Introduction to the BEBPA HCP conference
May 22-24, 2023, we attended BEBPA’s 11th annual Host Cell Protein (HCP) conference in Dubrovnik, Croatia. Nestled beside the beautiful Adriatic Ocean and surrounded by Croatian mountains, Dubrovnik is renowned as the filming location for Kings Landing in the popular Game of Thrones series.
While we had limited time to enjoy the beach and the old town, the conference was a remarkable experience. The organizing committee had once again put together an exciting conference program of high-level scientific talks, audience polls, and panel discussions on topics related to Host Cell Protein analysis by ELISA, LC-MS, and related technologies. Each session was chaired by experienced persons in the field, facilitating high-level discussions and interaction with the virtual audience.
A large audience of skilled HCP experts from the biopharmaceutical industry, service providers, and regulatory agencies attended the hybrid conference in person and by virtual participation. Once again, the hybrid format was handled exceptionally well by the organizers.
3 top insights from the conference:
- The new US Pharmacopeia General Chapter <1132.1> on Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry is open for public comments until July 31, 2023. It is an opportunity for stakeholders to provide feedback and contribute to developing this important chapter.
- New methods for handling potentially problematic HCPs are emerging: Detection using sensitive LC-MS techniques, analyzing the ELISA’s coverage of problematic HCPs by advanced immunocapture LC-MS techniques including immunoaffinity-chromatography (IAC)and ELISA-MS™, and risk assessment for evaluating the potential risk presented by individual HCPs present in drug substances. These methods enable a more cohesive strategy for HCP clearance, process control, comparability, and product purity using LC-MS methods.
- Comprehensive CMC strategies are necessary for HCP clearance and control using combinations of well-characterized ELISAs using LC-MS methods (regardless of commercial kit, platform ELISA, or product-specific ELISA) and quantitative LC-MS. The combined technologies enable improved HCP clearance through process development, tighter control of process consistency in PPQ runs, and better HCP documentation for regulatory filings from INDs to BLA market license applications.
Key topics and take-home messages
Each session started with questions for the audience. The most interesting polls and their results were:
- How long did the development of an HCP assay take – immunogen generation through validation? 12-18 months: 30%, 18-24 months: 25%, >2 years: 45%.
- What are the main shortfalls of the HCP ELISA you are currently using? Coverage is too low: 3%, Coverage is unknown: 8%, LOQ not acceptable: 2%, Unknown if problematic HCPs are measured: 26%, Running out of antigen reagents: 22%, Bridging to new reagents or kits: 26%, Lack of dilutional linearity: 13%.
- Which ELISA do you use for late-stage development and commercial phase? Process-specific ELISA (custom for one project): 40%, Platform ELISA (custom for one organization): 40%, Commercial ELISA: 20%.
These survey results reveal some of the challenges of HCP ELISAs the industry faces and some of the solutions adopted. The time required to develop a process-specific or platform ELISA assay is a particularly significant concern – almost half of the participants have spent more than two years developing an ELISA assay.
Workshop 1: Advanced MS Topics
Two industry experts, Ying Zhang (Sarepta Therapeutics) and Kevin Van Cott (University of Nebraska-Lincoln), led a panel presentation on the new US Pharmacopeia General Chapter <1132.1> Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry. They focused on sample preparation, quantitation of HCPs by LC-MS/MS, data analysis, reporting of HCP results, and the integration of MS and HCP ELISA results for improved product and process understanding. A discussion followed on how MS results in the context of HCP ELISA results may facilitate a better understanding of the product and process.
During the workshop, Kevin Van Cott highlighted the common industry practice of using methods and instruments “off label” and “pushing them to the limit.” In other words, in HCP analysis, we are utilizing methods, instruments, and software developed for other purposes, typically proteomics or drug characterization, and pushing them to their maximum potential to achieve higher sensitivity. Therefore, we must perform enough HCP analyses to gain proficiency and a thorough understanding of the instruments to optimize their performance and obtain the most accurate results. In addition, it is essential to incorporate good controls that resemble the HCPs in a sample and ensure they undergo the same workflow. With the new USP chapter, there will be better guidance for implementing quantitative LC-MS workflows for HCPs, and likely also regulatory expectations that biologics developers are using best practice LC-MS methods.
Workshop 2: ELISA & Critical Reagent Development
Industry experts in HCP reagent development – Denise Krawitz (CMC Paradigms LLC), Robert Hooper (Rockland Immunochemicals), Stefan Sommerschuh (BioGenes GmbH), and Eric Bishop (Cygnus Technologies) – hosted a workshop and discussed best practices for HCP reagent generation, including how to prepare the Mock immunogen and raise antibodies, how to control critical reagents, and how to bridge assays with new reagent lots. A new topic this year was ELISA detection of potentially problematic HCPs evaluated by LC-MS methods, including the ELISA-MS™ method.
Workshop 3: Phase-Specific HCP Strategy
This workshop was led by Fengqiang Wang (Merck), Oliver Anderka (Novartis Pharma AG), and Stefanie Wohlrab (Roche Diagnostics GmbH) and covered transition and bridging between different immunoassays (e.g., generic to process-specific); potential purposes of using LC-MS (troubleshooting, process development, comparability, drug substance characterization, coverage determination of immunoassay reagents); opportunities and risks of including LC-MS in the development toolbox for HCP characterization; phase-appropriate use of LC-MS for HCP characterization; phase-appropriate acceptance limits and/or overall control strategy for individual HCPs; control of single HCP: LC-MS vs. single-HCP ELISA; and informed risk assessment and decision-making through well-characterized process and product. The workshop participants were organized into small groups and collaboratively worked on actual case examples. This interactive approach proved highly effective, fostering an environment where learning and knowledge-sharing thrived. Our joint conclusion was that an effective HCP control strategy requires expertise from multiple areas: upstream and downstream process development, analytical scientists specializing in ELISA and LC-MS HCP analysis, CMC managers, and regulatory professionals.
Workshop 5: Risk Assessment
Christina de Zafra (Seagen), and Denise Krawitz (CMC Paradigms LLC), Fengqiang Wang (Merck) delivered an excellent introduction to acceptable risks and risk assessment framework of HCPs in biopharmaceutical products. They explored two critical questions: What risks are associated with dosing patients with host cell protein impurities? What defines an acceptable risk?
Identifying each protein impurity and its approximate amount is essential for evaluating its potential risk for patients. The speakers shared their expert insights and experience addressing risk assessment in this context. However, the answer is complex: it depends on many factors, and you must evaluate the individual HCP and patient population. These factors include the severity of the disease, the (potentially immunocompromised) state of the patient, the dosage of the biopharmaceutical and the HCP, the frequency of dosage administration, and the existing knowledge from clinical trials and animal toxicity studies involving the specific HCP.
You can find further information in the paper: Host cell proteins in biotechnology-derived products: A risk assessment framework, Biotechnol Bioeng. 2015 Nov;112(11):2284-91, by de Zafra et al.
Session 1: HCP Profiling During Process Development
Analyzing CHO Host Cell Proteins: Impact of Cell Age and of Digest Conditions
Kelvin Lee, Professor & Director, University of Delaware
This presentation focused on issues related to HCP analysis, including how HCP profiles change as a function of cell age over 30, 60, and 90 doublings and the impact of sample processing conditions before SWATH LC-MS-based HCP analysis. The CHO protein expression and SWATH LC-MS analysis were very reproducible, and most HCPs do not change with age. They quantify the proteins using intact spike-in protein standards. A small subset of HCPs, which may be difficult to remove, demonstrate age-dependent expression. Comparisons of standard and native digest conditions might impact the assessment of HCPs in a sample.
HCP Case Study in Biosimilar Development: Effect of Different HCP Populations on Comparative Forced Degradation Studies
Jon Valgeirsson, VP ARD, Alvotech
A comparative forced degradation study compared the stability of a monoclonal antibody (mAb) biosimilar to its originator using thermal, photolytic, low pH, high pH, agitation, and oxidation stressors. The biosimilar exhibited greater stability than the originator under acidic conditions. HCP characterization by LC-MS performed by Alphalyse revealed the presence of a well-known low-pH activated protease (Cathepsin) in the originator, which was absent in the biosimilar. Cathepsin L is known to degrade mAbs with the pattern observed in the forced degradation results.
Analysis of Host Cell Proteins in Protein A Eluates of mAb-based Therapeutic Proteins
Sherin Panikulam, PhD Student, Univ. of Applied Sciences & Arts NW Switzerland
This interesting PhD research project investigates HCP correlation and various causes of HCPs co-purifying with mAbs. Commonly appearing HCPs in mAbs are chaperone complexes, ribosomal proteins, and translational proteins. Sherin divides high-risk HCPs into three categories based on the severity of their effect: Immunogenic or biological responses (patient safety concerns), mAb fragmentation or aggregation (drug quality concerns), and excipient degradation (shelf-life concerns). Once completed, this project may demonstrate methods to remove even more HCPs from mAbs purified on Protein A columns.
Session 2: Implementation of HCP ELISAs: Commercial, Platform, and Process-Specific Assays
HCP Control Strategy — Experiences from an 'Off Gel Road'
Margarita Sabater, Genmab
Margarita presented a holistic HCP control strategy for the Chemistry, Manufacturing, and Controls (CMC) of mAb products. She focused on different approaches to gain a thorough understanding of HCP clearance through residual HCP testing and evaluation of commercial CHO kits. The presentation also covered ELISA reagent characterization and coverage analysis of 7 commercial ELISA kits using ELISA-MS™ performed by Alphalyse and the combined use of LC-MS and ELISA for PPQ runs. This comprehensive CMC HCP control strategy using a combination of commercial ELISA kit, ELISA-MS coverage analysis, and LC-MS and ELISA for process consistency has been presented to and accepted by different regulatory agencies for biological license applications. Margarita reported that the FDA, MHRA, and EMA all had accepted Genmab's use of a commercial HCP ELISA kit with HCP coverage documented by ELISA-MS™ analysis. In other words, all authorities approved the ELISA-MS™ coverage method. EMA also requested a 2D-DIGE analysis, whereas the Canadian authorities requested a process-specific HCP ELISA assay.
Session 3: Alternatives to ELISA for HTP and Routine HCP Testing
Scientists from Boehringer Ingelheim, Novo Nordisk, Sanofi, and Merck presented several different analytical methods as alternatives for routine HCP testing, including Gyrolab, Bio-layer Interferometry, and Luminex. These are emerging technologies that are starting to be used and explored.
Session 4: Methods for Detection of Low Abundance HCPs
Assessing Methods for Detecting Low Abundant HCPs
Thomas Wärner, Boehringer Ingelheim Pharma GmbH & Co. KG
It is well known that some HCPs with very little abundance can strongly impact product quality. Understanding how HCPs co-purify with the product ("hitchhiker" HCPs) and how to detect those HCPs is a prerequisite for developing a suitable HCP depletion process. Thomas outlined his research on methods for detecting low-abundant HCPs by MS/MS. He compared how three methods (hexapeptide columns, partial digest, and immunoaffinity-chromatography (IAC)) enrich HCP levels in samples. IAC and native digest identified most HCPs. As always, HCP levels and species depend on the product and can vary between mAb products.
Identification and Absolute Quantification of a Problematic HCP with Lipase Activity to Support Therapeutic Protein Development
Sook Yen (Kelly) E, Senior Scientist, Regeneron Pharmaceuticals
Early detection of HCPs during the drug development cycle is key to mitigating the impact of problematic HCPs. HCP detection and analysis methods include native digest, immuno-purification combined with LC-MS, and a combination of untargeted (IDA) and targeted (PRM/MRM) approaches.
Sook Yen presented a case study of a high-risk lipase HCP, liver carboxylesterase (CES), which caused PS80 degradation in stress studies. The concentration of CES in the drug substance (DS) was initially at 43 ppm, but subsequent processes reduced it to 10 ppm and eventually 0.3 ppm. To measure CES and other problematic HCPs (ceramidase, lysosomal acid lipase, cathepsin D, PLBL2, and TIMP1), they established an MRM assay, although several isoforms in the mAb product complicated the assay setup. They also used a recombinant protein form of the enzyme to achieve absolute quantification. Interestingly, native digest and analysis on an optimized process batch did not detect liver carboxylesterase, which correlates with the absence of PS80 degradation.
Compared to other methods, the current LC-MS/MS-MRM technique demonstrated more than a 10-fold improvement in sensitivity, enabling accurate quantification of problematic HCPs at sub-ppm to ppb levels during drug development.
Session 5: Guidance
HCP Impurity Control Covering Various Product Classes
Erika Friedl, Senior Quality Expert, Paul-Ehrlich Institut
Regulatory agencies review all types of HCP assay and will accept them if supported by sufficient data. It emphasizes the significance of providing comprehensive and reliable information to ensure successful regulatory submissions.
Platform assays utilizing antigen/antibody combinations are emerging and expected to become more prevalent in HCP analysis. However, while the agencies have observed the use of generic assays for monoclonal antibodies (mAbs) and vaccines, their implementation has experienced delays due to insufficient comparability packages. This underscores the importance of comprehensive documentation and thorough comparability studies to establish the validity and reliability of these assays.
Finally, Erika showed typical product HCP limits but could not specify universal limits; Appropriate HCP limits are decided on a case-by-case basis, considering factors such as the product, its intended use, HCPs of concern, and the associated risks.
USP Standards to Support Host Cell Protein Analysis by Mass Spectrometry
Niomi Peckham, Director, Pipeline Development, United States Pharmacopeia
The new US Pharmacopeia General Chapter <1132.1> on Residual Host Cell Protein Measurement in Biopharmaceuticals by Mass Spectrometry is open for public comments until July 31, 2023. Stakeholders are encouraged to provide feedback and contribute to developing this important chapter.
Additionally, Niomi announced that the US Pharmacopeia would release its first reference standard protein for HCP analysis in the third quarter of 2023. This reference protein, known as PLBL2, will serve as a valuable tool for ensuring accuracy and comparability in HCP analysis. Furthermore, in the fourth quarter of 2023, six peptides for two protein targets will be made available. These peptides will play a crucial role in supporting HCP analysis by enabling accurate identification and quantification of specific protein targets.
Session 6: HCP Critical Reagent Characterization Methods
Comprehensive Characterization of In-House ELISA Host Cell Protein Reagents by IAC & LC-MS
Suli Liu, Principal Scientist, Biogen
Suli presented a comprehensive and detailed characterization case of a Biogen mAb. She explained the differences between coverage method principles and emphasized the wide range of coverage % obtained with each method – meaning the reagent coverage % is method dependent. The case showed how two highly abundant HCPs, each above 100 ppm, were shockingly not detected by the ELISA but only detected and quantified by LC-MS. The two HCPs were potentially problematic as HCP 1 is known to regulate lipid breakdown, and HCP 2 was a chaperone protein assisting in protein folding and degradation.
ELISA Reagent Characterization Using Advanced LC-MS Methods
Ejvind Mørtz, COO, Alphalyse
The HCP impurity assay intends to show the consistency of the downstream purification process, comparability of the batches, and documenting the HCP impurity level of the purified drug substance. The talk showed how two advanced LC-MS methods could properly characterize the ELISA antibody reagents, the Mock antigen used for immunization, and the HCP standard. ELISA-MSTM and quantitative HCP LC-MS analysis can demonstrate the fit-for-purpose of reagents.
The focus of the reagent characterization should not be on the Coverage percentage – it should be on which HCP impurities the assay measures in the bioprocess and drug product. Is the HCP standard presentative for the bioprocess? Are potential problematic HCPs being measured? These are key questions in ELISA reagent characterization that mass spectrometry methods can answer.
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