LC-MS has become an increasingly valuable analytical approach in the analysis of host cell protein (HCP) for biotherapeutic products. There are unique challenges in the application of LC-MS for HCP analysis of new modalities, including adeno-associated virus (AAV) products. The presentation discusses how to leverage our existing knowledge and expertise from biologics using LC-MS to overcome these challenges and generate comprehensive HCP profiles for AAV-based gene therapy products.

Regulatory authorities are increasingly requesting orthogonal LC-MS data for residual protein characterization and documentation. Often, available ELISAs do not have sufficient HCP coverage for the new manufacturing process and complex product. The presentation will present and discuss experiences from using quantitative LC-MS analysis on more than 300 projects, with case examples from vaccines, viral vector therapies, method validation, and GMP release testing.

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs still show significant HCP copurification. Based on our investigation of 30-ish mAb molecules, we proposed a novel mechanism for HCP copurification; i.e., mAb-clustering strengthens interactions between mAb with HCPs. Breaking mAb-clusters effectively prevents HCP copurification.

• Only small subset (10%) of mAbs exhibited significant HCP copurification issue
• High-HCP mAbs share many common HCPs and similar copurification mechanism
• HCP copurification correlates with mAb's self-association propensity
• mAb-clustering strengthens interactions between mAb with HCPs
• Breaking mAb-clusters effectively prevents HCP copurification

For production of high-quality medicines, efficient removal and control of impurities during product development and lifecycle is mandatory. As critical quality attributes, HCP impurities are covered by specific guidelines. It is often challenging meeting the regulatory expectations regarding HCP removal, characterization, and control throughout the product lifecycle. Therefore, HCP case studies will be presented to highlight and mitigate the hurdles affecting the quality, efficacy, and safety of medicinal products.

Host cell proteins (HCPs) are process-related impurities monitored during expression of biotherapeutics. HCPs must be well-characterized, monitored, and reported. The most widely used method for monitoring HCP is an enzyme-linked immunosorbent assay (ELISA), due to its high-throughput, sensitivity, and selectivity. We report the development of a robust HEK293 total HCP ELISA which demonstrates superior sensitivity and precision for process clearance and DS samples over all existing assays.

Problematic HCPs in extremely low quantities in biological drug products poses a major challenge towards their identification and characterization. We developed a highly sensitive, fast and high-throughput method to monitor the presence of hydrolytic activities in samples generated during bioprocessing. By utilizing a custom-designed surrogate substrate combined with a well-established and robust detection platform, the method provides a rapid electrochemiluminescence-based readout of the hydrolytic impurity status in a given sample.

Each biotherapeutic production host cell line has its own unique signature of expressed HCPs. Even within the same cell line, upstream culture condition change can influence HCP profile and abundance. ELISA is the gold standard for total HCP quantitation. However, individual HCP information can’t be captured in the global HCP quantitation approach. Therefore, an LC-MS/MS workflow is established for in-depth and comprehensive understanding of the HCPs in our manufacturing processes.

Increases in mAb titre has been achieved mainly by cell culture feed improvement and cell line engineering to increase cell density and specific mAb productivity. This has caused a higher accumulation of host cell proteins (HCPs) in the supernatant. Herein, a population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in bioreactors to predict HCP dynamics. The PBM then served as a platform for generating operating strategies that optimise antibody titre and increase cost-efficiency while minimising impurity levels. 

Residual HCPs in biopharmaceuticals can impact product quality and safety. Mass spectrometry has become an increasingly common approach for identification and quantitation of HCPs to support risk assessment and process development, as well as characterization and quality control. In response to stakeholder needs, USP is developing standards and tools to support the quality and consistency of HCP measurements. The presentation will provide an overview of the proposed general chapter <1132.1> Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry and an update on development of physical reference materials to support identification and quantitation of HCPs of particular concern.

• Comparing a platform assay against a second assay (e.g., commercial kit) for relevance and prospective uses
• Strategies for transitioning from a commercial kit to process-specific or platform assay (antibody coverage, bridging, phase appropriateness) 
• Immunoassays for commercial phase DS manufacturing
• ELISA test results for individual HCPs on DS release specs​

• Why is there a need for LC-MS as a release method? ​
• What types of products/biologics would it be particularly relevant for?
• What are the major challenges using LC-MS as release test? And how could these be overcome?
• What will the new USP chapter and USP standards for HCP Analysis by Mass Spectrometry lead to?
• What are relevant product release specifications CoA – total HCP amount? Individual amount of problematic HCP? Amount of Top20 HCPs?
• Should animal ethics be a concern? Would you choose LCMS instead of ELISA that requires immunization and use of experimental animals?

A new method is developed to quantify HCPs at sub-ppm levels with ProteoMiner enrichment, coupled with limited digestion, followed by targeted analysis with nano liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%–111% of the theoretical value, and inter-run and intra-run precision within 12% and 25%, respectively.

During drug manufacture, large volumes of samples are submitted for host cell protein (HCP) quantitation to assess purification efficiency and ensure patient safety. Therefore, it’s beneficial to have methods with enhanced throughput. Here, we describe development of a custom HCP method that incorporates automated sample preparation with automated immunoassay and data analysis, resulting in a higher throughput assay that produces high-quality data needed to support gene therapy product development.

Host cell proteins (HCP) are co-produced into cell culture harvest and must be removed to acceptable low levels by downstream process due to their potential impact on product quality, safety, and efficacy. The suitability of the analytical method (i.e., stringency) used to detect and quantitate these impurities is typically related to the development cycle at which the biologics is. This presentation will discuss the bridging and validation strategy used to support HCP analytical method change in a late phase drug development, to meet an accelerated timeline for regulatory filing.

The presentation shows our strategy for LC-MS/MS based HCP characterization workflows to enable fast support of process specific development. Three case studies will be discussed, including 1) using quantitative MS methods to support process-specific method development for PRDX1 clearance, when the specific ELISA is not available, 2) supporting downstream process development using a robust high-load LC-MS/MS method, and 3) identification of low-level polysorbate degradation enzymes in the final purification pools.

Host cell proteins (HCPs) are one of the process-related impurities that may cause issues with the safety and stability of biotherapeutics. This presentation will cover recent analytical method innovations in sample preparation and mass spectrometry detection, to achieve in-depth characterization for low abundant HCPs. In addition, comprehensive HCP profiling by LC-MS coupled with proteomics analysis could help understand the HCP abundance change and its impact on various biomanufacturing processes.

Host cell proteins (HCPs) are process-related impurities that have the potential to impact patient safety and product efficacy. Even with state-of-the-art downstream processing to remove HCPs, some make their way through the process and copurify with the biopharmaceutical product. High-risk HCPs that make it through the process should be evaluated. Methods used to detect, quantitate, and monitor high risk HCPs will be presented along with several case studies.

Modalis delivers its CRISPR Guide Nucleotide Directed Modulation (CRISPR-GNDM) therapy via a single AAV vector. Cells used in the production of recombinant AAV contain host cell proteins (HCPs) that can contaminate drug products. Regulatory bodies expect these impurities be reduced to the lowest levels possible. Modalis utilizes an automated workflow evaluating HCP reduction across clarification, concentration, and purification steps, and ensures HCP levels at CDMOs align with internally developed processes.