Host Cell Protein (HCP) Detection Methods and Their Principles

HCP (Host Cell Proteins) refer to protein impurities from the production cells in the process of biological product production. For example, when recombinant proteins are produced using mammalian cells (such as CHO cells), in addition to the target protein, there may be some proteins from the CHO cells themselves in the final product. These HCPs may affect the stability, potency, or safety of the product, so they need to be detected and controlled.

 

Below are the commonly used methods for HCP detection and their principles.

 

1. ELISA (Enzyme-Linked Immunosorbent Assay)

(1) Principle: Specific anti-HCP antibodies are fixed on the enzymatic plate. HCPs in the test sample bind to the antibody, then enzyme-labeled secondary antibodies that bind to HCPs are added. By measuring the activity of the enzyme, the content of HCPs can be indirectly measured.

(2) Advantages: Sensitive, can quantify the total amount of HCPs.

(3) Disadvantages: High-quality anti-HCP antibodies are required, may not detect all HCP impurities.

 

2. Mass Spectrometry

(1) Principle: Uses a mass spectrometer to detect and identify proteins in the sample.

(2) Advantages: Can identify specific types of HCPs, high resolution.

(3) Disadvantages: High technical requirements, high cost, complex data processing.

 

3. Immunoblotting (Western Blot)

(1) Principle: Proteins are separated by gel electrophoresis, then transferred to a membrane, and the target HCPs are detected using specific anti-HCP antibodies.

(2) Advantages: Can identify and quantify specific HCPs.

(3) Disadvantages: Can only detect HCPs that react with the used antibodies.

 

4. Two-Dimensional Gel Electrophoresis (2D-PAGE)

(1) Principle: Proteins are separated by gel electrophoresis in two directions, one dimension by isoelectric point, and the second dimension by molecular weight, forming a two-dimensional map of proteins.

(2) Advantages: Can observe the distribution of all proteins in the sample.

(3) Disadvantages: High technical requirements, can't directly quantify.

 

In the development and production of biological products, multiple methods are usually combined to detect and control HCPs to ensure the quality and safety of the product.