Host Cell Protein Analysis

by 1D SDS, 2D PAGE, or LC MS/MS

Do you know all the protein contaminants in your therapeutic protein product?

When proteins from one organism are expressed recombinantly in another organism, the purified protein always contains small amounts of contaminating proteins from the expression organism. These Host Cell Proteins (HCP's) in medicinal products are process-related impurities that must be identified and evaluated qualitatively and quantitatively.

Alphalyse offer identification and relative quantification of individual HCP's by gel electrophoresis and mass spectrometric analysis.

HCP contaminant profiles in manufacturing process
HCP profiles of individual drug substance batches
Regulatory documentation of pre-clinical toxicology batches and clinical batches

1D electrophoresis and HCP Identification by Mass Spectrometry

1D SDS PAGE combined with sensitive silver staining of the gel, followed by protein identification using mass spectrometric peptide mapping, offers an attractive supplement to the ELISA Host Cell Protein assay.
This analysis does not require time-consuming production of antibodies nor development of a process-specific antibody based ELISA assay.

The 1D SDS PAGE visualize the individual HCP bands and allows batch to batch comparison of the relative amounts. The protein bands are cut out from the gel, digested with trypsin and the peptide mixture analysed by MALDI MS/MS. A Mascot database search provides the identity of the individual HCPs in the protein band. The database contains the majority of protein sequences from common bacterial, plant and mammalian expression organisms, e.g. Yeast, E.coli, sf9 Insect cells, Rice, CHO cells.

: Silver-stained 1D gel with 3 drug substance batches.The protein bands were cut out and identified by mass spectrometric peptide mapping and database searching as drug substance (DS) or host cell protein (HCP).

1D SDS PAGE with silver staining and mass spectrometric protein identification are standard techniques that are applied off-the-shelf on a weekly basis by Alphalyse.

2D Gel Electrophoresis and HCP Identification

2D PAGE provides an even more detailed analysis of individual HCPs than 1D SDS PAGE.

If the HCP level is very low, it is also possible to load a higher protein amount on the 2D gel compared to the 1D gel.

However, the 2D PAGE also has certain limitations, for example limitations in pI and MW range, and that hydrophobic membrane proteins are not observed.

LC MS/MS analysis for Host Cell Protein Identification

In the discovery phase complex Host Cell Protein patterns may also be analysed by LC MS/MS on a QTOF or OrbiTrap mass spectrometer, with database searching of tryptic peptides from the digested drug substance batch.

Once the HCP's are identified they may be quantified individually by Multiple Reaction Monitoring (MRM) on a quadrupole mass spectrometer, using label-free quantitation or relative iTRAQ quantitation.