A Pichia pastoris-based platform for large-scale production of functional recombinant human serum albumin with therapeutic efficacy.

Human serum albumin (HSA) is critical for treating liver cirrhosis with ascites. However, plasma-derived HSA (pHSA) faces limitations in supply and carries potential infection risks. To address the urgent need for a therapeutically equivalent alternative, we established an efficient expression system for recombinant HSA (rHSA) in Pichia pastoris. We purified rHSA via a three-step chromatography purification process, yielding approximately 260 mg per 1.5 L of culture. Comprehensive analyses confirmed its structural equivalence to pHSA. Mass spectrometry revealed a molecular weight of 66,530.8 Da, while circular dichroism showed a 36.9% α-helical content. Furthermore, isothermal titration calorimetry demonstrated comparable drug-binding affinities for naproxen and warfarin. In a CCl₄-induced rat model of liver cirrhosis with ascites, rHSA displayed significant, dose-dependent therapeutic effects. Notably, high-dose rHSA achieved efficacy statistically equivalent to pHSA. It reduced ascites volume by 50.1%, improved liver index by 33.5% and restored colloid osmotic pressure along with liver function markers (ALT, AST, and bilirubin). This study provides the first in vivo evidence confirming the therapeutic equivalence and dose-effect relationship of P. pastoris-derived rHSA compared with pHSA. These findings establish a scalable, cost-effective, and safe platform for rHSA production, supporting its potential as a viable clinical substitute for plasma-derived albumin.