Analysis of Pichia pastoris components in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric detection

Analytical Chemistry
W OhtaniT Ohmura

Abstract

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...Continue Reading

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Citations

Jan 21, 2000·FEMS Microbiology Reviews·J L Cereghino, J M Cregg
Nov 2, 2011·Proceedings of the National Academy of Sciences of the United States of America·Yang HeDaichang Yang
May 5, 1999·Therapeutic Apheresis : Official Journal of the International Society for Apheresis and the Japanese Society for Apheresis·K KobayashiK Yokoyama
Jan 11, 2012·Molecular Aspects of Medicine·Gabriella FanaliPaolo Ascenzi
Feb 9, 2006·Biologicals : Journal of the International Association of Biological Standardization·Kaoru Kobayashi
May 7, 2013·Biochimica Et Biophysica Acta·Zhen ChenDaichang Yang
Aug 28, 2021·International Journal of Molecular Sciences·Vibhor Mishra, Richard J Heath

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