B-060 A Recombinant Human Albumin Produced in Thermothelomyces heterothallica (C1) as a High-Purity Alternative for Diagnostic Assays

Abstract Background Human serum albumin (HSA) is commonly used in diagnostic assays as an alternative to bovine serum albumin (BSA), casein, and other blockers to minimize cross-reactivity. HSA may contain plasma-derived molecules such as vitamins, hormones, growth factors, or antibodies (IgG), which could limit its applicability in certain assays. To circumvent these limitations, we developed a novel recombinant human albumin (rHA) using Thermothelomyces heterothallica (C1) and purified it using a scalable proprietary process. This work presents the first comprehensive comparative analysis of this rHA against several commercially available HSA products, assessing its primary sequence, post translational modifications (PTMs), molecular weight (MW), purity, and IgG content to determine its suitability for diagnostic applications. Methods The primary sequences and PTMs, including phosphorylation, oxidation, isomerization, and deamidation of the rHA and HSA were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins were denatured, reduced, alkylated, and digested with trypsin. The resulting peptides were separated by LC and their masses determined by MS/MS. Protein sequences and PTMs were verified by comparison to theoretical MS data. Molecular weight analysis was conducted using native size exclusion chromatography-mass spectrometry. This method separated native monomer from oligomers and other impurities, followed by determination of monomer intact mass by MS. Albumin purity was assessed by polyacrylamide gel electrophoresis (PAGE). Samples were run on a reduced PAGE gel alongside a 4-point low protein standard curve. Impurities in the rHA or HSA samples were quantitated using densitometry against the standard curve with Phoretix 1D software. IgG was quantitated using a human IgG-Fc enzyme-linked immunosorbent assay (ELISA) kit (Fortis Life Sciences). 10 mg/ml solutions of commercially available HSA and novel rHA were serially diluted, run on the human IgG-Fc ELISA, and quantitated against an 8-point IgG standard curve (0-500 ng/ml). Results The rHA and HSA exhibit primary sequences consistent with theoretical expectations and show limited PTMs. Intact mass analysis reveals that the products do not contain glycosylation, as the intact masses matches their theoretical MW (66,472 ± 40 Da). rHA has a purity of 98.6%, consistent with the commercially available HSA products, which range from 94.5% to 99.8% purity. Notably, all commercially available HSA products contain varying levels of IgG, from 10 ppm to 1400 ppm, while rHA is IgG-free. Conclusion This study demonstrates that rHA produced in C1 and purified using a scalable proprietary process is analytically comparable to commercial HSA products in terms of intact mass, sequence identity, PTM profiles, and purity. With the added advantage of being IgG-free, this rHA retains all the benefits of native HSA while potentially offering greater uniformity, lower cross-reactivity risk, and improved assay sensitivity. These findings highlight rHA as a reliable and effective alternative to commercially available HSA products, making it well-suited for diagnostic assays that require high purity, consistency, and the absence of plasma-derived components.