Abstract
The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.
Highlights
Human serum albumin (HSA) is the most abundant protein component of human plasma
As a consequence of our recent studies showing marked differences between two recombinant human serum albumins (rHSA) expressed in yeast and Asian rice with respect to hexose modification of lysine and arginine residues, thermal stabilities and interactions with liposomal membranes [3], a more complex biophysical investigation involving commercially available OsrHSA from four suppliers with four lots of OsrHSA from a single supplier (Sigma-Aldrich) was carried out
Analysis of rHSA Products by size exclusion chromatography (SEC), capillary electrophoresis (CE) and reversed-phase high-performance liquid chromatography (RP-HPLC) SEC showed the main peak eluting at approximately 17.3–17.4 minutes for all samples with relative quantities of injected sample for this peak ranging from 66.6% for OsrHSA-sig-C to 99.4% for ScrHSA (Figure 1, Table S1)
Summary
Human serum albumin (HSA) is the most abundant protein component of human plasma. HSA has been used for the treatment of hypoalbuminemia due to severe burns (up to 10 g/dose) [5,6] and for chronic liver cirrhosis, and has been proposed as a treatment for Alzheimer’s disease [7]. This protein has been used in nanoscale drug delivery systems such as Abraxane (130 nm albumin nanoparticle for the delivery of Paclitaxel) and Albuferon (an interferon a-2b/albumin fusion protein) [8,9]. The complex nature of the protein’s surface allows it to function as an excipient, preventing protein aggregation and adsorption to glass vials [10,11]
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