Analysis of Pichia pastoris components in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric detection
- PMID: 9450369
- DOI: 10.1021/ac970596h
Analysis of Pichia pastoris components in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric detection
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 showed that the PPC EIA and the AE-PAD are useful methods for the purity analysis of biopharmaceuticals produced from yeast.
Similar articles
-
Extraction and purification of recombinant human serum albumin from Pichia pastoris broths using aqueous two-phase system combined with hydrophobic interaction chromatography.J Chromatogr A. 2012 Jul 6;1245:143-9. doi: 10.1016/j.chroma.2012.05.041. Epub 2012 May 15. J Chromatogr A. 2012. PMID: 22658659
-
In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time.Pharm Res. 2001 Dec;18(12):1775-81. doi: 10.1023/a:1013391001141. Pharm Res. 2001. PMID: 11785700
-
Physicochemical and immunochemical properties of recombinant human serum albumin from Pichia pastoris.Anal Biochem. 1998 Feb 1;256(1):56-62. doi: 10.1006/abio.1997.2480. Anal Biochem. 1998. PMID: 9466797
-
Summary of recombinant human serum albumin development.Biologicals. 2006 Mar;34(1):55-9. doi: 10.1016/j.biologicals.2005.08.021. Epub 2006 Feb 7. Biologicals. 2006. PMID: 16464610 Review.
-
The development of recombinant human serum albumin.Ther Apher. 1998 Nov;2(4):257-62. doi: 10.1111/j.1744-9987.1998.tb00118.x. Ther Apher. 1998. PMID: 10227751 Review.
Cited by
-
Cell engineering and molecular pharming for biopharmaceuticals.Open Med Chem J. 2008 May 14;2:49-61. doi: 10.2174/1874104500802010049. Open Med Chem J. 2008. PMID: 19662143 Free PMC article.
-
Recombinant protein expression in Pichia pastoris.Mol Biotechnol. 2000 Sep;16(1):23-52. doi: 10.1385/MB:16:1:23. Mol Biotechnol. 2000. PMID: 11098467 Review.
-
A chloroplast transgenic approach to hyper-express and purify Human Serum Albumin, a protein highly susceptible to proteolytic degradation.Plant Biotechnol J. 2003 Mar;1(2):71-9. doi: 10.1046/j.1467-7652.2003.00008.x. Plant Biotechnol J. 2003. PMID: 17147744 Free PMC article.
-
Production of human serum albumin by sugar starvation induced promoter and rice cell culture.Transgenic Res. 2005 Oct;14(5):569-81. doi: 10.1007/s11248-004-6481-5. Transgenic Res. 2005. PMID: 16245148
-
Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture.Int J Mol Sci. 2021 Aug 5;22(16):8411. doi: 10.3390/ijms22168411. Int J Mol Sci. 2021. PMID: 34445120 Free PMC article. Review.
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources