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Host cell protein detection gap risk mitigation: quantitative IAC-MS for ELISA antibody reagent coverage determination
- Source :
- mAbs, article-version (VoR) Version of Record
- Publication Year :
- 2021
- Publisher :
- Informa UK Limited, 2021.
-
Abstract
- Host cell proteins (HCPs) must be sufficiently cleared from recombinant biopharmaceuticals during the downstream process (DSP) to ensure product quality, purity, and patient safety. For monitoring of HCP clearance, the typical method chosen is an enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-HCP antibodies obtained from an immunization campaign. This polyclonal reagent is a critical factor for functionality and confidence of the ELISA. Therefore, it is important to ensure that the pool of ELISA antibodies covers a broad spectrum of the HCPs that potentially could persist in the final drug substance. Typically, coverage is determined by gel-based approaches. Here, we present a quantitative proteomics approach combined with purification of HCPs by immunoaffinity chromatography (qIAC-MS) for assessment of ELISA coverage. The cell culture fluid (CCF) of a mock fermentation and a recombinant monoclonal antibody product were characterized in detail to investigate whether the HCPs used for immunization of animals accurately represent HCPs that are relevant to the process. Using the qIAC-MS approach, the ELISA antibody coverage was determined for mock fermentation and product CCF, as well as several different DSP intermediates. Here, the use of different controls facilitated the identification and quantification of HCPs present in the polyclonal reagent and those that nonspecifically bound to IAC material. This study successfully demonstrates that the described qIAC-MS approach is not only a suitable orthogonal method to commonly used 2D SDS-PAGE-based analysis for evaluating ELISA antibody coverage, but that it further identifies HCPs covered as well as missed by the ELISA, enabling an improved risk assessment of HCP ELISA.
- Subjects :
- medicine.drug_class
education
Immunology
Quantitative proteomics
ELISA coverage
Enzyme-Linked Immunosorbent Assay
CHO Cells
immunoprecipitation
Monoclonal antibody
Protein detection
law.invention
Cricetulus
proteomics
Affinity chromatography
law
Report
medicine
Animals
Humans
Immunology and Allergy
Host cell protein
mass spectrometry
Chromatography
biology
Chemistry
IAC
qIAC-MS
Antibodies, Monoclonal
affinity purification
HCP
biopharmaceuticals
Recombinant Proteins
Polyclonal antibodies
Reagent
biology.protein
Recombinant DNA
Antibody
immunoaffinity chromatography
Subjects
Details
- ISSN :
- 19420870 and 19420862
- Volume :
- 13
- Database :
- OpenAIRE
- Journal :
- mAbs
- Accession number :
- edsair.doi.dedup.....09eebd0c83dbffb04baa8ce6ce8d45f3