B-105 A New Recombinant Human Albumin Produced in Thermothelomyces heterothallica (C1) Demonstrates Comparable Surface Binding to Native Serum Albumin

Abstract

Background Human serum albumin (HSA) and bovine serum albumin (BSA) are commonly used as a blocking agents in immunoassays to prevent non-specific surface binding, thereby reducing background signal and increasing assay sensitivity. However, these native albumins present challenges such as donor dependency, lot-to-lot variability, and contamination with plasma-derived molecules (e.g., vitamins, hormones, growth factors, antibodies/Ig), which can affect assay performance and reproducibility. Recombinant human albumin (rHA) offers a potential alternative by providing the same binding and blocking characteristics as native albumin without the limitations. A new rHA produced in Thermothelomyces heterothallica (C1) and purified using a scalable proprietary process may mitigate these challenges. This study aims to evaluate the amount of binding on different surfaces of this new rHA compared to native albumins to determine the viability of rHA as a surface blocker in immunoassays. Methods The amount of albumin binding to different surfaces was assessed using a bead binding assay. Here, albumin (rHA, HSA, BSA) was incubated with commercially available Dynabeads: hydrophilic (carboxylic acid or amine functionalized) and hydrophobic (tosylactivated). The amount of protein bound to the beads 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. Results All tested albumin products demonstrate 10-fold more protein binding to the hydrophobic beads than either of the hydrophilic beads, highlighting the chemical similarities of albumin from different sources. For both hydrophilic bead types, all tested albumins demonstrated statistically the same amount of binding, ranging from 0.10-0.23 µg and 0.19-0.23 µg for the carboxylic acid and amine beads, respectively, with rHA at 0.15 ± 0.06 µg and 0.23 ± 0.07 µg. The hydrophobic beads had a much larger range of albumin binding, from 1.4-3.7 µg, with rHA being at the lower end of the range at 1.4 ± 0.2 µg, but statistically the same as BSA and two HSA products. The large differences in binding of the native albumins showcase the variability of natively derived products from different producers. Even with these slight differences, these data demonstrate rHA has comparable surface binding chemistry to native albumins. Conclusion This new rHA produced by C1 and purified in a scalable process binds surfaces comparably to commercially available native albumin products on all surfaces tested. These results support the use of rHA as an alternative to native albumins in diagnostic applications, providing effective surface binding without the limitations associated with plasma-derived products. Further studies will investigate the impact of rHA on immunoassay consistency and sensitivity as well as using rHA within other areas of diagnostics, such as enzyme stabilization.