Synthesis and characterization of polyethylene glycol polyacrylamide copolymer (PEGA) resins containing carbohydrate ligands. Evaluation as supports for affinity chromatography

Abstract

The PEGA resin, a beaded polyethylene glycol dimethylacrylamide copolymer, was evaluated as an affinity support for the purification of carbohydrate-binding macromolecules, namely, the cation-independent mannosyl phosphate receptor (CI-MPR) and a polyclonal antibody directed against a Streptococcus Group A oligosaccharide. Two polyethylene glycol (PEG) derivatives, a di-acryloylated PEG 1900 derivative or a longer di-acryloylated PEG 4000 derivative, were used as cross-linkers. The longer cross-linker was synthesized in four steps from polyethylene glycol 4000. The mannosyl 6-phosphate (M6P)-containing immunoaffinity columns were prepared through the inverse suspension radical copolymerization of the corresponding allyl glycoside with acrylamide and the PEG cross-linker. The resin with the shorter cross-linker (PEG 1900 derivative) had a 6.3% molar cross-linking while that with the longer cross-linker (PEG 4000 derivative) had a 3.8% molar cross-linking. For the Streptococcus Group A trisaccharide-containing immunoaffinity columns, three PEGA affinity supports bearing free amino groups were prepared and subsequently substituted with a trisaccharide activated as its squarate adduct. While one resin contained the shorter cross-linker PEG 1900 and had a 3% molar cross-linking, the other two resins contained the longer cross-linker PEG 4000 with a molar cross-linking of 5% and 3%, respectively. In affinity chromatographic studies, the M6P-containing columns were ineffective in retaining the cation-independent mannosyl phosphate receptor (CI-MPR, ~ 215kDa), whereas antibody (~ 150kDa) retention was observed with two of the three Streptococcus Group A trisaccharide-containing immunoaffinity columns. Key words: PEGA resins, immunoaffinity supports, carbohydrate ligands, antibody purification.