Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Improved 1D MS-Based HCP Platform with Single-Digit PPM Sensitivity, Enhanced HCP Coverage and Shorter Turnaround Time

• Speaker: Christina Bell, Caprion Biosciences Inc.
• Abstract: Biological drugs produced from host cell expression systems inevitably contain host cell protein (HCP) impurities whose identity and presence may determine whether the drug is accepted by regulatory agencies. In conventional HCP analysis, immunoassays based on polyclonal antibodies raised against the host cell system are used to provide HCP measurements in aggregate. Due to increased regulatory expectations for deeper HCP characterization however, mass spectrometry (MS)-based analyses are increasingly used as a complementary and supplementary method to achieve in-depth, comprehensive identification and quantitation of residual HCP content.Basic (1D) LC-MS/MS workflows can provide sensitivity in the 10 ppm range, whereas extensive peptide pre-fractionation using 2D-LC-MS/MS workflows may be used to obtain single-digit ppm sensitivity. However, fractionation prior to LC-MS/MS analysis is a time-consuming and intensive process, requiring intensive quality controls to ensure reproducibility.To address the need for single-digit ppm MS-based HCP analysis with higher throughput and shorter turnaround time, an optimized 1D HCP workflow was developed though systematic optimization of the various assay workflow steps. Optimization of sample processing and digestion conditions resulted in a shorter processing time and improved digestion efficiency. Optimization of the chromatography conditions and implementation of the new MS technology, the timsTOF Pro from Bruker, enabled a 4- to 5-fold increase in assay sensitivity with enhanced HCP coverage and data quality.
• Contributing Authors: Christina Bell, Laetitia Cortes, Vanessa Diniz Atayde, Rudolf Guilbaud, Michael Schirm and Lorella Di Donato
• (Day 2: Applications of Mass Spectrometry to Identify and Quantify HCPs Session)

Streamlined, Vendor Neutral Data Processing for Host Cell Proteins by Mass Spectrometry

• Speaker: Eric Carlson, Protein Metrics Inc.
• Abstract: Residual host cell proteins (HCPs) are contaminants in biotherapeutics, which may pose safety or stability risks. HCPs are typically found at low levels in highly purified proteins and need to be monitored per regulatory guidelines. Most biotherapeutics are recombinantly expressed in Chinese Hamster Ovary (CHO) cells, and thus the HCPs monitored in these samples originate from the CHO host cells or the media source used in cell culture (mostly bovine). Another category of biotherapeutics are those that are derived from human plasma. For human plasma-derived products, immunogenicity is not usually a concern, unless the HCPs are in modified form, such as aggregation or oxidation. Instead, the primary concern is the biological function of the HCPs. HCP detection and measuring is particularly challenging for plasma proteins, mainly due to the highly glycosylated proteins found in humans. Here, we present a mass spectrometry and analysis workflow to identify and quantify host cell proteins for plasma-derived products.Since mass spectrometry analyses for Host Cell proteins (HCPs) have advanced in recent years, so have expectations about the ability to provide data to non-experts.
  Mass spectrometry vendors have developed solutions that are generally unique to their own platforms, with acquisition and fragmentation techniques that are unique to their own instruments. There are different strategies for label-free quantitation, different ways of determining which peptides should be included for quantitation, and differences in the processing of data. Additionally, each vendor has their own software package, none of which import data from the rival vendors, and all based on dedicated proteomics packages that were historically used for complex mixtures of proteins, rather than for biotherapeutic products. High individual expertise is generally required to process this data, and just the data processing alone is time-consuming for an expert in that mass spectrometry vendor’s tools. It is still unusual for analysts to have cross-platform expertise. Furthermore, major focus points of HCP studies by the vendors have been to study the mechanics of the process, rather than relate the results to existing, orthogonal techniques, because it can be so time-consuming. The re-use of software tools was often simply expedience, and great effort has been expended on the necessary optimization of chromatography and consumables.Here we present software that processes all mass spectrometry vendors’ data from accurate mass instruments in a coherent and uniform manner. All vendor data can be processed in the same way, irrespective of mode of acquisition or ionization. Furthermore, there are tools to cope with the different strategies desired by end users to quantify: the user has the ability to choose how to normalize the data, whether specific peptides are chosen, whether to use one or multiple charge states, and even flag when fewer than the desired number of peptides per protein are found. Templates may be stored and re-used, and user roles can be set to only allow data viewing and prevent unwarranted reprocessing.We show that despite having diverse raw data sets, an organization can harmonize a non-targeted, mass spectrometry-based HCP analysis method across geographies, laboratories, and even users.
• (Day 3: Innovations in Mass Spectrometry for HCP Analysis Session and Poster)

Automated, Optimized Sample Preparation for LC-MSMS Analysis of Host Cell Proteins

• Speaker: Amy Claydon, Thermo Fisher Scientific
• Abstract: During the manufacture of recombinant biotherapeutics, host cell proteins (HCPs) are released and consequently require removal from the final drug product. HCPs are a critical quality attribute (CQA) due to their unknown effects on product safety and efficacy. Additionally, consistency in the HCP profile is reliant on consistent manufacturing processes, therefore changes in the HCP profile can be an advanced warning for drug product QC.
  There is a growing realization that, despite it being the method of choice for regulatory purposes, traditional ELISA approaches to HCP analysis can only provide limited information e.g. bulk quantitation of immunologically reactive HCPs and no individual protein identification. They also rely on protracted development of high-quality standards and reagents specific to each assay. LC-MSMS can quantify a protein-protein basis as well as identify the individual HCPs present. This enables monitoring of individual HCPs during purification stages and can indicate potential for harmful effects due to the presence of specific HCPs. Any lot-to-lot variability, or effects from version changes, can also be assessed. However, HCP analysis by LC-MSMS is not without its complications. The major challenge is to overcome the huge dynamic range (up to six orders of magnitude) to detect low ppm concentrations of residual HCPs within the biotherapeutic.
  To successfully implement a LC-MS approach for HCP analysis into routine discovery, a workflow that is easy to use, fit for purpose and that satisfies regulatory requirements is required. Automating sample preparation increases robustness while minimizing the necessary analyst time. Optimized sample preparation specific for HCP analysis is also essential to maximize the extractable data from each sample. Various strategies for sample preparation prior to LC-MS HCP analysis have been used within the current literature, including overloading of HPLC columns to improve HCP limits of detection in the MS, as well as non-denaturing digests to keep the mAb intact while the easier to digest HCPs are proteolyzed. Automated sample preparation solutions including Thermo Scientific™ SMART Digest™ and ImmunoAffinity kits, have been assessed and compared to more traditional in-solution digestion approaches, with a focus on maintaining simplified and automated processes as much as possible. Samples were analyzed using a Thermo Scientific™ Vanquish™ Horizon UHPLC coupled to a Thermo Scientific™ Q-Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer using both DDA and DIA to maximise sample information.
• Contributing Authors: Amy Claydon¹, Thomas Buchanan¹, Philip Widdowson¹, Andrew Williamson², Yue Xuan³, David Horn⁴, and Rowan Moore^{2
  1} Thermo Fisher Scientific, Runcorn, UK; ² Thermo Fisher Scientific, Hemel Hempstead, UK; ³ Thermo Fisher Scientific, Bremen, Germany; ⁴ Thermo Fisher Scientific, San Jose, CA, USA
• (Poster)

HCP Risk Assessment

• Instructor: Carl Co, Biogen, Christina de Zafra, Amgen, Markus Haindl, Roche, and Nisha Palackal, Regeneron Pharmaceuticals
• Abstract: (Pending)
• (Day 1, Half Day Workshop: Track 2)

Host Cell Protein ELISA Reagents: A Case Study Comparing Platform and Process Specific Reagents for Testing of a Viral Vaccine Product

• Speaker: Saskia Crowe, Janssen
• Abstract: A crucial step in Host Cell Protein ELISA development is the generation of critical reagents and specifically the choice of antigen to be used for antibody generation. As described in USP1132, an upstream process specific assay for phase III and beyond may be preferred over a commercial or platform method, especially if the cell culture process is significantly changed from the platform process. However, a commercial or platform method could still be justifiable provided the assay reagents are demonstrated to be applicable to the process HCPs. A study case focusing on HCP reagents for an adenovirus-based vaccine product will be discussed. In this example, HCP reagents from a platform kit and reagents from an up-stream process specific method generated from different processes are compared through an extensive characterization package. This characterization work includes MS based protein profiling, MS based spectral counting, 2D-DIGE and Antibody Affinity Extraction methods. Results indicate that both sets of HCP antigen are representative of the process HCPs and that both sets of antibodies show similar levels of coverage. From this package we conclude despite major differences in the cell culture process both sets of reagents are equally suitable for measuring Host Cell proteins in our viral vaccine products confirming that process specific reagents aren’t necessarily superior and platform reagents may be used for late phase and beyond.
• (Day 1: Analysis of HCP’s for Gene Therapy and Viral VaccinesSession)

High Throughput Mass Spectrometry-Based Proteomic Screening for Host Cell Contaminants

• Speaker: Sophia Doll, Max Planck Institute of Biochemistry
• Abstract: An increasing number of proteins are applied as therapeutics and a whole industry is based on producing them for clinical application. One of the most common protein production systems for therapeutic drugs like antibodies is the Chinese Hamster Ovary (CHO) cell line, resulting in high protein yields. Host cell proteins (HCPs), however, can easily contaminate the recombinant protein drug during purification. While HCP levels are kept at a minimum, even traces of HCPs can potentially trigger adverse events like immune reactions. Thus, it is critical to improve the understanding of HCP impurities in biopharmaceuticals.
  To this end, we used an unbiased mass spectrometry (MS) based discovery approach to characterize the proteomes of the host cell CHO and two therapeutic antibodies. Using a ‘loss-less’ high-pH reversed-phase fractionator (Kulak et al., 2017), we generated a deep library of the potential CHO cell contaminants including more than 6000 proteins. After this first characterization, we developed a fast screening method that would allow us to analyze purified proteins in high-throughput for HCPs. Therapeutic antibody samples were prepared in a 96-well format in only four hours using a fully automated sample preparation method with the Agilent BRAVO robot (Geyer et al., 2016). We also integrated a new liquid chromatography, called Evosep One (Bache et al., 2018) into our proteomic workflow to achieve uninterrupted high-throughput. Herein, we compared different gradient lengths for the coverage of HCPs and reproducibility, allowing the proteomic measurement of 60, 100 or even 200 samples per mass spectrometer per day. Using the recently developed data-acquisition method termed “BoxCar” (Meier et al., 2018) enabled us to further increase proteomic depth in single-shot injections.
  In conclusion, we developed a high-throughput and highly reproducible screening platform that can be easily applied in an industrial setting for the fast and rapid characterization of HCPs in pharmaceutical antibodies.
• (Day 2: Applications of Mass Spectrometry to Identify and Quantify HCPs Session)

An Efficient Workflow for Identification and Monitoring of Host Cell Proteins During Monoclonal Antibody Bioprocessing

• Speaker: Catalin Doneanu, Waters Corporation
• Abstract: Even after sophisticated purifications steps, low levels (1-100 ppm) of host cell proteins (HCPs) remain in the final purified drug substance. Some of the HCPs may cause immunogenic reaction in humans, therefore it is critical for patient safety that HCPs be identified and quantified. The analytical methods typically used for HCP quantification are based on immunoassays (ELISA), but ELISA cannot guarantee proteome-wide coverage. In recent years, LC/MS-based assays have been adopted as orthogonal techniques to ELISA for HCP analysis due to their flexibility and potential for full proteome-wide applications. Here we describe an efficient analytical scale LC/MS workflow that allows the identification and quantification of HCPs during mAb purification in a CHO cell line. The first step of the HCP identification and quantification workflow is the HCP discovery assay employing data-independent MSE acquisition using 90 min peptide separations. Following data processing with Progenesis QI for proteomics 4.2, HCPs are identified by a proteome-wide database search. In addition, LC/MS data can be assembled into spectral libraries, containing peptide precursors, charge states and retention times. In the second step of the HCP workflow, additional HCP samples derived from the purification of the same biopharmaceutical are analyzed by higher–throughput HCP monitoring assays using MSE acquisitions with 30 min peptide separations. Each purified sample can be analyzed by LC/MSE followed by searching against the spectral library for HCP identification and quantification. The HCP workflow described above was tested for identification of HCPs from the NIST mAb and then applied to identification and monitoring of HCPs from a different monoclonal antibody. The antibody product, initially purified by Protein A affinity chromatography, was further purified by SCX (strong cation exchange) chromatography using various elution conditions. All mAb samples were digested with trypsin using a protocol designed to deplete the quantity of antibody-derived peptides, while retaining the HCP-derived peptides [1]. Seven HCPs were identified across 5 mAb preparations following a CHO database search using Progenesis QIP 4.2 software. The same software was also used to build a spectral library of CHO HCPs including peptide retention time, precursor m/z and MS/MS fragmentation spectra for facilitating subsequent HCP identification and quantification in other mAb preparations. Our results show that HCPs can be confidently identified, quantified and monitored in biopharmaceutical samples using the 1D LCMS HCP workflow.
• Contributing Authors: Catalin E. Doneanu¹, Malcolm Anderson¹, Alex Xenopoulos², Romas Skudas², Ying Qing Yu¹, Asish Chakraborty¹, Mark Bennett¹, and Weibin Chen^{1
  1} Waters Corporation; ² EMD Millipore Corporation
• References: 1.Huang L, Wang N, Mitchell CE, Brownlee TJ, Maple SR, De Filippis MR Anal Chem 2017, 89, 5436
• (Poster)

Host Cell Protein (HCP) Quantification for AAV Gene Therapy Products

• Speaker: Fabien Dorange, ImmunXperts
• Abstract: AAV vectors are highly promising tools for treating monogenic diseases and have been recently enrolled in a large number of clinical trials. As a biopharmaceutical product, AAV vectors are manufactured using a host cell system and characterization of the product includes testing for impurities including Host cell Proteins (HCP). For our AAV vectors, HCPs are derived from the HEK293 cell line used for the production.
  For HCP detection and quantification, a traditional sandwich-type ELISA is currently used. New technologies based on immunodetection have been evaluated for HCP monitoring and a comparison between these methods are presented. HCP coverage was established using the 2D-DIGE western Blot method for reagent characterization.
  Mass Spectrometry was performed as orthogonal method in order to complement immunodetection method with the identification of HCPs in Drug Substance product and to support process development.
• Contributing Authors: Fabien Dorange and Christine Le Bec
• (Day 1: Analysis of HCP’s for Gene Therapy and Viral VaccinesSession)

Identification and Quantification of HCPs in Biopharmaceutical Products

• Presenter: Henry Fisher, Covance
• Abstract: With the steady increase of industry regulators requesting submissions to include data detailing HCP quantification by techniques orthogonal to ELISA, LC‐MS is becoming the technique of choice for many pharmaceutical companies. An LC‐MS based approach offers significant advantages, allowing identification of specific HCPs. A single LC‐MS workflow can also be applied to multiple sample types expressed from any cell line without the need for re‐qualification or the use of less informative ELISA kits and reliance on the consistency of component reagents. A data rich LC‐MS approach is ideal for facilitating in‐process development decisions and for downstream HCP monitoring.To enable safer and faster drug development and to fulfil the needs of the growing market, Covance has developed a pragmatic approach for routine HCP analysis by 1D LC‐MS. Here we present evaluation of two workflows; a process development support workflow where HCPs are identified and roughly quantified using spiked standards and the Huang mAb depletion methodology; with testing of alternative MS experiments MSe and SONAR. Using in‐process samples associated with an antibody biosimilar development program, we demonstrate removal of identified HCPs at various stages of the product purification process, with potential to facilitate subsequent targeted absolute quantification.
• (Poster)

Health Authority Panel: Short Presentations, Pre-Loaded and Open Questions

• Speakers: Erika Friedl, Paul-Ehrlich-Institute and Alexey Khrenov, US Food & Drug Administration
• Abstract: (Pending)
• (Day 3: Safety and Tox Risks Associated wtih Host Cell Protein Impurities Session)

Western Blot Assessment of Polyclonal Anti-Host Cell Protein Antibody Production

• Presenter: Tobey Gooding, Covance
• Abstract: The most critical part of developing an ELISA for measuring Host Cell Protein (HCP) impurities in Biotherapeutics is generation of specific antibodies with appropriate recognition of the total population of HCPs potentially present in the product. The generation of the antiserum is dependent upon the sum of the individual biological responses (i.e., antibodies) of multiple animals to the antigen. Response is monitored throughout the program qualitatively by 1D (one dimension) Western Blot and adjustments are made to the antigen (immunogen) as needed to yield the broadest antigen recognition. Ultimately, the highest quality antisera from multiple animals are pooled. The quality of the final product is demonstrated through evaluation of coverage which is typically by 2D (two dimension) Western Blot or more recently by mass spectrometry. The BioCMC Group at Covance was tasked with development of a process capable of supporting this type of assessment. This capability will be illustrated through discussion of a typical antibody reagent production program.
• (Poster)

Precise and Efficient Coverage Analysis for HCP ELISA Assay Validation Using 2D DIBE™

• Presenters: Joe Hirano, GE Healthcare Life Sciences and David Chimento, Rockland Immunochemicals
• Abstract: The most widely used method to monitor host cell protein (HCP) levels in biologics is enzyme-linked immunosorbent assay (ELISA), which uses a set of specially designed polyclonal antibodies to detect as many HCPs as possible. To validate the efficacy of the ELISA, it is critically important to determine the proportion of HCPs that the anti-HCP antibodies detect by coverage analysis. HCP coverage can vary greatly between different samples and processes and therefore it is vital to determine coverage for each new process. The most accepted method to calculate the percentage of proteins that the HCP antibodies detect is 2D electrophoresis and subsequent Western blotting. Additional orthogonal methods such as mass spectroscopy are used to a lesser extent and can be used to confirm the 2D validation. A major weakness of conventional 2D analysis is difficulty in the alignment of protein spots between the gel and the blot. Imprecise alignment could lead to false or incorrect interpretation of the antibody coverage and performance.In order to overcome the technical challenges in generating accurate and robust 2D coverage data, we have previously proposed an enhanced anti-HCP antibody coverage analysis method, 2D differential in-blot electrophoresis (2D DIBE). Here we discuss the advantages of using 2D DIBE compared to the traditional ECL detection method using the antibodies from a recently released CHO K1 HCP ELISA kit (Amersham™ HCPQuant CHO). The overall efficiency of coverage analysis is greatly improved using 2D DIBE which enables the measurement of total protein and detected protein from a single membrane and the resulting omission of a time-consuming alignment of the 2D spot patterns.
• Contributing Authors: Andrew Hamilton¹, Joe Hirano¹, Phil Beckett², Yu-Hung Huang³, Narumi Furuuchi³, Karin Abarca Heidemann³, Sonja Voordijk⁴, and Gerard Bouchet^{5
  1} GE Healthcare Bio-Sciences AB, Uppsala, Sweden; ² GE Healthcare Life Sciences, Marlborough, MA, USA; ³ Rockland Immunochemicals Inc., Limerick, PA USA; ⁴ Geneva Bioinformatics (GeneBio) SA, Geneva, Switzerland; ⁵ SIB Swiss Institute of Bioinformatics, Geneva, Switzerland
• (Poster)

Impact of Host Cell Protein on Safety of Intravitreally-Dosed Biologic

• Speaker: Francois Huet, Novartis
• Abstract: Intravitreal injection of therapeutic antibodies into the eye has proven to be a remarkable success for treatment of diseases such as neovascular age-related macular degeneration and diabetic macular edema. However, ocular inflammatory reactions in the nonclinical testing of these proteins have been frequently observed, and to a lesser extent in humans as well. We have found that the vitreal space of the eye in nonclinical species is particularly sensitive to the purity of the therapeutic protein. Early preparations of candidates has at times resulted in inflammation, whereas later more purified preparations have had fewer findings. This may also explain the relatively rare incidence of ocular inflammation in human subjects administered more refined GMP quality material. Here we will discuss one program where initial large animal studies with a monoclonal antibody fragment identified severe inflammatory reactions that correlated with high levels of host cell protein in the preparation. In later stages of manufacturing development, host cell protein was reduced by an order of magnitude; this was accompanied by an elimination of the inflammatory reaction and advancement of the protein into the clinic.
• Contributing Authors: Tim MacLachlan and Vito Sasseville
• (Day 3: Safety and Tox Risks Associated wtih Host Cell Protein Impurities Session)

Mass Spectrometry Provides HCP Identity and Validates ELISA Results

• Speaker: Jared Isaac, Cygnus Technologies
• Abstract: A broadly-reactive HCP ELISA should be used during the purification processes to ensure removal of HCPs and to demonstrate process consistency and final DS purity. Regulatory authorities are requesting biopharmaceutical companies employ orthogonal methods to demonstrate antibody coverage to individual HCPs and provide a comprehensive assay qualification package to ensure the HCP ELISA used by a sponsor is fit for this purpose.
  Antibody Affinity Extraction (AAE), a novel method developed by Cygnus Technologies, is used to determine antibody coverage and reactivity to those HCPs that co-purify with DS. AAE is more predictive of the anti-HCP antibody performance in the HCP ELISA and facilitates identification of individual downstream HCPs. While ELISA is the gold standard for monitoring HCP levels, it does not provide information about what HCPs are present in the DS. Identification of HCPs by mass spectrometry (MS) is a powerful orthogonal method to ELISA. However, one of the limitations of MS is that IgG drug substances often mask HCPs by a factor of 10^4 – 10^6. To improve MS sensitivity, AAE can be used as a sample preparation method to enrich HCPs and eliminate most of the DS in a sample.
  In this talk, we will present data showing HCP identification by MS before and after AAE. In addition, we will discuss the integration of orthogonal methods for comprehensive HCP analysis that provide data throughout DS purification to inform process development teams of how to modify their purification processes to improve DS purity. Enrichment of HCPs by AAE and detection by MS is a powerful orthogonal method to ELISA. Biopharmaceutical companies that integrate MS with ELISA data provide comprehensive quality control data for regulatory agencies.
• Contributing Authors: Jared Isaac, Eric Bishop
• (Day 2: Improved Methods for HCP Antibody Coverage Determination Session)

Antibody Affinity Extraction Enables Identification of Host Cell Proteins by Mass Spectrometry

• Presenter: Jared Isaac, Cygnus Technologies
• Abstract: A broadly-reactive HCP ELISA should be used during the purification processes to ensure removal of HCPs and to demonstrate process consistency and final DS purity. Regulatory authorities are requesting biopharmaceutical companies employ orthogonal methods to demonstrate antibody coverage to individual HCPs and provide a comprehensive assay qualification package to ensure the HCP ELISA used by a sponsor is fit for this purpose. Antibody Affinity Extraction (AAE), a novel method developed by Cygnus Technologies, is used to determine HCP antibody coverage and reactivity to those HCPs that co-purify with drug substance (DS). AAE is more predictive of the anti-HCP antibody performance in the HCP ELISA and facilitates identification of individual downstream HCPs. While ELISA is the gold standard for monitoring HCP levels, it does not provide information about what HCPs are present in the DS. Identification of HCPs by mass spectrometry (MS) is a powerful orthogonal method to ELISA. However, one of the limitations of MS is that IgG drug substances often mask HCPs by a factor of 104 – 106. To improve MS sensitivity, AAE can be used as a sample preparation method to enrich HCPs and eliminate most of the DS in a sample. In this poster, we will show that AAE followed by MS is a powerful HCP enrichment method to verify ELISA quantification results and provide the identity of unknown HCPs. ELISA and MS are independent methods that can provide data throughout biopharmaceutical drug purification to inform manufacturers of how to modify their manufacturing processes and drug substance purity.Take home message:
  Enrichment of HCPs by AAE and detection by MS is a powerful orthogonal method to ELISA. Biopharmaceutical companies that integrate MS with ELISA data provide comprehensive quality control data for regulatory agencies.
• Contributing Authors: Alla Zilberman, Jared Isaac, Eric Bishop, Ken Hoffman
• (Poster)

An Improved Method for Simultaneous Detection and Quantitation of >4000 Host Cell Proteins

• Speaker: Eric Johansen
• Abstract: Here we present improvements upon existing LC-MS methodology for detecting and quantifying multiple individual host cell proteins (HCP) simultaneously. This technique provides individual protein identities, rather than the total sum abundance of HCP, which is a limitation of current ELISA-based methods. The high sensitivity and parallelization of quantitative measurements made possible by Data-Independent Acquisition (DIA) make it a useful solution for the quantitation of multiple low abundance CHO-HCPs in biotherapeutics.
  This methodology uses an in-house CHO-HCCF spectral library containing >4000 proteins to process DIA quantitative analysis of process samples. To date, this methodology has successfully identified HCP impurities across process pools for ten different programs at Abbvie Stemcentrx. We have verified detection and quantitation of proteins as low as 0.6 ppm in abundance relative to therapeutic product (w/w).
  Evaluating the data from each of these analyses has revealed that out of the 4000+ proteins in the library, less than 100 proteins were detected in any process pool after Protein A purification. After subsequent polishing steps that number is reduced to less than five.
  Here we present a targeted workflow, using a commercially-available heavy-labeled CHO secretome spike-in standard (Sigma) in conjunction with recombinant Chinese hamster, mouse, and rat proteins (with Chinese hamster homology) from the list of detected HCPs. First: calibration curves are generated by serial dilution of the recombinant “HCPs” against a constant concentration of MAb (not grown in CHO). An equivalent amount of the Heavy-labeled CHO secretome is spiked into each cal. curve sample and any product/process sample to be interrogated. Second: Samples are digested and data is acquired for both cal curve and process samples using DIA. Data from DIA runs can be processed using heavy-light chromatogram transition ratios just as with traditional MRM-based absolute quantitation. Additionally, the DIA approach provides a relative quantitation of all proteins regardless of whether they were among those with cal curves, as has been demonstrated previously.
• (Day 3: Innovations in Mass Spectrometry for HCP Analysis Session)

BioPhorum Development Group – A Multi-company Collaboration; Views on “High Risk” Host Cell Proteins

• Speakers: Marisa Jones, GSK and Nisha Palackal, Regeneron Pharmaceuticals
• Abstract: Host cell proteins (HCPs) are process-related impurities that may co-purify with biopharmaceutical drug product. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, difficult to purify and/or degrade both product molecules and excipients. Why should the biopharmaceutical industry worry about these high-risk host cell proteins? What approach could be taken to deal with these high-risk HCPs? To answer these questions the BioPhorum Development Group (BPDG) HCP Workstream initiated a collaboration among its 24-company team with the goal of industry alignment around high-risk HCPs. A sub team was formed, in which the members performed literature searches and discussed the information available around this topic. The BPDG HCP Workstream team members were surveyed, which led to team discussions and insight into a list of problematic frequently seen HCPs. These HCPs can be classified based on available information into different risk categories that could be beneficial to the biopharmaceutical industry.
• (Day 3: Safety and Tox Risks Associated wtih Host Cell Protein Impurities Session)

SILuCHOP: An Internal Standard for Quantification of CHO HCPs by Bottom-Up LC-MS

• Presenter: Yue Lu, MilliporeSigma
• Abstract: Residual presence of host cell proteins (HCPs) in recombinant therapeutic products has considerable clinical safety risks associated with a potential immunological response in patients. Although traditional methods, such as anti-HCP-ELISA, are well accepted for quantification, these methods may underestimate the amount of HCPs. Not all HCPs provoke the same immune responses during antibody generation. Further, interaction of low abundance HCPs with high abundance therapeutics may limit detection of HCPs with ELISA’s antibodies. To enable a more comprehensive characterization and quantification of HCPs, we have developed a SIL-CHOP (stable isotope labeled Chinese hamster ovary proteins) internal standard spanning a wide dynamic range of abundance and demonstrate its utility in identification and quantification of HCPs in downstream process development samples via LC-MS.
  Methods
  CHO-K1 cells were cultured in serum-free medium enriched with 13C615N4-Arg and 13C615N2-Lys. The proteins from host cell culture fluid were isolated and the total protein concentration was determined using BCA assay. For characterization of SIL-CHOP, the sample was reduced, alkylated and digested with trypsin. The digested peptides were fractionated in a spin column format using high-pH reversed-phase SPE. Eight resulting fractions were analyzed using low pH C18-nano-LC-MS/MS. The isotopic incorporation was calculated to be greater than 98%. The data was searched against the Uniprot Cricetulus Griseus database and relative abundance of each protein was calculated based on ion intensities. The SIL-CHOP was subsequently spiked into downstream process development samples and the amount of each HCP was determined in each sample.
  Preliminary data
  Using LC-MS/MS analysis and database search of high and low pH fractionation, over 2370 proteins were identified with false discovery rates of 1% at the peptide level and 2% at the protein level. A spectral library was generated using Skyline software and relative abundances of individual proteins were calculated using a label-free quantification algorithm in Scaffold software. As a preliminary study, we investigated the amount of HCPs in Protein A purified SigmaMAb, a commercially available antibody standard expressed in CHO cells. SIL-CHOP internal standard was spiked into SigmaMAb at 100, 1000, and 10,000 ppm. An amount of 10 ug tryptic digests were loaded on the nano-LC column, separated using 150 min gradient, and analyzed by MS/MS. Several major host cell proteins, such as Clathrin and Clusterin, were identified in SigmaMAb. The amount of each individual HCP was estimated by comparison of extracted ion chromatograms of unlabeled sample HCP peptides and corresponding labeled peptides from SIL-CHOP internal standard. Further we will investigate the amount and type of residual HCP in a monoclonal antibody after each of five downstream purification processes by LC-MS using SIL-CHOP.
  Novel aspect
  This development of an SIL-CHOP internal standard advances identification and quantification of HCPs in biotherapeutics by LC/MS.
• Contributing Authors: Yue Lu, Pegah R Jalili, Zhiyun Cao and Kevin Ray; MilliporeSigma, St. Louis, MO
  Rong-Rong Zhu; MilliporeSigma, Bedford, MA
• (Poster)

Replacement of Detergent Triton X-100 in HCP ELISA for a Marketed Product

• Speaker: Stanislav Mandelc, Novartis/Lek
• Abstract: In-house developed project specific HCP ELISA for a marketed product included Triton X-100 as a detergent in sample dilution, washing and blocking buffers. In June 2017, European Chemicals Agency (ECHA) has included Triton X-100 in the Authorization List (Annex XIV) under EU REACH regulation due to the endocrine disrupting properties of its degradation products. As a consequence, the use of Triton X-100 in the EU will be prohibited from sunset date (January 4th 2021) onwards unless authorization is granted. For this purpose, an alternative buffer without Triton X-100 had to be selected, tested for critical parameters identified in corresponding risk assessment and finally replaced for the HCP ELISA assay. A complete package of documents compiled based on the results led to a successful approval of the change from health authorities.
• (Day 1: Which HCP Assay is Most Suitable Session)

Detection of A Host Cell Protein Impurity-Hamster Phospholipase B-Like 2 (PLBL2) in Therapeutic Monoclonal Antibodies

• Presenter: Sheetal Mehta, Bristol Myers Squibb
• Abstract: The wide variability in abundance and diversity of Host Cell Proteins (HCPs) are persistent challenge during the development of downstream purification processes for therapeutic Proteins. The physicochemical properties of the therapeutic Protein may have a significant effect on the clearance of HCPs that interact and co-purify with the therapeutic Protein. In most processes, the majority of HCPs are cleared during Protein A chromatography step. Some HCPs bind to the therapeutic Protein body during purification and appear at higher levels in the purified product compared to the in-process steps. PLBL2 is an example of one such HCP that interacts and co-purifies with a number of Therapeutic Proteins. Using commercially available kit from My BioSource, PLBL2 level was measured in BMS portfolio molecules. The detection and quantification of PLBL2 enables in-process monitoring of PLBL2 clearance during process development and trends process performance. In addition to the potential immunogenic tendencies PLBL2 plays a role in analytical and formulation development, as certain Lipases are known to impact product quality through PS80 degradation which in turn exposes IgG to potential aggregation and fragmentation. The establishment of PLBL2 method supplements our current approach of monitoring total HCP by ELISA and strengthens our impurity control strategy to ensure product safety and efficacy. This presentation will focus on PLBL2 method development and its applications in Biologics FIH molecules.
• Contributing Authors: Sheetal Mehta, Lei Zhang and Tapan Das
• (Poster)

Different Assay = Different Result. Evaluating HCP Assays and How to Choose the Right One

• Speaker: Emily Menesale, Biogen
• Abstract: HCP levels are highly dependent on the anti-HCP antibodies and HCP antigen standard. Therefore, different HCP assays will give different measured HCP levels. Here, we present an evaluation of several commercial HCP kits and in-house HCP assays on the same process intermediate and product samples. This presentation will demonstrate that different HCP kits give different HCP values. It will also show that higher HCP numbers does not equate to a better HCP assay. We will discuss the challenges in choosing an HCP assay and present our strategy for assessing these kits, including the use of orthogonal methods to support our justification of the most appropriate assay.
• (Day 1: Which HCP Assay is Most Suitable Session)

HCP Coverage by Immunocapture and LC-MS

• Presenter: Ejvind Mortz, Alphalyse
• Abstract: HCP Coverage is typically measured in “% Coverage” by spot counting in 2D PAGE Western blots of a mock cell lysate or an early process sample. However, the number of protein spots in a gel is difficult to correlate to the quality of an ELISA.
  We have developed an improved HCP coverage analysis based on ELISA antibody immunocapture followed by LC-MS/MS protein identification. The analysis provides a list of individual HCPs that are recognized by the ELISA antibodies. The list of HCPs covered may be compared to all the proteins identified in the starting material to calculate a “% Coverage” to assess the overall coverage. The list of HCPs covered may also be compared to the actual HCPs identified in the purified drug substance.
  We will present results from different immuno-affinity matrices, and discuss important analytical parameters, including antibody specificity, non-specific binding to affinity matrix, and calculation of HCP Coverage.
  We foresee that the improved analysis will aid ELISA development substantially; A) for selection of the ELISA antibodies with the broadest coverage of in process samples, B) for documenting that the ELISA antibodies can detect the HCPs present in the purified drug substance, and C) for fit-for-purpose validation of the ELISA for the specific bioprocess and drug substance.
• (Poster)

Analytical Method Development for Enzyme Control in Biocatalytic Syntheses of Small Molecule APIs

• Presenter: Alice Newman, Merck & Co., Inc.
• Abstract: Biocatalytic routes are becoming much more common in the synthesis of small molecule APIs due to increased selectivity and specificity. These biocatalytic routes present a number of unique analytical challenges not found in more traditional small molecule synthetic chemical routes. For example, APIs that are being developed for both oral and parenteral delivery require different levels of control for residual protein in the final API. Parenteral drugs also require the control of residual DNA in the final API to less than 10 ng per dose. Here, we will discuss our strategy in setting these specifications and the analytical control methods we have in place to ensure the specifications are met. Each of the process enzymes also requires the development of an enzyme activity assay to a) ensure quality of the enzyme batch from the vendor, b) release the enzymes for GMP use, and c) confirm there is no active enzyme remaining in the final API. These activity assays require additional capabilities and GMP considerations (e.g., GMP instrumentation, method validation, etc.), beyond the standard small molecule analytical workflow. This poster will aim to provide a holistic view of biocatalytic analytical challenges that may be encountered for small molecule APIs, including technical aspects and control strategies.
• Contributing Authors: Alice Newman, Erica Schwalm, Claire Ouimet, Yuan Jiang, Junyong Jo, Simon Hamilton
• (Poster)

Host Cell Protein Impurities and T Cell Epitopes in Antibody-Based Therapeutics: Analysis by Mass Spectrometry and Evaluating the Risk for Unwanted Immunogenicity by In Vitro Assays

• Speaker: Sofie Pattyn, ImmunXperts
• Abstract: (Pending)
• (Day 3: Safety and Tox Risks Associated wtih Host Cell Protein Impurities Session)

Which HCPs Does Your HCP-ELISA Cover? Analysis by Immunocapture and LC-MS

• Speaker: Rikke Raaen Lund, Alphalyse
• Abstract: HCP Coverage is often evaluated by subjecting a mock cell protein lysate to 2D-PAGE and Western blotting with the ELISA antibodies. The analysis provides a “HCP Coverage percent” estimated by comparing protein spots observed in the 2D-gel to the spots detected in the Western blot. This has been the only techniques with the required sensitivity and separation power to analyze complex protein mixtures. However, the technique has several limitations for evaluating the specificity of ELISA antibodies; A) ELISA is performed on the native HCP antigens, whereas the antigens are reduced and denatured on the 2D-Western blot membrane, B) The 2D-PAGE image analysis comparing the total protein gel stain with a Western blot immunostaining is very subjective and difficult to perform reproducibly.We are developing an improved HCP coverage analysis based on immunocapture with ELISA polyclonal antibodies followed by LC-MS/MS protein identification. The analysis is performed on the native HCP antigens and provides a list of individual HCPs that are recognized by the ELISA antibodies.The analysis is being developed in collaboration with a team of university experts with excellence in immunoaffinity purification and ELISA, and with biopharma collaborators providing ELISA antibodies as well as mock and process development samples. We will present results from different immuno-affinity matrices, and discuss important analytical parameters, including antibody specificity, non-specific binding to affinity matrix, and calculation of HCP Coverage.
  We foresee that the improved analysis will aid ELISA development substantially; A) for selection of the ELISA antibodies with the broadest coverage of in process samples, B) for documenting that the ELISA antibodies can detect the HCPs present in the purified drug substance, and C) for fit-for-purpose validation of the ELISA for the specific bioprocess and drug substance.
• (Day 2: Improved Methods for HCP Antibody Coverage Determination Session)

Methodological Challenges of Host Cell Protein Antibody Characterization

• Presenters: Mirko Sobotta, Boehringer Ingelheim Pharma GmbH Co. KG and Pia Paarmann, BioGenes
• Abstract: The reliable assessment of Host Cell Protein (HCP) antibody performance is challenging. This case study exemplarily presents the development and characterization of HCP ELISA antibody reagents for quantification of process-related impurities derived from a Chinese hamster ovary production cell line (CHO). The selection of a suitable capture antibody mixture for HCP ELISA development not only depends on its ELISA characteristics but also on the antibody coverage. Coverage is conventionally determined by 2D Western blotting. However, 2D Western blot-based coverage results strongly depend on the methodological/technical details of the performed analysis. The informative value of 2D Western blot analysis can further be limited due to the denaturing assay conditions, thereby potentially underestimating coverage performance. For comprehensive and reliable immunological reagent characterization under almost native assay conditions (similar to ELISA), orthogonal coverage assessment can be additionally performed employing Immuno Affinity Chromatography (IAC) followed by 2D difference gel electrophoresis (2D DIGE). In the current case study, the HCP population was fractionated and used for individual immunizations in order to improve immune response towards weakly immunogenic low molecular weight (LMW) HCPs. The reactivity of the resulting capture antibody mixtures was tested in ELISA, by 2D Western blotting and IAC/ 2D DIGE revealing significant differences depending on the antibody composition itself as well as the analytical method used.
• Contributing Authors: Dr. Mirko Sobotta (Boehringer Ingelheim), Dr. Pia Paarmann (BioGenes), Stefan Sommerschuh (BioGenes)
• (Poster)

Compiling a Method Toolbox to Improve Detection of Host Cell Proteins

• Speaker: Martha Stapels, Sanofi Corporation
• Abstract: Host cell proteins (HCPs) are process-related impurities that are monitored to evaluate purification processes and ultimately ensure product efficacy and safety. Mass spectrometry (MS) is a highly sensitive technique used to identify HCPs, as it facilitates detection of individual HCPs down to 1 ppm, depending on the type of instrument that is used. However, several factors, such as sample complexity and suppression of HCP peaks by product peaks, can make the detection of certain HCPs difficult. To address the challenges of identifying hard-to-detect HCPs, our group has compiled several MS workflows which can be used orthogonally to one another to search for and identify HCPs that are below the detection limit of our typical high-throughput workflow. These workflows include sample preparation for enrichment of HCPs as well as targeted methods. A comparison of the utility of each method as well as the results obtained by different techniques on the same samples will be shown.
• Contributing Authors: Martha Stapels, Helena Awad, Michelle Busch, Joanne Cotton, Fateme Tousi, and Karen Lee
  Biologics Development, Sanofi Corporation
• Day 2: Applications of Mass Spectrometry to Identify and Quantify HCPs Session

New Immunoassay Kits for HCP Analysis Using Gyrolab® Automated Systems

• Speaker: Ann-Charlott Steffen, Gyros Protein Technologies
• Abstract: While scaled down models and high-throughput testing in bioprocess development generate large numbers of samples for analysis, HCP immunoassays remain largely manual in many companies, with high assay variability and low productivity.
  Gyrolab® immunoassay systems in combination with ready to use kits for CHO-HCP detection using the gold standard 3G reagents from Cygnus Technologies are increasingly used by biopharma companies to increase analytical productivity. A four-log assay dynamic range, matrix insensitivity and software support for analysis of dilutional linearity and spike recovery data minimizes the manual labor workload associated with HCP immunoassays. Gyrolab systems automate the analysis and yield faster, highly reproducible results for CHO-HCP impurity testing of many bioprocess samples in one day. Here we present data generated for new assay kits, optimized for use in Gyrolab systems, for determination of host cell proteins impurities from E. coli and HEK 293 as well as quantitation of viral vectors for new gene therapy products.
• Contributing Authors: Ann-Charlott Steffen; Hannah Litwin
• (Poster)

Improving HCP ELISA Using Ella – An Automated Microfluidic Platform

• Presenter: Kathleen Van Manen-Brush, Glaxo Smith Kline
• Abstract: Chinese Hamster Ovaries (CHO) are a widely used mammalian cell line to produce therapeutic proteins. CHO cells, while producing Drug Substance (DS), result in Host Cell Proteins (HCPs). HCPs are process-related impurities that are derived from a host cell expression system that may copurify with the product. Therefore, it is important during biopharmaceutical development that the process removes HCPs. The most common technique used to quantify these HCPs is an Enzyme-Linked Immunosorbent Assay (ELISA). ELISA is an enzymatic immunoassay that uses antigen-antibody reactions to quantify an analyte of interest at very low concentrations. In general, ELISAs take 5-7hrs to complete, are highly labor intensive and result in high failure rates. Ella is a fully automated immunoassay technology that uses microfluidics and Glass Nano Reactors (GNRs) to perform an ELISA in 75 minutes, with high quality data and low failure rates. Ella’s microfluidic technology and automated washes eliminates analysts from adding each individual antibody step manually. The antibodies and antigens are captured on three distinct GNRs in each microfluidic channel, which translates to triplicate high quality data points per well. Here we present data generated by Ella that quantitates CHO HCPs with precision, accuracy, sensitivity and has comparable trends to our In-house CHO HCP ELISA.
• Contributing Authors: Jacob Zeitler¹, Marisa Jones¹, Paul Younge², and Samantha Willis \^{2
  1} Glaxo Smith Kline, Billerica, MA; ² Protein Simple
• (Poster)

Cleaning Up the CHO Cell: Reducing the HCP Load

• Speaker: Bjorn Gunnar Voldborg, NNF Center for Biosustainability
• Abstract: Using proteomics and targeted genome engineering, we have rationally reduced the amount of secreted host cell proteins to improve protein secretion and facilitate downstream processing.
• (Day 3: Process Impact on HCP Profile Session)

HCP Coverage and Similarity Analysis with Advanced 2D Gel-Based Approaches

• Speaker: Sonja Voordijk, GeneBio
• Abstract: HCPs are critical process-related impurities for biotherapeutics produced through recombinant technology. The workhorse method for assessing HCP clearance through the product purification steps is ELISA. Still, however sensitive, selective and high throughput, ELISA doesn’t provide proteome-wide coverage and its reagents must be carefully characterized and validated. The use of orthogonal techniques such as 1D/2D SDS-PAGE and Western blot, and LC-MS, is therefore inescapable.While advances in mass spectroscopy-based techniques have received considerable attention, methods based on 2D gel electrophoresis have been branded as outdated while still being recommended by regulatory bodies. Many link the technology with traditional silver stained gels and chemiluminescent Western blots where finding correspondences between spots is often more a gambling game than real science. Combined with a number of fundamental mistakes often made at the image acquisition level, the resulting antibody coverage or antigen similarity unsurprisingly lacks reliability.Yet, a number of important developments such as 2D difference in blot electrophoresis (2D-DIBE) and antibody affinity extraction followed by 2D difference in gel electrophoresis (2D-DIGE) have opened the door to considerably more reproducible coverage and similarity analysis. We will discuss the principles, weaknesses and advantages of each of these methods. We will also illustrate how appropriate imaging equipment and acquisition parameters can safeguard the high sensitivity and reliability that these methods can potentially achieve. Finally, we will show how to take 2D gel-based coverage analysis beyond a single measurement of percent coverage or similarity, to zoom in on differences and similarities at the individual protein level.
• ( Day 2: Improved Methods for HCP Antibody Coverage Determination Session)

Mass Spectrometric Evaluation of Process Influences on Host Cell Protein Patterns in Biopharmaceuticals for Setting Up a HCP Control Strategy

• Speaker: Thomas Waerner, Boehringer Ingelheim Pharma GmbH & Co. KG
• Abstract: For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g. HCP-ELISA). The capacity of the immunoassay to detect a broad range of relevant HCPs should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry. In particular mass spectrometry has become a valuable tool to identify and quantify HCP in complex mixtures. We obtained a similar HCP pattern in different cell culture fluid harvests. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in- process control and release analytics, and mass spectrometry for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products.
• (Day 3: Process Impact on HCP Profile Session)

Mass Spectrometric Evaluation of Upstream and Downstream Process Influences on Host Cell Protein Patterns in Biopharmaceutical Products

• Presenter: Daniel Waldera-Lupa, Protagen Protein Services GmbH
• Abstract: For production of mAbs, biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are characterized regarding influence of upstream and downstream process (USP/DSP) parameters on critical quality attributes (CQAs), which must be monitored strictly by adequate control strategies. One process-related CQA is the content of host cell protein (HCP), which is typically analyzed by immunoassays. The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal methods. In particular, mass spectrometry has become a valuable tool to identify and quantify HCP. Here, we evaluate USP and DSP parameters of different biopharmaceutical products and different process variants on the HCP pattern by shotgun MS and ELISA.
  First, the HCP patterns in DSP samples of two significantly different USP large scale productions of mAb7 were analyzed by shotgun MS analysis. Condition 1 represents the platform process under target upstream parameters. For condition 2, the feed rate was changed so that the cells were cultured under slightly starving conditions and the level of one essential ion was reduced. The overlap and the number of HCPs decrease in the line of the DSP. When counting the HCP detected in both conditions as a mean, from 652 HCPs in CCF samples (100%), the number was reduced in Protein A eluate to 300 (46%), in polishing A to 17 (3 %) and polishing B to 13 (2%). This is accompanied by decrease of the HCP level quantified by ELISA. It is important to note that MS is capable of monitoring depletion throughout the process for every HCP in parallel, whereas by ELISA, an individual assay would have to be established for each individual HCP.
  Next, we analyzed HCP in drug substance (DS) of six different mAb products. The number of total HCP detected in our six different DS samples ranged from 33-63 different protein species. Ten different HCP were detected in a minimum five out of six different mAb products. Notably, two of these ten HCP, namely Peroxiredoxin-1, and GRP78 precursor, have also been detected in another recent study, which indicates presence of “common” HCP in DS of multiple mAb products. The number of detectable HCP is strongly dependent on instrument sensitivity. Detection of HCP with low abundance is still critical and the risk of false positive detection exists. Therefore, we conclude additional validation of detected HCP peptides in DS for example by using heavy isotope synthetic peptides is crucial.
  Mass spectrometry represents a valuable tool for USP and DSP optimization by monitoring multiple HCPs in parallel.
• Contributing Authors: Daniel Michael Waldera-Lupa¹, Heiner Falkenberg¹, Martin Vanderlaan², Thomas Schwab³, Roland Moussa³, Thomas Flad³, and Thomas Waerner^{3
  1} Protagen Protein Services, Otto-Hahn-Straße 15, 44227 Dortmund, Germany
  ² Consultant, 1626 14th Avenue, San Francisco, California, USA
  ³ Boehringer Ingelheim Pharma GmbH & Co. KG, Analytical Development Biologicals, 88397 Biberach, Germany
• (Poster)

An Improved CHO HCP Detection System for Biopharmaceuticals

• Speaker: Mindy Wan, KBI Biopharama
• Abstract: KBI has applied a unique strategy to develop an improved CHO HCP detection system for biopharmaceuticals. This strategy has yielded a platform ELISA assay with broad applications for a diverse range of recombinant protein therapeutics expressed in various CHO cell lineages and purified by various approaches. KBI’s CHOP ELISA method exhibits superior dilution linearity and matrix flexibility for detecting CHOPs present in purification process intermediates and final products. The reagent used in this ELISA shows >70% coverage for a pooled null harvest generated from several CHO cell lines. KBI’s polyclonal antibody library can be used to tailor customized HCP assays within shortened timelines. Orthogonal technology(ies) are also applied to provide key supporting data for demonstrating the performance capability of ELISA applications.
• (Day 1: Which HCP Assay is Most Suitable Session)

Taking Up the Cudgels for the Coverage of Anti-HCP Reagents

• Speaker: Michael Wiedmann, Roche
• Abstract: Host cell proteins (HCPs), a major class of process related impurities during manufacturing of
  biopharmaceuticals, are generally considered a critical quality attribute (CQA), whose substantial clearance is routinely monitored by enzyme-linked immunosorbent assays (ELISA).
  The performance of these assays primarily relies on the capability of the employed anti-HCP
  antiserum for recognizing a high proportion of HCPs. Testing the coverage of HCP specific
  Antibodies by 2D Western Blot (WB) analysis has revealed a considerable number of non-covered HCP spots that are particularly pronounced in the low molecular weight region, which in turn raises questions if these HCPs can be adequately detected by immunoassays. Based
  on the characterization by comprehensive methods, we demonstrate here that the non-detected HCPs
  can ascribed to practical constraints of the WD approach rather than the actual absence of individual antibodies.
• Contributing Authors: Christina Seisenberger, Markus Haindl, Ulrich Mohn, Rudolf Vogel, Michael Wiedmann, Stefanie Wohlrab
  Pharma Development, Roche Diagnostics GmbH, Penzberg, Germany
• (Day 2: Improved Methods for HCP Antibody Coverage Determination Session)

Advanced LC-MS Approach for Characterization of HCPs and Delivery Vehicle (Viral) Proteins in Gene Therapy Products

• Speaker: Shiaw-Lin (Billy) Wu, BioAnalytix, Inc.
• Abstract: Host cell proteins (HCPs) in recombinant protein products have been characterized increasingly by LC-MS based approaches as an orthogonal standard for typical ELISA detection. However, HCPs derived from human cell lines in typical gene therapy products are more complex than typical recombinant biologics produced in CHO or E. coli cell lines. Multiple viral proteins co-exist in gene therapy products, increasing complexity compared to most biologic drugs expressed as single proteins in host cells. We have developed advanced LC-MS approaches to determine the identity of HCPs while providing useful information about respective levels in gene therapy product samples. Both in-gel and in-solution LC-MS methods were developed along with a further annotated database (specific for viral protein produced cell line) with quantitation capability for determining relative HCP quantities as well as the viral protein types with related splice variants. The HCP method was applied for analysis of multiple GMP Lots with related purification step samples. In addition, viral proteins and related truncations were also identified. In HCP identification, tens of relatively high abundance HCPs (>100 ppm), with several HCPs (>1000 ppm), could be seen. The level of HCP (by LC-MS) was estimated to be approximately 2x higher than the level estimated by ELISA. The LC-MS approach was used to determine the identity of HCPs while additionally providing useful information on their respective levels in drug product samples. In addition to HCP identification, the LC-MS method has the potential to be incorporated into typical assessment approaches for concurrently monitoring key HCPs as well as related viral product quality attributes (i.e. for lot-to-lot comparability).
• (Day 1: Analysis of HCPs for Gene Therapy and Viral Vaccines Session)

Quantitative Analysis of Host Cell Proteins by LC-MS/MS

• Speaker: Ying Zhang, Pfizer
• Abstract: Host cell proteins (HCPs) are residual protein impurities expressed along with the desired recombinant protein therapeutic. Failure to remove residual HCPs sufficiently during downstream processing can affect efficacy and safety of the therapeutic drug, or even induce adverse effects to patients. Biopharmaceutical companies utilize an enzyme-linked immunosorbent assay (ELISA) to monitor levels of total HCP at each step of the purification process and in final drug substance. ELISA is considered the gold standard for efficient HCP analysis/release testing because it affords broad coverage and reliable quantitation in a rapid manner. However, the specificity and quantitation for particular HCPs in ELISA may suffer due to incomplete coverage by the anti-HCP antibodies. In recent years, biotherapeutics development laboratories have established liquid chromatography-tandem mass spectrometry (LC-MS/MS) as an orthogonal approach to ELISA, providing qualitative and quantitative information on individual HCPs in the downstream process. To assess the accuracy and precision of quantitation in the LC-MS/MS method, we spiked 5 different protein standards into 3 different monoclonal antibodies (mAbs) at 6 different levels. All spiked standards showed linear responses. However, the quantitation trendlines for the spiked proteins varied between individual mAbs. We also investigated our LC-MS/MS method alongside published sample preparation protocols for both fully purified and in-process samples to better understand the advantages and disadvantages toward enhancing the reliable detection and quantitation of residual HCPs.
• Contributing Authors: Ying Zhang, Natalia Kozlova, Matt Thompson and Jason Rouse, Analytical Research and Development, Pfizer Inc.
• (Day 3: Innovations in Mass Spectrometry for HCP Analysis Session)

Evaluation of Antibody Coverage with the Combination of Immunoaffinity Chromatography and Mass Spectrometry

• Presenter: Yu Zhou, BioMarin
• Abstract: The clearance of residual host cell proteins (HCPs) is usually monitored by enzyme-linked immunosorbent assay (ELISA). The most critical component of an HCP ELISA is its suitability, which is primarily defined in terms of reagent (antibodies) coverage against the total upstream HCP population. The antibodies must have suitably comprehensive coverage (immunoreactivity) against the HCP impurities to enable effective monitoring of process clearance. HCP immunoreactivity (coverage) is commonly assessed by gel-based techniques such as two dimensional (2D)-Western blot or two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) with the subset recognized by the ELISA reagent. However, due to incomplete HCP resolution, incomplete transfer to the blotting membrane, and the use of denaturing conditions that may destroy native epitopes, antibody coverage can be underestimated.We have developed a strategy to evaluate the antibody coverage with the combination of Affinity Extraction with a polyclonal anti-HCP antibodies and mass spectrometry (AE-MS). Antibodies were covalently immobilized on the immuno affinity column through oxidized carbohydrates and it provided oriented covalent attachment. This eliminates the loss of antibody activity experienced in primary amino coupling at or near the antigen binding site. Then the antigen pool was loaded on the column, after washing, the bound HCPs were eluted for detection by MS. HCPs were identified by a high-resolution ESI-Orbitrap Velos Pro mass spectrometer (Thermo Scientific). Proteome Discoverer (Thermo Scientific) and Progenesis (Waters) software and MascotTM (Matrix Science) and SEQUEST (Thermo Scientific) search engines were used to search against the CHO-K1 UniProt database with 1% false discovery rate (FDR) threshold and at least 2 unique peptides for protein identification.This approach resulted in a significant increase in antibody coverage compared to the antibody coverage obtained using 2D-Western blot. LC-MS/MS approach also provided each individual HCP’s MW, pI, and level of response to the antibodies. Our AE-MS based approach offers not only comprehensive antibody coverage, but also provides characteristic information of host cell proteins which is useful for product specific purification process development.
• Contributing Authors: Yu Zhou, Meghna Patel, Geuncheol Gil, and Sushmita Mimi Roy
  BioMarin Pharmaceutical Inc., Novato, CA, USA
• (Poster)