A tandem mass spectrometric approach to the identification of O‐glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in Pichia pastoris

Abstract

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.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris

“…The mannosylated impurities in insulin analog, insulin glargine, have been previously characterized [6]. A similar approach was taken for the characterization of these impurities in recombinantly produced insulin where we enriched the mono-mannosylated and di-mannosylated insulin which are insulin molecules attached with one and two mannose moieties, respectively.…”

“…A similar approach was taken for the characterization of these impurities in recombinantly produced insulin where we enriched the mono-mannosylated and di-mannosylated insulin which are insulin molecules attached with one and two mannose moieties, respectively. This was achieved by fractionating the corresponding peaks in the analytical RP-HPLC peak [6] and pooling multiple runs of the same.…”

“…Purified insulin was solubilized in 0.01 N HCl at a concentration of 1 mg/ml, followed by adjustment of the pH to 8.0 using Tris Buffer. Finally the enzyme Endoproteinase Glu-C (1 mg/ml) was added to the protein solution in the enzyme to protein ratio of 1:50, and the reaction mixture was incubated at 37°C for 4 h [6]. The peptide mass fingerprint was generated using analytical RP-HPLC in tandem with ESI-MS Agilent 1100 LCMS) to identify each chromatographic peak.…”

“…In the first step, mannose from dolichol monophosphate-activated mannose is transferred to Serine or Threonine residues of secretory proteins [4]. The number of mannose residues added further can vary from 5 in Candida albicans and 2-3 in Pichia pastoris (P. pastoris) to 13 in Saccharomyces cerevisiae (S. cerevisiae) [5][6][7]. In mammals, other sugars such as galactose, fucose, N-acetylglucosamine, glucose and sialic acid are also added to the O-linked carbohydrate chain.…”

Biosimilar Insulins

“…For example, it was reported that insulin glargine expressed in Pichia pas toris (a yeast)-that normally is not glycosylated-had 3 sugar molecules (mannose) attached to it as the result of a different manufacturing process employed by 1 manufac turer of recombinant insulins. 6 The publication refers to an experimental glycosylated product; however, also a pub lished analysis of the glargine marketed by Biocon showed that this is glycosylated. 7 The issue is that even "minor" differences/changes in the manufacturing process may have considerable (potentially clinically relevant) effects on the biological effects induced by such insulins.…”