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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.

In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute. Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable. In this context, disulfide linkages play a significant structural role and their determination is of paramount importance. As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest. The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing. Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined. The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.