B-168 Purified Recombinant Human Albumin Produced in Thermothelomyces heterothallica (C1) Demonstrates Effective Blocking and Comparable Surface Binding to Other Recombinant and Native Albumins

Abstract

Recombinant and native human serum albumins (rHA and HSA) are widely used as blocking agents to prevent nonspecific binding of enzymes, antibodies, and other biomolecules to surfaces. HSA is primarily used in diagnostic assays to reduce background signals and improve assay sensitivity, though assay performance can be affected by lot-to-lot variability and plasma-derived molecules. Reflecting cost, rHA is more often used as a surface coating for clinical and bioprocess applications. rHA produced in Thermothelomyces heterothallica (C1), purified using a scalable proprietary process may provide a cost-effective alternative that maintains comparable surface binding and blocking capabilities. This study evaluates C1 rHA binding across surfaces and its effectiveness as a surface blocker and stabilizer relative to other human albumins. The amount of surface-bound albumin was assessed using a bead binding assay. Albumin was incubated with Dynabeads: hydrophilic (carboxylic acid or amine functionalized) and hydrophobic (tosylactivated). Bead-bound protein was determined by heating the coated beads in a denaturing buffer and quantifying via densitometry on an SDS-PAGE gel against a standard curve. Statistical analysis was performed using GraphPad Prism, with data presented as the mean of at least three independent replicates with standard deviations. A two-tailed t-test was used to determine statistical significance at the 95% confidence interval. Demonstration of C1-derived rHA as a blocker and stabilizer was evaluated by monitoring enzyme activity in polypropylene tubes. Albumin was added to solutions of amylase, alkaline phosphatase, and lactate dehydrogenase. Initial and subsequent activity was measured in triplicate over a 14-day period using ABX Pentra reagents on the Pentra C400 clinical chemistry analyzer. Samples containing albumin were compared to control samples without albumin to assess rHA influence on enzyme activity over time. On hydrophilic beads, C1-derived albumin demonstrates equivalent amounts of binding compared to native and recombinant albumins, ranging from ranging from 0.10-0.23 µg and 0.18-0.27 µg for the carboxylic acid and amine beads, respectively, with C1-derived rHA at 0.15 ± 0.06 µg and 0.23 ± 0.07 µg. Hydrophobic beads reflected a broader range of albumin binding, from 1.4-3.7 µg with C1-derived rHA at 1.4 ± 0.2 µg, statistically the same as half the other native and recombinant products tested. Additionally, C1-derived rHA shows effective blocking of a hydrophobic polypropylene surface through measured enzyme activity over time. Activity in solutions without C1-derived rHA decreased to nearly zero within 24 hours, whereas solutions containing rHA maintained initial activity ± 10% over three weeks. Recombinant human albumin produced in Thermothelomyces heterothallica (C1) demonstrated binding comparable to commercially available native and recombinant albumin products on both hydrophilic and hydrophobic surfaces. Although binding is slightly lower in some cases, further studies confirmed its effectiveness as a surface blocker and stabilizer, as enzyme activity was preserved over time in the presence of C1-derived rHA, while activity declined rapidly in its absence. These findings support C1-derived rHA as a reliable alternative for surface blocking and coating applications, offering comparable performance while reducing variability and production cost limitations associated with existing albumin products.