Summary of recombinant human serum albumin development
Introduction
With a plasma content of 42 ± 3.5 g/l, human serum albumin (HSA) is the major protein component of human plasma. It consists of a single non-glycosylated polypeptide chain of 585 amino acids forming a heart-shaped molecule with molecular weight of 66.5 kDa [1] and comprising three domains [2]. Produced in the liver, HSA is largely responsible for maintaining normal osmolarity in the bloodstream but also functions as a carrier for numerous small molecules. Clinically, HSA is used to treat severe hypoalbuminemia and traumatic shock, with usual dosage in excess of 10 g/dose.
To date, HSA has been produced by fractionation of human plasma. As the source of the blood can vary, there is the potential risk of HSA contamination by blood-derived pathogens. Human plasma is also in limited supply, especially in Japan, with only about one-third of Japanese-produced albumin obtained from domestic plasma. The development of an alternative, industrial method of preparation would therefore greatly assist in the general movement toward self-sufficiency in blood and blood products. Although expected to play an important role, recombinant DNA technology faces a number of problems in the large-scale production of pharmaceutical-grade rHSA, which is unlike other blood proteins and other recombinant proteins in having a structurally complex molecule, a low unit price, and finally a large market volume.
In the present paper, we review the development of rHSA production by Pichia pastoris. As the clinical dosage of rHSA is very high when compared to other pharmaceutical products prepared using recombinant DNA technology, our emphasis will be placed on the character and purity of rHSA.
Section snippets
Expression systems
The non-glycosylated feature of HSA has made it possible to screen a wide range of host organisms for correct structure and high productivity potential. With the aim of establishing an rHSA-producing strain for industrial use, studies have been performed of secretion systems using Bacillus subtilis [3], Saccharomyces cerevisiae [4], Hansenula polymorpha [5] and Kluyveromyces lactis [6]. With B. subtilis, however, incorrect processing of rHSA was reported and improvement by gene manipulation
Purity requirement of rHSA
Secreted rHSA is purified from the culture broth of P. pastoris by a combination of several chromatographic and membrane filtration techniques [10]. It should be noted that as the clinical dosage of HSA often exceeds 10 vials (125 g of HSA), an extremely high degree of purity is required; immunologically active contaminants in particular must be completely removed. Methods such as sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) or high-performance liquid chromatography
Structure of rHSA
Unlike enzymes or cytokines, HSA possesses no biological activity. The similarity of rHSA and pdHA was evaluated by the structural analyses listed in Table 1.
HSA is a globular monomeric protein with a relative molecular weight of 66.5 kDa and containing 35 cysteinyl residues present as 17 disulfide bridges, with one free sulfhydryl group present at Cys34. The presence of these disulfide linkages in rHSA was tested using mild Edman degradation followed by isocratic analysis of the
Antigenicity of rHSA
Antigenicity tests on rHSA were conducted to ensure the safety of the purified product and immunological identity with pdHA. Antisera were obtained from immunized rabbits sensitized with rHSA or pdHA emulsified in Freund's complete adjuvant. As shown in Fig. 4, all immunoprecipitated lines between rHSA or pdHA and anti-rHSA sera fused with one another. Anti-rHSA sera absorbed by pdHA did not form any immunoprecipitated lines with rHSA. The absorbed anti-rHSA sera also indicated negative
Phase I clinical trial
To evaluate the safety of rHSA, 25 normal volunteers were subjected to intravenous administration for 3 consecutive days of either 50 ml of rHSA (12.5 g rHSA/body) or progressively increased doses of rHSA in a bolus injection (5–100 ml; 1.25–25 g rHSA/body). No abnormalities relating to the administration of rHSA were observed during the trial in physiological tests (i.e. blood pressure, pulse, body weight, electrocardiogram fundus examination), subjective and objective symptoms, hematological
Conclusions
In the pre-clinical tests, rHSA produced using the yeast P. pastoris was intensively evaluated from the viewpoint of identity to pdHA. In the clinical studies, emphasis was placed on the safety aspects of rHSA administration. rHSA appeared to be both virtually identical to pdHA and safe.
In addition to the similarity to pdHA, other advantages are associated with this method of rHSA production. These include:
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elimination or reduction of the potential risk of viral and other unknown factors (e.g.
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