Host Cell Protein monitoring in process development – using mass spec

Alphalyse
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 2 clinical batches of a recombinant vaccine protein.

We separated proteins by 1D SDS PAGE and visualized 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, 8 specific HCPs were identified.

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 time-consuming development of antibodies.

Advantages of Mass Spec orthogonal method

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

Applications

  •  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) were first loaded on a 1D SDS-PAGE gel and stained with sensitive silver stain. Then the gel was overloaded with DS for detection of very small amounts of contaminating proteins. We cut out protein bands for analysis by MALDI mass spectroscopy to identify contaminating host cell proteins. Eight specific HCP’s were identified and marked HCP1-HCP8, as well as product-related variants, marked DS.

Reference:

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

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