BEBPA Presents:

Host Cell Protein Conference

May 17-19, 2016
Lisbon, Portugal
Register for the Conference HERE

The morning workshops are brought to you based on popular demand. Attendees from last year’s conference requested that primers be included as part of our program. These are designed for “non-experts” to allow you to understand the technology of your colleagues. Therefore if you are an expert in immunoassays sign up for the LC-MS course and vice versa. Here is your chance.

Speakers

Posters

Workshops

Abstracts (Alphabetical by Speaker)

State-of-the-Art Proteomics Methods for Characterization of Host Cell Proteins

  • Speaker: Dörte Becher, Ernst-Moritz-Arndt University Greifswald
  • Abstract: During the last decade, a rapid development of methods and techniques has been observed in the field of proteomics. Several methods for global proteome expression analysis have been established to analyze proteomes of prokaryotic and eukaryotic cells. Due to the high sensitivity, 50-80% of predicted proteins of a cell can be analyzed in a quantitative manner. Here, 2D gel-based, as well as gel-free methods, contribute significantly for characterization of the expressed proteins.
    These methods, initially developed for protein expression analysis, can be used to characterize host cell proteins in biopharmaceutical products. We will present a study in which we conducted a comparison of different gel-based and gel-free approaches. Advances and limitations of the different methods will be discussed.
    References:
    1. Otto A, et al. Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis. Nat Commun. 2010, 1(9):137
    2. Maass S, et al. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics. Anal Chem. 2011, 83(7):2677-2684
    3. Buescher JM, et al. Global network reorganization during dynamic adaptations of Bacillus subtilis metabolism. Science. 2012, 335(6072):1099-103
    4. Bonn F, Bartel J, Büttner K, Hecker M, Otto A, Becher D. Picking vanished proteins from the void: how to collect and ship/share extremely dilute proteins in a reproducible and highly efficient manner. Anal Chem. 2014, 5;86(15):7421-7.
    5. Maaß S, Becher D. Methods and applications of absolute protein quantification in microbial systems. J Proteomics. 2016,pii: S1874-3919(16)30018-5.
  • (Day 2: Proteomics Approaches to HCP Analysis: Core Technologies Session)

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Identification and Quantification of mAb Host Cell Protein Impurities Down to 1 ppm: An Inter-Laboratory LC/MS Study.

  • Speaker: Scott J. Berger, Waters Corporation
  • Abstract: Host cell protein (HCP) impurities present at low ppm levels in protein biopharmaceuticals are challenging to detect and more challenging to identify within the encompassing biotherapeutic matrix. Multiple separation dimensions in an HCP LCMS discovery workflow are clearly beneficial to achieve the chromatographic resolution of these low level impurities, while enabling the high sample loading to overcome the dynamic range inherent to this complex analysis. Our laboratory developed and previously published a novel 2D (High pH/Low pH Reversed Phase) peptide digest separation coupled to MSE based data independent MS/MS detection approach for HCP identification down to sub-50 ppm levels [Doneanu et. al. mAbs, 2012, 4, 24-44.]. This poster details three improvements to this methodology required to nominally achieve 1 ppm (1 ng HCP/mg mAb) HCP identification levels from purified mAb samples: Use of a Charged Surface Hybrid (CSH) UPLC chemistry enabled 5-fold higher peptide loading, to raise more peptides above assay detection limits; the use of ion mobility MS based HDMSE acquisition workflows provided additional peptide resolution, for clean detection of lower level components; and, the use of optimized fragmentation energy profiles based on peptide ion mobility drift profile improved fragment ion spectra, for superior HCP peptide identification. The robustness of this second generation HCP discovery methodology was demonstrated by a cross-laboratory (n=3) study using the new NIST mAb standard as a common sample for the analysis. This approach was also used to identify HCP profiles from innovator vs. biosimilar Infliximab products. These results demonstrate that LC/MS assays are able to attain comparable sensitivity to traditional HCP assays (e.g. ELISA), while offering unique advantages of individual HCP identification and quantification.
    Authors:
    Catalin Doneanu1, Scott J. Berger1, Brad Williams2, Malcolm Anderson3 Matthew Lauber1, Asish Chakraborty1, and Weibin Chen1,
    1Waters Corporation, Milford, MA, USA
    2Waters Corporation, Beverly, MA
    3Waters Corporation, Manchester, UK
  • (Poster)

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Identification of HCP in Purification Steps by Antibody Affinity Enrichment and LC-MS/MS

  • Speaker: Eric Bishop, EMD Millipore
  • Abstract: Host cell proteins (HCPs) are biomanufacturing process-related impurities. HCPs can induce immune responses (especially for high-dose and high-frequency drugs). As a result, regulatory agencies require that HCPs in the final drug have to be closely monitored and accurately quantified. The FDA guidance on biosimilars state that “The sponsor should characterize, identify, and quantify impurities in the proposed product and the reference product, to the extent feasible”.
    Host cell proteins are the most challenging impurity to remove, partly because of their diverse biophysical/chemical nature and wide range of concentrations. Enzyme-linked immunosorbent assay (ELISA) quantification of HCPs have been the most common method used to guide the purification process development. One of the limitations of ELISA is that this assay does not reveal HCP identities and characteristics. Knowing the identity and quantity of individual HCPs in each purification step can better guide purification scientists to develop more effective downstream process for HCP removal.
    In our study, a monoclonal antibody (mAb02) produced in CHO was purified with the common mAb purification platform, which contains protein A, low pH viral inactivation and cation/anion exchange chromatographic steps. Samples collected from each purification step were treated with antibody affinity enrichment (AAE) chromatography to deplete the abundant mAb02 and enrich HCPs. Nano-LC-MS/MS identified individual HCPs in each purification step and HCPs that can impact product quality. The combination of AAE and nano-LC-MS/MS proteomic approach significantly increases sensitivity and confidence in HCP identification in final drug substance. This approach can be used to demonstrate HCP similarity between innovator reference standard and biosimilar’s products.
    Authors:
    Rong-Rong Zhu1, John Boyle1, Matthias Joehnck1, Eric Bishop2, Ken Hoffman2 and John Leszyk3
    1 EMD Millipore; 2 Cygnus Technologies and 3 UMass Medical School)
  • (Day 1: HCP Immunoassay: Core Technologies Session)

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Identification and Quantification of HCPs by Mass Spectrometry.

  • Speaker: Daniel Chelsky, Caprion Proteomics
  • Abstract: Mass spectrometry can be used to obtain a nearly comprehensive assessment of host cell proteins without the use of antibodies, clearance of the drug substance or any prior knowledge of the protein contents. A method will be described that includes an initial phase of protein digestion with trypsin, followed by peptide fractionation by two orthogonal methods to ensure good coverage. Each fraction is analyzed by LC-MS/MS to identify peptides and their parent proteins and to determine relative quantification in each sample. This approach has been used to monitor process improvements, batch uniformity, the effect of different host strains or media, scale-up or to compare production sites. The analysis can be further refined by developing a targeted multiple reaction monitoring (MRM) assay for absolute quantification of proteins of interest with the aid of isotopically labeled reference peptides and calibration curves. In addition to higher sensitivity, precision and accuracy of the MRM assay, protein concentration can be determined in ppm. At least two peptides are monitored for each protein to demonstrate clearance as well as the final concentration in the drug substance. Case studies will be presented for both the discovery and absolute quantification approaches.
    Caprion Proteomics, 1455 Adams Dr.
    Menlo Park, CA 94127, USA 650-350-0358.
  • (Day 2: Proteomics Approaches to HCP Analysis: Core Technologies Session)

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HCP Analysis of a Small Drug-Protein in Process Sample by Combining Platform Immunoassays and Mass Spectrometry

      • Speakers: David Chimento, Rockland Immunochemicals and Ejvind Mørtz, Alphalyse
      • Abstract: Among various evaluation methods to detect host cell protein (HCP) impurities, 2D-gel based separation of drug samples followed by Western blotting with anti-HCP antibodies offers visual confirmation of immunodetected proteins. In addition to these visual results, ELISA based assays provide a valuable high throughput tool to determine product purity in terms of total HCP content relative to the drug protein itself. Mass spectrometry based orthogonal methods, such as geLC-MS/MS enables identification of individual host cell proteins, and provides exact database entry name as well as amino acid sequences for each identified HCP.
        Here, we combine a high coverage platform immunoassay with highly sensitive geLC-MS/MS for HCP analysis of both a purified drug protein and the corresponding null cell lysate. Detection of HCPs was performed using a combination of methods to allow maximum coverage and information details on each HCP. Western blot and ELISA assays were performed utilizing generic HCP antibodies generated by immunization of host cell proteins fractionated by size. For geLC-MS/MS, proteins were separated by 1D-PAGE and the drug protein was isolated in separated gel fractions, allowing host cell protein identification in the drug substance sample. All gel fractions were analyzed by nLC-MS/MS.
        Combining the strengths of ELISA and MS orthogonal HCP detection methods enabled a convenient high throughput detection strategy for drug product impurities with capabilities and the ability to visualize, quantify and identify host cell proteins in the samples.
        Authors:
        David Chimento, Carl Ascoli, Maheen Sayeed, Karin Abarca Heidemann (Rockland Immunochemicals Inc., Limerick, PA, USA)
        Rikke Raaen Lund, Marie Grimstrup, Ejvind Mørtz (Alphalyse A/S, Odense, Denmark)
      • (Day 1: Developing an Integrated Assessment Using LC-MS/MS & Immunoassays Session)

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Comparison of Host Cell Proteins Profiles Between an Innovator and a Biosimilar Monoclonal Antibody.

      • Speaker: Catalin Doneanu, Waters Corporation
      • Abstract: Sensitive detection and quantification of residual HCPs represents a critical step in the design of robust, well-controlled manufacturing processes that yield high-quality biopharmaceuticals. LC/MS-based methods are becoming a routine approach for HCP analysis where residual HCPs can be detected, identified, and quantified directly.
        Here we present the results obtained in a comparison study using a generic HCP assay used for identification and quantification of HCPs in two highly-purified monoclonal antibodies, an innovator therapeutic mAb (Infliximab, 21 mg/mL) and a biosimilar product (Inflectra, 10 mg/mL). The HCP assay relies on: 1) proteolytic digestion, 2) two-dimensional chromatographic separation (high pH/low pH) by RP/RP UPLC, 3) high-resolution ESI-mass spectrometric detection coupled with ion-mobility separation (IMS) of peptide precursors, 4) drift-time specific fragmentation of peptide precursors using a fixed collision energy and 5) database searching for HCP identification and quantification.
        Two HCPs (epidermal growth-factor like protein 8 and WD repeat containing protein 37) were identified in both samples with concentrations ranging from 10 to 100 ppm.
        These findings were subsequently validated by acquiring “pure” MS/MS fragmentation spectra for targeted HCP peptide precursors isolated using the quadrupole filter (~ 2 Da mass window), separated from co-eluting isobaric precursors using ion mobility and fragmented with an optimized CE in the transfer-cell of a QTOF instrument.
        These results indicate that LC/MS assays are now able to attain comparable sensitivity to traditional HCP assays (e.g. ELISA), while offering the unique advantage in providing unambiguous HCP identification.
        Authors:
        Catalin Doneanu1, Ying Qing Yu1, Asish Chakraborty1, Alain Beck2 and Weibin Chen1,
        1Waters Corporation, Milford, MA, USA
        2Institute de Recherche Pierre Fabre, Centre d’Immunologie, Saint-Julien-en-Genevois , France
      • (Day 2: Putting It All Together: Integrated Quality Strategy for HCPs using Available Assay Technologies Session)

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European Regulatory Scientific Perspective on HCP Testing: Focus on Monoclonal Antibodies

      • Speaker: Joerg Engelbergs , Paul Erhlich Institute
      • Abstract: (Pending)
      • (Day 2: Putting It All Together: Integrated Quality Strategy for HCPs using Available Assay Technologies Session)

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Lightning the Black Box of HCP Immunoassays – Alternative Immunization Strategies in Combination with Innovative Characterization Tools

      • Speakers: Olaf Stamm, Charles River Laboratories, and Thomas Flad, Protagen Proteome Services
      • Abstract: Immunoassays for host cell protein (HCP) quantitation require high quality polyclonal antisera which are generated by immunization of common animal models such as rabbits, goats and sheep. The challenge in HCP antisera development is the large number of proteins contained in the antigens and their heterogeneity. The antigens can differ widely with respect to size, physio-chemical properties and immunogenic profile. There are multiple ways to address this challenge and the following presentation is focused on a case study using an alternative immunization models (chicken) in combination with a set of state of the art methods for antisera characterization ranging from high resolution 2D gel electrophoresis, affinity chromatography and different mass spectrometry applications. Available tools from proteomics can generate significant intelligence with respect to the specificity of HCP-Immunoassays. In consequence, a polyclonal antiserum based Immunoassay must not be considered as a black box anymore.
      • (Day 1: HCP Immunoassay: Core Technologies Session)

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Establishing a Consistent, Phase Appropriate Approach to Intergrating ELISA & LC-MS/MS Data During Product Development

      • Speaker: Susan Flor, Genentech
      • Abstract: (Pending)
      • ( Day 2: Putting It All Together: Integrated Quality Strategy for HCPs using Available Assay Technologies Session)

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HCP Case Studies for Blood Products – European Regulatory View

      • Speaker: Erika Friedl, Paul Ehrlich Institute
      • Abstract: Process-related impurities such as host cell proteins (HCPs), derived from the manufacturing process of recombinant biopharmaceuticals, could impact patient safety by induction of immunogenicity and anaphylactic acute reactions. Therefore, the capacity of the production process to efficiently eliminate HCPs, as well as an appropriate technology for the quantification of HCPs, are important measures to ensure patient safety. In addition, a suitable quality control strategy should be defined for the in-process and the release testing of HCPs.
        The development of potent HCP assays exhibiting high sensitivity and specificity and thereby covering a large range of process-specific HCP species is a challenging task. With emphasis on recombinant blood products, the European regulatory expectations will be discussed, highlighting the current practice in view of the novel requirements based on the newly developed guidance documents. Regulatory expectations focusing on the development and the validation of HCP assays will be covered. In addition, acceptable HCP specification limits for blood products, based on a risk-based approach, will be addressed. The different stages during the manufacturing process covering the clinical development phases up to the marketing authorization will be looked at concerning the different requirements (process-specific versus generic assays). The suitability of alternative quality control approaches, validation of process capacity to adequately eliminate HCPs versus routine release testing, will be further reviewed.
      • (Day 1: Developing an Integrated Assessment Using LC-MS/MS & Immunoassays Session)

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Antibody HCP Coverage Assessment: How to Calculate It?

      • Speaker: Zhong-Hua Gao, ZymoGenetics, Inc.
      • Abstract: Host cell protein (HCP) level in drug product is typically considered as a critical quality attribute and, therefore, controlling the level of HCP is essential. Quantitation of HCP is commonly achieved by anti- HCP polyclonal antibody-based immunoassays. Regulatory authorities typically require proof of sufficient HCP coverage by the antibody used in the assay prior to initiation of human clinical trials. Two Dimensional Difference Gel Electrophoresis (2-D DiGE) followed by fluorescent western blot is a standard approach to determine the HCP reagent coverage. However, the details of the procedures used to calculate reagent coverage are not well defined, and the decision of which method to use is often arbitrary. After surveying the available literature, 3 different calculation methods were identified and evaluated in our lab. Antibody 2D-DiGE coverage experiments were performed on E. coli and CHO HCP samples and the antibody coverage was calculated using the 3 different procedures. The results indicate that coverage values vary significantly depending on the calculation methodology used. A consensus may be necessary to harmonize a standard calculation method across the industry.
        Authors:
        Zhong-Hua Gao, John Fann, Karen De Jongh, Robert Mallett
        Department of Biologics Process Development, ZymoGenetics, Inc., a Bristol-Myers Squibb Company, Seattle, WA USA
      • (Poster)

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Post Approval Process Changes: How Closely Do the HCPs Have to Match?

      • Presenter: Feny Gunawan, Roche-Genentech
      • Abstract: (Pending)
      • (Poster)

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Tutorial: Key Concepts in the Development of Immunoassays for the Analysis of HCPs

      • Speaker: Eric Bishop, Cygnus Technologies
      • Abstract: This workshop will cover the steps involved in the development of immunoassays for HCP analysis. Choice of immunogen, standard, species to immunize, purification of antibodies and choices in assay format will be covered. Common mistakes and how to avoid them will be reviewed based on the instructor’s 20-years of experience. Cygnus Technologies is a leading global supplier of both assay kits based on platform reagents, and custom development of anti-HCP assays. Assay qualification for specific applications will be discussed.
      • (Workshop 2)

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Polysorbate Degradation by Phospholipase A2 in CHO Derived Products

      • Speaker: Lihua Huang, Eli Lilly and Company
      • Abstract: Decreases of intact polysorbate content were observed while evaluating the long-term storage stability of CHO derived, purified monoclonal antibodies. It was determined that polysorbate had been enzymatically degraded; therefore, studies were performed to identify and characterize the protein(s) responsible. Polysorbate degrading activity was enriched from CHO media leading to the identification of Group XV phospholipase A2 Isomer X1 by LC-MS/MS. Recombinant phospholipase A2 was expressed, purified and conformational integrity confirmed against a phosphatidylcholine substrate. Incubation with PS-20 and PS-80 resulted in hydrolysis of both monoester and higher order PS-20 and PS-80 but a much slower rate was observed for higher order PS-80. Endogenous phospholipase A2 was detected and quantitated at less than 1 ppm in three purified antibodies while phospholipase A2 was not detected (or less than 0.1 ppm) in a fourth antibody. Furthermore, antibodies with detectable quantities of endogenous phospholipase A2 demonstrated polysorbate hydrolysis whilst antibody without did not
        Authors:
        Lihua Huang, Troii Hall, Stephanie L. Sandefur, Christopher C. Frye and Tammy L. Tuley
        Bioproduct Research and Development, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
      • (Day 1: Developing an Integrated Assessment Using LC-MS/MS & Immunoassays Session)

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Single-Digit PPM Level Identification of Host Cell Protein Contaminants using a Simple One-Hour Data-Independent LC/MS Acquisition Method and CHO Spectral Library

      • Speaker: Eric Johansen, Stemcentrx
      • Abstract: A robust, reliable, and high-performance LC-MS method using SWATH Acquisition is presented here as an innovative and efficient technique for detecting and quantitatively monitoring multiple individual HCPs simultaneously. This technique provides individual protein identities, rather than a number for the total sum amount of Host Cell Antigen, which is a limitation of current ELISA-based methods. The high sensitivity and maximal parallelization of quantitative measurements made possible by SWATH acquisition makes it an invaluable solution for the quantitation of multiple low abundance CHO-HCPs in biotherapeutics.
        This methodology consists of two pieces – first, the one-time generation of a CHO-HCP spectral library, and second, the high-throughput SWATH quantitative analysis of process samples. The HCP spectral library was generated by comprehensive proteomic analysis of a mixture of three host-cell conditioned fluid (HCCF) samples with a simplistic three-dimensional separation strategy prior to MS. First dimension of separation included a size-based fractionation (100KD cutoff Amicon Ultra centrifugal filter), after which both fractions were subjected to tryptic digestion. The second dimension was offline high-pH reverse phase separation using SPE cartridges (SOLA HRP, Thermo Fisher) with ten varied %ACN elutions. The third dimension was running each fraction using a one-hour HPLC gradient (low pH, 1 mm by 150 mm CSH Column, Waters) and analysis with traditional data-dependent MS and MS/MS acquisition. These off-the-shelf separations require no complicated multi-dimensional LC devices or nano/microflow capabilities, and successfully detected 4373 proteins from 75,412 distinct peptides. This is consistent with current expectations for the abundance of media secreted CHO proteins (BEBPA HCP Conference 2015). The search results from these runs form a comprehensive MS/MS spectral library for use in all subsequent SWATH analyses.
        Following spectral library generation, a single one-hour LC gradient is used with SWATH data-independent acquisition for detection and quantitative monitoring of multiple HCPs in samples. SWATH data acquisition is performed using a Sciex TripleTOF-6600 system on development samples with known biotherapeutic protein abundance, and then, the data is mined post-acquisition for evidence of HCP presence using the comprehensive CHO derived MS/MS spectral library. The quantitative information from the SWATH data file is correlated with protein identities in the spectral library, and relative abundance of each detected HCP is scored and reported. Method validation tests have demonstrated that this technique can detect and quantitate proteins as low as 0.6 to 2 ppm in abundance relative to therapeutic product (w/w). We have also used this method in-house to evaluate ligand-binding based HCP tests, as well as to evaluate product purification campaigns.
        The SWATH data-independent acquisition workflow captures chromatographic ion data of every peptide fragment, for every peptide present within a given sample. Due to the unbiased nature of the technique, this method can detect HCP contamination regardless of process-specific procedures. This data can then be retroactively mined to test for the existence of any peptide or protein by pulling out quantitative extracted ion chromatograms of these proteins.
      • (Day 2: Proteomics Approaches to HCP Analysis: Core Technologies Session)

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Protein Expression Profiling of Different CHO Cell Lines: Striking Similarities

      • Speaker: Denise Krawitz, Genentech
      • Abstract: Host cell proteins (HCP) are a complex process related impurity often monitored with multi-analyte immunoassays, like ELISA. In order to use platform ELISAs to monitor HCPs in multiple products, we must demonstrate that the HCP population does not vary significantly with cell culture changes. Proteomics studies of different cell lines grown under different culture conditions suggest that the vast majority of proteins expressed are the same between cell lines.
      • (Day 1: HCP Immunoassay: Core Technologies Session)

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Evaluation of a CHO platform HCP ELISA

      • Speaker: Frieder Kröner, Novartis Pharma AG
      • Abstract: Host cell protein (HCP) impurities on individual process steps can vary quantitatively and qualitatively between different biopharmaceuticals expressed in Chinese hamster ovary (CHO) cells. In the present study, we have evaluated if these differences can be covered by a single platform HCP assay, or if the use of HCP assays specifically developed for different upstream processes is necessary to sensitively detect HCP impurities.
        Process intermediates from several biopharmaceuticals produced in CHO cells were compared, revealing differences in HCP levels between different production processes. To investigate if the observed differences require for process-specific HCP assays, a platform assay was compared to several process-specific assays. In a combinatorial testing it was found that the platform assay was able to accurately quantify non-cognate HCP standards. The platform assay performed at least equally well as the process-specific assays in monitoring HCP removal during downstream processing. In addition, the immunoreactivity of anti-HCP antibody reagents was compared by western blotting, and differences in the HCP composition of the assay standards were quantified by 2-D DIGE.
        Overall, the results reveal a high degree of similarity of the platform and process-specific HCP assays and assay reagents. This supports the notion that a platform assay is widely applicable, whereas process-specific HCP assays might offer an advantage only in limited cases.
        Authors:
        Frieder Kröner1, Darja Obrstar2, Thomas Millward1, Lea Bojic2, Oliver Anderka1
        1 Novartis Pharma AG, Basel, Switzerland
        2 Sandoz Biopharmaceuticals, Mengeš, Slovenia.
      • (Day 1: HCP Immunoassay: Core Technologies Session)

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An Update on the New Chinese Hamster Reference Genome – 2016

      • Speaker: Kelvin Lee, University of Delaware
      • Abstract: The advent of short-read second generation sequencing technology has enabled a rapid pace of genome sequencing. Such technologies rely heavily on bioinformatics algorithms to assemble and annotate the genome of interest. In 2013, two versions of the Chinese hamster genome were made available using short read technology. In one version, higher sequence coverage enabled the assembly and annotation of the genome; whereas in the other version, isolation of individual chromosomes generated information content useful for studying individual chromosomes. However, there are a number of inconsistencies related to these draft genomes which may be due in part, to the short read technology. To help the community move forward, an international consortium has pursued the use of long-read third generation sequencing technology (Pacific Biosciences) to facilitate Chinese hamster genome assembly and annotation support via a crowdfunding effort. The CHO community has also established and optimized technologies for identifying and quantifying the whole CHO proteome and secretome to complement the genome sequencing effort. The newly annotated genome will further increase our understanding of the CHO physiological pathways. This presentation will discuss the latest findings from novel recent approaches including a discussion of various assembly methods and their outcomes.
        Authors:
        Maddy MacDonald1, Kelley Heffner2, Deniz Baycin Hizal2, Michael Betenbaugh2, Kelvin Lee1
        1 University of Delaware
        2 Johns Hopkins University
      • (Day 2: Proteomics Approaches to HCP Analysis: Core Technologies Session)

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TBD

      • Speaker: Laureen Little, Principal Consultant, Quality Services
      • Abstract: (Pending)
      • (Day 2: Surveys Session)

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Case Study in HCP Assay Design – Impacts of Anti-HCP Antibody Purification and Assay Format

      • Speaker: Xiaohui Lu, Biogen
      • Abstract: Case Study in HCP Assay Design – Impacts of Anti-HCP Antibody Purification and Assay Format
        HCP immunoassays are the workhorse for HCP testing. Even though the same sandwich immunoassay concept is used, the exact designs of the HCP immunoassays, including assay format and the choice of the antibodies, can vary from one to another. In this case study, anti-HCP antibodies were either purified with Protein-A column only, or further purified with an affinity column prepared with the matching HCP antigens. The two antibody preps were characterized using western blotting and showed no significant difference in performance. However, very different HCP results were generated with these antibodies. Additionally, performing the HCP immunoassay in traditional stepwise format or a semi-homogenous format also gave significantly different results. These differences couldn’t be compensated by increasing the antibody concentrations in the assay. Overall, choice of anti-HCP antibody purification and assay format can make a qualitative difference in the HCP assay results, and may have broad implications in HCP testing and control. Besides considerations for historical practice and convenience factors, the impacts on assay performance should be carefully evaluated before finalizing the HCP assay design.
        Authors:
        Xiaohui Lu, Emily Menesale, and Svetlana Bergelson
        Analytical Development, Biogen, 225 Binney Street, Cambridge, MA 02142
      • (Day 1: HCP Immunoassay: Core Technologies Session)

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Small HCPs in a 12 kDa Protein Drug Analyzed by geLC-MS/MS

      • Speaker: Rikke Raaen Lund, Alphalyse A/S
      • Abstract: The content of low molecular weight host cell proteins (HCPs) in purified protein drugs is often difficult to evaluate, due to their low immunogenicity and poor ability to be visualized in gel-based total protein stains. The proteome of commonly used expression organisms, such as E. coli and Chinese Hamster cells, contains 30-40% proteins with a molecular weight below 20kDa, and these are easily missed in both in gel separations, Western blots and ELISA quantitation of the total HCP-content. To provide unbiased analysis of small as well as larger HCPs, we introduce the use of a mass spectrometry-based orthogonal method, well known from proteomics, called geLC-MS/MS.
        Here, we analyze a purified protein drug, a 12 kDa protein produced in E. coli, as well as the corresponding null cell lysate, using geLC-MS/MS to obtain high coverage of small HCPs. Proteins were separated by 1D-SDS-PAGE and stained by Coomassie Brilliant Blue. The staining allows isolation of the high concentration protein drug in separate gel fractions, leading to more protein identifications in the fractions without the protein drug. The gel lane was divided into eight continuous fractions, all analyzed by nLC-MS/MS. This analysis identified 152 host cell proteins in the purified protein drug and 553 proteins in the null cell lysate all at high confidence. Further, this analysis also provided exact database entry names as well as amino acid sequences for each identified HCP. High percentages of the identified HCPs were smaller than 20kDa: 37% in the purified protein drug and 46% in the null cell lysate. The sensitivity of the method was estimated by parallel analysis of the purified protein drug with two spiked-in standards, these standards were both identified as low as 50ppm.
        Interestingly, the host cell proteins that were identified in the protein drug as well as in the null cell lysate covered the entire pI range and over 99% of the Mw range of the total E. coli proteome. This observation shows that the developed geLC-MS/MS method has no inherent limitations with respect to pI or Mw during HCP identification.
        Authors:
        Rikke Raaen Lund, Marie Grimstrup and Ejvind Mørtz (Alphalyse A/S, Odense, Denmark)
        Karin Abarca Heidemann and Maheen Sayeed (Rockland Immunochemicals Inc., Limerick, Pennsylvania)
      • (Poster)

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Development of a Sensitive ELISA Platform Assay to Quantify Host Cell Protein

      • Speaker: Guojie Mao, Lonza Biologics plc
      • Abstract: Host cell proteins (HCPs) are host cell-derived impurities derived from biological manufacturing process. HCPs can pose risks to patient safety and product efficacy. Therefore, residual HCPs should be measured and monitored in the final biopharmaceutical products.
        ELISA is one of the most commonly used immunoassays by the industry to quantify the HCPs impurities. Lonza has developed a new HCP ELISA assay to measure HCP impurities. In this ELISA, sheep polyclonal HCP antibody was raised against cell lysate from pooled mock transfected null cells from representative Lonza process. The HCP antibody was purified using a novel two-step affinity strategy. Total IgG was purified by Protein A and combined with an HCP-specific affinity purification step. The resulting immunocoverage of the antibody was 71% (as assessed by spot-matching using a two-dimensional (2D) western blot), which confirmed the efficiency of the optimised purification parameters. The antibody concentration, buffer system and assay detection system were optimised during ELISA development.
        The final sensitive and user-friendly HCP ELISA was evaluated according to ICH guidelines. The method was tested on six occasions for each of four mAb products. The accuracy, intra-assay precision, intermediate precision and linearity were each determined and met or exceeded all quality targets defined for the method prior to development. The working range of this ELISA was determined as from 2ng/ml to 80ng/ml. The LOQ was defined as 2ng/ml with acceptable spike recovery at multiple levels on bulk purified products. Specificity against Protein A and CHO DNA, which are major impurity sources together with HCP, was also demonstrated.
        After validation, the HCP ELISA is to be served as a platform assay to monitor the HCP impurity during the bio-purification process and final bulk drug substance, particularly compatible to the Lonza manufacturing process. An effective framework has also been established for development of high-performance HCP ELISA methods for individual production cell lines.
        Authors:
        Guojie Mao*, Alison Smith, Gemini Shah, Helen Emmines, Chris Lloyd, Neema Antony, Steve Flatman and James Graham
        Lonza Biologics plc, 228 Bath Road, Slough, UK, SL1 4DX
      • (Poster)

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Host Cell Proteins: The Hidden Side of Biosimilarity Assessment

      • Speaker: Kazutoshi Mihara, JCR Pharmaceuticals Co., Ltd.
      • Abstract: One major concern with biosimilars is that small differences compared with reference products might lead to unforeseen immunogenicity, thus affecting patient safety and drug efficacy. Differences could be due to either post-translational modifications of the therapeutic protein and/or to traces of impurities from the manufacturing process. The results presented in this communication illustrate the efforts to assess “biosimilarity” of a biosimilar candidate to a reference product for a specific group of process-related impurities, the host cell proteins (HCP). Extensive characterization of HCP in the drug substance of a biosimilar candidate revealed the identity of HCP copurifying with the protein of interest and guided process development to improve overall HCP clearance in the downstream process. The data presented illustrate the challenge of matching the reference product on either quantitative or qualitative aspects of HCP impurities.
        Authors:
        Kazutoshi Mihara1, Yae Ito1, Yukichi Hatano1, Yoshikazu Komurasaki1, Atsushi Sugimura1, Marisa Jones2, Haiyan Liu2, Shing Mai2, Oscar Lara-Velasco2, Lin Bai2, Amol Ketkar2, Michael Adams2, Tohru Hirato2 and Roxana Ionescu2
        1Research Institute, JCR Pharmaceuticals Co., Ltd., Kobe 651-2241, Japan
        2R&D Platform Technology and Science, GlaxoSmithKline, King of Prussia, Pennsylvania 19406
        3R&D Platform Technology and Science, GlaxoSmithKline, Stevenage, UK
      • (Poster)

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Improved Identification of Host Cell Proteins in a Protein Biopharmaceutical by LC-MS/MS with the ProteoMiner Enrichment Kit

      • Speaker: Harriet Mörtstedt, Swedish Orphan Biovitrum (Sobi)
      • Abstract: Background: Host cell proteins (HCPs), which are contaminating proteins present in protein biopharmaceuticals, can be identified by enzymatic digestion and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). HCPs are often present at very low levels (
      • Aim: The aim was to evaluate the usefulness of the ProteoMiner Enrichment Kit (Bio-Rad Laboratories) for enrichment of individual HCPs to increase the number of identified HCPs.
      • Methods: A recombinant protein (Protein X) produced in Chinese hamster ovary (CHO) cells and with a total HCP content of 2µg/mg Protein X (determined by ELISA) was spiked with four standard proteins at varying concentrations (10-500 ppm). The sample was split into two aliquots; one that was prepared with the ProteoMiner Enrichment Kit and one control, where the enrichment procedure was omitted. The ProteoMiner Enrichment Kit was combined with the ProteoMiner Sequential Elution Large-Capacity Kit eluting the proteins into four fractions. The samples were trypsin digested overnight and analyzed with LC-MS/MS on a Q-TOF instrument (Synapt G2-S). The resulting data was processed using the software Protein Lynx Global Server and searched against a database containing the protein sequences for the Chinese Hamster proteome, human Protein X, and the spiked standard proteins. Proteins detected by at least 2 matching peptides were considered identified. Multiple reaction monitoring (MRM) on a triple quadrupole instrument (QTRAP6500) was then applied to obtain an estimate of the relative protein abundance of identified and spiked proteins (ng protein/mg Protein X) in the samples.
      • Results: With the untargeted LC-MS/MS method, Protein X and eight HCPs were identified in the four fractions. Only Protein X was identified in the non-enriched control sample. When the targeted MRM method was applied, all eight HCPs were detected in at least one of the fractions and seven of them were detected in all fractions as well as in the control. In the most enriched fraction a 400-fold enrichment of individual HCPs was observed. The spiked proteins were detected down to 100 ppm with the untargeted LC-MS/MS method and down to 50 ppm with the targeted MRM-method.
      • Conclusion: The results clearly showed that the ProteoMiner Enrichment Kit combined with the sequential elution kit enriched the HCPs in the fractions enabling identification of several HCPs and spiked proteins down to 100 ppm.
      • Authors:
        Harriet Mörtstedt, Åsa Brunnström, Per-Olof Edlund, Katarina Sjöberg and Agneta Tjernberg
        Drug Design and Development, Sobi, Stockholm, Sweden
      • (Poster)

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Practical Application of Orthogonal HCP Testing: How to Interpret & Use the Data

      • Speaker: Ned Mozier, Pfizer
      • Abstract: Many companies are now using, or beginning to apply, LC-MS/MS testing in a new application for detection and monitoring of residual host cell proteins (HCP). These impurities are also tested by the more established immunoassay that was reviewed in the USP guideline. Both approaches have value, and each has unique benefits and drawbacks. What appear to be contradictory results numerically can be understood if one considers the manner in which these methods detect and report results. Used properly, in combination, they are orthogonal and increase the power of the control strategy in our industry. For example, ELISA is simple, robust, QC-friendly, and useful for both drug substance release and for demonstrating clearance of HCP throughout processes. LC-MS/MS, on the other hand, though requiring more time and cost, has the benefit of identifying individual HCP species. Identification affords a more intelligent risk assessment and a tool for bioprocessing and removal of HCP, where necessary. Case studies are provided where both data sets are shown to be complimentary. Discussed will be how this tandem approach enables a more powerful control strategy and means to characterize processes within and across a portfolio.
      • (Day 2: Putting It All Together: Integrated Quality Strategy for HCPs using Available Assay Technologies Session)

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LC-MS Host-cell Protein Detection using Data-Independent SWATH-MS to Reach Sub-PPM Level Detection

      • Speaker: Milla Neffling, Sciex
      • Abstract: Introduction: Analysis of the HCPs of Biotherapeutics by MS provides a way to supplement and improve assays developed with other techniques. Earlier studies relied on proteomics techniques, and required some pre-knowledge of the HCP complement and considerable expertise. We present routine methodology for unbiased and comprehensive HCP analysis. A standard instrument platform is used to detect and quantify a number of contaminant proteins in the presence of highly abundant product protein with unambiguous evidence for identity and concentration. Routine chromatography provides a robust methodology that is complete within a few hours and demonstrates unsurpassed sensitivity. The approach presented here offers time and cost saving as compared to other currently promoted LC-MS HCP assays to truly enable high-throughput detection and monitoring of HCPs.
        Methods: MAb IgG1were spiked with a commercial protein mixture at relative concentrations between 10 and 0.6 ppm. LC (Eksigent 425 in micro mode): 60-minute RP LC gradient at 10 µL/min. MS: (SCIEX 6600), MS-based identification (Library): data dependent method, TOFMS survey scan followed by 20 MS/MS spectra of 35 msec each and ProteinPilotTM Software. HCP Detection: Triplicate SWATHTM acquisitions, using variable window width to produce high-resolution MSMS chromatograms of every fragment ion of every precursor between m/z 400 and 1000.
        Preliminary Results: Quantitative analysis was performed with SWATH™ in PeakView® Software. For each of the model HCPs and mAb, 3-4 peptides were used for SWATH quantitation. Despite up to a 100,000 fold difference in abundance between the product and the contaminants, the quantitation of both HCPs and antibody was highly reproducible. The lowest detection limits reached were 0.7ppm. These results indicate extremely high quantitative reproducibility, independent of protein identity, suggesting that this approach should be broadly applicable for the unbiased quantitation of HCPs down to below ppm levels.
        Novel Aspect: Reproducible HCP quantitation down to sub- ppm in a single 60 minute gradient without using nanoflow or multidimensional chromatography.
        Authors:
        Milla Neffling; Eric Johansen; and Justin Blethrow
      • (Poster)

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Generation and Characterization of E. coli HCP Reagents of a Platform Immunoassay

      • Presenter: Romain Pizzato, Sanofi Pasteur
      • Abstract: (Pending)
      • (Poster)

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Evaluating the Need for a Process Specific Host Cell Protein (HCP) Assay in Escherichia coli (E. coli) Expressed Recombinant Proteins

      • Speaker: Chandrima Palit, ZymoGenetics
      • Abstract: Expression of recombinant proteins in Escherichia coli (E. coli) typically results in formation of inclusion bodies (IBs) and requires solubilization and refolding to recover the protein of interest. As a result, the strategy for generating a representative host cell protein (HCP) antigen requires careful consideration because null strains not expressing the protein of interest do not typically generate inclusion bodies. Experiments were conducted to assess suitability of HCP assays using antibodies prepared against a whole cell lysate and against proteins solubilized and refolded from E. coli for proteins expressed in IBs.Host Cell Protein (HCP) levels in downstream process intermediates originating from E. Coli inclusion bodies were measured using a commercially available ELISA kit. The HCP antigen, and resulting anti-HCP antibody, used in this kit originated from whole cell lysates. HCP antibodies in this kit failed to detect any host cell proteins in purified bulk drug substance (BDS). The recognition of HCPs in an early process stream intermediate by the commercial HCP antibody was assessed using two dimensional difference gel electrophoresis (2D DiGe). While the commercial antibody showed fairly good coverage, lack of recognition for a certain population of HCP was confirmed. In contrast, ELISA using polyclonal antibodies generated against HCPs prepared from a “mock” refold process (fermentation, solubilization and refolding from E. coli strain containing plasmid without DNA sequence for protein of interest) detected HCPs throughout the purification process, with good dilution linearity, including recognition of HCPs in purified BDS. Additionally, 2D DiGE analysis using the antibody reagent generated against the “mock refold” revealed higher HCP coverage. These results indicate an IB process specific HCP assay is needed and demonstrates the importance of using HCP reagents that reflect the production process.
      • (Poster)

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Image Analysis Used to Minimize Inter-User and Inter-Lab Variation of Results from Measuring HCP-Antibody Coverage by Comparing Features Between 2D Gel and 2D Western Blots

      • Speaker: Kelly Parkin, TotalLab Ltd.
      • Abstract: The current advice is to use the potential of 2D gels in resolving complex protein mixtures as a way to improve characterizing HCP-antibody profiles. 2D gels have previously been considered to be difficult to run and analyze. Recent laboratory and in silico techniques have dramatically improved gel running and ease of analysis but there are sources of variation to be controlled and minimized in order to exploit the analytical power of 2D gels and 2D Western blots.
        SpotMap has been specifically developed for the purpose of analyzing HCP coverage for antibody product and process characterization. It addresses the challenge of comparing significantly different spot patterns and calculate percentage coverage of 2D gel vs 2D Western blot. SpotMap offers an objective solution for quantitatively determining HCP antibody coverage.
        We demonstrate the reproducibility of relative coverage results using SpotMap to analyze a process specific antibody. To achieve this two pairs of a 2D gel vs a 2D Western Blot were analyzed. We compared the results to look at inter-user and inter-site variability and how SpotMap software reduces the subjective decisions required during image analysis.
        Finally, we look at where SpotMap fits within the wider process of optimizing upstream gel running and image capture for 2D gels and 2D Western blots. The net result is to provide tactics you can deploy to increase the objectivity, reproducibility and reliability of results.
        Authors:
        K. Parkin, C. Moncada, A. Gilmore, M. Sayeed, P. Lavery
      • (Poster)

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Proteomics-Based LC-MS Approach For Assessing Host Cell Protein Impurities in a Complex Biological Product

      • Speaker: Pavlo Pristatsky,
      • Abstract: Conventional approaches to measure host cell protein (HCP) impurities involve the use of anti-host cell antiserum in a plate-based ELISA platform. Inherent to this approach is the need for a high quality immunoreagent with broad coverage against the host cell proteins and an appropriate reference standard. Generation of these reagents is not trivial and can require a long lead time. An alternate approach that relies on LC-MS is presented. Identification of host cell impurities along with measurements of relative abundances can be determined using a proteomics approach. In contrast to traditional ELISA-based approaches, the proteomics-based method does not rely on either immunoreagents or a reference. Here we present the methodology and the assessment of the analytical parameters such as precision, linearity and accuracy based on a case study where this approach was used to characterize the host cell protein impurities in a live viral vaccine candidate. Additionally, the approach was used during process optimization to increase the clearance of host cell proteins and bovine serum proteins present in the medium.
        Authors:
        Colleen Price, Larry Dick, Josef Vlasak, Tim Culp, Richard Peluso, Pavlo Pristatsky and Van M. Hoang
      • (Poster)

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Insights on the Identification of Host Cell Proteins by Nano-LC-MS/MS Bottom-Up Mass Spectrometry

      • Presenter: Lleana Rodreguez Leon, NovoNordisk
      • Abstract: Co-purification of host cell proteins (HCPs) together with the protein therapeutic (PT) is highly undesired due to its potential negative effect on the PTs, e.g., on protein stability and/or safety. The current gold standard methodology for the global quantification of HCPs is an ELISA based assay with antibodies raised against an HCP pool. The result of this assay is a global quantitation (in ng/mg or ppm) of HCP content on the sample. However, the identity or nature of HCPs cannot be determined. Knowing the nature of the HCP is a relevant piece of information which could, in potential, distinguish between harmless or harmful individual HCPs.
        With the latest advances in mass spectrometry (MS) instrumentation, the analysis of HCPs by LC-MS/MS has become a promising complementary strategy. LC-MS/MS combined with well establish quantitative proteomics workflows has the advantage of providing identity and quantity of individual HCPs. The drawback is that the large amount of data generated in single LC-MS/MS runs requires sophisticated but reliable bioinformatics tools.
        Here we present a case study of the investigation of HCPs in a protein therapeutic from a mammalian expression system. In this study we have applied two different bioinformatic platforms, including licensed in house Mascot. Top 3 strategies for quantitation, in combination with the use of a well characterised internal standard (UPS2, Sigma), was applied for relative and absolute quantitation. Pitfalls of using specific but incomplete databases will be also discussed.
        Authors:
        Ileana Rodríguez León, Finn Matthiesen and Johan Henrik Faber
        Global Research Unit, Novo Nordisk A/S, Denmark.
      • (Poster)

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Mass Spectrometry as a Powerful Tool for Host Cell Protein Analysis

      • Speaker: Anke Schnabel, Protagen
      • Abstract: Developing and producing recombinant biopharmaceuticals in mammalian cells requires unambiguous monitoring of HCPs impurities. Immunoassays are still the method of choice for release testing, for which it is recommended to demonstrate the suitability of antisera in a QM-regulated environment to meet regulatory demands. However, peptide analysis by MS has been proven to be a powerful tool by providing complementary data for HCP characterization. We show that MS supports the data interpretation of methods for antisera characterization, like high resolution 2D gel electrophoresis combined with immunoblotting to demonstrate antisera coverage, and anti-CHO affinity chromatography to specify the antigen coverage. Here we introduce an approach to increase coverage of HCP antisera by HCP-GAPexSM aligning advantages of high-resolution 2D-PAGE with mass spectrometric sensitivity and specificity. Furthermore, peptide analysis by MS offers orthogonal solutions for detection and monitoring of residual HCPs. The advantage compared to immunological methods is the unbiased discovery of HCP impurities to reveal the HCP identities. By using label-free MS quantification the removal of HCPs during downstream processing was monitored and demonstrated the suitability of the technique for quantitative analysis of trace impurities and thus elucidating effectiveness of individual downstream processing steps.
        Authors:
        Anke Schnabel, Heiner Falkenberg, Anja Dommermuth, Gerhard Koerting, Anna Lendzian, Roland Moussa, Olaf Stamm, Thomas Flad
      • (Poster)

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Image Analysis Used to Minimize Inter-User and Inter-Lab Variation of Results from Measuring HCP-Antibody Coverage by Comparing Features Between 2D Gel and 2D Western Blots

      • Speaker: K. Perkin, TotalLab Ltd.
      • Abstract: The current advice is to use the potential of 2D gels in resolving complex protein mixtures as a way to improve characterizing HCP-antibody profiles. 2D gels have previously been considered to be difficult to run and analyze. Recent laboratory and in silico techniques have dramatically improved gel running and ease of analysis but there are sources of variation to be controlled and minimized in order to exploit the analytical power of 2D gels and 2D Western blots.SpotMap has been specifically developed for the purpose of analyzing HCP coverage for antibody product and process characterization. It addresses the challenge of comparing significantly different spot patterns and calculate percentage coverage of 2D gel vs 2D Western blot. SpotMap offers an objective solution for quantitatively determining HCP antibody coverage.We demonstrate the reproducibility of relative coverage results using SpotMap to analyze a process specific antibody. To achieve this two pairs of a 2D gel vs a 2D Western Blot were analyzed. We compared the results to look at inter-user and inter-site variability and how SpotMap software reduces the subjective decisions required during image analysis.Finally, we look at where SpotMap fits within the wider process of optimizing upstream gel running and image capture for 2D gels and 2D Western blots. The net result is to provide tactics you can deploy to increase the objectivity, reproducibility and reliability of results.
      • (Day 2: Putting It All Together: Integrated Quality Strategy for HCPs using Available Assay Technologies Session)

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Monitoring of HCPs Throughout a Complete Bioprocess Using Mass Spectrometry & Implications for Continuous Processing

      • Speaker: Mark Smales, University of Kent
      • Abstract: (Pending)
      • (Day 2: Putting It All Together: Integrated Quality Strategy for HCPs using Available Assay Technologies Session)

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Development of a Specific Host Cell Protein Assay for the Plant-Based Expression System Physcomitrella patens

      • Speaker: Stefan Sommerschuh, BioGenes
      • Abstract: Plant-based expression systems, such as Physcomitrella patens, have emerged only recently as an alternative to established recombinant protein production platforms (e.g. bacterial cell lines, yeast cell lines or mammalian cell lines) and, therefore, generic HCP ELISAs are usually not available for such cell lines. For the development of a specific P. patens HCP ELISA appropriate Mock-HCP material was produced and used for the immunization of rabbits. The purified antibodies were tested by 2D analysis, resulting in a coverage of 91 %, and used for ELISA development. The preliminary working range of the assay was set between 0.1 and 5 ng/mL and an acceptable intra-assay precision with CV Authors:
        Stefan Sommerschuh, Paulina Dabrowska-Schlepp, Claudia Geserick, Nicole Gliese, Mathias Knappenberger, Holger Niederkruger and Andreas Schaaf
      • (Poster)

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Lightning the Black Box of HCP Immunoassays – Alternative Immunization Strategies in Combination with Innovative Characterization Tools

      • Speakers: Olaf Stamm, Charles River Laboratories, and Thomas Flad, Protagen Proteome Services
      • Abstract: Immunoassays for host cell protein (HCP) quantitation require high quality polyclonal antisera which are generated by immunization of common animal models such as rabbits, goats and sheep. The challenge in HCP antisera development is the large number of proteins contained in the antigens and their heterogeneity. The antigens can differ widely with respect to size, physio-chemical properties and immunogenic profile. There are multiple ways to address this challenge and the following presentation is focused on a case study using an alternative immunization models (chicken) in combination with a set of state of the art methods for antisera characterization ranging from high resolution 2D gel electrophoresis, affinity chromatography and different mass spectrometry applications. Available tools from proteomics can generate significant intelligence with respect to the specificity of HCP-Immunoassays. In consequence, a polyclonal antiserum based Immunoassay must not be considered as a black box anymore.
      • (Day 1: HCP Immunoassay: Core Technologies Session)

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Comparison of Five New Commercial Kits Specific for CHO-HCP Developed for the Gyrolab™ Platform

      • Speaker: Ann-Charlott Steffen, Gyros Protein Technologies
      • Abstract: Host cell protein (HCP) levels in drug products are critical to product quality since HCPs may pose a serious risk to patient safety. The challenge is to accurately quantify the complex mixture of HCP impurities.
        Generic immunoassays commonly used to measure HCP impurities are based on polyclonal antibodies raised against host cell proteins from non-transfected cell lines. How well a particular HCP assay recognizes all proteins will depend on how well the antibodies match the HCP composition, their abundance, and their affinities for each host cell protein. The reactivity of the antibodies will depend on how the antigen was prepared, the method of immunization, and the purification process.
        Gyros has developed a range of five ready-to-use Gyrolab™ kits to measure HCP impurities from Chinese Hamster Ovary (CHO) cells, based on commercially available antibody preparations.
        We have evaluated the five kits using samples from a bioprocess in CHO cells as well as pharmaceutical grade drug products produced in CHO cells. We found in that for both sets of samples the different kits showed significant differences in ability to detect HCP.
        Access to several CHO-HCP kits increases the probability of selecting a generic CHO-HCP assay that is suitable for a given process/drug product.
        Authors:
        Ann-Charlott Steffen, Cecilia Bill, Gunnar Ekstrand and Mats Inganäs
        Gyros Protein Technologies, Uppsala Science Park, SE-75183 Uppsala, Sweden
      • (Poster)

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Tutorial: Effective Use of Mass Spectrometry in the Analysis of HCPs

      • Speaker: Kevin Van Cott, University of Nebraska-Lincoln
      • Abstract: In recent years, the use of mass spectrometry (MS) methods in HCP analysis has grown. While MS methods have not matured to the point of being used as release assays, they have the potential to complement and clarify the information obtained by HCP ELISAs and other analytical techniques. The target audience for this tutorial is scientists with little to no direct experience in MS analysis. The objectives of this tutorial include the following:
        • Introduce the participants to standard MS technologies and methods that are used in HCP analysis
        – Basic MS instrument design and capabilities
        – Sample preparation methods and MS data acquisition modes
        – Protein identification from database search programs
        – Qualitative vs. Quantitative MS methods
        • Highlight the strengths and advantages of MS methods in HCP analysis
        • Acknowledge the limitations of MS methods in HCP analysis
        After completion of this tutorial, the participants should be familiar with basic MS technology and terminology, they should understand how to critically review MS results, and they should be able to envision how MS methods can complement other HCP analysis methods to produce a comprehensive understanding of the purity of their products.
        Kevin Van Cott, Ph.D.
        Associate Professor
        Department of Chemical and Biomolecular Engineering
        Nebraska Center for Mass Spectrometry
        University of Nebraska-Lincoln
        Lincoln, NE 68588
      • (Workshop 1)

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In-Depth, Quantitative (Host Cell) Protein Fingerprinting of Adenovirus-Based Vaccines by Mass Spectrometry

      • Speaker: Annemiek Verwilligen, Janssen Infectious Diseases & Vaccines
      • Abstract: The recombinant viral vectors employed in Janssen’s AdVac® vaccine platform are harmless adenoviruses. These are modified to genetically encode proteins of viral origin so-called transgenes which are expressed in the human cell upon administration. Adenoviruses are nonenveloped and consist solely of 14 capsid (structural) proteins and DNA.
        In analytical terms, manufactured recombinant adenovirus-based vaccines constitute a mixture of adenovirus capsid proteins and a small portion of residual host cell proteins (HCPs). For batch release of such vaccines, information on protein identity and quantity of both protein constituents is required. Currently, such tests are laborious and time consuming and are biased towards the much higher abundant adenovirus proteins due to the large variation in dynamic range.
        To overcome these challenges, we have developed a fast all-in-one semi-quantitative mass spectrometry-based approach. The method combines filter assisted digestion, C18 RP-UPLC separation directly coupled to ESI-Q-TOF MSE data acquisition. This allowed us to confirm the presence of all 13 adenovirus capsid proteins with ample sequence information and at the same time identify a significant amount of HCPs in a single 3h analysis. Additionally, relative quantitation based on signal intensity further refined the method to estimate the ratio of adenovirus protein to HCPs.
        The obtained method is currently employed in characterization to gain product knowledge on multiple properties of the adenovirus vaccine, such as virus identity confirmation, evaluation of HCP co-purification from the vaccine manufacturing process and assessment of batch-to-batch variation. We envision the method should be embedded in targeted quantification of frequently co-purifying HCPs to aid in the development of FACS and ELISA platforms.
        Because of the short execution time of only three hours and the deep coverage, this assay exemplifies the importance of mass spectrometry in the characterization of complex (adeno)virus based vaccines.
        Authors:
        Annemiek Verwilligen, Dayar van der Steeg, Jonathan Knibbe, Arjen Scholten, Harold Backus, Johan Nijenhuis
        Janssen Infectious Diseases and Vaccines, Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands
      • (Day 2: Proteomics Approaches to HCP Analysis: Core Technologies Session)

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Quality Aspects of Host-Cell Protein Assays

      • Speaker: Thomas Waerner, Boehringer Ingelheim Pharma GmbH & Co.
      • Abstract: Host-cell proteins (HCPs) are never completely depleted during the production process of biopharmaceuticals. This is due to different HCP properties and technical downstream limitations. Although only rare published cases exist that might indicate clinical incidents referring to HCPs, the remaining HCPs should be sufficiently characterized to ensure patient safety. Only if critical ELISA components such as reference material and antibodies are generated adequately and characterized sufficiently, the ELISA technique is a suitable method to quantify a broad spectrum of HCPs in test samples.
        This talk provides assistance for setup of an HCP control strategy over a product’s lifetime by addressing ELISA characterization aspects, including 2D antibody coverage, HPLC – ELISA combination, and about setting a design space for a “platform ELISA”. The intention is to give support to generate necessary data to fulfill regulatory requirements.
      • (Day 1: Developing an Integrated Assessment Using LC-MS/MS & Immunoassays Session)

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Bridging the Gaps: A Case Study Using Upstream Process-Specific Assays to Monitor Residual HCPs Identified by 2D-LC-MS in a CHO-produced Therapeutic Antibody

      • Speaker: Fengqiang Wang, Merck Research Laboratories
      • Abstract: Host cell proteins (HCPs) in biological drugs are often monitored by a sandwich immunoassay to ensure they are removed to an adequate low level that will not jeopardize product efficacy and patient safety. For monitoring residual HCPs in CHO-produced monoclonal antibodies (mAbs), commercial kits targeting generic CHO cell line HCPs are commonly used during early phase product development. At late phase of development, HCP control strategy often changes from commercial assays (or other generic assays) to process-specific assays to improve antibody coverage to HCPs specific to the production cell line and manufacturing process. During the switch, extensive characterization of product-specific HCPs is required and the antibody coverage to product-specific HCPs by anti-HCP antibodies from both the commercial kits and process-specific assay needs to be evaluated for assay qualification and risk assessment of the HCP control strategy. Meanwhile, the identification of product-specific HCPs in drug substance by 2D-LC-MS creates knowledge gaps on the effectiveness of using immunoassay solely for HCP monitoring. Here, using mAb A HCP assay development as a case study, we elaborate on the strategy for HCP control during early and late phase product development, the gaps of knowledge created by the identification of product-specific HCPs in mAb A drug substance using 2D-LC-MS, and the steps taken to fill the gaps with an upstream process-specific HCP assay. This study indicates the importance of HCP characterization by orthogonal methods and the developing of a process-specific assay for HCP monitoring when commercial assays do not provide sufficient antibody coverage for process-specific HCPs.
      • (Day 1: Developing an Integrated Assessment Using LC-MS/MS & Immunoassays Session)

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New Approaches for Analyzing HCP Coverage

      • Speaker: Stefanie Wohlrab, Roche-Penzberg
      • Abstract: Host cell proteins (HCPs), as the most prominent kind of process-related impurities, are critical quality attributes (CQAs) in the development of protein-based pharmaceuticals. HCPs are undesirable in the final product as they may act immunogenic and potentially exhibit biological activity. Means to quantify HCPs generally use highly sensitive enzyme-linked immunosorbent assays (ELISA). The performance of this assay is tightly linked to the quality of applied reagents like HCP antigens for calibration and anti-HCP antibodies. Therefore, multiple orthogonal methods are being applied to investigate quality and suitability of these critical assay reagents. Here, we describe a workflow, including methods like 2D gel electrophoresis and mass spectrometry, to analyze HCP coverage providing reproducible results.
      • (Day 1: Developing an Integrated Assessment Using LC-MS/MS & Immunoassays Session)

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A Mass Spectrometric Libraray for HCP Quantification, Identification & Clearance

      • Speaker: Chris Yu, Genentech
      • Abstract: (Pending)
      • (Day 2: Proteomics Approaches to HCP Analysis: Core Technologies Session)

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