Antibody Affinity Extraction Enables Identification Of Host Cell Proteins By Mass Spectrometry

Host cell proteins (HCP) constitute a major group of impurities for biologic drugs produced using cell culture technology. Even at nanogram per milligram concentrations of HCP to drug substance (DS), HCPs can elicit undesired immune response, interfere with drug safety and efficacy, or impact DS stability.

A broadly reactive HCP ELISA should be used during the purification processes to ensure removal of HCPs and to demonstrate process consistency and final DS purity. Regulatory authorities are requesting biopharmaceutical companies employ orthogonal methods to demonstrate antibody coverage to individual HCPs and provide a comprehensive assay qualification package to ensure the HCP ELISA used by a sponsor is fit for this purpose.

Antibody Affinity Extraction™ (AAE™), a novel method developed by Cygnus Technologies, is used to determine antibody coverage and reactivity to those HCPs that copurify with DS. AAE is more predictive of the anti HCP antibody performance in the HCP ELISA and facilitates identification of individual downstream HCPs.

While ELISA is the gold standard for monitoring HCP levels, it does not provide information about what HCPs are present in the DS. Identification of HCPs by mass spectrometry (MS) is a powerful orthogonal method to ELISA. However, one of the limitations of MS is that IgG drug substances often mask HCPs by a factor of 10 4 10 6 . To improve MS sensitivity, AAE can be used as a sample preparation method to enrich HCPs and eliminate most of the DS in a sample.

Enrichment of HCPs by AAE and detection by MS is a powerful orthogonal method to ELISA. Integration of orthogonal methods for comprehensive HCP analysis provides data throughout DS purification to inform process development teams of how to modify their purification processes to improve DS purity as well as ensures comprehensive quality control data for regulatory agencies.

Objectives:

1. To illustrate the superiority of AAE to traditional 2D Western blot for HCP antibody coverage analysis
2. To demonstrate the power of integrated AAE and MS for HCP identification in process samples and final drug substances