Why and what are HCP ELISAs?
Biopharmaceutical companies have a duty to make sure their products meet safety regulations. Optimising purification steps and monitoring the presence and quantity of host cell protein (HCPs) throughout by enzyme-linked immunosorbent assays (ELISA) are two key ways to do this.
ELISAs are the workhorse of HCP quantitation, but they aren’t perfect. Several biologics in recent years have shown immunoreactions in late clinical trials (eg, Sandoz-Novartis Omnitrope™, IPSEN™ Coagulation Factor IX and Genentech™ Lebrikizumab). As a result, 2016 and 2017 saw the US and EU pharmacopeia updated to include coverage assays as best practice for risk management for any future safety issues.
Coverage assays provide a means to validate the ELISAs used in HCP quantitation. They enable you to estimate the percentage of HCPs that can be detected, or ‘covered’, by ELISA, and direct you towards filling any gap.
A coverage assay can be quite straightforward, often just 2D gel electrophoresis/Western blot (Fig 1A). Comparing the colorimetric anti-HCP Western blot to a total protein stain, and identifying the matching spots, provides an estimate of antibody-to-HCP coverage.
This approach provides good resolution of individual HCPs as a 2D map, but has several challenges including denaturation of HCP antigens, which might not represent native antigen in ELISA, variability in electrophoresis and transfer efficiency between gels, reliability of matching spots between blot and gel, consistency between users in judging coverage and time required for experimental preparation and data analysis.
The US pharmacopeia proposes using an immunoaffinity approach, followed by differential gel electrophoresis (DIGE) for checking coverage. DIGE is an established multiplexing variation of 2D gel electrophoresis/Western blot (Fig 1B) that enables you to separate and image up to three samples simultaneously, simplifying workflows and improving the reliability of spot analysis. In this case, the method labels the column chromatography fractions: input (all HCPs), eluate (HCPs covered by anti-HCP antibodies), and flow-through (HCPs missed by antibodies), with different fluorophores. These fractions can run on a single 2D gel, benefiting from multiplexed fluorescence imaging and objective analysis by software.
A simpler way of gaining some or all the benefits of DIGE without changing approach is provided by 2D DIBE: differential in blot electrophoresis (Fig 2) which takes the immunodetection method used in Western blotting and combines it with the fluorescent tagging and imaging of DIGE. The resulting assay format retains the high sensitivity of a primary/secondary antibody system, and the ability to multiplex using fluorescent dyes. When switching from a colorimetric Western blot, taking this approach allows you to run one gel rather than two.
Reducing the number of gels saves time and reagents and removes any variation from imperfect duplicate gels and transfers. Using a single sample of HCPs for detecting both total protein and anti-HCP immunoreacted spots also removes the risk of bias that could result from HCP loading differences between two gels.
The 2D DIBE workflow proposed here is no more time-consuming or challenging to set up than the existing standard 2D gel electrophoresis/Western blot coverage assay. The HCP samples need to be labelled with a fluorescent dye and the existing secondary antibody muse be swapped for a fluorescently-tagged antibody.
Thus, the 2D DIBE/DIGE provides:
- Sensitivity: Sub-nanogram order of total HCP detection
- Accuracy: Resolution of a 2D map of HCPs
- Speed: Fewer gels needed compared to colorimetric Western blot
- Objectivity: Quantitative analysis of fluorescent signal by Melanie software
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