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A novel approach to evaluate ELISA antibody coverage of host cell proteins-combining ELISA-based immunocapture and mass spectrometry.
Pilely, Katrine; Nielsen, Solveig B; Draborg, Anette; Henriksen, Maiken L; Hansen, Søren W K; Skriver, Lars; Mørtz, Ejvind; Lund, Rikke R.
Afiliação
  • Pilely K; Alphalyse A/S, Odense, Denmark.
  • Nielsen SB; Alphalyse A/S, Odense, Denmark.
  • Draborg A; Alphalyse A/S, Odense, Denmark.
  • Henriksen ML; Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
  • Hansen SWK; Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
  • Skriver L; Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
  • Mørtz E; Savara ApS, Hørsholm, Denmark.
  • Lund RR; Alphalyse A/S, Odense, Denmark.
Biotechnol Prog ; 36(4): e2983, 2020 07.
Article em En | MEDLINE | ID: mdl-32087048
Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme-linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D-Western blot or immunoaffinity-purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA-based immunocapture with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS): ELISA-MS. ELISA-MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti-Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species-specific isotype control antibody. We propose that ELISA-MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA-MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ensaio de Imunoadsorção Enzimática / Proteínas / Anticorpos Monoclonais Limite: Animals / Humans Idioma: En Ano de publicação: 2020 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ensaio de Imunoadsorção Enzimática / Proteínas / Anticorpos Monoclonais Limite: Animals / Humans Idioma: En Ano de publicação: 2020 Tipo de documento: Article