A well-developed, broadly reactive, and qualified enzyme-linked immunosorbent assay (ELISA) is the gold-standard method for ensuring removal of host cell proteins (HCPs) and demonstrating process consistency and final drug substance (DS) purity. Regulatory guidelines require sponsors to use orthogonal methods for demonstrating antibody coverage of individual HCPs and provide a comprehensive assay qualification package to ensure that the ELISA is fit for purpose. In a June 2022 webinar, Eric Bishop (head of R&D and custom development services at Cygnus Technologies) discussed best practices in HCP immunoassay qualification and bridging necessitated by critical reagent changes.

Bishop’s Presentation
After many years of performing 2D Western blotting for coverage analysis, Cygnus Technologies concluded that the method is not predictive of how antibodies would perform in an ELISA. So the company launched Antibody Affinity Extraction (AAE) technology in 2014 — and since then has incorporated it with mass spectrometry (MS) into HCP analytics.

HCP ELISAs can measure ng/mL levels of HCPs in the presence of ng/mL levels of product. They require no special expertise and are easy to transfer. The method is limited in that results are semiquantitative. And results can vary with reagent changes. It can take 12 months to generate new reagents for a new HCP ELISA. Such changes are inevitable over time, and not all of them require the same level of characterization to implement. Critical reagent changes come when an antibody or standard antigen has been fully depleted and requires replacement.

Bishop identified three steps to follow when transitioning to use of new reagents: orthogonal characterization, assay qualification, and bridging. He highlighted coverage analysis using the AAE method, with antibodies (the same as in the HCP ELISA) coupled to a solid support. With a sample loaded, the AAE column is washed until a background signal is achieved, then eluted. Results compare the starting material (all HCPs that could end up in a product) with the eluate (all immunoreactive HCPs).

Many companies have transitioned from large-format 2D silver staining to MS detection for coverage analysis. The results appear as a “virtual” 2D image that’s color-coded to reflect matching spots, coverage percentage, and identified HCPs with molecular weights and isoelectric points. Bishop said his company has worked to demonstrate AAE specificity with MS identification with results proving that the procedure specifically removes HCPs for superior coverage assessment.

In an assay-kit bridging case study, Bishop showed how good dilution linearity indicates that the antibodies in an assay fit a cell line’s HCPs and process samples well. Coverage analysis follows to demonstrate that antibodies are broadly reactive to HCPs in a process.

To characterize CHO HCP antibodies for F550 and F550-1 kits, Cygnus scientists fractionated samples using the AAE technique, then used an LC-MS analysis to confirm the results. Bishop showed how color coding made the virtual gel images more useful than 2D silver stains, facilitating identification of patterns in missed spots. The same data could show whether antibodies in the two kits recognize the same proteins (a 96% overlap in this case). “Very strong data demonstrate that the F550-1 antibody is a suitable replacement for the F550 antibody.”

With dilution linearity established, the need remained to demonstrate coverage, precision, and range. Bishop defined bridging as “testing representative samples from a purification process in both assays at the same time.” It helps to determine the differences and similarities between two HCP ELISAs. For bridging studies, Cygnus maintains a sample library to use in assessments before launching new kits or reagents. Such sample sets also help in trending how assays perform over time. He also suggested maintaining a set of controls to run on every assay.

Changing immunoreagents alters the originally validated specificity of an ELISA for each product regardless of whether measured levels are the same. The standard operation procedure (SOP) must be revalidated and updated before a new ELISA is used.

Questions and Answers
How do you identify HCP molecules that cannot be recognized by anti-HCP antibodies? The only effective way to identify those that were missed is by combining AAE and MS analysis.

How do you handle the HCPs in DS with regard to reagent change? The HCP specification is based on manufacturing capability. Test reactivity of the antibody with HCPs in the DS (e.g., by AAE-MS) before your bridging study. If you have retained DS samples, then you can test them with the current and new assays to understand the effects of reagent/assay change and then update your specifications accordingly.

Do you provide a specific HCP reactive protein list from AAE-MS analysis for specific kits? We do. When we do AAE-MS coverage analysis, we generate lists of all proteins identified in both the pre- and post-process samples.

Find the full webinar online at www.bioprocessintl.com/category/webinars.