B-017 A Purified Recombinant Human Albumin Produced in Thermothelomyces heterothallica (C1) Better Mimics Native Human Serum Albumin than Recombinant Albumin from Other Expression Systems

Abstract

Recombinant human albumin (rHA) is widely used as an alternative to plasma-derived human serum albumin (HSA) in diagnostic and bioanalytical applications. However, rHA produced in different expression systems can exhibit varying post-translational modifications (PTMs), impurity profiles, and free thiol content, which may affect functional similarity to native HSA. In this study, we describe a novel rHA produced in Thermothelomyces heterothallica (C1) and purified using a scalable proprietary process. We present an analytical comparison of C1-derived rHA to native HSA and rHA products produced in yeast (Saccharomyces cerevisiae and Pichia pastoris) and rice (Oryza sativa), focusing on PTM profiles, molecular weight (MW), purity, free thiol content, and residual endotoxin. The primary sequences and PTMs of C1-derived rHA and native HSA, including oxidation, deamidation, isomerization, and phosphorylation, were experimentally analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins were denatured, reduced, alkylated, digested with trypsin, and analyzed by LC-MS/MS. Sequences and PTMs were confirmed by comparison to theoretical MS data. Comparative PTM data for yeast- and rice-derived rHA were obtained from published literature. Molecular weight analysis of C1-derived rHA and native HSA was performed using native size exclusion chromatography–mass spectrometry, enabling separation of monomeric albumin from higher-order species and impurities, followed by intact mass determination. Albumin purity of all rHA products and native HSA was assessed by reduced polyacrylamide gel electrophoresis (PAGE) with densitometric quantitation against a low-protein standard curve. Free thiol content was determined for all samples using a thiol-reactive Ellman’s assay targeting the functional Cys34 residue. Endotoxin abundance was determined for all samples using a Limulus Amebocyte Lysate (LAL) assay. C1-derived rHA and native HSA exhibited primary sequences consistent with theoretical expectations and minimal non-native PTMs. Intact mass analysis confirmed the absence of glycosylation, with observed masses matching the theoretical MW (66,472 ± 40 Da). Literature reports indicate that rHA produced in rice has higher levels of arginine and lysine glycation than native HSA, while rHA products from S. cerevisiae and P. pastoris contain higher levels of other non-native PTMs such as acylation relative to native HSA. Purity analysis showed that C1-derived rHA exhibited consistently high purity (99.9% and 98.9%), outperforming rice-derived rHA (97.8% and 97.6%) and matching or exceeding yeast-derived rHA (98.8% for Saccharomyces cerevisiae, 99.9% for Pichia pastoris). Experimentally, C1-derived rHA demonstrated free thiol content of the functional Cys34 residue intermediate between yeast- and rice-derived rHA products, with yeast-derived rHA showing higher free thiol levels and rice-derived rHA showing lower levels. Residual endotoxin levels across all rHA products were extremely low, below 1 EU/mg. This study demonstrates that rHA produced in Thermothelomyces heterothallica (C1) more closely mimics native HSA than recombinant albumins produced in yeast and plant expression systems. Based on experimentally determined intact mass, and endotoxin levels, together with literature-supported PTM comparisons, C1-derived rHA exhibits superior native-like characteristics. These findings position the novel C1-derived rHA as a recombinant albumin with enhanced functional equivalence, consistency, and purity for diagnostic and bioanalytical applications.