Host Cell Protein monitoring in process development – using mass spec

Oct 2. 2009

Mass spectrometry-based Host Cell Protein identification and quantification

The Mass Spec Host Cell Protein (HCP) assay is applied for monitoring product purity and consistency of the pre-clinical tox batch and two clinical batches of a recombinant vaccine protein.

We separated proteins by 1D SDS PAGE and visualized them by silver staining. Then we digested the protein bands with trypsin and the proteins identified by MALDI MS/MS peptide mapping and database searching. In total, we identified eight specific HCPs.

The protein identities and information about physical properties, such as pI, Mw, and hydrophobicity, can guide further protein purification and process development for phase III. An advantage compared to ELISA-based HCP assays is that the MS HCP assay provides the identities of the contaminating HCPs. Also, it does not require the time-consuming development of antibodies.

Advantages of Mass Spec orthogonal method

  •  Immediate solution
  •  Alternative to ELISA when not available
  •  Identity for each HCP
  •  The relative quantity of HCPs
  •  Complementary with Western blot
  •  Fast assay setup in 2 weeks


  •  PAT compliant
  •  Monitor process development,
  •  Compare pre-clinic TOX batches and clinical batches

Learn more about Mass Spec Host Cell Protein analysis

Mass spec - Host Cell Protein Assay Principle

Mass Spec Host Cell Protein Assay Principle

Figure 1: The drug substance [DS] from different cGMP batches (samples 1-3) was first loaded on a 1D SDS-PAGE gel and stained with sensitive silver. Then the gel was overloaded with DS to detect tiny amounts of contaminating proteins. We cut out protein bands for analysis by MALDI mass spectroscopy to identify contaminating host cell proteins. Eight specific HCPs were identified and marked HCP1-HCP8, as well as product-related variants, marked DS.


Mortz et al.: “Proteomics technology applied to upstream and downstream process development of a protein vaccine,” Bioprocess International 6, p36-43, 2008.

More posts about Analysis of Biopharmaceuticals
  • Host Cell Protein monitoring in process development – using mass spec

  • Identification of small host cell proteins (small Mw HCPs)

  • Determine the Molar Extinction Coefficient of protein in 3 small steps

  • Why you need accurate concentration determination of protein standards

  • What is SWATH mass spectrometry and its advantages?

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

Newsletter Subscribe to The Alphalyse Newsletter and get more
information on protein analysis methods and applications
© 2022 Alphalyse, Inc.