to overlapping peptide pools representing the putative epitopes and to the whole HCP proteins. PLBL2 and 5 additional CHO HCP impurities will be evaluated in the EpiVax IVIP (In-vitro Immunization Protocol) T cell assay. We expect to publish the results and methods so as to enable drug developers to reduce the risk of immunogencity that can be associated with HCP.
Conclusion
In silico and in vitro analysis of identified HCPs within product preparations for potential immunogenicity is useful for pre-determining the degree of immunogenic risk associated with HCPs. Our process to investigate the in-vitro immune response of HCPs will enhance the development of ISPRI-HCP, providing a better understanding of HCP impurities in biologics, enabling drug developers to improve the safety and efficacy of biologics.
References
• 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.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.
Margarita Sabater, Genmab A/S
Title: HCP Control Strategy – Experiences from an “off gel” road
Abstract: Selection of assay type and the appropriate reagents to demonstrate HCP control through the life cycle of the product is of outmost importance, in view of the potential safety issues and product stability associated with residual HCPs.For this purpose, we present a holistic HCP control strategy that encompasses a set of approaches for the understanding of process HCP clearance, method for testing of residual HCP and risk assessment of residual HCP in final product, including:• Understanding HCP reduction throughout process, and process consistency runs using quantitative LC-MS
• Characterizing HCP profiles for product comparability
• Process changes – impact on HCP profile
• Evaluation of commercial CHO kits fit for product – 11 different antiserum reagents were screened using process specific HCP samples by an off-gel technique to support the choice of HCP ELISA.
• ELISA coverage and reagent characterization using non-gel-based methods, i.e. LC-MS – Experiences and discussion points with coverage analysis results obtained using a range of different techniques are presented.
• Experience from licensing application for European and US markets with a control strategy containing both ELISA and LC-MS characterization.
Nicklas Skjoldager, Novo Nordisk
Title: The Use and Implementation of Bio-layer Interferometry for Host Cell Protein Detection
Abstract: Host cell proteins (HCPs) are inevitable impurities in biologic drug production and a decrease through the purification processes must be documented. To be able to report a reliable HCP value at low concentration, the analytical assay must be not only sensitive, but also resistant to matrix effects.Bio-layer interferometry (BLI) is a quick and versatile technique, which is known for being less impacted by matrix effect than e.g. ELISA. Here we report a customized BLI HCP assay to be more sensitive than its ELISA counterpart, and at the same time being less impacted by matrix effects and turbidity of samples, using in-house antibodies.From the same batch of rabbit polyclonal antibodies, two pools were labeled with biotin and fluorescein, respectively. The assay was designed as a multi-step quantification sandwich, using pre-coated streptavidin biosensors for immobilizing Ab-biotin. In the final step AEC (3-Amino-9-ethylcarbazole) was used as signal enhancement by generating a precipitate on the biosensor tip. The quantitative range of the standard curve was optimized to approx. 0.5–200 ng/mL, depending on the specific assay. HCP samples from various purification steps could be quantified including low concentration samples, exceeding the performance of ELISA.The BLI method is a semi-automatic ‘walk-away’ assay reducing hands-on time, giving results within 2–3 hours, thus making it suitable for fast decision making in early drug development and potentially as an at-line quality procedure in the production. In conclusion, the developed BLI method serves as a serious alternative to the traditional HCP ELISA with equal or better sensitivity.
Stefan Sommerschuh, BioGenes GmbH
Title: Case Study – Challenges and Solutions in Specific HCP ELISA Development for the Physcomitrium patens (Moss) Plant-Based Expression System
Abstract: Together with common bacterial and mammalian host cell expression systems, plant-based expression systems are becoming increasingly important for recombinant drug manufacturing in the pharmaceutical industry.
The enzyme-linked immunosorbent assay (ELISA) is the gold standard method for host cell protein (HCP) monitoring for product development and batch release testing. Specific HCP ELISAs are based on the polyclonal antibodies raised by immunizing a suitable animal species (e.g., rabbits or goats) with a process-related mock material. The mock antigen represents a broad spectrum of potential HCP impurities, but does not include the drug substance. In this case study, we present the successful development of a process-specific HCP ELISA for the plant-based production cell line Physcomitrium patens (Moss), including comprehensive sample and reagent characterization. We place a focus on the cross-reactivity testing of the generated antisera and derived purified antibodies, as the presence of drug substance-specific antibodies was indicated by the analytical data after HCP immunization. Cross-reactivity of the HCP antibodies was most likely caused by the glycosylation of the drug substance expressed in Physcomitrium patens. Immune adsorption against the immobilized drug substance was used for the depletion of cross-reactive antibodies. The resulting antibody preparation was tested for proper HCP measurement. Coverage analysis by 2D Western blot and Immunoaffinity Chromatography (IAC)/2D DIGE demonstrated excellent suitability. A process-specific HCP ELISA in a kit format with high sensitivity (working range of ~1.5 – 100 ng/mL) was then developed, which is currently being used for HCP detection in the novel drug manufacturing process. The case study demonstrates how intrinsic hurdles during plant HCP ELISA development can be handled successfully.
Jon Valgeirsson, Alvotech
TBA
Martin Voigtmann, Boehringer Ingelheim RCV GmbH & Co KG
Title: Using GyroLab for HCP Analysis from Microbial Derived Samples
Abstract: Boehringer Ingelheim’s Process Science department in Vienna develops and establishes microbial derived production processes, including the analytical portfolio to evaluate the quality of our products. The understanding of HCPs in our process has significantly become more important. Therefore, we introduced the high throughput HCP assay based on the GyroLab system to support process development activities. The system is a miniaturized automated immunoassay on a CD platform. The advantages of the method are a broad dynamic range (3-4 logs), its high sensitivity (ng/mL) and high precision (CV < 10%), as well as the fast assay time (16 HCP samples with 5 dilutions in approximately 1.5 h (excluding sample preparation)). Of particular note, the system works by a flow-through mode, which reduces incubation times and minimizes matrix effects.In the talk, we want to point out our experience with the system on several examples. This includes,
• The establishment of an in-house HCP Assay in comparison to the ready to use HCP-assay by Gyros
• The comparison to the corresponding ELISA HCP data
• The comparison to HCP data from mass spectrometry analysis
• Our system suitability tracking concept for Gyrolab HCP assays
The Gyrolab HCP assay is part of our routine analytical panel for process development, and we used it for over 2000 samples in the last 2 years. We are keen to share our knowledge with you.
Thomas Waerner, Boehringer Ingelheim Pharma GmbH & Co. KG
Title: Assessing Methods for Detecting Low Abundant HCPs
Abstract: Host cell proteins (HCPs) are process-related impurities that must be adequately cleared from recombinant biopharmaceuticals during the downstream process to ensure product quality, purity, and patient safety. It is well known that some HCPs with very little abundance have a strong impact on product quality. Understanding how HCPs escape the purification process and the detection of those HCPs is a prerequisite for developing a suitable HCP depletion process. Here we assess our research on methods for detection of low abundant HCPs by MS/MS and compare 3 different methods (Hexapeptide -, partial digest and immunoaffinity-chromatography) to enrich HCP levels in samples.
Todd Wright, Seagen
Title: A Multi-Angled Approach for Host Cell Protein Population Analysis Provides a Holistic View of Upstream Process Influence
Abstract: Host cell protein (HCP) expression depends, in part, on differences during the upstream process, including CHO cell lineage, viability, and cell age. Even so, similarities of ~80% of HCP identities have been reported in literature between individual null runs where antibody was not generated. We performed a cross-comparison of HCPs identified by LC-MS/MS in harvest cell culture fluid (HCCF) from different antibody and upstream processes. Using identity alone, unique HCP populations were observed in each HCCF sample. HCPs shared between separate processes were further contrasted through incorporation of relative HCP abundance. Upstream processes with noteworthy differences also exhibited variation in HCP relative abundance, whereas similar upstream processes demonstrated close agreement in HCP relative abundance. We followed this comparison with analysis of HCPs present in each individual process to those characterized in an HCP ELISA standard. A predominance of HCPs identified in each antibody process sample was also detected in the assay standard. Furthermore, when abundance of HCPs shared between process sample and standard was evaluated, minimal differences in overall HCP abundance rankings were observed. These results suggest that relative abundance along with identity, applied to LC-MS/MS results and orthogonal methods such as 2D DIGE, may provide additional insight when evaluating HCP populations with different upstream processes.
2023 HCP Poster Abstracts
Christian Albers, Bruker Daltonics GmbH & Co. KG
Title: Fast and Robust Quantitation of Host Cell Proteins withSub ppm Sensitivity using dia-PASEF and VIP-HESI
Abstract: Introduction:
Host Cell Proteins (HCPs) are residual host-derived proteins which are present in biopharmaceutical drug preparations, such as monoclonal antibodies, and can be detrimental to the drug quality or efficacy. Most HCPs are typically removed in the final product using a combination of purification steps, and HCP clearance is monitored throughout the pipeline. Mass spectrometry has emerged as an alternative to the more established ELISA method for clearance monitoring due to several benefits such as sample flexibility and the ability to identify individual HCPs. Here we apply parallel accumulation – serial fragmentation combined with data-independent acquisition (dia-PASEF) in a robust high throughput approach, demonstrating HCP detection and quantitation with sub ppm sensitivity.Methods:
NISTmAb 8671 (Merck) was spiked with either Universal Proteomics Standard 1 (UPS) 1 or 2 (Sigma) and digested with trypsin using native digest method. UPS1 (equimolar) was spiked at 1 in 50 for generating the spectral library, UPS2 (dynamic) was spiked at 1 in 100 for high throughput library screening.
Peptides were separated using an Elute UHPLC interfaced with a timsTOF Pro via a VIP-HESI ion source and analysed by PASEF (spectral library creation, 150 min gradient) or dia-PASEF (library screening, 15 min gradient).
dia-PASEF data were acquired using 25 Da isolation windows offset across the mobility range in a 2 sec cycle and searched against the spectral library using Spectronaut.Results:
dia-PASEF data were acquired using a 15-minute gradient in triplicate and 36 out of 48 UPS2 proteins were identified and quantified in all 3 dia-PASEF runs, spanning six concentration levels.
A regression line was calculated from this data and used for calculating the amounts of NISTmAb HCPs present in the sample. In total, 140 NISTmAb HCPs were identified and quantified in all replicates between 0.03 ppm and 158 ppm.
The quantitation and identifications correlate and overlap very well with previously published data whilst extending the overall coverage of NISTmAb HCPs. Mobility data is recorded for all peptides and is used as an additional identification metric, reducing validation efforts.Conclusion:
The used dia-PASEF acquisition mode combined with the VIP-HESI source provides a robust analytical flow platform for high throughput HCP screening with sub-ppm sensitivity and high coverage.
Emilie Bodin, Agro-Bio
Title: When is a Process Specific HCP ELISA Assay Required for HCP Monitoring ? Case Study on the Impact of Cross-Reactivity Against the Drug
Abstract: A critical aspect of the bioproduction processes is the removal of host cell proteins (HCPs) to appropriate levels in order to minimize risk to patient safety and drug efficacy.Regulatory authorities require these HCPs to be monitored and quantified using a gold-standard method: the ELISA sandwich. To quantify HCPs, a generic HCP kit is usually used during each phase of the bioprocess, but from Phase III, it is strongly recommended to develop a process specific HCP ELISA kit. Another scenario may require to go with a process specific kit: when the general kit is no longer well adapted, because of a deficient coverage or, as discussed here, because of the cross reactivity against the drug substance.As described in the guideline: “any cross-reactivity of the anti-HCP antibodies with the product may compromise the test method and yield biased results. Therefore, any contamination of the HCP antigen with product must be avoided to prevent the generation of anti-product antibodies.” (Guideline FDA Residual Host Cell Protein Measurement in Biopharmaceuticals).Indeed, for a generic HCP kit, afterwards it will be mandatory to demonstrate that the kit is suitable for specific process by testing ELISA parameters. One of the aspects of ELISA testing is the general antibodies’ cross-reactivity against the drug substance.In this poster, we present several cross-reactivity tests against the drug substance with two analysis methods: ELISA testing and Western Blot. We expose the protocol (ELISA testing: – MRD Determination (Minimum Required Dilution) in the drug buffer; – Dilution linearity; – Specificity), and the results achieved in various projects. Also, a focus is made on when we detected cross reactivity against the drug with generic antibodies: how to develop a process-specific ELISA in a highly risky context of cross reaction of the generated anti-HCP antibodies?
Companies: Agro-Bio & BioCytex, Stago Group
Main Authors: Lucille Lassalle and Lionel Cambrils
Shao-Yung (Eric) Chen, Seer Inc.
Title: Comprehensive Identification and Quantitation of Host Cell Proteins in Monoclonal Antibody Products Utilizing a Fully Automated Multi-Nanoparticle Proteomics Methodology
Abstract: Host cell proteins (HCPs) are residual impurities from host expression systems used to produce biopharmaceuticals and can contaminate the final drug product. The presence of HCPs in biopharmaceuticals can have several negative consequences, including immunogenicity, reduced efficacy, and even safety issues for patients. For instance, the presence of HCPs could trigger an immune response, leading to allergic reactions or even anaphylaxis for patients. Furthermore, HCPs can interfere with the desired therapeutic effect of the drug, or even cause adverse effects. Therefore, it is critical to detect and quantify HCPs during the development and manufacturing of biopharmaceuticals to ensure that the final product is safe and effective for patients. This is typically done using immunoassays, which can detect general HCP levels within a sample. More recently, liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical tool that can be used for HCP analysis, providing high sensitivity, specificity, and accuracy in identifying and quantifying individual HCPs. Compared to traditional HCP detection methods, such as ELISA, MS-based analysis offers several advantages. MS-based analysis can detect a broader range of HCPs and provide more accurate and quantitative results. It also eliminates the need for specific antibodies, which can be a limitation in traditional detection methods. With that being said, MS-based HCP analysis also has its challenges, such as the complexity and heterogeneity of the sample matrix, as well as the requirement for sophisticated instrumentation and expertise in data analysis.Here, we explore the performance of the Proteograph™ platform, a standardized, automated multi-nanoparticle-based proteomics workflow coupled with conventional nano-flow liquid chromatography with a state-of-the-art mass spectrometer (nanoLC-MS/MS). To benchmark performance against a community standard, we analyzed the NIST monoclonal antibody (NIST mAB, RM 8671) reference material. We have investigated the performance of the Proteograph workflow with LC-MS/MS, using relatively short 60 min sample-to-sample Data-Independent Acquisition (DIA), as well as 120 min sample-to-sample Data-Dependent Acquisition (DDA) MS strategies. The LC-MS/MS data was analyzed using Proteograph™ Analysis Suite (PAS) software, using MSFragger and DIA-NN search engines where we evaluated peptide yield, HCP identification performance against traditional direct digests, and reproducibility of the Proteograph workflow.Overall, our results demonstrate a significant performance advantage of the Proteograph workflow for analysis of HCPs relative to traditional HCP LC-MS approaches, including >4X improvement in identified HCPs. The workflow was able to readily quantify problematic HCPs such as Fructose-bisphosphate aldolase A, granulin, and Protein disulfide-isomerase A6. The Proteograph assay also exhibited high technical reproducibility with more than 700 proteins detected across triplicate DIA runs and pearson correlation of 0.965 for HCP quantification across replicate analysis. Along with these performance gains, the Proteograph platform offers a substantial convenience advantage without the need for conventional pre-processing, sample manipulation schemes, large sample quantities, or extended LC-MS analysis often required to study the wide dynamic concentration ranges of proteomes inherent to these complex biopharmaceuticals.
Contributing Authors: Shao-Yung (Eric) Chen, Max Mahoney, Paul Pease, Aaron S. Gajadhar
Marius Felix, Boehringer Ingelheim Pharma GmbH & Co.KG
Title: Tackling HCPs Responsible for Polysorbate Degradation: Using Activity-Based Protein Profiling (ABPP) to Generate an Activity Map of the CHO Cell Culture Fluid Proteome with Different Probes
Abstract: The vast majority of host cell proteins (HCPs) originating from the production cell lines of biopharmaceuticals is removed during the downstream process. However, there is a certain risk that some HCPs are still present within the final product and may adversely affect product quality even at very low abundance. Along these lines, enzymatic degradation of polysorbates used to stabilize therapeutic antibodies has been observed by different pharmaceutical companies.To tackle this challenge quite some effort has been put in the development of highly sensitive LC-MS workflows to determine the identity of remaining HCPs. This effort apparently comes along with the need to discriminate HCPs being active (regarding polysorbate degradation) from inactive HCPs. Again, mass spectrometry provides a powerful tool to address this issue by applying Activity-Based Protein Profiling (ABPP): This technique is based on a bifunctional probe which is on the one hand able to form a stable covalent bond with the catalytic serine of e.g. lipases/esterase and on the other hand contains an affinity tag to enrich bound enzymes prior to enzymatic digestion and relative quantification by LC-MS.We used ABPP to generate an activity profile of CHO cell culture fluid at two different probes. Besides the commercially available probe ActiveXTM, we also applied a second in-house synthesized probe which has been shown to merely completely inhibit polysorbate degradation when spiked into final product.From the roughly 900 proteins detected in CHO cell culture fluid more than 50 proteins could be enriched by at least a factor of 100. Additionally, partly different proteins could be enriched by the two different probes.On the basis of this data, the criticality of enzymes recently published in connection with polysorbate degradation can be assessed and serve as guidance e.g. for upstream and downstream development.
Joe Hirano (1), Cytiva
Title: A Generic ELISA Kit for CHO HCP Quantification Assay Development and Validation
Abstract: This Amersham™ HCPQuant™ CHO (supernatant) Kit includes an HCP ELISA and
antibody reagents designed to sensitively and robustly quantitate HCP contamination
within therapeutics manufactured in Chinese hamster ovary (CHO) cell lines. The
purified antibodies react strongly with supernatant proteins derived from CHO-K1 and
CHO-S backgrounds. The complete HCP solution facilitates method optimization and
data submission throughout process and product development.
Joe Hirano (2), Cytiva
Title: PrismA ELISA™ kit – Quantification of a Process Related Impurity
Abstract: The PrismA ELISA™ kit is an enzyme-linked immunosorbent assay designed to measure residual ligand leakage when using MabSelect PrismA™ chromatography resin and Fibro™ PrismA chromatography adsorbers. The kit is used during the development and manufacture of monoclonal antibody (mAb) biopharmaceuticals.
Xavier Homo-Prault, ANAQUANT
Title: HCP Profiler: Another Way for HCP Identification and Quantification in DSP Evaluation by LC-MS/MS
Abstract: Nowadays, a growing number of therapeutics are produced in cellular systems. When biopharmas develop a biological drug, they will likely have to perform a bioprocess optimization, with the ultimate goal to increase production yield. However, getting a better yield requires USP/DSP modifications, potentially impacting the product purity or efficacy due to change in the content of process-related impurities, such as host cell proteins (HCPs). Consequently, a thorough characterization of all the changes performed by LC-MS/MS coupled to the READYBEADS™ technology is of high interest in order to improve correctly the DSP. Indeed, HCP ELISA kits allow for a global HCP quantification only, and do not permit to identify and quantify HCPs individually at once. Here, a DSP optimization lasting over months with different steps evaluation was described.
Sabrina Liberatori, Lonza Biologics R&D
Title: An Agile Analytical Workflow to Monitor Residual Host Cell Proteins and Facilitate Decision-Making in Purification Process Development
Abstract: Regulatory agencies have designated residual host cell proteins (HCPs) a critical quality attribute of biotherapeutics, as they could potentially affect their safety and efficacy. In particular, proteases and lipases have been reported to have an impact on product quality and shelf life either by direct degradation or by impacting formulation.HCP-ELISA is the gold standard for measuring total HCP content. However, it is becoming increasingly clear that, in combination with HCP-ELISA measurement of total HCPs, mass spectrometry-based methods could provide the identity information required to monitor the content of specific “high-risk” HCPs.The proposed work reports an agile LC-MS/MS approach to monitor specific HCPs in in-process samples and drug substance, which has been successfully used to inform the downstream process decision-making and to minimize the content of unwanted HCPs in final product.
Guojie Mao, Lonza Biologics plc
Title: Optimization of Two Dimensional Difference Gel Electrophoresis for Host Cell Protein Analysis
Abstract: Host cell proteins (HCP) are process related impurities in biopharmaceutical products that should be monitored during manufacturing. HCP populations vary in different manufacturing processes or cell lines. CyDye® fluor labelled two dimensional difference gel electrophoresis (2D DIGE) is one popular method used to compare HCP differences. This study describes an assessment of alternative fluorescent dye (Alexa Flour) for 2D DIGE. Multiple assay parameters including sample buffer optimisation, sample loading methods (manifold cup or strip-holder) and the choice of flurorecent dyes to label HCP were investigated.The fluorescent dye labelling efficiency was compared to silver staining to confirm the suitability of the 2D method. For labelling the same HCP sample, Alexa Flour dyes showed mis-colocalisation of HCP spots during 2D DIGE analysis which requires further optimisation before used for 2D DIGE.The optimised 2D DIGE with Cydye® fluor showed a sensitive and robust approach to separate the HCP populations, and provide informative comparison of HCP profile changes during manufacturing process development.
Griffin Moran, Novo Nordisk A/S
Title: Automation of Host Cell Protein Analysis by Mass Spectrometry: Increased Speed and Reproducibility, from Sample Preparation to Report
Abstract: Detection of host cell proteins (HCPs) present in a drug product is a requirement from regulatory authorities during the development of biopharmaceuticals. Traditionally, HCP detection is performed using Enzyme-Linked Immunosorbent Assay (ELISA). However, ELISA has a long development time, does not provide protein identity, and is cell-line specific. HCP analysis by mass spectrometry (HCP-MS) is a trending technique due to its speed of method development and project flexibility, the ability to assist the downstream development process, and the improved risk assessment enabled by sensitive and accurate HCP identification. HCP-MS can however be a labour-intensive, time-consuming process with limited reproducibility, and may become a bottleneck for complete characterisation and rapid production of biopharmaceuticals.
Starting from a complex proteomics workflow, we have developed an analytical setup in which each step was optimized and automated to improve robustness and save time. Initial changes involved improvements to the chromatographic stages; namely, replacing custom-made nano-HPLC columns with an off-the-shelf solution (Evosep®). This not only increased the throughput from approximately eight samples per day to a minimum of 30 — thereby meeting the requirements of higher analytical speed — but also delivered the improved reproducibility associated with advanced and standardised chromatographic solutions. To match the increased LC capacity of our HCP assay, sample content normalisation and sample preparation were transferred to a liquid handler to further increase the speed and reproducibility. The data analysis of the mass spectrometry raw files was automatised with a vendor-neutral client-server solution to increase processing speed, standardise processing, and improve reproducibility. A subsequent automated reporting step compiles the data from various sources and generates the final report.As a proof-of-concept, we investigated technical replicates to evaluate the overall reproducibility while defining the gains in efficiency. The reproducibility of the liquid handler for sample preparation was tested across a range of Evosep LC gradients (30, 60, 100, 200, 300 samples per day) in at least five replicates. A combination of simple and complex protein samples was analysed, including samples extracted during process development that contained different levels of HCPs. Customised data analysis visualisations facilitated evaluation of results and enabled conclusions to be drawn with confidence.
This optimized setup and workflow have significantly increased the capacity and reproducibility of the MS-based HCP assay through automation of the analytical procedure, replacing custom-made products with off-the-shelf solutions, and automating data analysis and subsequent reporting. Here we present the gains in efficiency that these improvements brought to the process.
Author: Carsten P. Sönksen, Ph.D., Novo Nordisk A/S, Bagsværd, Denmark
Co-Authors: Griffin Edward Moran, Jan Bruun Andersen
Acknowledgements: Amy Claydon, Arnd Brandenburg, Markus Stepath (Genedata AG)
Sapphire Sloan, BioPhorum
Title: High Risk Host Cell Proteins – Establishing a Comprehensive Control Strategy
Abstract: The BioPhorum Development Group is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals.The Host Cell Protein & Other Bio-residual Impurities Workstream is a working group of highly regarded subject matter experts who have been working together within BioPhorum since 2014.The main goal of the HCP workstream is to aim for industry alignment by building a common understanding of agency requirements for HCPs through benchmarking and gap analysis of Regulatory guidance.As a member led consortium, key topics are chosen and members then volunteer to form a working sub team to action value adding deliverables to aid and influence industry alignment.
Kevin Van Cott, University of Nebraska-Lincoln
Title: Evaluation of Search Engines and Results Curation Options for HCP Analysis
Abstract: (The goal of HCP data analysis is to identify low-abundance proteins from peptide MS/MS spectra that have low signal intensities. This presents a challenge for search engines that were designed for standard proteomics. It also changes the calculus of how search engines should be evaluated for their appropriateness in HCP data analysis: instead of evaluating search engines for total number of peptides or proteins identified (as they generally are for conventional proteomics data), they need to be evaluated at the limits of their operating ranges, for peptides present at low parts-per-million levels (relative to the product protein’s peptides) and for HCPs with low sequence coverage.New search engines have been developed over the years, with each claiming superior performance compared to predecessors. However, there are little to no formal reports of how these search engines perform with HCP data. In previous BEBPA HCP conferences, several presenters have stated that using more than one search engine for HCP data is needed for reliable results. There are two aspects to this rationale: (1) each search engine has its strengths and weaknesses, and a single search engine should not be trusted to be comprehensive, and (2) if multiple search engines identify an HCP, the confidence in that HCP truly being present in the sample is higher.This brings up some important questions that have not been directly addressed in the HCP field: what is the best way to do HCP data analysis, keeping in mind that we are searching for peptides close to the limits of detection for the MS instrument?• A perfect search engine may not exist, but do all search engines have serious blind spots?
• Are multiple search engines required to cover up those blind spots?
• Are any of the 9 freeware search engines included with SearchGUI adequate for HCP analysis?
• What about the expensive commercial search engines – are they just as flawed as any of the freeware search engines?
• What about HCP results curation? If no search engine is perfect, the final judgement must be provided by the expert analyst. What are the best options for results curation: freeware or a commercial product?To begin answering these questions, we took two HCP datasets and searched them with the 9 freeware search engines in SearchGUI (combined with PeptideShaker) and compared the results with a commercial search engine from Protein Metrics (Byonic). Both datasets have multiple HCPs that span ~3 orders of magnitude in abundance (~5 – ~5,000 ng/mg), so there is a diverse representation of HCP peptides in this study. MS data were searched against the same FASTA database files using the same general search parameters (digestion specificity, modifications, m/z tolerances, etc.). A 1% False Discovery Rate (FDR) was used in all search engines. HCP results from SearchGUI were exported into Skyline for curation using the default confidence thresholds. Byonic results were exported into Protein Metrics’ Byologic for curation. Every candidate peptide was manually inspected to confirm that the MS1 data were clear with no interference, the XIC peak was clearly integrated, and the MS/MS spectra were of high quality to justify a true positive designation. HCP acceptance required at least 2 peptides/protein. The total number of HCPs (and their abundance) and the total number of peptides for each HCP (and their abundance) will be reported for each search engine. By using PeptideShaker, Skyline, and Byologic, we were able to pinpoint where potential HCP peptides failed to meet confidence thresholds during the process and pinpoint what causes differences in reportable HCP results among these search engines.)
Sonja Voordijk, SIB (Swiss Institute of Bioinformatics)
Title: Impact of Cell Line and Culture Harvest Method on CHO HCP Profiles
Abstract: Two-dimensional differential in gel electrophoresis (2D-DIGE) was used to compare CHO HCP profiles from 10 different CHO CHP reference standards. The mock in process samples were derived from null-strain cultures lacking the gene for recombinant protein expression and were comprised of variable processes that represented both supernatant and lysate harvest methods. The CHO HCP reference standards included three frequently used CHO lineages (CHO K1, CHO DG44 and CHO S) and included two standards for which CHO lineage and harvest method is unknown.Our findings show that differences amongst CHO cell lines had less impact on the CHO-derived HCP populations than did the specific bioprocess methods. This observation should be taken into consideration when creating the HCP references standard used for CHO HCP ELISA development or when selecting a commercial CHO-ELISA kit. The HCP standard must be representative of the biomanufacturing process to generate fit-for-purpose anti-CHO HCP antibodies and thus selection of the appropriate harvest method might have a higher impact on obtaining required HCP coverage than the choice of the CHO cell line variant.
Contributing Authors: S. Voordijk1, G. Bouchet2, D. Walther2, A. Hamilton2, V. Barbié2, D. Chimento2, and J. Hirano2
1 SIB Swiss Institute of Bioinformatics, Geneva, Switzerland; 2 Cytiva, Uppsala, Sweden; 3 Rockland Immunochemicals Inc., Limerick, PA, USA