CHO HCP Assay
The CHO HCP assay is an analytical method for the quantitative detection of impurity proteins originating from CHO cells. CHO cells are widely used in the production of recombinant proteins, monoclonal antibodies, and cell therapies due to their high protein expression capacity, well-established culture systems, and efficient human-like post-translational modification capabilities. However, alongside the expression of target proteins, CHO cells also secrete a substantial number of endogenous, product-unrelated proteins. These host cell proteins (HCPs) are often difficult to completely eliminate during downstream purification and may persist in the final formulation, posing potential immunogenicity or toxicity risks. As a result, major regulatory authorities require quantitative monitoring of CHO HCPs to assess removal efficiency and ensure product safety. The CHO HCP assay is not only an integral component of biopharmaceutical quality control but also a critical step for ensuring the safety, consistency, and stability of biological products. From a methodological perspective, the CHO HCP assay typically employs immunoassays based on antibody-antigen recognition, with enzyme-linked immunosorbent assay (ELISA) being the most commonly used technique. ELISA relies on polyclonal antibodies to achieve broad-spectrum recognition of CHO-derived proteins and utilizes colorimetric reactions to quantify the concentration of HCPs in the sample. Owing to its high level of standardization, high throughput, and excellent sensitivity, ELISA has been widely adopted for the CHO HCP assay. With the advancement of proteomics, liquid chromatography–mass spectrometry (LC-MS) is emerging as a complementary or alternative approach, enabling the identification and quantification of individual HCP species. LC-MS facilitates the investigation of batch-to-batch variability in HCP profiles and supports risk assessment and process optimization.
One of the major challenges in the CHO HCP assay is achieving a balance between broad detection coverage and sufficient antibody reactivity. CHO cells produce a complex array of proteins with diverse structures, and although polyclonal antibodies offer wide-ranging reactivity, they may fail to adequately detect low-abundance or structurally atypical proteins. This can result in underestimation of total HCP levels. Therefore, assessment of antibody coverage is a crucial aspect of assay validation. Common approaches include two-dimensional electrophoresis coupled with Western blotting (2D-Western blot) and mass spectrometric identification following immunoaffinity enrichment. These techniques help ensure that the assay captures the majority of relevant HCP species with sufficient sensitivity and specificity.
In terms of sample preparation, the CHO HCP assay must account for potential interference from formulation components. High concentrations of the target protein, surfactants, or stabilizers may obscure HCP signals or interfere with antibody binding specificity. Thus, sample pretreatment steps such as appropriate dilution, buffer optimization, and removal of interfering substances are critical for reliable measurement. The use of well-characterized standard curves, suitable positive and negative controls, and optimized assay conditions forms the basis for generating robust and reproducible results in the CHO HCP assay.
Leveraging a high-sensitivity immunoassay platform in combination with proteomics-based strategies, MtoZ Biolabs offers customized HCP detection services tailored to specific product types and production stages. Our capabilities encompass end-to-end assay development, method validation, and data interpretation. We are committed to providing scientifically rigorous and efficient solutions to support the establishment of robust quality control systems and enhance the competitiveness of our clients’ products.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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