Solid Phase Pegylation of Hemoglobin

Abstract

A solid phase conjugation process was developed for attachment of polyethylene glycol to hemoglobin molecule. Bovine hemoglobin was loaded onto an ion exchange chromatography column and adsorbed by the solid medium. Succinimidyl carbonate mPEG was introduced in the mobile phase after the adsorption. Pegylation took place between the hemoglobin on the solid phase, and the pegylation reagent in the liquid phase. A further elution was carried out to separate the pegylated and the unpegylated protein. Analysis by HPSEC, SDS-PAGE, and MALLS demonstrated that the fractions eluted from the solid phase contained well-defined components. Pegylated hemoglobin with one PEG chain was obtained with the yield of 75%, in comparison to the yield of 30% in the liquid phase pegylation. The P(50) values of the mono-pegylated hemoglobin, prepared with SC-mPEG 5 kDa, 10 kDa and 20 kDa, were 19.97, 20.23 and 20.54 mmHg, which were much closer to the value of red blood cells than that of pegylated hemoglobin prepared with the conventional method.

Solid‐Phase Protein Modifications: Towards Precision Protein Hybrids for Biological Applications

“…Proteins bound to the column close to the active center are PEGylated only on the opposite face, thus preventing reactions near the active site (Figure 3a). Several solid‐phase matrices commonly employed for PEGylation are ion exchange, [48–50] heparin sepharose, [51,52] nickel affinity, [53] and hydrophobic interaction (HIC) resins [54] . Proteins successfully PEGylated using the SP approach include recombinant and native members of the fibroblast growth factor (FGF) family, [51,53,54] hemoglobin, [48] recombinant interferon α‐2a, [49] human serum albumin (HSA), [50] staphylokinase (SAK), [50] recombinant human keratinocyte growth factor, [52] lysozyme, [54] enhanced green fluorescent protein (eGFP), [55] and immobilized protein cages [56] …”

“…Capitalizing on the multiple surface charges of the native proteins, ion‐exchange resins were employed in the earliest examples of SP PEGylation to adsorb the proteins of interests onto a suitable cationic or anionic column, and thereafter, a circulating solution of PEG reagent is applied through the column over a defined time [48–50] . Upon conjugation, the bound PEG shields the protein resulting in a weaker interaction with the resin, and a separation of PEGylated and native proteins is thus feasible using a linear‐gradient elution.…”

“…In contrast to PEGylation in solution, no further purification is necessary. Using this approach, PEGylation of hemoglobin, an iron‐containing oxygen‐transport metalloprotein, was achieved by reaction of the lysines on the exposed face with succinimidyl carbonate monomethoxy poly(ethylene glycol) [48] . While liquid phase PEGylation led to a mixture of mono‐, di‐, and multi‐PEGylated hemoglobin, solid‐phase showed consistent degree of PEGylation with six PEGs per protein for PEG5 000 and five PEGs per protein for PEG10 000 and PEG20 000 due to the hindrance conferred by the SP matrix.…”

Perspectives, Tendencies, and Guidelines in Affinity-Based Strategies for the Recovery and Purification of PEGylated Proteins

“…Table 2 displays, for the most part, relative bioactivities greater than 50%, whereas in unplanned PEG conjugations, the achieved bioactivity is only 40% or even null. In general, solid-phase PEGylation has improved yields for the mono-PEGylated protein in comparison to liquid-phase PEGylation [44,46]. Furthermore, on-column PEGylation represents an attractive integration of operations obtaining a desirable product with less downstream steps [47] in less time and with guaranteed biological activity.…”

Solid‐phase preparation of protein complexes

“…Another approach to the implementation of solid-phase protocols in protein conjugation has been reported by Hu and coworkers ; the authors used an ion exchange matrix to temporarily immobilize bovine haemoglobin aiming to obtain dimers of the protein; in this case glutaraldehyde was used as linker. The same group has also reported a method for haemoglobin pegylation requiring immobilization of the protein on ion exchange columns (Suo et al, 2009). Here, we present a different solid-phase approach for the preparation of protein complexes where a special attention is paid to preserve the immune reactivity of the single components.…”