Host cell protein analysis mass

spectrometry

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.

2D-DIGE silver stain of a harvested product 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

Overview methods of HCPs methods

Technology platform

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)

Summary

Identification / quantification of HCPs

Report

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.

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