A multi-mode chromatographic method for the comparison of the N-glycosylation of a recombinant HIV envelope glycoprotein (gp160s-MN/LAI) purified by two different processes.
The glycosylation pattern of a recombinant gp160s-MN/LAI variant of human immunodeficiency virus type 1 (HIV-1) was studied in relation to two alternative purification techniques one of which involves an immunoprecipitation step. For analysis a multi-mode high performance liquid chromatography (HPLC) method which combines gel permeation chromatography on the RAAM 2000 GlycoSequencer, weak anion exchange chromatography and normal phase chromatography was developed and profiles were obtained for the fluorescently-labelled glycans released from the two gp160s-MN/LAI preparations. Charged glycans accounted for 77 and 80% of the total glycans for the IAP- and SP-purified samples, respectively. The acidic character of these glycans was mainly due to the presence of sialic acids. However, following sialidase treatment, residual charged glycans were still found. No differences were found in the glycan distributions of the two gp160s-MN/LAI preparations either in their degree of sialylation or in their relative proportion of each separated structure. Although this did not reach statistical significance, a lower proportion of large glycan structures regardless of their charge status was found on the gp160s-MN/LAI prepared by the procedure which involved an immunoaffinity chromatography step.
Laboratoire de Chimie Analytique, Faculté de Pharmacie, Châtenay-Malabry, France.
M
M
Taverna
T T
TT
Nguyet
C
C
Valentin
O
O
Level
T
T
Merry
H V
HV
Kolbe
D
D
Ferrier
D000818:Animals; D002845:Chromatography / Q000379:methods*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006031:Glycosylation; D019302:HIV Envelope Protein gp160 / Q000737:chemistry* / Q000302:isolation & purification; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D011134:Polysaccharides / Q000032:analysis*; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D019302:HIV Envelope Protein gp160; D011134:Polysaccharides; D011994:Recombinant Proteins