The BEBPA HCP meeting 2018 was this time held in Europe – more precisely in beautiful Dubrovnik, Croatia. The conference was again very well planned with a great mix of talks from industry, academia and regulatory authorizations.
The BEBPA HCP meeting is the perfect place to get the full picture about current best practice for HCP analysis and get insights about future developments.
My colleague Rikke Lund and myself have below prepared a short summary about the key topics and take-home messages from this meeting.
(Click to download as PDF document)
For our part, it is great to see that most HCP analysis now includes mass spectrometry (MS). Either directly for HCP identification and quantification, or indirectly in the qualification of ELISA reagents and coverage analysis. Erika Friedl, from Paul Erlich Institute and EMA advisor, told that during the last year they have seen a great increase in MS data as part of new filings. So, anybody involved in HCP analysis must include MS as part of their HCP analysis strategy.
The use of mass spectrometry for HCP analysis includes:
- HCP risk assessment
- ELISA coverage analysis
- Evaluation of generic, template and process specific ELISA
- An orthogonal method to ELISA
HCP risk assessment:
Currently, HCP risk assessment is mainly based on drug specific parameters. For instance, patient groups, diseases, developmental stages, route of administration, treatment regime and frequency of treatment.
But the increasing amount of detailed information obtained from mass spectrometric analysis makes it possible to perform risk assessment in a whole new way, looking at individual host cell proteins. Especially, phospholipases have attracted a lot of interest. This is due to their known degradation of polysorbate material often used in buffers for biologicals. It is very clear from the conference that the added information obtained by mass spec analysis makes it possible to perform a much better risk assessment. As we gather more information about individual HCP’s there will possibly be a list of HCP’s that are known to be harmless and also a list of HCP’s that are unwanted at almost any level.
ELISA coverage analysis:
Several presentations provided detailed information about how to show ELISA coverage. The industry still uses 2D Western and 2D DIGE extensively. But there is a huge need to get better and more reliable data to verify the coverage of a given ELISA. One talk from Scott Henry (Seattle Genetics) gave an example of how to use mass spec to identify HCP covered by ELISA. The method, he applied, included an affinity purification step on beads followed by MS analysis. When using immunoprecipitation, you should ensure that all HCPs are eluted of the antibodies. Further, bead-based coverage analysis requires a careful selection of controls. This is because some unspecific binding is seen.
The current limit for identification and quantification is around 5-10 ppm for individual HCP. But several innovative methods were able to get sub ppm level. Lihua Huang (Eli Lilly) presented a method for antibodies based on a minimal mAb digest procedure that gave HCP quantification as low as 0.1 ppm. Digesting the sample without reduction and alkylation leaves most antibody material undigested. Only the HCP’s turns into peptides which drastically increases the sensitivity of the analysis.
Also, for the first time a MS method using label free quantification was shown by Alphalyse to be absolute using a SWATH LC-MS approach. They have fully automated the method based on 1D micro flow chromatography combined with mass spec analysis using Data Independent Acquisition mode. This makes the analysis very precise and robust. The method is further being developed towards validation.
From Regulatory Authorities:
One of the key questions at the conference was if it is possible to use a generic ELISA as a release assay.
The regulatory guidelines allow the use of generic assays. But examples of using uncharacterized generic ELISAs provided surprises when the product went into phase 3 and gave much higher level of HCP’s than anticipated. This resulted in a delayed project because they had to go back and optimize the whole downstream process. Oppositely, some examples also showed the use of well characterized generic ELISAs. So, the take home massage is that you can pretty much use any ELISA. You should just document the efficiency of the assay to cover the HCP’s in your drug sample.
Overall the BEBPA HCP meeting 2018 was yet another excellent meeting facilitating high level presentations and discussions. We increased our understanding of this emerging field and the need for pushing improvements in orthogonal method & technologies. We look forward to contributing to the BEBPA Host Cell Protein conference in 2019 in Los Angeles.
Rikke Lund and Thomas Kofoed, Alphalyse