ELISA-Entwicklung GmbH & Co. KG

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     Range of Services
     Assay Development
     Assay Validation
     Sample Measurement
     Protein Purification
     Antibody Production
     Anti Drug Antibody, ADA



Host Cell Protein (HCP)

Challenges of Monitoring Host Cell Protein Impurities

Recombinant protein pharmaceuticals have to be purified to a high degree from host cell components to avoid adverse drug effects like allergic or anaphylactic reactions. Typically a multianalyte enzyme-linked immunosorbent assay (ELISA) is the method of choice, using polyclonal antibodies to fulfil the following features:
-  A simple validated method
-  High sensitivity to find trace amounts in a final drug substance
-  Exact reproducible quantification
-  Recognition of a wide number of epitopes present in the impurities

Options to increase HCP recognition

  • Utilisation of Polyclonal antibodies instead of monoclonal
  • Usage of a mix of antibodies of different host species (e.g. goat and rabbit),
  • Protein A purified antibodies instead of specifically HCP purified antibodies for additional measurement of epitopes with low antigenity,
  • Streptavidin coated plates instead of direct antibody coating to reduce antibody denaturation,
  • Mix of differently conjugated antibodies to biotin and HRP (antibody linked via lysine and via cysteine),
  • Test performance with only one incubation step to reduce antigen loss of low-affinity antibodies

Principle of a HCP ELISA

  • an excess of streptavidin coating reduces well to well variation and unspecific background
  • indirect antibody coating via streptavidin avoids AB binding site denaturation which occurs during direct AB coating
  • indirect AB coating increases sensitivity by reducing steric hindrance
  • direct AB-enzyme conjugation allows a 1-step incubation assay protocol to reduce dissociation of low affinity AB
  • using a combination of Cys- and Lys-linked AB increases antigen binding

Optional established methods supporting HCP assay development

  • Organization and supervision of antibody production
  • Antiserum screening, purification and conjugate syntheses
  • 1D and 2D electrophoresis with silver staining for HCP visualization
  • Western blotting for demonstration of antibody binding and specifity
  • Assay development, validation, documentation and test kit production
  • Technology transfer and adaptation at customer lab including training of responsible persons
  • Adaptation to automatic systems if desired