A critical task in biologics manufacturing is the control of process-related impurities such as host cell proteins (HCPs), which co-purify with the drug substance and are known for adverse effects (e.g., immunogenicity). For decades, the method of choice to detect host cell proteins has been an immunoassay based on polyclonal antisera raised against a population of HCPs. However, there are some inevitable risk factors (e.g., antigens, animals, etc.) associated with this approach, and the final selectivity and sensitivity of the HCP assay strongly depends on the preparation work and materials used at the start.

Combining tools from proteomics, bioinformatics, protein synthesis and immunology, effectively minimizes black box elements, provides targeted enhancement of coverage, and maximizes the performance of HCP assays.

PPS is offering services in:

1) Testing and demonstration of specificitiy of generic and specific HCP assays

2) HCP-GAPex™ – Development of HCP assays with improved sensitivity and selectivity

3) Identification of HCPs by shotgun proteomics techniques as orthogonal method to immunoassays

 

Demonstration of specificitiy

For demonstration of specificity high resolution 2D PAGE of respective antigens (e.g. whole cell lysate, DSP fraction) is performed. Total HCPs are visualized by sensitive silver staining. In parallel a second 2D PAGE gel is subjected to Western blotting to visualize all HCPs recognized by  respective antisera. These patterns are compared and visualized by overlay. Ideally, all HCPs visualized by silver staining are also recognized in the Western blot experiment, which would mean there are no detection gaps of the used antiserum. If detection gaps occur, we recommend usage of our HCP-GAPex™ services.

Application example for qualification of HCP antiserum:

CHO cell lysate analyzed by high resolution 2D PAGE and Western Blotting (overlay: blue-silver stain, yellow-Western Blotting)

 

HCP-GAPex™ Services

HCPs not recognized by respective antisera used in immunoassay based release tests comprise significant risks for patients. HCP-GAPex™ addresses these detection gaps by following services:

 

  • Proteins, which represent detection gaps can be unambiguously identified by a combination of high-resolution 2D Western Blotting with subsequent mass spectrometric analyses
  • Identified proteins are screened for immunogeneic peptides, which are basis for design and production of protein specific immunogens
  • Protein specific antibodies recognizing previous HCP gaps are combined with antiserum in order to obtain maximum detection coverage

 

HCP-GAPex™ is a combined service of PPS in our strategic alliance with Charles River Labs -  Biopharmaceutical Services (CRBS).

For more information also have a look at our recent HCP-Webinar.

 

Identification of HCPs by shotgun proteomics techniques as orthogonal method

Significant improvements in mass spectrometry have allowed us to use the technique as a complementary method to immunoassays and gain more detailed intelligence on individual HCP patterns and species. For identification of HCP during DSP and in the final product we combine solution digestions and separation techniques such as nano-RP-HPLC or ion exchange chromatography with online analysis on highly sensitive mass spectrometers such as Orbitrap, Xevo or Maxis. This approach facilitates comparison of individual identified HCPs in the field of production or DSP process optimization or Biosimilar development.

 

Your contact for ICH Q5E compliance at Protagen Protein Services GmbH

Thomas Flad

Dr Thomas Flad

Director Business Development
Our business development team will be happy to assist you with your project.

Phone: +49 (0) 231 9742-6100
Email: salesproteinservices@ProtagenProteinServices.com