Immunogenic Host Cell Protein Analysis

Host Cell Protein

Host cell proteins (HCPs) are a major class of process-related impurities (PRIs) that arise during biopharmaceutical manufacturing. Whether it is HCP in the cell culture harvest before the downstream purification process, or residual HCP in the drug substrate or formulation after the purification process, the efficacy, quality, and shelf life of the drug can be affected.

Fig.1 Workflow for identifying HCPs in supernatants.¹

The primary problem with residual HCPs as exogenous proteins is that they trigger an immune response in patients when administered. Immune responses to exogenous proteins occur through a T cell-dependent pathway, where proteins are uptaken, digested, and presented by antigen-presenting cells, then recognized by T cells, followed by further activation and maturation by B cells expressing complementary antibodies. Although the scale of immunogenicity of CHO HCP is smaller than that of non-mammalian organisms such as E. coli or yeast, the sequence differences between CHO and human proteins are significant. In addition to direct immunogenicity, HCPs have the potential to induce and enhance anti-drug antibodies or induce immune responses to endogenous proteins. Creative Biolabs offers a range of HCP analysis services and kits to ensure the quality and safety of your biologics.

HCP Analysis Methods

Creative Biolabs offers, but is not limited to, the following HCP analysis methods:

• ELISA
  Enzyme-linked immunosorbent assay (ELISA) is commonly used to measure total HCP concentration, the most commonly used of which is polyclonal antibodies produced by injecting HCPs from empty cell lines into animal models.
• 1D/2D-PAGE
  1D polyacrylamide and 2D polyacrylamide gel electrophoresis (1D/2D-PAGE) have been widely used to study HCP kinetics at various stages of the biotherapeutic recombinant protein production workflow.
• LC-MS/MS
  2D-PAGE binds to LC-MS/MS which can identify specific HCPs, and Western blotting can also be performed using anti-HCP antiserum or specific HCP antibodies.
• Protein Isoelectric Point
  The protein isoelectric point determination method is a commonly used analytical method to determine the isoelectric point of a protein. By determining the isoelectric point of proteins, we can help us understand the charge properties of proteins, so as to better analyze and control HCPs. Commonly used methods for determining the isoelectric point of proteins include Isoelectric Focusing (IEF) and Isoelectric Point Electrophoresis (IEP).
• Others
  Novel detection techniques, such as proximity ligation assay (PLA) and biosensor technology, are also being used to detect HCPs.

In addition to being used alone to detect HCP, the combination of these techniques can be more effective. If ELISA is combined with 2D-PAGE and western blotting is used, the coverage of antibodies in the ELISA can be verified, which can help improve the accuracy of the ELISA method. 2D-PAGE can also be combined with LC-MS/MS to identify specific HCP components and help identify high-risk factors.

Based on years of continuous research in the fields of proteomics and genomics, Creative Biolabs has become more and more knowledgeable about HCP. Creative Biolabs offers customized HCP analysis services. In addition, we develop specific kit products for specific host cells and process nodes to ensure the quality and safety of your biologics. If you have a need for HCP analysis, please contact us now.

References

1. Mazziotta, C.; et al. Cancer biology and molecular genetics of A3 adenosine receptor. Oncogene. 2022, 41(3): 301-308.
2. Park, J.H.; et al. "Proteomic analysis of host cell protein dynamics in the culture supernatants of antibody-producing CHO cells." Scientific reports. (2017) 7 44246.

For Research Use Only | Not For Clinical Use