Antibody Affinity Extraction (AAE™) For Host Cell Protein Identification

Antibody Affinity Extraction (AAE™), a type of immunoaffinity chromatography, is an advanced orthogonal method designed to assess coverage of a polyclonal antibody to host cell protein (HCP) mixture present in a given process as well as HCP antibody reactivity to downstream, process-specific HCPs that may co-purify with a drug substance. This method was developed by Cygnus Technologies in 2013 to overcome the analytical deficiencies and limitations of older, traditional 2D Western Blot (2D WB) and 2D-DIBE orthogonal methods used to assess coverage of polyclonal antibodies to total host cell protein. For lack of better methods, 2D WB has been traditionally performed from large format two-dimensional gel electrophoresis (2D PAGE) gels with protein transfer to membranes for 2D WB comparison to silver stain. Due to the sensitivity limitations of 2D WB, coverage can only be estimated on upstream, harvest samples where the concentration of most HCPs are still within the sensitivity limitations of various staining methods. Because of the recognized sensitivity limitations of 2D WB, the conventional acceptance criteria are that >50% of the total HCP should be reactive and that the antibody must recognize HCP in all quadrants of a 2D PAGE gel. Due to the inherent sensitivity limitations of 2D WB, such as (1) loading capacity, (2) destruction of native epitopes by harsh sample treatment, (3) failure to transfer some HCPs out of the gel, (4) HCPs bound to the membrane such that antibody binding is sterically inhibited, (5) difficulties in aligning the dissimilar PAGE gel to a WB membrane images, and (6) poor specificity, 2D WB significantly underestimates true antibody coverage to upstream HCPs. More importantly, 2D WB does not predict how that antibody will quantitatively react to the most important HCPs which are those that co-purify with the drug substance.