Table 4: Host Cell Protein Analysis Facilitator: Annemiek Verwilligen, Janssen Vaccines & Prevention Scribe: Sarah Rogstad

Table 4: Host Cell Protein Analysis

Facilitator: Annemiek Verwilligen, Janssen Vaccines & Prevention

Scribe: Sarah Rogstad, CDER, FDA

Scope: Major hurdles in the detailed analysis of host cell proteins (HCPs) have been lowered by the introduction of mass spectrometry in this field supplying knowledge on protein abundance and identity. Sensitivity increases of LCMS based methods in recent years have subsequently increased HCP data output enormously. Particularly of interest for this topic are best strategies for HCP analysis and best practices for data interpretation and reporting. The focus of this round table will be on recombinant protein and peptide products, whereas viral vaccines will be considered out of scope. As this round table will focus on strategic approaches, overly technical details will be also be considered out of scope. This round table aims to discuss the technical aspects of obtaining, interpreting, and reporting HCP data.

Questions for Discussion: 1. What methods do you use to address the abundance difference between HCPs and API (i.e. sample clean-up, chromatography, and/or fragmentation approaches)? How do these methods differ with different product types and cell lines? 2. At which steps are you assessing HCPs (i.e. final product, in process samples, reagents)? 3. How do you handle large HCP datasets? What types of data interpretation work best? How do you interpret highly sensitive HCP data (i.e. what is the relevance of low level HCPs)? How do you report HCP data (i.e. individual HCP abundances or total protein content as with ELISA)?

Discussion Notes: 1. What methods do you use to address the abundance difference between HCPs and API (i.e. sample clean-up, chromatography, and/or fragmentation approaches)? How do these methods differ with different product types and cell lines? • Major sample preparation steps used included immunoprecipitation (IP), 2D gels, and gel band digests. • A recent approach that several participants used was recently published by Eli Lilly. This approach skips denaturation when digesting a mAb sample in order to preferentially digest HCPs as the mAbs are too stable to digest when non-denatured and are removed from the sample prior to analysis. • This method has resulted in ten-fold higher HCP recovery levels and was found to give complementary results to those using IP. A trade-off between a higher percent recovery for fewer proteins and a lower percent recovery for potentially more proteins was discussed. • A potential trend toward not enriching for HCPs was mentioned. The group acknowledged that sample preparation would be different for non-mAbs, but most discussion was focused on mAbs. • Major chromatographic approaches included 1D LC and conventional flow rate UPLC. • Nano-spray was discussed, but micro-spray was agreed to generally be more robust. • A variety of scanning techniques were used including MRM, DIA (MSE and SWATH), and DDA (top 10 or top 20). Often these techniques were used in parallel to gain better coverage of all HCPs. • As a side note, some concern was noted about the possibility of using MS for HCPs in a QC environment as consistent results may be difficult to obtain using these approaches.

2. At which steps are you assessing HCPs (i.e. final product, in process samples, reagents)? • Most participants assess HCPs throughout the development and production processes. • MS analysis of HCPs is used for process understanding by starting with assessment of the cell culture supernatant as well as the antigen and continuing throughout the purification process to identify possible impurities in the final product. • MS was also sometimes performed for HCP analysis of clinical batches. While some groups wanted to ensure low levels of changes in HCPs between batches, others questioned the necessity and cost of such testing.

3. How do you handle large HCP datasets? What types of data interpretation work best? How do you interpret highly sensitive HCP data (i.e. what is the relevance of low level HCPs)? How do you report HCP data (i.e. individual HCP abundances or total protein content as with ELISA)? • For quantitation, most agreed that absolute quantitation was not necessary for HCPs. Many use the Hi3 approach for relative quantitation based on total protein. • For software, some groups have created their own software browsers while others use SQL queries but noted that filtering and sorting can be challenging. Other groups use commercially and/or freely available software such as MaxQuant and Skyline.

Additional Discussion:

• Comparison with ELISA – Most participants agreed that ELISA and MS are complementary approaches for HCP analysis and can be used orthogonally. Many groups use MS in case ELISA might miss a protein or to test whether or not ELISA catches proteins. High ELISA results were noted to lead to MS testing in order to identify specific HCPs. Most groups found good correlation between ELISA and MS results. Participants noted that ELISA has the advantage of being higher throughput than MS. • Risk Assessments – The group agreed that a risk assessment of HCPs is important to consider. Questions arose around the biological relevance of HCPs, which HCPs have what type of effects, how to know which HCPs may be an issue, and what the target levels of HCPs should be. Resulting suggestions included creating a list of problematic HCPs to follow and using tools like CHOppi to predict immunogenicity (although this may not be very accurate). The group noted that target levels and biological relevance will be dependent on the specific product, dose, and route. Additional concerns included HCPs affecting antibody stability through aggregation or degradation and HCP-related PS80 degradation.