HCP Coverage Analysis

The successful development of an HCP ELISA requires evaluating the coverage of the generated polyclonal antibodies (pAbs) to ensure effective detection of a broad range of host cell proteins (HCPs). According to recent EMA and FDA regulations, BioGenes highly recommends the usage of orthogonal methods for reliable characterization of the developed antibody reagents. 

BioGenes uses various methods to assess antibody coverage:

We are a specialist for high-resolution 2D fluorescent Western Blotting: The HCP mock material and/or an early downstream processing (DSP) sample are fluorescently labeled and subjected to high-resolution 2D PAGE and subsequent Western Blotting. Fluorescent immunodetection is performed with the selected capture antibody and a fluorophore-conjugated detector antibody.  % Coverage is calculated as the ratio of HCP species detected by the pAb divided by the total number of HCPs detected on the Western blot membrane. This technique is a widely accepted standard method. 

Immunoaffinity chromatography (IAC) in combination with 2D DIGE is an orthogonal method for coverage analysis under non-denaturing conditions. The capture antibody will be immobilized and HCPs will be extracted from the sample of interest. IAC allows for enrichment of low-abundant HCPs on the capture column, resulting in a broad dynamic range of detection. 2D DIGE is afterwards performed for visualizing and comparing HCP spot patterns between the eluted sample fraction and the input sample. 

MS-based analysis is highly valuable for gaining insights into the identity of HCPs present in a given sample. Input samples and eluted sample fractions after IAC may be analyzed by MS methods to determine coverage and to collect additional protein information. BioGenes offers IAC in combination with MS analysis for coverage analysis in collaboration with our experienced partners. 

Regulatory Guidelines

Drug impurities originating from the manufacturing process, like host cell proteins (HCPs), possess the potential to have adverse effects on the overall excellence, safety, and effectiveness of a biological drug item. Specifically, the guidelines ICH Q6B, Q8(R2), and Q11 delineate these sorts of impurities and emphasize the requirement for accurate surveillance and the minimization of HCPs throughout gradual downstream processing (DSP), until they reach minimal levels.

Detailed directives and general information concerning the quantification of remaining host cell proteins in biopharmaceuticals are accessible here:

• Ph. Eur. 9.1., 4041 (04/2017) Pharmacopoeia E. 2. 6.34 Host cell protein assays. EUROPEAN PHARMACOPOEIA. 2017; 91: 4041–17.
• U.S. Pharmacopeia Monograph <1132> Residual host cell protein measurement in biopharmaceuticals. Second supplement to USP38-NF33, United States Pharmacopeia: Rockville, MD, 2016.
• ICH Q6B. Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. European Medicines Agency: London, UK, 1999.