A solid-phase adsorption method for PEGylation of human serum albumin and staphylokinase: preparation, purification and biochemical characterization

Abstract

A solid-phase adsorption method was developed to circumvent the disadvantage of the conventional liquid-phase PEGylation, i.e. the heterogeneity of the PEGylated products. The model proteins, human serum albumin (HSA) and staphylokinase (SAK), were adsorbed on the ion exchange chromatography media, followed by PEGylation with succinimidyl carbonate (SC)-mPEG5K and salt elution. Since PEGylation with SC-PEG5K alters the positive charge of the proteins, Q-Sepharose Big Beads and DEAE Sepharose Fast Flow were used for adsorption of HSA and SAK, respectively. Size exclusion chromatography and SDS-PAGE studies demonstrated that solid-phase PEGylation of proteins generate monoPEGylated proteins with the yield of 35-47%. Circular dichroism and intrinsic fluorescence studies showed that solid-phase PEGylation led to little conformational change of the proteins. Solid-phase PEGylation resulted in 35% loss in the biological activity of SAK, which is lower than the liquid-phase PEGylation (70%).

Microspheres for Solid-Phase Organic Synthesis

“…For example, the yield of the modified product is relatively low in the method based on IEC media (25). The mono-PEGylated protein was not well-separated from the multi-PEGylated protein in the SERC method (31).…”

“…Protein-NH 2 Suo's research group has established a solid-phase modification method for PEGylation of human serum albumin (HSA, 66 kDa) and staphylokinase (SAK, 15 kDa) (25). Q Sepharose Big Bead is a cross-linked agarose coated with quaternary ammonium ligand.…”

Microspheres for Separation of Bioactive Small Molecules

“…It is one of the most commonly used techniques for the purification of PEGylated biomolecules (9,(13)(14)(15)(16)(17)(18)(19)24), especially in large-scale production. In addition, high performance IEC is also successfully applied to the subfractionation of PEG-proteins on the basis of their degree of PEGylation and positional isomerism (24)(25)(26).…”

Current advances in the non‐chromatographic fractionation and characterization of PEGylated proteins

“…PAGE, which has been used mainly for the analysis of the PEGylated products during and after reaction, is probably the most widely used non‐chromatographic technique at this time. Although advances have been made regarding the characterization of PEGylated derivates using this technique,14, 15, 44, 45 it is necessary to explore alternative staining techniques to distinguish free PEG and PEGylated proteins in one step. On the other hand, no reports have been found that indicate the use of electrophoresis in two dimensions for the analysis and characterization of PEGylated products, so it may be interesting to carry out research in this area in order to determine the potential of this technique for the present application.…”

“…Regarding this liquid–liquid fractionation strategy it has been reported that one single grafted chain is enough to promote a selective fractionation towards a specific system phase 11. Also, the use of polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (CE) has been reported for the analysis of PEGylated proteins 12–15. PAGE has been widely used for the characterization of the PEGylation reaction.…”