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Batch-to-batch N-glycosylation study of infliximab, trastuzumab and bevacizumab, and stability study of bevacizumab
  1. Ana Planinc1,2,
  2. Bieke Dejaegher2,3,4,
  3. Yvan Vander Heyden2,4,
  4. Johan Viaene2,4,
  5. Serge Van Praet5,
  6. Florence Rappez5,
  7. Pierre Van Antwerpen1,2,
  8. Cédric Delporte1,2
  1. 1Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Brussels, Belgium
  2. 2Joint Research Group ULB-VUB, Brussels, Belgium
  3. 3Laboratory of Instrumental Analysis and Bioelectrochemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Brussels, Belgium
  4. 4Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Faculty of Medicine and Pharmacy, Center for Pharmaceutical Research (CePhaR), Vrije Universiteit Brussel (VUB), Brussels, Belgium
  5. 5Pharmacy, CHU Saint-Pierre, Brussels, Belgium
  1. Correspondence to Ana Planinc, Faculty of Pharmacy, Laboratory of Pharmaceutical Chemistry and Analytical Platform of the Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Bld du Triomphe, Campus Plaine, CP 205/05B, Brussels B-1050, Belgium; aplaninc{at}ulb.ac.be

Abstract

Objectives Infliximab, trastuzumab and bevacizumab are among the most frequently prescribed therapeutic proteins, and like most other therapeutic proteins, are glycosylated. As differences in glycosylation may significantly change the safety and efficacy of therapeutic glycoproteins, it is extremely important to control N-glycosylation consistency. In the first part of this study, the batch-to-batch consistency of the N-glycosylation of infliximab, trastuzumab and bevacizumab was analysed. In the second part, the consistency of the N-glycosylation of bevacizumab stored in polycarbonate syringes (for off-label drug use) for 3 months was examined.

Methods N-glycans were (i) enzymatically released using peptide-N-glycosidase F, (ii) reduced, and (iii) analysed using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry. Mass spectrometry data were interpreted using principal component analysis combined with two-way analysis of variance and Tukey post hoc tests. The biological activity of infliximab and trastuzumab was examined using enzyme-linked immunosorbent assays.

Results The results of both studies make important contributions to the field of hospital pharmacy. All batches of the studied therapeutic glycoproteins (infliximab, trastuzumab and bevacizumab) varied considerably (especially in galactosylation), while the N-glycosylation of bevacizumab remained unchanged during 3-month storage.

Conclusions Threshold values for batch-to-batch N-glycosylation variations should be established and batch-to-batch glycosylation consistency should be regularly tested. In our study, samples with significantly different N-glycosylation profiles showed no significant variations in biological activity, suggesting that the differences are probably not therapeutically significant.

  • BIOANALYSIS

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