Abstracts (Alphabetical by Speaker/Instructor/Presenter)

Control of Host Cell Proteins throughout Product Lifecycle

• Speaker: Alexey Khrenov, OTAT/CBER/US FDA
• Abstract: Host Cell Protein (HCP) impurity is a critical quality attribute for biological products because it can affect product quality, efficacy and safety. Safe level of HCP is established prior to product licensure, and the manufacturing process should demonstrate the ability to remove HCP to below safe level. Control strategy for HCP should include appropriate analytical methods, reference materials, and well-justified acceptance limits. It is common for manufacturers to make changes to the HCP assay due to reasons such as changes in the manufacturing process, depletion of critical reagents, or requests from regulators. Establishing the suitability of a new HCP assay is a challenging task, which will involve the qualification of critical reagent(s) and possibly multiple bridging studies. In addition to the validation of the assay, the acceptance criteria of the release specification and in-process controls should also be re-evaluated using proper statistical analysis and adjusted if necessary. In this presentation, I will discuss my perspective on the current regulatory expectations on establishing control strategies for HCP, as well as how to maintain control of the level of HCP when changing the assay. Case studies illustrating the challenges associated with assay replacement will be discussed. My comments are an informal communication and represent my own best judgement. These comments do not bind or obligate FDA.
• (Day 1: Regulatory Trends Session)

Case Study: Using Our HCP Analysis Suite to Select the Most Appropriate ELISA for a Late Stage Program

• Speaker: Margaret Lin, Genentech
• Abstract: We present a case study reviewing a suitability assessment of an existing HCP ELISA test method on the control system of a late stage program. This program had previously undergone several process changes with relatively consistent levels of HCPs measured by the existing test method. As part of the assessment, the program was assessed using the current internal test method, a platform HCP ELISA, which resulted in higher observed HCP levels and dilutional-non-linearity. Moreover, CHOP levels using the internal test method measured distinctly different HCP levels between 3 modified processes. From these observations, extensive characterization was performed to better understand the differences between the two test methods and select the test method that best ensures patient safety.
• (Day 1: Assay and Reagent Development Session)

Transitioning a Late Stage Gene Therapy Program from a Commercial Kit to a Platform, In-House Immunoassay

• Speaker: Cullen Mason, Biogen

• Abstract: Sandwich immunoassays, most commonly enzyme-linked immunosorbent assays (ELISAs), are the workhorses of HCP detection and quantitation for biological samples. The antibodies used in these assays are generated by immunizing animals with harvest material generated from host cell lines. Commercially available assays may be used for HCP quantitation up until process validation; however, once a program advances to phase III and beyond, the assay reagents need to be specific to the process HCPs. Transitioning a program from a commercial kit to an in-house platform or process specific method requires the completion of a bridging study which demonstrates assay suitability and supports assay replacement. A case study will be presented to demonstrate how one such late stage gene therapy program was transitioned from a commercial assay to an in-house platform assay.
• (Day 2: Gene Therapy Session)

USP Standards to Support Host Cell Protein Analysis

• Speaker: Diane McCarthy, USP
• Abstract: Residual host cell proteins (HCPs) in biotherapeutic products can pose a risk to patients and to the quality of the product and therefore must be monitored and controlled. Over the past several years, the use of mass spectrometry to identify and quantitate host cell proteins has become increasingly common, but results can be difficult to compare across different laboratories and organizations. This presentation will provide an overview of USP efforts to support consistency of mass spectrometry-based HCP analysis through development of documentary standards outlining best practices for HCP analysis and development of new physical standards to support identification and quantitation of high risk and high abundance HCPs.
• (Day 1: Regulatory Trends Session)

Residual Analysis of cGMP Adeno-Associated Virus and Lentivirus for Human Gene Therapy

• Speaker: Michael Meagher, St. Jude Children’s Research Hospital
• Abstract: (Pending)
• (Day 2: Gene Therapy Session)

Case Study – Comparison of Orthogonal Methods for Reliable HCP Coverage Determination

• Speaker: Pia Paarmann, BioGenes GmbH
• Abstract: Host cell protein (HCP) coverage analysis is one of the most crucial parts of the characterization/ qualification of HCP antibodies. Broad coverage of the given HCP spectra is required to confirm reliable impurity measurement with the corresponding ELISA methods. Conventionally, antibody coverage has been determined by means of 2D Western Blotting. Recently, different orthogonal methods have emerged to support coverage analysis and to overcome the natural limitations of the Western Blot approach. Moreover, technical equipment for performing 2D electrophoresis as well as the available staining methods and evaluation software have been further developed in order to provide improved sensitivity and precision of coverage determination. Consequently, the interpretation of HCP antibody coverage values has become rather complex. The results are highly method-dependent and may significantly vary between different approaches.In this case study, we compared 2D fluorescence Western Blotting and Immuno-AffinityChromatography (IAC), followed by 2D difference gel electrophoresis (2D DIGE) for characterization of HCP antibody coverage on a Chinese Hamster Ovary (CHO) cell line derived sample.By direct comparison, we aim to support the interpretation of absolute percentage coverage values considering the methodological and technical details, as well as the advantages and disadvantages of the respective analytical method.
• Contributing Authors: Dr. Pia Paarmann (BioGenes), Stefan Sommerschuh (BioGenes)
• (Day 1: Assay and Reagent Development Session)

Combinatorial HCP Analysis and Characterization using LC-MS/MS-MS: Gene Therapy

• Speaker: Lake Paul, BioAnalysis, LLC
• Abstract: The utilization of the combinatorial approach not only to quantitate the Host Cell Proteins (HCP) but also Post-Translational Modifications (PTMs) in the Gene Therapy Space can be accomplished through a robust LC-MS/MS-MS protocol. A single point absolute quantitation scheme can be employed to evaluate both the levels of HCPs and detected PTMs on AAV systems. Refinement of the initial database searches along with manual validation can provide confidence in the HCPs and PTMs. This combinatorial approach aids with mitigation strategies in regards to HCPs and elevated PTMs (e.g. oxidation and deamidation) from a single LC-MS/MS-MS experiment thereby reducing experimental times, costs, and labor.
• (Day 2: Gene Therapy Session)

HCP Profiling in Commercial mAbs and Biosimilars

• Speaker: Rikke Raaen Lund, Alphalyse
• Abstract: What HCPs are present in approved mAb products on the market? How do the HCP profiles compare between different commercial mAbs? What are the levels of HCPs of potential concern for product quality and patient safety? How do Biosimilar HCP profiles compare to Originator?
  We will present HCP profiles of 15 originator mAbs and biosimilars, expressed in CHO and murine cell lines. The HCP content is analyzed using an optimized workflow for identification and absolute quantification of total and individual HCPs in purified mAbs. The workflow is based on automated, high throughput sample preparation and LC-MS analysis. Analysis performance will be demonstrated on the NISTmAb reference (IgG1 expressed in murine cells), including native digest providing sub ppm level HCP detection.
• (Day 2: ID/Management of ID’d HCP’s Session)

Development and Qualification of an Absolute Quantification Workflow for an Immunogenic HCP

• Speaker: Veronika Reisinger, Novartis
  

  

• Abstract: ELISA is the standard method for HCP detection but is usually not capable to identify and quantify single HCPs. Therefore, LC/MS-based methods where residual HCPs can be detected, identified, and quantified individually, are more and more routinely used for process design and testing of final drug substance.For tracking of one specific HCP, an absolute quantification workflow based on LCMS/MS was developed and qualified regarding different parameters like linearity or repeatability. The method was successfully applied to support process optimization as well as increase knowledge on the final drug substance.
• (Day 2: ID/Management of ID’d HCP’s Session)

Generation of High-Coverage Anti-CHO-HCP Antibodies: Is Chicken as Good as Promised?

• Speaker: Christina Seisenberger, Roche Diagnostics GmbH
• Abstract: Host cell proteins (HCPs), constituting the major part of process-related impurities during manufacturing of protein-based biopharmaceuticals, are generally considered a critical quality attribute (CQA) due to their potential immunogenicity and impact on product stability. Depletion of these HCPs is routinely monitored by enzyme-linked immunosorbent assays (ELISA), whose performance primarily relies on the capability of the employed anti-HCP antibodies to cover HCPs featuring diverse molecular properties. Coverage, in this context, refers to the ratio of HCPs recognized by the HCP ELISA relative to the total population of HCPs.Besides on the characteristics of the immunogen itself, generation of appropriate anti-HCP antibodies also depends on the applied immunization protocol and the selected animal. For instance, immunogenic responses may hinge on the phylogenetic distance between the HCP origin (usually CHO proteins) and the immunized host. As a consequence, the immunization of phylogenetically less related species (e.g. chicken) hold great promise to improve HCP coverage relative to conventional approaches. Putting this theory to the test, a comparative study of anti-CHOP HCP antibodies originating from several host species will be presented here.
• Contributing Authors: Christina Seisenberger, Stefanie Wohlrab, Ulrich Mohn, Michael Wiedmann, Markus Haindl
• Affiliation: Pharma Development, Roche Diagnostics GmbH, Penzberg, Germany
• (Day 1: Assay and Reagent Development Session)

Improving Product Quality and Enabling Patient Risk Assessment by LC-MS/MS Analysis of Host Cell Proteins in Inhalation Products

• Speaker: Lars Skriver, Savara Pharmaceuticals
• Abstract: Controlling HCP’s in biopharmaceuticals is critical for the patient’s safety. HCP measurements and HCP characterization are pivotal factors in this control. We present our strategy to develop a platform HCP ELISA for release testing of an E. coli expressed protein drug substance and the use of LC-MS/MS orthogonal technology to select the best HCP antigen preparations for HCP antibody development. The use of this LS-MS/MS technology for HCP characterization and evaluation of HCP process clearance will be discussed. Manufacturing process consistency and robustness is documented by identification of the same top 4-5 HCP species in drug substance batches produced during development and in commercial scale. The knowledge of HCP identity and amounts present in drug substance batches will be used in discussion of patient safety issues for an inhaled drug product.
• (Day 2: ID/Management of ID’d HCP’s Session)

Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation

• Speaker: Andreas Zerr, Lonza AG
• Abstract: We developed a plate reader-based assay to detect lipolytic activity in biopharmaceutical DS and DP, and, also in partly purified protein containing solutions as intermediate pools of the downstream purification process. The assay was optimized by variation of key parameters, e.g., the concentration of buffer components, the assay pH, and substrate and inhibitor concentration. In addition, we tested if typical excipients (e.g., polysorbate) and their degradation products have an influence on the assay readout, e.g. by product inhibition, quenching and auto‐hydrolysis. We conclude that the assay can support the optimization of the downstream purification process in order to remove HCPs with lipolytic activity.
• (Day 2: ID/Management of ID’d HCP’s Session)