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Insights from the 2024 BEBPA HCP Conference
May 30, 2024 – By Thomas Kofoed, Ejvind Mørtz, Rikke Raaen Lund, and Victor Chrone.
Introduction
May 14-16, 2024, we attended BEBPA’s 12 annual Host Cell Protein (HCP) conference in Maryland, US.
We had been looking forward to this conference for a long time. It was an opportunity to share the outcome of the work and research that we, and our colleagues at Alphalyse, have performed for the last 20+ years.
We also enjoyed the exciting conference program with high-level scientific talks, audience surveys, and panel discussions on topics related to Host Cell Protein analysis, including ELISA, mass spectrometry (MS), risk-assessment, polysorbate-degrading HCPs, and regulatory requirements. A diverse audience of experienced HCP scientists from biopharmaceutical companies, service providers, and regulatory agencies attended the hybrid conference in person and by virtual participation.
Top 5 insights from the conference
1. The new US Pharmacopeia General Chapter
For obvious reasons, the new US Pharmacopeia General Chapter <1132.1> on Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry was a big focus point for the 2024 BEBPA HCP Conference.
The first draft of the chapter was posted for public comments last year, recognizing LC-MS/MS as a powerful orthogonal approach. The USP received more than 100 comments, which have since been reviewed by the expert panel and changes approved by the expert committee. Comments not added to the chapter will be included with an explanation. The chapter and commentary will be available on the USP website on December 1st, 2024, and become official on May 1st, 2025.
Niomi Peckham, Director Biologics Pipeline Development at the United States Pharmacopeia, presented an overview of the new chapter, which will include sections on sample preparation (denatured digestion, product protein depletion, or HCP enrichment), chromatographic separation, and mass spectrometry analysis; methods for HCP quantitation (relative to product protein, spiked-in proteins, and spiked-in peptides), method validation, system suitability, and best practices for reporting ELISA vs. LC-MS/MS data. The chapter will also include a case study on low-abundance HCP detection to show normal variation in analyst and instrument performance.
Additionally, Niomi mentioned that the US Pharmacopeia offers analytical reference materials for individual high-risk HCPs: A purified protein for PLBL2 is already available, and 6 stable isotope-labelled proteins targeting Clusterin and Lipoprotein Lipase (LPL) will be released in the summer of 2024.
The new chapter was also one of the topics for the third day’s in-person interest group led by Fengqiang Wang, Merck & Co., Ying Zhang, Sarepta Therapeutics and Ned Mozier, retired, Pfizer.
Ying went through a detailed description of the methods described in the chapter. Quantification methods was a key topic during both the interest group debate and the conference at large, and the new USP chapter will include three options for quantifying against a reference, using 1) peptides from the drug substance, 2) added stable isotopic (SIL) peptides, or 3) added intact standard protein(s).
It was a general observation that MS can be quantitative, but how the quantification is performed and against which reference standard must be thoroughly explained. The new USP chapter also includes a detailed description of the difference between ppm obtained with ELISA and MS.
2. Product stability and polysorbate-degrading host cell proteins
Polysorbate degradation of monoclonal antibody (mAb) products was a hot topic this year, with several presentations and a poster. It was noted that even though the industry tends to refer to PS-degrading enzymes as lipases since the first ones discovered were PLBL2 and LPL, most polysorbate-degrading enzymes are, in fact, not lipases. Instead, several presenters used the term Polysorbate-Degrading Enzymes (PSDE).
The two recurrent questions were: 1) What causes polysorbate degradation, and 2) how do we apply mass spectrometry to understand this problem in more detail?
In her presentation titled Assessing and Mitigating Particle Risks in Drug Products: A Prospective Approach to Identifying and Characterizing Polysorbate-Degrading HCPs, Inn Yuk, Senior Director, Genentech, shared some of the cross-functional work done at Genentech and Roche to identify and characterize polysorbate-degrading enzymes to assess and mitigate particle risk in the drug product. Over a dozen review papers have been published on polysorbate degradation in the last ten years, and about half of the audience at the convention had directly worked on issues related to polysorbate degradation.
It is challenging to identify polysorbate-degrading HCPs because they are likely to be present in the product at trace levels yet still have a sufficiently high enzymatic activity to cause issues. Genentech used two methods to identify these HCPs: A retrospective and a prospective approach.
The retrospective approach examined a mAb showing polysorbate degradation and particle formation. Concentrating the HCPs and depleting the mAb protein made it possible to detect three HCPs (PPT1, LAL, and LPL) that degrade polysorbate. However, the retrospective examination is inconvenient because it is used only after issues have occurred and only identifies 1-2 new HCPs of concern per study.
Instead, Genentech took a prospective approach to identify HCPs capable of degrading polysorbate. They picked 20 HCPs with known or suspected hydrolytic activity (hydrolases acting on ester bonds) and tested them again with Polysorbate 80 and 20. All enzymes showed lower reaction rates in Polysorbate 80 than in Polysorbate 20. Different enzymes also had different substrate specificity, with some preferring monoesters and others higher-order esters.
The conclusion presented by Inn was that the easiest way to minimize polysorbate degradation and particle formation is to either reduce the product concentration or increase the polysorbate concentration, along with knockout in the host cells and tracking individual HCPs through bioprocessing.
Marius Felix, Post-Doc, Boehringer Ingelheim Pharma GmbH, also focused on polysorbate degradation in his presentation on Developing a High-Sensitivity Method for Detection and Quantification of Host Cell Proteins in Biopharmaceuticals by Mass Spectrometry: A Company Perspective on an Industry-Wide Challenge.
Like many other pharmaceutical companies, Boehringer has experienced challenges with HCPs which have the potential to cause critical problems in late stages of the pharmaceutical development and could ultimately result in project termination.
The challenge when using mass spectrometry for HCP analysis is the low amounts of HCPs and the high dynamic range (up to 6 orders of magnitude) which influences detectability, especially in samples from the downstream process. This can be mitigated by decreasing the dynamic range through sample preparation (native digest or other methods of mAB depletion or HCP enrichment) or by increasing the sensitivity of the mass spectrometer.
In one case, the team at Boehringer examined a mAb that was being degraded by an unknown HCP. mRNA sequencing showed expression of 20 HCPs of potential concern, but only 2 were found in the HCCF. Using a PRM MS assay, it was determined that cell age did not correlate with increased expression of these proteases. This enabled the choice of a new cell line with a low expression of the proteases, i.e. a greater chance to deplete them during the downstream process, for subsequent development phases, as well as the option of doing a knockout.
Divya Chandra, Principal Scientist, Merck & Co. Inc., also spoke about strategies for controlling polysorbate-degrading HCPs and ensuring robustness of the manufacturing process and HCP clearance for biologics in Merck’s pipeline in her presentation on Novel Approaches and a Holistic Process – Analytical Control Strategy for Host-Cell Protein Clearance and Control During Biologics’ Production.
The outlined strategies for HCP control included cell line development (knockout/knockdown, alternative cell lines), upstream (media, feed, environmental factors), downstream (chromatography, filtration, loading, resin/buffer conditions), and formulation (materials, inhibitors, alternative surfactants).
In one case study, the team at Merck used a holistic approach for the downstream process of a mAb in commercial process development, explicitly addressing lipase clearance and polysorbate degradation risk to ensure the stability of the drug product over its desired shelf life. After concentration, the product showed significant polysorbate degradation in short-term stability studies.
Among the tools used by Merck were ELISA, LC-MS, LC-MRM, and an Enzymatic Activity Assay for quantitation and identification of total HCP and individual HCPs of concern.
Finally, our own Victor Chrone et al. from Alphalyse brought a scientific poster on how to use an MRM MS-assay to screen for polysorbate degrading proteins.
Proactively identifying and addressing polysorbate-degrading host cell proteins (HCPs) in mAb products can prevent costly and time-consuming late-stage changes to the manufacturing process.
For this reason, Alphalyse has developed a highly sensitive, reproducible mass spectrometry (MS)-based Multiple Reaction Monitoring (MRM) assay for detecting polysorbate-degrading HCPs in mAb products.
You can download the poster here:
3. Patient safety and risk assessment
With the increased use of mass spectrometry and the details we get from this analysis it is of course relevant to discuss risk assessment.
Fengqiang Wang, Principal Scientist, Merck and Co. Inc., and Ying Zhang, Director, Sarepta Therapeutics, opened their presentation on the BioPhorum Development Group – A Multi-company Comprehensive Guidance to Assess Clinical Safety Risks of Individual HCPs, Incorporating Regulatory Considerations and Industry Experience by quoting Aristotle: "The more you know, the more you know you don't know."
The speakers focused on the challenge and necessity of HCP risk assessment. The BioPhorum Development Group's primary goal is to improve biologics' process development by developing industry strategies, best practices, and regulatory requirements for HCP control strategy. The Clinical Risk Assessment team was started in 2020 to move the focus from product quality and shelf life to patient safety by developing a patient-centric risk assessment framework for different phases of development, curating prior experience with HCPs in marketed products to identify ways of mitigating their impact.
Petr Obrdlik, Associate Director, Novartis, in his very interesting and relevant presentation titled From Identification of a Potentially Critical HCP to IMPD and Beyond: Analytical Perspective, described a case study of a high-risk HCP (CCL2/MCP1) encountered during early product development of a recombinant human protein in CHO, and how the team handled this HCP before and after IMPD submission.
CCL2/MCP1 has been connected to adverse patient effects in other drugs. Using their own in-house ELISA, the team at Novartis had seen a good reduction of the HCP of concern, but their CMO found a thousand times more ng/mg of the HCP. They suspected either a ‘jackpot’ effect or interaction between the HCP and drug product in one of the ELISAs. There was no CCL2-specific ELISA in the public domain, so using a semi-quantitative LC-MS assay, they measured CCL2 at 400 ppm in three different batches.
The team submitted for an IMPD and included a protocol to monitor potential effects of CCL2 as a side effect in patients (they have not observed any adverse patient responses to CCL2 so far), committing to performing HCP-ELISA and LC-MS under non-GMO conditions (because they do not have LC-MS under GMP conditions in-house). They received some very good advice from the health authorities, and Petr strongly recommends dialogue with the regulatory authorities if encountering similar challenges.
Post-IMPD, they developed a CCL2-specific ELISA for process optimization and release testing, and also developed a knockdown host cell line with significantly reduced CCL2 in the purified drug product.
Lessons learned from this case include using a generic HCP-ELISA as an internal golden standard, using LC-MS much earlier for HCP identification and risk mitigation, as well as and during research to development transitions, and to use an alternative HCP-ELISA to check for blind spots.
4. Regulatory perspectives
This year’s BEBPA conference was also attended, as always, by representatives from the regulatory authorities.
In a Q&A session dedicated to questions from the audience to Alexey Khrenov, Branch Chief, CBER, FDA, and Erika Friedl, Senior Quality Expert, Paul-Ehrlich-Institut, EMA, they once stressed that no specific number is required for HCP analysis. It is a misconception – and is not mentioned in any regulatory requirement – that an HCP content of less than 100 ppm, or even 10 ppm, is required for and/or guaranteed to result in regulatory approval.
Rather, the critical factor is consistency: Data generated during development must correlate with regulatory data.
When using ELISA, it is essential to document that the standard fits the samples. It is preferable to develop an in-house standard instead of using the standard supplied with commercial ELISA kits.
Alexey also recommended retaining data and samples from historic samples for bridging and comparability studies.
In her presentation titled HCP Control Strategies and Regulatory Requirements, Erika furthermore spoke about impurities and critical quality attribute evaluation, problematic HCPs and their impact, regulatory guidance, orthogonal methods, and regulatory views on assay requirements, specifications, comparability approaches, and lifecycle control.
Erika highlighted the increased importance of MS data during MAA, stating that they are becoming standard in licensing applications. LC-MS/MS data is used for identification and quantitation of HCPs, for product development and characterization, during manufacturing process changes and site transfers. Erika said that she has not seen MS data used for quality control and release yet.
In addition, Erika stated that licensing applications should include the immunogenicity profile (determined by in silico prediction tools) and risk assessment of Top X(X) impurities – the level of detail depends on the amounts of HCPs (ppm) present. HCP hitchhikers in the final drug substance might require development of specific assays to control them. If platform or generic assays are used, comparability data will be required.
5. AI and deep machine learning can enhance LC-MS analysis
One of the most anticipated presentations was from Victor Chrone, Bioinformatics Scientist, Alphalyse A/S, who presented What You Can Learn from HCP Analysis of 500 Projects Using LC-MS.
We are seeing an industry shift from measuring only the total HCP amount to focusing on individual HCPs of concern, guided by the upcoming USP General Chapter <1132.1>. LC-MS enables tracking, identifying, and monitoring individual HCPs across process samples and between projects, generating a large amount of data. At Alphalyse, this data is comparable because we use the same 7 intact protein standards for quantification, according to USP Chapter 1132.1, adjusting for the natural variability of mass spectrometry technology.
Using a robust and reproducible quantitative LC-MS/MS assay and intact protein standards to measure 2000+ samples, Victor has constructed a comprehensive SQL database with MS data on HCPs and contaminants coupled with metadata on drug substance, host cell organisms, buffer composition, purification levels, expected amounts, process, etc.
Using the database to determine which HCPs are most frequently identified in client samples, Alphalyse has set up an assay to detect polysorbate-degrading HCPs in mAb products. The database is also used for QC tools to measure MS instrument performance using artificial intelligence (AI).
Take-home message
So, at the end of the 12th BEBPA HCP Conference, we could again look back at a conference with a very high scientific level and fruitful discussion among a very engaged audience.
The new USP chapter will add even more focus on using mass spectrometry to monitor HCPs. The potential is that with commercially available reference standards (proteins and peptides) we will have a mutual reference, making it possible to compare results within a given project and between projects, laboratories and companies.
Want to know more about our experiences and learnings at BEBPA – and other conferences?
Follow us on LinkedIn: Alphalyse | CEO Thomas Kofoed | COO Ejvind Mørtz | Head of Characterization Department Rikke Raaen Lund | Bioinformatics Specialist Victor Chrone.
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