Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Enhanced Peptide Mapping with a PASEF Enabled Ultra High Resolution QTOF

• Presenter: Christian Albers, Bruker Daltonik GmbH
• Abstract: Peptide mapping by LCMS is always a compromise in between acquiring high quality and dynamic range MS1 data for quantitation, high quality MS2 data for unambiguous assignment of heterogeneities and high sensitivity to minimize the risk of low abundance forms going undetected.
  A newly developed ultra high resolution QTOF – the timsTOF Pro – harnesses the potential of a dual Trapped Ion Mobility Spectrometry (TIMS) cell to simultaneously increase ions utilization and MS2 speed. Together with the Parallel Accumulation Sequential Fragmentation workflow (PASEF) this enables high sequencing speed of over 100 Hz without losing sensitivity by synchronizing the quadrupole isolation mass window with the elution time of the specific peptide packages from the TIMS funnel.
  These hardware enhancements can benefit the detection of isobaric peptides as separation in the mobility cell prior CID fragmentation reduces the occurrence of chimeric spectra. The enhanced sensitivity also enhances the general quality of MS2 spectra simplifying the data review tasks and the identification of low concentration proteins such as HCPs.
  Using the NISTmab reference material as the basis for this case study, the capabilities of this novel instrument to support the development of monoclonal antibodies were evaluated in the context of high speed peptide maps as well as longer methods for the sensitive detection of low abundance heterogeneities.
• Contributing Authors: Christian Albers², Guillaume Tremintin¹, Detlev Suckau², and Stuart Pengelley^{2
  1} Bruker Corporation, Billerica, MA; ² Bruker GmbH, Bremen, Germany
• (Poster)

Identification and Quantification of HCPs by Mass Spectrometry

• Presenter: Christina Bell, Caprion Biosciences Inc
• Abstract: As a Critical Quality Attribute (CQA), the residual host cell protein (HCP) in a drug substance (DS) must be characterized to ensure the desired product quality [ICH Q8]. Gel-free, label-free mass spectrometry (MS) is playing an increasingly greater role in characterization of HCPs by enabling identification and monitoring of low level HCPs. We have established robust workflows for the characterization and monitoring of HCPs. For identification and relative quantification of HCPs, samples are subjected to trypsin digestion, extensive fractionation to reduce the complexity of the tryptic peptide mix due to the presence of the DS in very high amounts, and high mass accuracy shotgun RPLC-MS/MS. PQCs (Process Quality Controls) are analysed alongside the samples as a measure of the instrument and analytical procedure performance. HCPs are identified by using an unbiased bioinformatics workflow with multiple search engines to match detected peptides against a custom process-specific HCP database (i.e., CHO, human, yeast and bacteria), and spectral counts provide a qualitative assessment of relative abundance of each HCP. The sensitivity of the method depends on the particular characteristics of the DS, but has been shown to identify spiked proteins in the presence of drug substance between 1-10 ppm. For absolute quantitation of HCPs, a multiplexed targeted LC-MRM/MS assay is developed using stable-isotope labelled peptides representing the specific HCPs to be monitored (2 surrogate peptides per HCP, 2 transitions per peptide). QC samples and calibration curves are run alongside study samples, and stable-isotope labelled peptides are spiked into all samples as internal standards. The sensitivity of detection is routinely achieved in the 1 ppm range. Examples of client studies will be presented, including monitoring HCP profiles to assess process changes, improvements or scale-up, to demonstrate batch uniformity, to understand clearance, and to demonstrate comparability of Biosimilars to Innovators.
• (Poster)

It’s all about the Quality of the Material, Developing Optimal HCP Immunoassay Critical Reagents

• Speaker: Jessica Campbell, Pfizer
• Abstract: Immunoassays are commonly used to detect and measure HCPs in therapeutic products, and a successful assay is directly dependent on the quality of the critical reagents used. We will present our HCP critical reagent strategy, including details on the purification of the anti-HCP polyclonal antibodies. These antibodies are enriched from antisera of animals immunized with a broad mixture of HCPs, but there is limited information regarding the best strategy for purification of these critical reagents. We compared anti-HCP antibodies generated from different purification procedures by evaluating purity, concentration, reagent coverage, and performance in an enzyme-linked immunosorbent assay (ELISA).
• Contributing Authors: Jessica Campbell, Phoebe Baldus, and Ned Mozier
• (Day 1: Anti-HCP Antibody Characterization Session)

Improvement of Host Cell Proteins Analysis Using Gyrolab Technology

• Presenter: Michael Chaplet, GSK
• Abstract: The monitoring of residual host cell proteins (HCPs) impurities is a major critical quality attribute (CQA) in the process development of biopharmaceutical recombinant proteins. Indeed, residual HCPs have the potential to impact product quality, safety, stability and efficacy, and therefore an efficient and suitable method is essential to assess their clearance through the different downstream purification process steps.
  Enzyme‐linked immunosorbent assay (ELISA) is usually seen as the ligand-binding assay of choice to support HCP testing due to its sensitivity and selectivity. In order to develop a suitable process-specific ELISA we carefully selected the reference standard material and we performed a full characterization of the specific immunotools (anti-HCP polyclonal antibodies) using ELISA and orthogonal methods like 1D and 2D-PAGE to assess antibodies coverage. In our assay development we identified distinct dilution dependence for samples of the last two purification steps. A hypothesis of this nonlinear behavior would be associated with antigen excess relative to available antibody for the particular set of HCPs present in the samples. In addition, the quantification range of the ELISA becomes limiting in reaching dilution where the result is independent of the samples dilution.
  We used the Gyrolab xP microfluidic automated workstation to perform the assay on each sample and we did not observed dilution dependent effects. The sample dilution linearity was achieved and we were able to quantify HCP levels for each particular purification step. Due to the affinity columns and the higher quantity of immobilized antibodies engaged in the assay, the Gyrolab xP Workstation demonstrated high potential for residual HCP testing in biologics compared to ELISA. Ongoing mass spectrometry experiments are being done as orthogonal method to complement data.
• Contributing Authors: Vincenzo Vinci, Isabelle Antoine, Dominique Ingels, Michel Plisnier, Claudia Magagnoli and Michael Chaplet
  GSK, Rixensart, Belgium
• (Poster)

Comparison of Common Approaches to Generation of AntiHCP Antibody Reagents

• Speaker: Murty Chengalvala, Covance
• Abstract: Despite recent modest advances in LC-MS/MS methodology, immunoassays continue to be a preferred method of testing for host cell protein (HCP) impurities in biotherapeutics. Anti-HCP antibody reagents used in these immunoassays have historically been generated using a wide variety of approaches – including various species, adjuvants and purification methodologies. The success and usefulness of these immunoassays heavily depend on the quality of these antibody reagents, yet the approaches to producing these critical reagents vary widely throughout the industry. We directly compared three main variations of these common approaches. Selection of host species, choice of adjuvant and the benefit of cascade immunization paradigms are analyzed by ELISA to assess titer and western blot to assess coverage.
• (Day 1: Generation of High Quality Anti-HCP Antibodies Session)

European Pharmacopoea HCP Chapter

• Speaker: Gwenaël Cirefice, EDQM-Council of Europe
• Abstract: (Pending)
• (Day 2: Health Authority Considerations Session)

Mass Spectrometry Brightening the HCP ELISA Results Black Box

• Speaker: Séverine Clavier, Sanofi R & D
• Abstract: Over the past few years, the use of mass spectrometry as an orthogonal method for HCP monitoring in biotherapeutic products has grown rapidly. Different approaches either exploratory or targeted have been developed to answer a growing number of questions and investigations: Could the decreased level of an excipient be due to an enzymatic degradation? Is this HCP increase due to one or several HCPs? Can we absolutely quantify this HCP of interest? Is our current downstream purification process allowing to get a “clean” drug substance? Is the Mock used for generation of our specific HCP Elisa kit representative of the HCP population that will be produced together with the monoclonal antibody? Where does the variation of response between two HCP ELISA kits come from?
  This presentation will be built on various case studies showing how HCP monitoring has evolved with the use of mass spectrometry towards a better understanding of HCP/mAb, HCP/purification stationary phases interactions and how this new information source is today modifying the biotherapeutic products development. Difficulties encountered for some HCPs detection and quantitation and some remaining challenges in the field will also discussed.
• Contributing Authors: Séverine Clavier, Dawid Bugnazet, Florence Cordier, Bruno Genet
  Structural Analysis, Characterization Service, BioAnalytics Department, Biopharmaceutics Development France, Sanofi R&D
• (Day 2: Mass Spectrometry as an Orthogonal Method Session)

Applying Platform Knowledge and Risk Assessment Tools to Define HCP Strategy for a Gene Therapy Program

• Speaker: Carl Co, Biogen
• Abstract: The detection and quantitation of HCPs for gene therapy programs presents new challenges to the HCP field, which has predominantly evolved using the CHO-based production platform. The utilization of non-standard host cell lines and fast to pivotal clinical trials of gene therapy programs make it challenging to generate process specific HCP assays. Using detailed characterization and different risk assessment tools, we present a strategy for HCP measurement in a gene therapy program.
• (Day 2: HCPs for Non-Antibody Products Session)

HCP Risk Assessment

• Instructors: Carl Co, Biogen; Marcus Haindl, Roche; Fengqiang Wang, Merck Sharp & Dohme; and Christina de Zafra, Amgen
• Abstract: (Pending)
• (Workshop 2-AM)

Design of Experiment (DOE)-Based Approach for Defining Critical Parameters in Host Cell Protein Analytical Method Development and Validation

• Speaker: Joachim Dietenberger, Boehringer Ingelheim
• Abstract: Host cell proteins (HCPs) are a complex mixture with potential impact to product quality and patient safety. Therefore, a reliable, validated analytical method showing a high sensitivity, selectivity and defined robustness/design space is mandatory, as well as, the characterization of critical reagents used, such as antibodies and antigens. A statistical approach for the development of an Enzyme-linked Immunosorbent Assay (ELISA) based analytical method used for quantification of HCPs is helpful to understand where critical parameters are and to reduce their effect/influence on accuracy and precision.
  Design of Experiment (DoE) is a commonly used systematic approach applied for product-/process development and is a key aspect of quality by design (QbD0. In recent times, the use thereof for analytical method development has increases continuously and the multivariable experimental design is gaining more and more importance as a powerful statistical tool providing high level of information with a reduced number of experiments.
  Following, DoE could also support the HCP ELISA method validation by using a predefined range of different factors. In this way, changes, such as formulation composition or buffer concentration will not require a further validation of the analytical method, as long as these changes are within the characterized and known design space.
  The focus of our current work is to apply a systematic, statistical approach for analytical method development and validation of HCP ELISAs by using DoE – in line with International Conference of Harmonization Q2(R1) and Q7 guidelines and general quality guidance USP chapter <1132> and Ph. Eur. 2.06.34.
• (Day 1: Reagent and Assay Lifecycle Management Session)

Data Independent Acquisition Modes for Identification, Quantification and Monitoring of Low-Abundance Host Cell Proteins During Monoclonal Antibody Bioprocessing

• Speakerr: Catalin Doneanu, Waters Corporation
• Abstract: A major portion of biopharmaceuticals today are produced by recombinant DNA technology using well-selected host cell systems. 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 provide 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 sensitivity. 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 LC/MS discovery assay described here relies on a novel data-independent acquisition mode recently implemented on a quadrupole/time-of-flight (QTOF) mass spectrometer, namely SONAR data acquisition. Instead of transmitting all peptide ions produced in the electrospray source, in SONAR mode, the quadrupole scans over the mass range of interest during the time required for recording a single mass spectrum by the TOF analyzer. Co-eluting precursor ions with different m/z are separated during the rapid quadrupole scan and their corresponding fragmentation spectra are acquired using an identical quadrupole separation. In this way, SONAR offers additional selectivity, by producing cleaner MS/MS spectra with less interferences.
  The first step of the HCP identification and quantification workflow is the HCP discovery assay performed in SONAR mode using extensive (90 min) peptide separations. Following data processing with Progenesis QI for proteomics 4.0, the HCPs are identified by a proteome-wide database search. In addition, SONAR MS/MS fragmentation spectra 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 resulted from the purification of the same biopharmaceutical, are analyzed by higher–throughput HCP monitoring assays employing data-independent MSE acquisition with 30 min peptide separations. The LC/MSE dataset is then searched against the spectral library for HCP identification and quantification at every step during biopharmaceutical purification. The HCP workflow described above was applied for identification and monitoring of HCPs from a purified monoclonal antibody. The antibody product, initially purified by Protein A affinity chromatography was further purified by SCX (strong cation exchange) chromatography using different elution conditions. All mAb samples were denatured, reduced, alkylated and digested with trypsin overnight. Two HCPs were identified across 5 mAb preparations following a CHO database search using Progenesis QIP 4.0 software. The same software was also used to build a database of CHO HCPs including peptide RT, 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 LC-MS HCP workflow with Progenesis QI for proteomics software.
• Contributing Authors: Catalin E. Doneanu¹, Alex Xenopoulos², Romas Skudas², Ying Qing Yu¹, Asish Chakraborty¹, and Weibin Chen^{1
  1} Waters Corporation; ² 2EMD Millipore Corporation
• (Day 2: Cutting Edge MS Technology for ID of HCPs Session)

Comparison of HCP LC-MS Workflow for Data Independent Acquisition of Host Cell Proteins

• Presenter: Fabien Dorange, Genethon
• Abstract: (Pending)
• (Poster)

Optimization of HCP LC-MS Workflow for Data Independent Acquisition of Host Cell Proteins

• Presenter: Lylia Drici, Novo Nordisk
• Abstract: Liquid chromatography coupled to mass spectrometry (LC-MS) has become over the last few years a technology of choice for protein analysis. The improvement in MS-based proteomics provides the identification and quantification of thousands of proteins in a single run. The shotgun approach yields to the identification of peptides generated from the proteolysis of complex protein mixtures. LC-MS analysis could become a complementary technique to the traditional ELISA method for HCPs detection and quantification.
  The challenges with analyzing HCPs in purification process samples are the great dynamic range of proteins as well as the complexity of the samples. Our focus is to develop a method that includes optimized sample preparation and a robust LC-MS analysis. Capillary flow liquid chromatography will be used for a better run-to-run reproducibility and increased robustness compared to nanoflow. To enable confident Data Independent Acquisition (DIA) for simultaneous identification and quantification, spectral library building will be based on the extensive fractionation of the samples followed by LC-MS analysis using Data Dependent Analysis (DDA) in order to confidently identify as many HCPs as possible. Routine identification and quantification of the HCPs will be carried out in the DIA mode using Thermo High Resolution (HR) MS instruments. DIA leads to fewer missing values in the dataset when compared to DDA mode as we are not limited by a predefined number of precursors.
  The coverage of the polyclonal antibodies used for HCP-ELISA is another objective that needs to be addressed. In fact, verifying that these antibodies are able to recognize all of the HCPs present in the sample is necessary for the ELISA validation step which is currently done by western blotting. We will develop an optimized immunoprecipitation procedure to enable LC-MS based HCP identification and hence determination of anti-HCP antibody coverage.
  The developed HCP-MS method will be used for both purification process optimization and for the validation of the antibodies used for HCP-ELISA.
• (Poster)

A Customizable, qPCR-Based Immunoassay Platform for Accurate Quantitation of Protein Impurities and Contaminants

• Presenter: Harald Ehlen, Thermo Fisher Scientific
• Abstract: Accurate detection, quantitation and removal of protein impurities and contaminants are critical in biopharmaceutical process development. The Applied Biosystems™ ProteinSEQ™ assay combines the specificity of antibody-protein binding with the sensitivity of quantitative PCR for accurate detection and quantitation of protein impurities and contaminants. The ProteinSEQ™ assay exhibits increased sensitivity, along with improved range, linearity and robustness. Representative data from a custom ProteinSEQ™ assay demonstrated a dynamic range of 0.04 to 20 ng/ml and a LOQ of 0.04 ng/ml.
• Contributing Authors: Darren J. Bauer, Harald Ehlen, Rita Shih and Darick Dayne
  Thermo Fisher Scientific, 35 Wiggins Avenue, Bedford, MA 01730 USA
• (Poster)

A Universal Quantification Standard Kit for Identification and Accurate Quantification of Host Cell Proteins

• Presenter: Quentin Enjalbert, Anaquant
• Abstract: In the context of the use of biotherapeutics, Host Cell Proteins (HCPs) can present a safety risk to patients. Therefore, HCPs identification and quantification must be monitored for consistency purpose. Currently, HCPs levels are typically measured with classical biochemical or immunological techniques, such as ELISA assays. Considering the time needed and the difficulty to develop specific antibodies, it appeared inconceivable to guarantee coverage of all every possible HCPs.
  In order to assay HCPs in biotherapeutics samples, mass spectrometry appears as an orthogonal tool to overpass limitations of other techniques. Also, stable isotope dilution strategy doesn’t appear judicious for high throughput quantification, label-free MS-based absolute quantification strategy was thus developed. This strategy consisted in performing titration curves with Escherichia coli protein standards accurately calibrated by the AAA-MS method. A 1/x linear regression was performed considering iBAQ values of each standard protein. Standards quantities were calculated from the best fit equation and plotted against added amount to assess accuracy and precision of the method. To go further, BSA obtained from the NIST was added in the sample as an internal standard. Once the performances benchmarked, all detected HCPs can be quantified in a single MS and label-free analysis, based on their iBAQ values. In spite of label-free aspect, performances assessment demonstrated highly accurate and precise results with high throughput potential. To go further we are currently testing the same approach with top3 strategy, to overpass protein standards limitations, and take advantage of peptides properties, particularly better stability. Associated with READYBEADS technology, it provides the first universal HCPs quantification kit. By adding one bead in the sample it becomes possible to quantify all HCPs present in the sample, in a single MS run.
• (Poster)

Host Cell Protein Analysis with Mass Spectrometry – The Power of Impurity Identification

• Presenter: Heiner Falkenberg, Protagen Protein Services GmbH
• 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 box 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.
  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.
  Additionally, isotopic labeled peptides were applied to quantify single host cell proteins within the processed drug substance. The use of internal standard combined with mass spectrometric LC-SRM (selected reaction monitoring) is a reliable method for absolute quantification. The robust nature of this approach offers the technical potential for validation and usage as GMP release testing for biopharmaceuticals to address the purity. However, multiple challenges need to be solved, to use MS within a release testing.
• (Poster)

Host Cell Protein Clearance in Downstream Purification Processes: Lessons Learned and Future Considerations

• Speaker: Jayme Franklin, Genentech
• Abstract: The clearance of host cell proteins (HCP) is a key consideration in the design of robust purification processes for biologically derived therapeutics. Bioprocess development activities rely heavily on historical knowledge as well as high throughput technologies to drive streamlined development. The advance in analytical technologies for the detection and identification of HCPs provides increased knowledge of HCP clearance in downstream purification processes but raises questions about how to utilize this information. The union of early HCP identification/characterization, high throughput purification technologies, and historical process knowledge must be shaped into a time-efficient workflow for the development of robust purification processes. This presentation will focus on the purification lessons learned from challenging to remove HCPs, the impact on current purification approaches and a look towards desired future state.
• (Day 1: Process Development Perspectives on HCP Monitoring Session)

Regulatory Aspects and the Pitfalls Regarding HCP Analysis and Documentation

• Speaker: Erika Friedl, Paul Erlich Institute
• Abstract: Host cell proteins (HCPs) are process-related impurities which could cause immunoreactivity in patients. In order to control these impurities, which are considered Critical Quality Attributes, specific guidance documents from the European Pharmacopoeia and the US Pharmacopoeia are in place. The strategies for HCP analysis and characterization are described. In addition to the regulatory framework for HCP assay development, these guidelines also allow for flexibilities to support product- and company-specific formats.
  Critical issues for interpretation and discussion are frequently related to the choice of the type of assay dependent on the development stage of the product, the way of setting specifications, the level of detail regarding the characterization of HCP species especially in view of the potent technology available and the documentation expected. These are only a few of an array of questions. Practical solutions for these HCP regulatory challenges are anticipated.
  Regulatory expertise will be shared to facilitate the interpretation of the regulatory framework. Case studies for recombinant blood products will be discussed to highlight the regulatory decisions taken.
• (Day 2: Health Authority Considerations Session)

Same but Different: A Case Study of HCP Comparability for an In-Licensed Project

• Speaker: Feny Gunawan, Genentech
• Abstract: This presentation will focus on a case study in which we evaluated the HCP comparability strategy for an in–‐licensed project. The challenges were two-fold: 1) to assess the HCP profile of in–‐house produced material in relation to the material produced by the previous company which was used in the pivotal trial; 2) to assess the suitability of the commercial HCP ELISA used by the previous company and compare it to the platform HCP ELISA used in–‐house. Various orthogonal methods were used to assess the HCP profiles of the two manufacturing processes, and to understand the differences in the HCP levels seen with the two different HCP ELISA methods. The strategies and considerations for overcoming these challenges will be discussed.
• Contributing Authors: Feny Gunawan, Nicole Liu, Don Walker Jr, Julie Nishihara, and Shelley Elvington
  Genentech, A Member of the Roche Group
• (Day 1: Reagent and Assay Lifecycle Management Survey)

Assessment of Platform Antibodies Over Process-Specific Antibodies for Estimation of Host Cell Proteins (HCPs)

• Presenter: Indranil Gupta, EDQM
• Abstract: (Pending)
• (Poster)

Protein-Centric ELISA Reagent Coverage Evaluation by Affinity Purification Mass Spectrometry

• Speaker: Scott Henry, Seattle Genetics
• Abstract: Downstream processing must adequately remove host cell proteins (HCPs) from recombinant therapeutics to ensure patient safety, product quality, and regulatory compliance. Typically, HCP levels are monitored using a polyclonal ELISA reagent that has sufficiently broad immunoreactivity (coverage) against the HCPs expressed by the production cell line. Gel-based methods that rely on optical comparison are utilized to evaluate coverage, and do not provide protein-specific definition of immunoreactivity. It is challenging to provide assurance that a given reagent can monitor all HCPs that persist downstream since ELISA reagents to do not provide 100% immunoreactivity. We describe protein-specific determination of ELISA reagent coverage by proteomic analysis following affinity purification using a polyclonal anti-HCP reagent (AP-MS). The resulting HCP identifications enable a highly focused assessment of ELISA reagent suitability relative to known downstream HCP impurities. The utility of this approach is illustrated by coverage evaluation against HCPs in a monoclonal antibody after Protein A purification, and against the HCPs present in a null cell harvest sample. Going forward, we envision using the AP-MS approach to help reconcile the results of ELISA and MS-based studies of HCP clearance. The AP-MS method also further enables a framework for HCP impurity analysis driven by protein-centric characterization of process impurities, in contrast to classical approaches that focus on evaluation of the total host cell proteome.
• (Day 1: Anti-HCP Antibody Characterization Session)

Efficient Coverage Analysis for HCP ELISA Assay Validation

• Speaker: Joe Hirano, GE Healthcare
• Abstract: The most widely used method to monitor the HCP levels in biologics is Enzyme-Linked ImmunoSorbent Assay (ELISA), which uses a set of polyclonal antibodies to detect HCPs as many as possible. These anti-HCP antibodies are critical reagents in the assay. The antibody coverage assay based on 2 Dimensional Electrophoresis (2DE) and subsequent Western Blot (WB) has been used to evaluate the performance of the anti-HCP antibodies. The result will be expressed as a % coverage of the antibodies to the total number of HCP spots separated by the 2DE (USP <1132> and Ph Eur 2.6.34).
  The challenges in the coverage assay are;
  1) Sensitive method to detect HCPs as much as possible
  2) High resolution to separate the complex mixture of HCPs
  3) Reliable comparison between total HCP & WB detection
  4) Time consuming in both experimental preparation and output data analysis
  We proposed an enhanced anti-HCP antibody coverage analysis method, 2D Differential In Blot Electrophoresis (2D-DIBE) in 2017 BEBPA HCP. The solution is based Fluorescent multiplexed methodology based on proven 2D-DIGE & WB technologies which enable simultaneous detection of total HCP & WB with image acquisition hardware and analysis software. In this presentation we focus on the technical aspect of 2D-DIGE and DIBE technology and how these solve the current challenges of the coverage assay.
  Joe Hirano, PhD
  GE Healthcare LifeSciences
  Björkgatan 30,
  751 84 Uppsala, Sweden
• (Day 1: Anti-HCP Antibody Characterization Session)

Title Coming Soon

• Presenter: Joe Hirano, GE Healthcare
• Abstract: (Pending)
• (Poster)

Title Coming Soon

• Presenter: Fabian Hosp, EDQM
• Abstract: (Pending)
• (Poster)

HCP Profiling and Lipase Monitoring for DNA Derived Biopharmaceuticals by LC/MS/MS

• Speaker: Lihua Huang, Eli Lilly
• Abstract: Residual host cell proteins (HCPs) in biopharmaceuticals derived from DNA technology can present potential safety risks to patients or compromise product stability. LC-MS has emerged as a powerful tool for residual HCP characterization and is used by the biopharmaceutical industry to support purification process development, together with a traditional ELISA. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs, especially for those related to product stability, such as lipases co-purifying with the molecule of interest. In this presentation, we describe simple and powerful methodologies to characterize residual HCPs in biopharmaceuticals by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics or parallel reaction monitoring approach. HCP spiking experiments demonstrated that our method could detect 0.5 ppm HCP with molecular weight > 60 kD, such as rPLBL2, by a shotgun proteomics or < 0.05 ppm by a parallel reaction monitoring approach.
• Contributing Authors: Lihua Huang, Ning Wang, Charles E Mitchell, Tammy Brownlee and Michael R De Felippis
• (Day 2: Mass Spectrometry as an Orthogonal Method Session)

Absolute Quantification of Host Cell Protein by SWATH LC-MS/ MS towards Method Validation and Batch Release Testing

• Instructors: Thomas Kofoed, Alphalyse
• Abstract: Mass spectrometry is an important orthogonal method to ELISA for detailed HCP analysis and identification of individual HCPs during process development. Mass spectrometry is not yet being used for GMP batch release testing due to complexity and difficulties in reproducible HCP quantification making it difficult to validate the method.
  Here we present a HCP analysis method based on a SWATH LC-MS/MS workflow using a new label free quantification algorithm. The final assay is fast, sensitive and provides reproducible identification and absolute quantification of individual HCP’s in the purified drug substance. The workflow is generic for small therapeutic proteins, mAbs, vaccines, and other protein biopharmaceuticals. We will present the new method to obtain accurate and absolute HCP quantification, and parameters critical for method validation, including reproducibility, linearity, LOD/LOQ and measurement range. Case examples will be shown on small therapeutic proteins, vaccines and mAbs.
• (Day 2: Cutting Edge MS Technology for ID of HCPs Session)

Title Coming Soon

• Presenter: Brian Kristensen, EDQM
• Abstract: (Pending)
• (Poster)

Making LC-MS for HCP Reliable: Challenges from Sample Preparation to Data Analysis

• Speaker: Nicolas Lebesgue, Novartis
• Abstract: LC-MS/MS has become an increasingly important orthogonal method for HCP analysis in biopharmaceuticals. Despite its sensitivity, LC-MS strategy needs to tackle the enormous dynamic range to permit robust identification of low abundant HCPs versus the high abundance of drug substance. Sensitivity and robustness are determined by multiple factors ranging from sample preparation over LC-MS instrumentation to data analysis. Here, we present and discuss factors, which are necessary to optimize, to obtain a reliable LC-MS analysis of HCPs. These general considerations are exemplified with product development applications.
• (Day 2: Mass Spectrometry as an Orthogonal Method Session)

Lipoprotein Lipase is a Difficult to Remove HCP: Studies from the 2018 Reference Genome

• Speaker: Kelvin Lee, University of Delaware
• Abstract: There is great interest in a better understanding of difficult to remove host cell proteins. We used proteomics to identify different classes of difficult to remove CHO host cell proteins. One of the proteins that was identified, lipoprotein lipase, may play a role in polysorbate degradation. We evaluated cell line development strategies to mitigate the expression of this protein and any resulting impact on polysorbate degradation. Analysis of the CHO and Chinese hamster reference genomes identifies a number of other lipases which may similarly impact product quality. We will discuss findings from the latest 2018 Chinese hamster reference genome in this context.
• (Opening Keynote Talk)

Looking for a Needle in a Haystack: Characterization and Quantification of the Host Cell Proteome in Drug Substances by a Novel 2D LC- MSe Approach

• Speaker: Benedetta Lombardi, Merck Serono S.p.A.
• Abstract: In the biopharmaceutical context, HCPs characterization has been classically conducted by bidimensional electrophoresis, which can provide a reference map of contaminating proteins generally present at the very beginning of the purification step (Cell harvest), but it is incapable of giving any sort of information on the nature of the proteins present and it is unable to detect low abundant HCPs.
  For what concerns HCPs quantification, it has always been conducted by immunodetection-based methods, particularly ELISA. Although ELISAs have been invaluable tools for the industry to monitor HCPs amount in process development and final product formulations, they suffer of important limitations, all largely dependent on the anti-HCP antibodies themselves.
  In recent years,many efforts have been made to develop analytical method able to provide a more comprehensive evaluation of HCPs, either at the qualitative and at the quantitative level.
  Liquid chromatography coupled to mass spectrometry (LC-MS/MS) represents an excellent tool to complement and, possibly, substitute ELISA in HCP characterization and quantification.
  Although mass-spectrometers based methods represents excellent approaches to analyze “standard” protein samples, the development of a mass-spec method to analyze HCPs in Drug Substances, still represents a challenge. In fact, you are facing an atypical sample, mostly composed by just a single protein (the DS, i.e. the haystack) and what you are interested in are those impurities representing, hopefully, less than 5% (the needles) of the total product.
  In the present study, the development and the validation of a novel analytical method based on multidimensional liquid chromatography coupled to high-resolution mass spectrometry was achieved. This novel method has been successfully applied to different DS, providing the full list of HCPs present in the formulation as well as their quantification.
  The application of Mass Spectrometry to the study of HCPs can lead to a completely new paradigm, where, for each DS (or IPC) there will bill be an associated Host Cell Proteome, i.e. the collection of all identified and quantified proteins for a particular manufacturing process. Through a deep bioinformatic evaluation of the Host Cell Proteome it would be possible to identify contaminating proteins raising specific safety issue and create a DS-specific fingerprint image, helping the constant monitor of product quality over time.
• References:
  Host Cell Protein Testing by ELISAs and the Use of Orthogonal Methods
  Zhu-Shimoni J, Yu C, Nishihara J, Wong R, Gunawan F, Lin M, Krawitz D, Liu P, Sandoval W, Vanderlaan M
  Biotechnology & Bioengeneering; Vol. 111, No. 12, December, 2014The Future of Host Cell Protein (HCP): Identification during Process Development and Manufacturing Linked to a Risk-Based Management for Their Control
  Bracewell D.G, Francis R, Smales M.C
  Biotechnology and Bioengineering, Vol. 112, No. 9, September, 2015
• (Day 2: Cutting Edge MS Technology for ID of HCPs Session)

Replacement of HCP Assay for a Marketed Product

• Speaker: Stanislav Mandelc, Sandoz/Novartis
• Abstract: During the lifetime of a product, the supply of critical reagents for HCP ELISA (HCP standard or anti-HCP antibodies) may become depleted. In such cases, new reagents need to be generated and a new assay may need to be developed and validated. Latest chapters of USP and PhEur provide basic guidelines how to proceed in case of changing HCP assay and/or reagents, however, details concerning the set of required activities and their extent remain unclear.
  Recently, replacement of HCP assay for a marketed product with a new assay received positive regulatory feedback. The activities performed included generation and characterization of new reagents (change of host animals for anti-HCP antibodies production from goat to rabbit), development of a new assay and comparability.
• (Day 1: Reagent and Assay Lifecycle Management Session)

Title Coming Soon

• Presenter: Giojie Mao, EDQM
• Abstract: (Pending)
• (Poster)

HCP Specification Setting

• Instructor: Ned Mozier, Pfizer
• Abstract: Justification of limits (or ranges) for host cell protein (HCP) methods used to test biopharmaceutical drug substances requires manufacturers to employ a broad understanding of bioprocess control, analytical method capabilities, and safety considerations. The phase of development is a factor, as the clinical experience with batches increases, and knowledge of the process capabilities at various scales of production become known. In many cases, test methods are also evolving, so it can be challenging to manage the data and confirm appropriate quality throughout product development. This workshop will begin with the general concepts of specification development and the application of various statistical tools, then transition to a summary of the unique problems associated with impurities and, finally, a thorough review of HCPs. Included in the workshop will be case studies and summaries of published literature, experience across various product types and expression systems, and a practical for how to determine limits. A range of scenarios will be discussed, along with a range of potential solutions based on HCP levels in various product types. Total HCP risk as well as individual, known, HCPs will be included in the discussion. The risk from updating or changing HCP assays during development or after commercialization will be shared.
• (Workshop 2-PM)

Frequent Challenges of Host Cell Protein ELISA Projects

• Presenter: Pia Paarmann, BioGenes GmbH
• Abstract: An important decision during Host Cell Protein (HCP) ELISA development concerns the choice of a suitable antibody for the immunodetection of a given HCP mixture. Affinity-purification of anti-HCP antibodies needs to be weighed against the use of a Protein A-purified total IgG fraction.
  Furthermore, sufficient coverage of the selected antibody, usually analysed by 2D electrophoresis/ Western blotting, is requested by authorities. The E. coli case studies presented here, address two different aspects: Firstly, we discuss the common advantages and disadvantages of an affinity-purified antibody and its corresponding total IgG fraction for HCP ELISA measurement of process samples. Secondly, the impact of 2D electrophoresis technical equipment on the representation and resolution of sample protein spots is detailed in the context of coverage determination.
• (Poster)

Assessing Immunogenicity Risk in Biotherapeutic Products and Process Development

• Speaker: Valarie Quarmby, Genentech
• Abstract: Every biotherapeutic has the potential to elicit unwanted immune responses, and these may compromise safety and efficacy. Immunogenicity risk ranking methods and tools are often used during lead selection and optimization to assess the likelihood that a biotherapeutic may be immunogenic. Some of these approaches may also be used to assess the impact of product variants or process related impurities on immunogenicity risk. This talk will review immunogenicity risk assessment systems in the context of process development and discuss host cell protein case studies.
• (Day 2: Health Authority Considerations Session)

What is Your HCP-ELISA Measuring? – Evaluated by SWATH-MS

• Presenter: Rikke Raaen Lund, Alphalyse
• Abstract: Product specific HCP-ELISA is widely used for batch-consistency and release test of recombinantly expressed protein drugs. During process development, before developing a product specific ELISA, generic HCP-ELISAs are frequently applied for semi-quantitative measurements of HCP content. The HCP-ELISA measures immuno-equivalents to “ng HCP/mg drug substance” and is highly dependent on the specificity of the polyclonal anti-HCP antibodies and on the HCP-standard used as reference. Therefore, different generic ELISAs will measure different HCP amounts and should be validated fit-for-purpose for the actual samples.
  HCP analysis by SWATH-MS has proven to be valuable for measuring HCP content of purified drug substance batches and for comparing in-process samples for protein purification process development. This analysis provides a detailed list of the HCPs identified in a sample including protein names, absolute amounts quantified by spiking in protein standards, accession numbers as well as theoretical pI and molecular weights of each individual protein.
  In the case described here, we have expanded the field of application of SWATH-MS HCP analysis to identify differences in the proteins measured by two different generic HCP-ELISAs. The HCP content of five in-process samples from an early stage drug production were measured by two generic HCP-ELISAs, and quite different outcomes between the two assays were observed. By combining the HCP-ELISA results with SWATH-MS analysis of HCPs, we were able to identify three specific HCPs covered by one assay and not the other. The results were furthermore supported by bead-based immunoprecipitation using antibodies from the two assays combined with LC-MS/MS.
• Contributing Authors: Anette Holck Draborg¹, Vyacheslav Akimov², Solveig Beck Nielsen³, Blagoy Blagoev², Søren Werner Karlskov Hansen¹, Ejvind Mørtz³, and Thomas Kofoed^{3
  1} Department of Molecular Medicine, University of Southern Denmark; ² Department of Biochemistry and Molecular Biology, University of Southern Denmark; ³ Alphalyse
• (Poster)

Characterization, Optimization and Application of an LC-MS based Workflow for HCP Identification, Quantification and Control

• Speaker: Michael Schrim, Caprion
• Abstract: The regulatory trend towards deeper characterization of host-cell protein (HCP) impurities has led to greater use of mass spectrometry (MS) to corroborate and supplement traditional immunoassays. MS-based assays are based on a bottom-up proteomic approach, and can be deployed relatively quickly without the need to develop antibodies, providing direct identification and quantification of proteins, including non-immunogenic and unknown proteins.
  MS-based HCP analysis involves several steps: [1] Sample processing (e.g. deglycosylation, denaturation, reduction/alkylation, digestion, desalting, and fractionation), [2] LC-MS analysis (e.g. unbiased (DDA/DIA) or targeted (SRM/MRM)), and [3] data analysis (e.g. database searches, quantification, curation and quality control). The way that each step is performed directly impacts the nature, quality, and reliability of the results.
  Thus, to demonstrate MS assay suitability for HCP analysis, we undertook a systematic approach to characterize, evaluate and optimize each workflow step. The results of these optimizations and case studies will be presented, showing unbiased and targeted approaches to HCP identification, quantification and control.
• Contributing Authors: Christina Bell, John Babetas, Laetitia Cortes, Lorella Di Donato, Rudolf Guilbaud, Stéphane Parent and Michael Schirm, Caprion Biosciences, Montréal, Canada
• (Day 2: Cutting Edge MS Technology for ID of HCPs Session)

Generation of High Coverage Anti-HCP Polyclonal Antibodies: Is Coverage of HCPs Impacted by Animals?

• Speaker: Christina Seisenbeger, Roche
• Abstract: Host cell proteins (HCPs) are process-related impurities and a potential critical quality attribute in the development of protein-based pharmaceuticals. Techniques to quantify HCPs generally include enzyme-linked immunosorbent assays (ELISA). HCP ELISAs are multi-analyte assays whose performance is tightly linked to the quality of the applied reagents like HCP antigen (HCP reference standard) and the corresponding target specific antibodies (antisera). The HCP-Antigen, used to produce polyclonal antibodies by immunizing a host species should contain a broad range of the representative HCP species of the biological manufacturing process. The immunogenicity of an individual HCP varies broadly making HCP quantification challenging. The polyclonal quality and immunogenic response as well as the ratio of HCPs recognized by the HCP ELISA relative to the population of HCPs (coverage) could be influenced by phylogenetical distance to CHO. Here, the influence of different host animals on the quality and coverage is examined. Choosing the most appropriate host animal is one important factor to produce CHOP specific polyclonal antibody reagents with high coverage over the entire range of HCPs.
• (Day 1: Generation of High Quality Anti-HCP Antibodies Session)

Development and Validation of a Platform ELISA for Quantitation of Host Cell Proteins

• Presenter: Anne-Sophie Sikora, UCB Pharma
• Abstract: The Host Cell Proteins (HCP) are complex mixtures of proteins produced by a host cell during production of a biotherapeutic. HCP levels must be monitored and controlled during the purification process as residual HCP may impact efficiency of the product and present a safety risk for patients. The most commonly used analytical method to monitor HCP levels is enzyme-linked immunosorbent assay (ELISA).
  Several generic HCP ELISA kits are commercially available. They allow the detection of a majority of HCP species but may not be able to detect a subgroup of proteins specific to a certain cell strain. These kits have the advantage of eliminating lengthy assay development time and are often used for early phase development.
  Nevertheless, for phase III and commercial, regulatory agencies suggest to company to develop ELISA specific for HCP of their own cell strain in order to obtain highly specific and sensitive assay. The development of such an assay has a cost. However, developing an HCP assay that can be used for all products derived from a particular cell strain when the upstream conditions are similar is a worthwhile investment for companies. We thus developed and validated a platform ELISA for quantitation of HCP.
  The poster will report the different steps of the development and validation of in-house platform assay for quantitation of HCP from generation of reagents to the ELISA method.
• UCB Pharma, Chemin du Foriest, 1420 Braine l’Alleud, Belgium
• (Poster)

The Challenges of Developing HCPs Methods for Vaccines – A Risk Based Approach to HCP Monitoring

• Speaker: Lee Smith, GreyRigge Associates, Ltd
• Abstract: While all host cell protein (HCP) determinations present substantial technical challenges, the production of a live viral vaccine product brings its own unique obstacles when attempting to estimate the quantity of residual HCP in a vaccine. We know that the regulatory authorities examine each product submission on a case-by-case basis. This allows specifications to ultimately be set that consider the level of patient risk addressing dose levels, the route & schedule of administration and the healthy patient population receiving the vaccine. This presentation will cover some of the journeys taken by organisations who began by working with commercial off-the-shelf (COTS) HCP immunoassays. It will reflect on the decisions taken to address assay deficiencies and how methods might evolve beyond COTS kits to construct unique HCP immunoassays that endeavour to broaden the number of HCPs detected in samples to an appropriate level. This will also include thoughts on the critical and challenging elements involved when attempting to generate suitable immunogens through generation of antigen, its characterisation and ultimately the generation of serum that provides broad and acceptable coverage. Aspects of discussions held with regulators on HCP levels during the product development process will also be included. Finally, thoughts will be expressed on how assays might best be developed and validated, including the generation, supply and distribution of critical regents and materials for up to and beyond product launch. This will address the questions around long-term supply of consistent & reliable immunoassays that span the commercial life of a vaccine.
• (Day 2: HCPs for Non-Antibody Products Session)

Advanced Mass Spectrophotometry Analysis of HCPs

• Instructor: Martha Staples, Genzyme
• Abstract: (Pending)
• (Workshop 1-PM)

Streamlined and Automated Immunoassays Adapted for Bioprocess Workflows Accelerate QbD Strategy Initiatives

• Presenter: Ann-Charlott Steffen, Gyros Protein Technologies
• Abstract: Bioprocess development has rapidly evolved to incorporate Quality-by-Design (QbD) principles that depend on deep scientific understanding about each process. Scaled down models and high-capacity testing generate large numbers of samples for analysis, increasing the demand for efficient and timely analytical support to monitor quantity and quality in products and processes. This necessitates reliable and robust analytical methods that can meet different challenges during development e.g. changing conditions and matrices, and the rigors of the regulated environment.
  These processes consist of many iterative stages, including selecting clones that must be optimized for product yield, and monitoring levels of impurities during downstream processing. Immunoassays play a major role in analytical support and there are clear advantages in quickly running multiple assays in parallel and on the same platform to support efficient data-driven decision-making.
  The newly introduced Gyrolab® xPand system automates affinity flow-through immunoassays to enable the rapid generation of highly reproducible results for multiple assays in parallel from the purification of a human IgG antibody produced using Chinese Hamster Ovary (CHO) cells. Delivering results in less than an hour, Gyrolab immunoassays are matrix insensitive for robust analysis from stage to stage, minimizing the risk of repeat analysis due to interference.
  Using this system, we were able to:
  • Automatically analyze the dilution linearity and spike recovery data required for assay qualification, and apply acceptance criteria according to current guidelines
  • Evaluate the specificities of several host cell protein (HCP) assays in a single run
  • Analyze IgG drug titer together with HCP and protein A impurities in 38 bioprocess samples from a single unattended run in less than four hours
  Combined with ready-to-use kits, Gyrolab xPand promises to streamline the implementation of QbD principles in bioprocess development and long-term monitoring.
• (Poster)

Beginning Mass Spectrophotometry Analysis of HCPs

• Instructor: Kevin Van Cott, University of Nebraska
• Abstract: (Pending)
• (Workshop 1 AM)

Knockout of Difficult-to-Remove and Troublesome CHO Host Cell Proteins to Create a Clean CHO Cell

• Speaker: Bjorn Gunnar Voldborg, NNF Center for Biosustainability
• Abstract: The Chinese hamster ovary (CHO) Cell Line Engineering department is addressing the need to obtain high yields and quality of protein biopharmaceuticals produced in optimized CHO cells through genome-scale-based methodologies. Host cell proteins (HCPs) are one of many process-related impurities generated during the production of biotherapeutic protein products. Although many HCPs are effectively removed in downstream purification processes, a small population of HCPs are particularly challenging. Because of the very high titers and demanding purity standards (typically 1-100 ppm HCP in final formulations), downstream processes generally account for the majority of production costs. It has been estimated to represent 50–80% of total mAb manufacturing costs. In production of a mAb, protein A chromatography is often employed as a generic first step, followed by two or three orthogonal polishing steps.
  Many studies have focused on identification of CHO HCPs that co-purify during mAb downstream processing, but few have removed troublesome HCPs by genome engineering. In this study, we use CRISPR/Cas9-mediated gene disruption to create a clean CHO cell line. HCP targets were selected based on three criteria: (i) abundance in the CHO supernatants analyzed by mass spectrometry, (ii) co-purification with mAbs, and (iii) negative impact on mAb product quality. In addition, we speculate that the removal of endogenous CHO proteins frees up “translational and metabolic power”, which can potentially be used by the cell to reach faster growth rates, higher cell density, and higher specific productivity.
  MiSeq analysis and targeted proteomics were used to check for indels and confirm the absence of our proteins of interest, respectively. We analyzed total HCP content of a 6-fold, 11-fold, and 14-fold knockout and characterized their growth in shake flasks and bioreactors. We observed a strong reduction (up to 60%) in total HCP content and improved viable cell density and viability. Purification of a monoclonal antibody (mAb) produced in these cell lines also led to a reduction in HCP content when measured at different steps during the purification process. A collaboration with KAIST Korea has recently been started to address the effect of deleting the targets that have been reported to have a negative impact on mAb quality. In parallel, a collaboration with the NNF Center for Biosustainability at the University of California has been started to analyze expression and proteomics data of our knockouts.
• Contributing Authors: Stefan Kol, NNF Center for Biosustainability, CHO core, Technical University of Denmark (DTU) stko@biosustain.dtu.dk
  Daniel Ley, NNF Center for Biosustainability, Quantitative Modelling of Cell Metabolism, DTU
  Tune Wulff, NNF Center for Biosustainability, CHO core, DTU
  Gyun Min Lee, Department of Biological Sciences, KAIST, Republic of Korea
  Nathan E. Lewis, NNF Center for Biosustainability at the University of California, CA, USA
  Helene F. Kildegaard, CHO Cell Line Engineering and Design, DTU
  Bjørn Voldborg, NNF Center for Biosustainability, CHO core, DTU
• (Day 1: Process Development Perspectives on HCP Monitoring Session)

Effect of Imaging Parameters on 2D-DIBE HCP Coverage Analysis

• Presenter: Sonja Voordijk, GeneBio
• Abstract: Characterization and testing of the anti-HCP antibody reagents used in HCP ELISA assays is commonly performed by 2D-PAGE separation followed by Western blotting. However, vastly different spot patterns often seen between independent gels and blots present serious challenges to coverage assessment. We showed earlier that the application of 2D Differential in Blot Electrophoresis (2D-DIBE) removes the need for subjective and laborious gel-to-gel matching, makes spot review easy, quick and less subjective through highly resolved spots, and allows for increased sensitivity.
  In this work, we use the dedicated Melanie Coverage software to look at the effect of image acquisition device, image resolution and image bit depth on spot detection sensitivity in 2D-DIBE images, and the impact on coverage. We show that choosing appropriate imaging parameters can significantly improve the sensitivity and so the value of 2D-DIBE based coverage analysis. We therefore recommend that image acquisition conditions are properly reported when publishing work.
• Contributing Authors: S. Voordijk¹, G. Bouchet², A. Hamilton³, K. Larsson³, B. Haldeman⁴, D. Walther², I. Xenarios², and J. Hirano^{3
  1} Geneva Bioinformatics (GeneBio) SA, Geneva, Switzerland; ² SIB Swiss Institute of Bioinformatics, Geneva, Switzerland; ³ GE Healthcare Life Sciences, Uppsala, Sweden; ⁴ GE Healthcare Bio-Sciences Corp., Marlborough, MA 01752, USA
• (Poster)

Challenges and Lessons Learned in Implementing an In-House CHO Platform HCP Assay for Process Characterization

• Speaker: Fengqiang Wang, Merck Sharp & Dohme
• Abstract: Platform HCP assay has been widely used in the biotech industry as the workhorse to monitor HCP clearance during biologics process development and drug substance release. To meet the needs for HCP monitoring in multiple GS-CHO-produced monoclonal antibodies (mAbs), an in-house GS-CHO platform HCP ELISA was developed and implemented for drug substance release testing after reagents and assay qualification. However, while implementing the same assay for process characterization, HCP readouts in one of the process intermediates (protein A) were much lower than the results measured by using a commercial HCP ELISA kit during bridging, which raised concerns over the platform assay’s sensitivity and coverage to HCPs in the process intermediates. An investigation was performed to address these concerns using multiple orthogonal approaches, including 1D/2D SDS-PAGE and Western blot, 2D-DIGE, SEC-HPLC coupled with ELISA, and LC-MS. Investigation results indicate that the platform HCP assay antibodies have broader coverage to HCPs than the anti-HCP antibodies used in the commercial kit, despite being less reactive than the commercial kit antibodies to certain hyper-immunoreactive proteins with very low abundance. In addition to the antibody coverage, platform HCP ELISA performance in process intermediates including precision, accuracy, and sample dilutional linearity were also evaluated and demonstrated superiority over the commercial kit. Overall, despite the numerical differences in HCP levels observed in protein A process intermediates, the platform assay’s suitability was demonstrated and implemented for process characterization.
• Contributing Authors: Fengqiang Wang, Dennis Driscoll, Yuetian Chen, Hans-Martin Mueller, Daisy Richardson
  Biologics Analytical Sciences, MRL, Merck Sharp & Dohme, 2000 Galloping Hill Road, Kenilworth, NJ 07033
• (Day 1: Process Development Perspectives on HCP Monitoring Session)

Tracking Host Cell Proteins While Biopharmaceutical Manufacturing: Advanced Methodologies to Improve Product Quality

• Presenter: Stefanie Wohlrab, Roche Diagnostics GmbH
• Abstract: During manufacturing of biopharmaceuticals prokaryotic and eukaryotic protein expression host systems generate a heterogeneous mixture of the desired therapeutic protein and a variety of endogenous host cell proteins (HCPs) with different physicochemical and immunological properties. HCPs, as the predominant class of process-related impurities are undesirable in the purified protein product as they are suspected to have an impact on patient safety. To detect small total quantities of HCPs against the background of protein product being present at huge excess highly sensitive and specific analytical methods with a wide dynamic range are required. Multi-analyte enzyme-linked immunosorbent assays (ELISA) capable to detect a majority of protein impurities are routinely used to quantify and monitor HCPs. The quantitative values obtained by HCP ELISA represent a cumulative parameter of all the immune–reactive HCPs present in a particular sample, unresolvable into individual HCP contaminants. Hence, mass spectrometric based methods provide a universal approach to identify and characterize HCPs without need for antibodies. The liquid chromatography tandem mass spectrometry (LC-MS/MS) technique is capable of addressing the complexity and dynamic range of HCP pools in bio-process samples, typically with a sensitivity in the lower ppm range. Here, we describe a straightforward method for performing sample processing from denaturation and reduction through elution and fractionation of purified peptides in a single reaction chamber.
• Contributing Authors: Stefanie Wohlrab¹, Regina Kufer¹, Michael Wiedmann¹, Martina Suessmair¹, Ingo Lindner², Don Walker², Christopher Yu², and Markus Haindl^{1
  1} Pharma Technical Development, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany;
  ² Analytical Development and Quality Control, Pharma Technical Development, Genentech Inc., 1 DNA Way, South San Francisco, CA94080, United States of America
• (Poster)

HCP Antibody Purification from CHO K1SV Antisera for Broad HCP Coverage from BMSCHO Cell Line

• Speaker: Lei Zhang, Bristol-Myers Squibb
• Abstract: Host cell proteins (HCPs) are a heterogeneous mixture of proteins from host cells that may be present in small quantities in biotechnologically produced therapeutics. The wide variability in abundance and diversity of HCPs are persistent challenges during the development of downstream purification process for therapeutic proteins. Enzyme-linked immunosorbent assay (ELISA) is widely used to measure residual HCPs in therapeutic products based on immunological binding between HCPs and the polyclonal antibodies (pAbs) used. These pAbs are typically generated by inoculating HCPs into animals. Subsequent purification and enrichment from antisera play a very important role in defining the quality of pAbs used in Immunoassays. In this study, antisera have been generated using HCPs from CHO K1SV cell line and the goal is to generate pAbs suitable for HCPs produced from a BMS proprietary CHO cell line. We compared HCPs from CHO K1SV and the BMS CHO cell lines using DIGE. We also compared different purification strategies: 1) Protein G affinity purification, and 2) HCP antigen affinity purification using antigens from different CHO cell lines. Western blot by 1D/2D have been used to evaluate quality of pAbs and to compare coverage from different purification schemes. This study demonstrated that pAbs generated using CHO K1SV HCP antigen and purified using BMS CHO HCP antigen purification are able to recognize a broad range of HCPs from BMS CHO cell line. The overall performance in an ELISA measuring range of HCPs during biological manufacture process.
• (Day 1: Anti-HCP Antibody Characterization Session)