Antibody Coverage Analysis Of E. coli HCPs In The BL21(DE3) Strain By AAE-MS™

To determine the suitability and similarity of Cygnus E. coli HCP ELISA Kits to quantify BL21(DE3) Host Cell Proteins (HCPs), antibody coverage analysis of the BL21(DE3) HCP antigen was performed using Cygnus Technologies’ Antibody Affinity Extraction and Mass Spectrometry (AAE-MS™) approach. AAE™ was performed using Cygnus’ commercially available anti-E. coli antibodies from three E. coli HCP ELISA kits: F410, F1020, and F1060. HCPs were identified in the Pre-AAE and Post-AAE samples by liquid chromatography/mass spectrometry (LC-MS). HCP coverage was determined by comparing the number of immunoreactive HCPs in the AAE elution fraction compared to the starting sample containing all BL21(DE3) HCPs. Prior to AAE extraction, 924 HCPs were detected in the BL21(DE3) HCP antigen sample, and the F410 and F1020 antibodies reacted with 836 and 829 proteins, respectively, indicating comparable performance. In contrast, the F1060 antibodies captured 923 proteins in the Post-AAE elution fraction, suggesting better coverage of the BL21(DE3) HCPs. Importantly, potentially high-risk HCPs were identified as immunoreactive with the Cygnus anti-E. coli antibodies. The antibody coverage of the BL21(DE3) host cell proteins was 90% for F410, 89-90% for F1020 and 99-100% for F1060.