Large scale purification of gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography

Abstract A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides G M3 (Neu5Ac) and G M3 (Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure G M3 (Neu5Ac) and G M3 (Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of G M3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two G M3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl- versus N-glycolylneuraminic acid).