Analysis of Pichia pastoris Components in Recombinant Human Serum Albumin by Immunological Assays and by HPLC with Pulsed Amperometric Detection

We have developed a recombinant human serum albumin (rHSA) from Pichia pastoris which expresses high levels of heterologous proteins. rHSA is used clinically in high concentration (approximately 250 mg/ml in a 50 mL vial). We had to consider not only proteins from host cells as impurities but also m...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 1998-01, Vol.70 (2), p.425-429
Main Authors: Ohtani, Wataru, Ohda, Toyoo, Sumi, Akinori, Kobayashi, Kaoru, Ohmura, Takao
Format: Article
Language:English
Subjects:
Analysis
Biological and medical sciences
Biotechnology
Chromatography, High Pressure Liquid
Drug Contamination
Fundamental and applied biological sciences. Psychology
Fungal Proteins - analysis
Health. Pharmaceutical industry
Hormones
Humans
Immunosorbent Techniques
Industrial applications and implications. Economical aspects
Organic chemistry
Other active biomolecules
Pichia
Production of active biomolecules
Proteins
Recombinant Proteins - chemistry
Serum Albumin - chemistry
Yeast
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Summary:We have developed a recombinant human serum albumin (rHSA) from Pichia pastoris which expresses high levels of heterologous proteins. rHSA is used clinically in high concentration (approximately 250 mg/ml in a 50 mL vial). We had to consider not only proteins from host cells as impurities but also mannan, which exhibits harmful effects on humans. However, the analysis of mannan in biopharmaceuticals produced from yeast has not been reported. Contaminating mannans in the final product were one important index to assess the clinical safety of rHSA. We have developed a highly sensitive enzyme immunoassay (EIA), utilizing an avidin−biotin system, for the detection of either the protein or mannan polysaccharide components from P. pastoris components (PPC) in rHSA. In addition, we used anion exchange chromatography with pulsed amperometric detection (AE-PAD) for monosaccharide analysis of glycoconjugates for the detection of mannan from PPC in rHSA. The detection limits of the EIA for PPC (PPC EIA) and the AE-PAD were 1 ng of protein/250 mg of rHSA and 180 ng of mannose/mg of rHSA, respectively. The mannan content in partially purified rHSA as determined by the AE-PAD was about same as the PPC content as determined by the PPC EIA. We showed that the PPC EIA and the AE-PAD are useful methods for the purity analysis of biopharmaceuticals produced from yeast.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac970596h