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Reliable Host Cell Protein (HCP) detection in biologics production

Enhanced Anti-HCP antibody coverage analysis to support HCP ELISA tests

Reliable Host Cell Protein (HCP) detection in Biologics production

We have seen the successes of biologics as new therapeutic solution for many diseases. Most production for biologics use host cells to express a target biological molecule as final drug product. The host cell continues to produce its own biological molecules together with the drug product. These host cell derived biological molecules are regarded as impurities and are subject to target substances to be removed during the downstream purification. One of the main impurities are host cell derived proteins and known as Host Cell Proteins (HCPs). The level of HCPs in biologics must be monitored carefully during the development to optimize the downstream purification and at the final production to make sure the quality and safety of the biological drug.

The most widely used and accepted method to monitor the HCP levels is an immunological assay, Enzime-Linked Immunosorbent Assay (ELISA), which uses a set of polyclonal antibodies to detect HCPs as many as possible. These anti-HCP antibodies are critical reagents in the assay and an appropriate evaluation is needed and is also requested by authorities.

The antibody coverage assay based on 2 dimensional (2D) electrophoresis and subsequent western blot has been used to evaluate the performance of the anti-HCP antibodies. In this method, the HCPs together with or without the final product are separated by 2D electrophoresis and the anti HCP antibodies are used at the subsequent western blot to measure how many HCP spots have been detected by the antibodies. The result will be expressed as a % coverage of the antibodies to the total number of HCP spots separated by the 2D electrophoresis.

The challenges in the coverage assay are;

  1. Sensitivity of total HCP detection in the 2D electrophoresis
  2. Gel to gel or blot to blot validations due to the electrophoresis and blotting
  3. Spot matching between 2D electrophoresis detection and immuno-detection (western blot)
  4. Time consuming process of evaluation and image data analysis to the scientists involved in the process development or in the monitoring Host Cell Protein (HCP) impurities in biopharmaceutical products.

We propose an enhanced anti-HCP antibody coverage analysis method, 2D Differential In Blot Electrophoresis (2D-DIBE). The solution includes:

  • Fluorescent multiplexed methodology based on CyDye™ pre-labelled and 2D separated proteins. This provide high sensitivities in total HCP detection.
  • In order to minimize gel to gel and blot to blot variation, the labelled proteins are transferred to a membrane (Western blot) that can be directly visualized and compared to the proteins detected by CyDye pre-labelled HCP-antibodies on the same membrane.
  • Multiplex fluorescence image acquisition – Capture both HCP antigen and anti-HCP antibody images from a single membrane with GE's laser based Amersham™ Typhoon™ or CCD based Amersham Imager 600. This avoid any mismatch issue due to the different detection mthods.
  • New Melanie™ Coverage software entirely and solely focused on coverage analysis, with a specific workflow and dedicated tools developed in collaboration with GE Healthcare and major companies in the biopharmaceutical arena. This makes investigators to spend shorter time to evaluate the data with more confidence.