A tandem mass spectrometric approach to the identification of O-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in Pichia pastoris.
Glycoforms of glargine expressed in Pichia pastoris were isolated by high-performance liquid chromatography and analyzed by a series of chemical and mass spectrometric methods for the identification of various glycoforms, glycosylation position, nature and structure of glycans. Reduction and alkylation, peptide mapping techniques were used to decipher the amino acid site at which glycosylation had taken place. Chemical methods were coupled with mass spectrometry techniques such as electrospray ionization and matrix-assisted laser desorption/ionization for identification of the glycosylation site.
Kannan
Vivekanandan
V
Research and Development, Biocon Limited, 20th KM Hosur Road, 560100 Bangalore, India. vivekanandan.kannan@biocon.com
Narayanaswamy
Pradeep
P
Gadamsetty
Deepak
D
Hazra
Partha
P
Khedkar
Anand
A
Iyer
Harish
H
D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D007004:Hypoglycemic Agents / Q000737:chemistry*; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry; D000069036:Insulin Glargine; D049528:Insulin, Long-Acting; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010843:Pichia / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
D006020:Glycopeptides; D007004:Hypoglycemic Agents; D007328:Insulin; D049528:Insulin, Long-Acting; D010446:Peptide Fragments; D010936:Plant Extracts; D000069036:Insulin Glargine