Host Cell Proteins (HCPs) are a heterogeneous, complex group of proteins that derived from the host cell. HCPs differ significantly in molecular mass, isoelectric point, hydrophobic properties, and structures. Figure 1 shows a harvested product pool of bacterial and mammalian fermentation by two-dimensional difference gel electrophoresis (2D-DIGE). Identification and quantification HCPs is very important in the development of biopharmaceutical products to ensure product purity and patient safety. Protease of HCPs can influence the protein pattern in culture and influence the purification process. In addition, HCPs can undergo post-translational modifications that make quantification and characterization more difficult. Moreover, high HCP variance results in different biochemical properties which could cause an immune response or allergic reactions even anaphylactic shock.
Figure 1 2D-DIGE silver stain of a harvested product
Although the host cells contain thousands of HCPs and that could potentially contaminate the final product and the host cells can be various organisms, such as, bacteria, yeast and cell lines derived from mammalian or insect species, there is no explicitly guideline that describes the selection of appropriate methods for characterization and quantification of HCPs and other impurities for biopharmaceuticals. Moreover, the regulatory agencies examine each protein drug on a case-by-case basis to determine appropriate maximum acceptable levels of HCPs and patient risk.
The selection of appropriate test methods for identification and/or quantification of HCP have been a concern during process development and manufacturing. The methods used for HCP analysis can be classified as either immune-specific methods, such as, western blotting and ELISA, or non-specific methods, such as, electrophoresis and MS. In general, a qualitative or semi-quantitative method would be sufficient for sample purification and for lot release. However, during process of drug development and characterization, the ideal method not only should have the capability to recognize all HCPs species, but also should be quantitative, high-throughput, time-saving and labor-saving.
A range of methods for the detection and characterization of HCPs are presented in Creative Proteomics (Table 1). Among these, ELISA, electrophoresis, and MS are the most common techniques and are sometimes they are used in combination for detailed characterization.
Table 1, Overview methods of HCPs methods
1D/2D - Silver stain/Coomassie blue/SYPRO Ruby/CyDyes/Autoradiography
1D/2D - Silver stain/Coomassie blue - HPLC - Mass spectrometry (Non-target,shotgun)
2D HPLC-mass spectrometry (Non-targeted, shotgun)
HPLC-mass spectrometry (target MRM)
Identification / quantification of HCPs
A detailed technical report will be provided at the end of the whole project, including the sample preparation, experimental procedure and instrument parameters.
The stain results, protein list (FDR<1%)/and concentration/amount will be reported in the report.