Host cells are the most commonly used key starting materials for the production of recombinant proteins, antibody drugs or vaccine drugs. The types of host cells used for the production of biological products mainly include bacterial cells (such as Escherichia coli), yeast cells (such as Saccharomyces cerevisiae) and mammalian cells (including Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK) cells), etc.
As non-target components in biological products, host cell proteins (HCPs) residues may cause immune responses in the body. At the same time, some HCPs with enzymatic activity may affect the composition of proteins in the final product and the stability of the product. They have an important impact on the quality, safety and efficacy of biological products and are considered to be critical quality attributes (CQA) of biological products. They need to be focused on in the release test of finished products and stock solutions and the process control of intermediate products. Studies have shown that even after complex purification processes, low concentrations (1~100 ppm) of host cell proteins may still remain in the final biological products. Therefore, establishing appropriate HCP detection methods will help control the quality of biological products and improve the safety of biological products.
In recent years, with the continuous advancement of cell engineering, cell line development, culture medium and culture condition optimization technology, the production capacity of cells has been continuously improved. However, with the improvement of process level, higher cell density and longer culture cycle also produce higher levels of process-related impurities, including HCPs. Therefore, it is very important to have a deep understanding of the technologies related to HCPs analysis and the behavior of these impurities in upstream and downstream processing. In addition, for the HCPs that remain in the biological products at the low ppm level after purification, especially the quality control of high-risk HCPs, how to achieve accurate qualitative and quantitative analysis is particularly critical for the quality control and process development of biological products.
Methods for determining residual host cell proteins in biological products mainly include enzyme-linked immunosorbent assay (ELISA) and immunoblotting based on specific antibodies, as well as non-immune specific methods represented by liquid chromatography-mass spectrometry (LC-MS). Currently, ELISA is the most commonly used method for HCP detection.
Table 1. Method for determining residual host cell proteins in biological products
| Method | Advantages | Disadvantages | Application |
|---|---|---|---|
| ELISA: ELISA utilizes antigen-antibody reaction to detect and quantify HCPs by producing a color change through a labeled enzyme-catalyzed substrate. | High sensitivity and specificity: Able to detect HCPs as low as ng/mL level. Simple operation: Suitable for large-scale screening with high automation. Quantitative analysis: Can provide accurate HCPs concentration data. |
Antibody dependence: Specific antibodies are required, and the preparation and validation of antibodies are costly and time-consuming. Incomplete detection: There may be a lack of suitable antibodies for some HCPs, resulting in missed detection. Cross-reaction: There may be cross-reactions of antibodies, affecting the accuracy of detection. |
Process development: Used to monitor HCPs levels in different process steps. Quality control: As a key detection method for final product release. |
| Mass Spectrometry: Mass spectrometry (MS) provides qualitative and quantitative information of HCPs by ionizing sample molecules and separating and detecting them according to their mass-to-charge ratio. | High sensitivity and resolution: Able to detect and distinguish a variety of HCPs. Wide coverage: No specific antibodies are required, suitable for unknown or newly discovered HCPs. Detailed information: Provides detailed information such as molecular weight and amino acid sequence. |
Complex operation: Difficult sample preparation and data analysis. Expensive equipment: Requires high-cost instruments and technical support. Long analysis time: Suitable for small-scale, detailed analysis, not suitable for high-throughput screening. |
HCPs Identification: Identify and quantify HCPs in complex mixtures. Antibody Development: Used to confirm and validate targets for ELISA antibodies. |
| SDS-PAGE: Proteins were separated according to molecular weight by SDS-PAGE and then stained with dyes to detect the total amount and distribution of HCPs. | Simple operation: low cost, low equipment requirements. Intuitive visualization: can directly observe the distribution and relative concentration of HCPs. |
Low sensitivity: can only detect HCPs at higher concentrations (μg/mL level). No precise quantification: difficult to provide accurate HCPs concentration. Limited resolution: difficult to distinguish HCPs with similar molecular weight. |
Preliminary screening: quickly evaluate the total amount and approximate distribution of HCPs. Process monitoring: used for process optimization and comparison between different batches. |
| Western Blot: Transfer the proteins separated by SDS-PAGE to the membrane, then use specific antibodies to detect the target HCPs, and detect the signal by chemiluminescence or color development. | High specificity: Able to detect specific HCPs. Qualitative and quantitative: Provide qualitative and relative quantitative information of specific HCPs. |
Complex operation: Long time, suitable for small-scale analysis. Antibody dependence: Specific antibodies are required, and the cost of antibody preparation and verification is high. Limited sensitivity: Limited by the sensitivity of the detection method and the antibody. |
Specific HCPs detection: Verify ELISA test results. R&D stage: Used for in-depth research on specific HCPs. |
| Capillary Electrophoresis: Separate proteins through the electric field in the capillary and detect HCPs based on the difference in electrophoretic mobility. | High resolution: Able to accurately separate different HCPs. High automation: High degree of automation in operation, suitable for high-throughput analysis. |
Expensive equipment: Requires high-cost equipment and maintenance. High technical requirements: Complex sample preparation and long analysis time. Limited sensitivity: May not be sensitive enough for some HCPs. |
Quality control: Used for detailed HCPs analysis. Process optimization: Used to optimize and monitor the production process of biological products. |
| HPLC: HCPs are separated by liquid chromatography columns, and detected and quantified according to different retention times. | High sensitivity and resolution: Able to separate and quantify different HCPs. High automation: The operation is highly automated and suitable for quantitative analysis. |
Expensive equipment: Requires high-cost instruments and technical support. Complex sample preparation: Long sample preparation time and slow analysis speed. Need to be combined with other detection methods: Such as MS, in order to accurately identify HCPs. |
Quality control: Used for detailed HCPs analysis and quantification. R&D stage: Used to optimize and monitor the production process of biological products. |
Explore Creative Biogene's Host Cell Protein Assay Kits
| Cat.No. | Product Name | Price |
|---|---|---|
| HCPAK-01 | E.coli HCP Assay Kit | Inquiry |
| HCPAK-02 | Pichia pastoris HCP Assay Kit | Inquiry |
| HCPAK-03 | CHO HCP Assay Kit | Inquiry |
| HCPAK-04 | HEK293 HCP Assay Kit | Inquiry |
| HCPAK-05 | SF9 HCP Assay Kit | Inquiry |
| HCPAK-06 | MDCK HCP Assay Kit | Inquiry |
| HCPAK-07 | NS0 HCP Assay Kit | Inquiry |
| HCPAK-08 | Vero HCP Assay Kit | Inquiry |