Immunoaffinity Chromatography (IAC) for HCP Coverage Determination

Immunoaffinity chromatography (IAC) for HCP coverage determination is a technique designed to assess antibody coverage of a polyclonal antibody (pAb) for a mixture of process-derived host cell proteins (HCPs) and to determine the pAb reactivity to process-specific HCPs to characterise the HCP coverage of those antibodies. HCP Antibody characterization is critical to ensure that the antibodies used to generate process-specific ELISAs cover as much of the HCP population in the product as possible. 

Combining IAC + 2D-PAGE Techniques

Using a combination of IAC and 2D-PAGE based techniques (2D Western blotting, 2D-DIGE or 2D-DIBE) enables the detection and quantification of very low levels of HCPs to perform extremely accurate coverage analysis to provide a comprehensive, detailed and efficient determination of ELISA HCP antibody coverage.

The first step in using IAC for HCP coverage determination involves using liquid chromatography specifically with bound candidate HCP antibodies for HCP analysis, sample purification and concentration, then the original HCP samples and the eluted HCP samples are further compared to each other and validated by 2D-PAGE based technologies.

Fig 1. Binding of the HCP sample to the antibody column and then elution and retention from the IAC chromatography column (Moser A C and Hage D S, 2010)

Employing a HCP antibody-based binding assay prior to 2D electrophoresis and imaging allows HCPs within your sample to specifically bind to the candidate anti-HCP antibody on the stationary phase, bound proteins can then be eluted and compared to the original sample, thus overcoming the limitations involved in the denaturation of the sample in traditional 2D-PAGE based coverage methods. The use of this orthogonal method allows for a comprehensive reagent characterization for immunoassays of HCP impurities in biological preparations and is now recommended by the United States Pharmacopeia Chapter <1132> for measurement of residual HCPs in biologics.

IAC purified samples can then be separated by 2D polyacrylamide gel electrophoresis (2D-PAGE) and analyzed using our SpotMap 2D HCP coverage analysis software either by comparison to a silver stained pre-IAC sample or by 2D difference gel electrophoresis (2D-DIGE) of pre-IAC and post-IAC samples labeled with cyanine 3 or 5 (Cy3, Cy5). Coverage is assessed by comparing the number of HCPs in the post-IAC elution fractions (as determined by staining with silver stain or Cy3) with those identified by 2D-PAGE in the starting sample (using silver staining or Cy5 staining)