From May 17-19 2021, the second virtual-only “BEBPA Host Cell Protein Symposium” took place. More than 120 experts and participants from biopharmaceutical companies, service providers, research organizations, and regulatory agencies joined this event. In the following, we would like to give a brief summary of the overall BEBPA conference impression, and the most important statements and opinions that were exchanged and discussed during this interactive event.
Although a great number of presentations by different speakers were given, and an even larger number of questions were asked by the audience, the main issues that are of concern for the HCP community, can be narrowed down to:
- How to make the best use of ELISA and complementary mass spectrometry (MS) for HCP monitoring and method validation.
- What are the regulatory and methodological hurdles to overcome when going forward with mass spectrometric approaches?
- How to make the best use of immunoassays during downstream process development, and in particular, how to address critical assay parameters such as HCP coverage. Which alternatives are there to under-performing commercial kits?
- How to avoid running into issues with reagent supply, and how to address a re-validation to regulatory bodies.
Mass spectrometry has undoubtedly found an entry into the field of HCP analysis and has established itself as a valuable method for providing information based on an orthogonal approach which fares well when compared to the traditional antibody-based analytical methods. Still, in the HCP community, the lingering question concerning the use of MS relates to how best to implement MS into the GMP-controlled environment during biopharmaceutical drug manufacturing. This has been addressed over a number of presentations, demonstrating the strengths and drawbacks of using MS as the sole method for HCP coverage analysis and detection.
During the BEBPA panel and roundtable discussions, one common opinion has emerged: the ELISA is still the gold standard for HCP impurity testing due to its key strengths, which are:
- the overall assay robustness and transferable assay standardization
- low requirements regarding the complexity of equipment and handling proficiency
- a semi-quantitative read-out is generated without the need of highly specific databases
- a fast performance and easy validation for applications under GMP regulations
- the ability to measure HCP in the range of ng/mL in the presence of drug substance at mg/mL concentrations
- the signal intensity accounts for the immunogenicity of individual HCPs (“immuno-dependency”), which can translate into an important message for highly immunogenic impurities that can potentially cause severe reactions in patients
The use of MS-based data for HCP characterization in drug substance specifications may be advantageous but is not required: It is up to the individual manufacturer to include or exclude MS data sets. Based on a survey of symposium participants, 43% of the participants do include MS data on HCP characterization in their documentation for regulatory agencies. It is also worth mentioning that the BEBPA audience still prefers ELISA over MS for HCP monitoring during process development (67% vs. 62%, multiple answering options were possible).
Despite the option of adding additional information on HCP species identification by MS, this technology still has considerably high demands with regard to handling proficiency and maintenance. Attempts to resolve issues regarding the standardization of MS approaches are currently being undertaken to reduce data inconsistency across different testing sites and equipment setups. Although only a few reference materials (such as the NIST antibody) are available globally, most MS technology users seem to rely on internal standards for data equilibration. Moreover, the dependency of MS approaches on established genome databases and the constant updating of those libraries is seen as a major hurdle towards the implementation of MS-based HCP monitoring along the drug development process in a GMP-regulated environment.
From a regulator’s perspective (as expressed by Erika Friedl), there is no limitation to the assay technique or method used for HCP monitoring, or how assay validation is achieved. In general, a successful Market Authorization Application (MAA) needs to fulfill 3 main criteria:
- Demonstration of the removal of HCP and precise description of the removal strategy,
- Identification of residual HCP in the final bulk drug, and
- Risk assessment of remaining HCP in final drug substance and immunogenicity assessment.
This general opinion has again been backed up by Denise Krawitz, a globally renowned expert in the HCP analysis field. She emphasizes that “There is no one perfect method for HCP analysis and coverage determination, but it’s more important to describe how your HCP coverage is actually performed, including a scientifically sound assessment of limitations and remaining risks”.
Still, the key word for explaining the best use of antibody-based immunoassay methods and MS technology for HCP monitoring and assay validation is “orthogonality”! This was reflected by the majority of the BEBPA’s attendees throughout the symposium. Here, the independence of methodological approaches is seen as the real advantage when using both methods in a back-to-back manner. The same holds true for immunoassay reagents characterization with a traditional visualization method, such as 2D analytics vs. MS-based characterization. With regard to 2D analytics, a great number of presentations reinforced the impression that the 2D Western Blot method will be (and already has been, in part) replaced by the Immunoaffinity Chromatography (IAC)-2D DIGE approach. This trend is expected to continue in the future. Unlike in earlier years, the determination of antibody coverage solely using IAC-2D DIGE is now widely accepted by customers and authorities. Since it not only provides higher coverage values, IAC-2D DIGE also gives a visual impression of the utilized samples, making IAC-2D DIGE now a favored and valued method. Data solely based on the Western Blot method was rarely presented, which demonstrates again that customers and authorities are well aware of its limitations and overall disadvantages.
The answer to the question: “When to use which HCP ELISA type?” has remained the same among the participants. Commercial (also known as generic) HCP ELISA and platform-specific HCP ELISA remain the preferred assay types for HCP monitoring during process development in early-to-mid stages of clinical trials (phases I and II). However, for late-stage clinical trials (phase III) and MAA, the use of process-specific HCP ELISA still dominates, despite an increased use of platform-specific HCP ELISA among biopharmaceutical manufacturers. When it comes to testing the suitability of commercial kits, it is generally accepted that the following holds true: Test as many different generic HCP assays as possible for your specific process, even though you may have a perfect match between your cell line and the cell line-background of the generic kit.
However, the antibodies used for appropriate HCP ELISA measurement should be well characterized and available for the lifetime of the product, if possible, to prevent reagent re-supply. In fact, this is also the approach preferred by regulatory bodies. If reagents’ changes have to be announced, critical antibody re-supply (including new antigen preparation and/or immunization) requires comprehensive bridging studies, including orthogonal methods such as coverage analysis and similarity testing of mock antigens. To avoid problems resulting from a lack of data comparability, it is advised to address these issues timely and to take care of proper assay transfer, including the performance of comparability studies.
Taken together, immunosorbent assays such as the ELISA are still seen as the most easy-to-use and easy-to-GMP-validate method across the biotherapeutic manufacturers’ landscape. Nevertheless, the use of sophisticated orthogonal methods for testing ELISA suitability for HCP monitoring, such as IAC-2D DIGE and LC-MS/MS, will become more and more important. Consequently, regulatory agencies will most likely make their decision for successful BLA filing dependent on the overall reasonability of a given data set. However, a change of reagents during established drug production is still considered critical, and something best avoided (for instance, by shifting HCP monitoring to a validated platform or process-specific immunoassay as early as possible during method development) if the manufacturer does not want to be confronted with laborious assay equivalence testing.
We hope you enjoyed our BEBPA 2021 review and look forward to seeing you again in exciting future meetings!
If you have further questions regarding your HCP monitoring approach or need advice on how to employ the best HCP detection strategy, please contact us directly.