Case study
HCP-ELISA coverage analysis without a null cell line
HCP-ELISA coverage analysis using immunocapture and LC-MS
Our client, a US-based developer of immuno-oncology biologics, required an HCP-ELISA with sufficient coverage for use during process development. However, 2D-PAGE and Western Blotting were not an option without a null cell line due to the relatively high concentration of drug substance in the production cell line samples obscuring a significant population of HCPs.
Immunocapture combined with mass spectrometry, known as ELISA-MS, does not require a mock sample but works with early and complex process samples. Furthermore, ELISA-MS uses less than 1 mg antibodies rather than methods based on affinity purification columns, which often need 10-15 mg ELISA antibodies.
Process
For ELISA-MS, ELISA antibodies are first immobilized. The early process sample is added as the antigen before washing off unbound proteins, cleaving the bound proteins, and performing LC-MS on the resulting peptides. The coverage analysis hereby mimics the ELISA conditions by immunocapture while using mass spectrometry to identify HCPs recognized by the HCP-ELISA. Also, a negative control with antibodies from another expression system is included as a quality measure.
Results
LC-MS detected 618 HCPs in the client’s early process sample. ELISA kit A captured 351 HCPs, corresponding to a coverage of 57%. For kit B, 300 HCPs were captured, resulting in a coverage of 50%.
Comparison of Host Cell Proteins in an early process sample covered by two different ELISA kits
The illustration below lists the 20 most abundant HCPs detected by LC-MS in the early process sample. Kit A detected 14 of them, while kit B only detected 12.
20 most abundant Host Cell Proteins detected in an early process sample bu two different ELISA kits
These data enabled the client to select a suitable HCP-ELISA with the broadest coverage of HCPs for their project. They also plan to use the coverage analysis results for IND filing.
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