Antibody Affinity Extraction (AAE) - An Alternative To 2D Western Blot In Establishing HCP Antibody Reactivity

A robust and broadly reactive host cell protein (HCP) ELISA is a critical component of monitoring purification process consistency as well as final drug substance purity. As such, regulatory agencies around the world have put measures in place to ensure the HCP ELISA used by a sponsor is fit for this purpose. The first step to demonstrate that an HCP ELISA is “fit for purpose” is to demonstrate that the antisera used in the ELISA is broadly reactive to the array of HCPs that are found in a given process. This generally means that the antibody must react to a majority (>50%) of the total HCP and that the antibody must recognize proteins in all quadrants of a gel. Traditionally, the regulatory expectation for the coverage assessment has been a large format 2D Western blot comparison to silver stain. In this method a sample is run on duplicate large format (20cm) gels. One gel is transferred to a membrane detected using the anti-HCP antibodies in a Western blot. The duplicate gel is silver-stained. The percent coverage is determined by aligning the 2 images and determining the number of spots detected by silver stain that have a corresponding Western blot reactive spot. Unfortunately, due to the severe limitations of 2D Western blot, the assessment of coverage using this method has proven to have very little predictive value in how well a HCP ELISA will perform in real world samples. These limitations include but are not limited to the destruction of conformational epitopes, harsh chemical treatment of the proteins, difficult alignment of a fixed gel and a blot, method sensitivities, poor specificity, etc. In our experience, these serious limitations may cause a very good antibody to appear to have low coverage, and conversely, a poor antibody may appear to have very good coverage. Cygnus has developed an alternative method to 2D Western blot analysis to assess coverage of polyclonal antibodies to an HCP population – the Antibody Affinity Extraction (AAE) method.

Additionally, we are using AAE to assess reactivity to HCPs that persist through the purification process. This was not a possibility using Western blot due to the high concentration of product protein limiting the amount of HCPs that could be loaded. AAE removes the majority of the product protein and enriches HCP to levels detectable by silver stain.

Objectives:

1. To illustrate the superiority of AAE to traditional 2D Western blot
2. To demonstrate the ability of AAE to assess the reactivity to HCPs that persist through the purification process