High throughput quantification of N-glycans using one-pot sialic acid modification and matrix assisted laser desorption ionization time-of-flight mass spectrometry

Abstract

Appropriate glycosylation of recombinant therapeutic glycoproteins has been emphasized in biopharmaceutical industries because the carbohydrate component can affect safety, efficacy, and consistency of the glycoproteins. Reliable quantification methods are essential to ensure consistency of their products with respect to glycosylation, particularly sialylation. Mass spectrometry (MS) has become a popular tool to analyze glycan profiles and structures, showing high resolution and sensitivity with structure identification ability. However, quantification of sialylated glycans using MS is not as reliable because of the different ionization efficiency between neutral and acidic glycans. We report here that amidation in mild acidic conditions can be used to neutralize acidic N-glycans still attached to the protein. The resulting amidated N-glycans can then be released from the protein using PNGase F, and labeled with permanent charges on the reducing end to avoid any modification and the formation of metal adducts during MS analysis. The N-glycan modification, digestion, and desalting steps were performed using a single-pot method that can be done in microcentrifuge tubes or 96-well microfilter plates, enabling high throughput glycan analysis. Using this method we were able to perform quantitative MALDI-TOF MS of a recombinant human glycoprotein to determine changes in fucosylation and changes in sialylation that were in very good agreement with a normal phase HPLC oligosaccharide mapping method.

Fig 1

(a) amidation of sialylated glycan, and (b) scheme of glycan analysis from glycoprotein using amidation and derivatization. Ah, Acetohydrazide

Fig 2

HPLC profiles of N-glycan from human IgG (a) unamidated and derivatized with 2-aminobenzoic acid (2-AA), and (b) amidated with acetohydrazide (Ah) and derivatized with 2-AA. MALDI-TOF MS spectra of N-glycan from human IgG (c) amidated with Ah and derivatized,with Girard’s T reagent (GT) and (d) amidated with Ah and derivatized with 2-AA.

Fig 3

(a) HPLC profile of unamidated and 2-AA derivatized N-glycan, and (b) MALDI-TOF MS spectrum of amidated with Ah and 2-AA-derivatized N-glycan of tg-FIX K45 d5 (4^th lactation stage). (c) Quantification comparison of major N-glycan of tg-FIX using between HPLC and MALDI-TOF MS.

Fig 4

MALDI-TOF MS spectra of amidated with Ah and 2-AA derivatized N-glycans from tg-FIX K45 (a) day10, (b) day 30, and (c) day 50. HPLC profiles of unamidated and 2-AA derivatized N-glycan from tg-FIX K45 (d) day10, (e) day 30, and (f) day 50. A solid star indicates fucosylated monosialylated and disialylated biantennary structures and an open star indicates the corresponding unfucosylated structures.

Fig 5

Relative percentage change of fucosylated N-glycan of tg-FIX during lactation. The summed percentages of fucosylated N-glycans of tg-FIX were obtained from (a) MALDI-TOF MS and (b) NP-HPLC profiles. The data were triplicate for each sample.

Fig 6

Relative percentage change of fully sialylated N-glycan of tg-FIX during lactation. The summed percentages of fully sialylated N-glycan of tg-FIX were obtained from (a) MALDI-TOF MS and (b) NP-HPLC profiles. The data were triplicate for each sample.