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Direct Site-Specific Glycoform Identification and Quantitative Comparison of Glycoprotein Therapeutics: Imiglucerase and Velaglucerase Alfa

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Abstract

Gaucher disease, the most common lysosomal metabolic disorder, can be treated with enzyme replacement therapy (ERT). Recombinant human glucocerebrosidase imiglucerase (Cerezyme®), produced in Chinese hamster ovary cells, has been used for ERT of Gaucher disease for 20 years. Another recombinant glucocerebrosidase velaglucerase alfa (VPRIV), expressed in a human fibroblast cell line, was approved by the US Food and Drug Administration in 2010. The amino acid sequence difference at residue 495 of these two products is well documented. The overall N-linked qualitative glycan composition of these two products has also been reported previously. Herein, employing our recently developed approach utilizing isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL electron transfer dissociation (ETD) hybrid mass spectrometer, the site-specific glycoforms of these products were identified with ETD and collision-induced dissociation (CID) spectra. The quantitative comparison of site-specific glycans was achieved utilizing higher-energy collisional dissociation (HCD) spectra with a NanoMate used as both a fraction collector and a sample introduction device. From the trypsin-digested mixture of these two products, over 90 glycopeptides were identified by accurate mass matching. In addition to those previously reported, additional glycopeptides were detected with moderate abundance. The relative amount of each glycoform at a specific glycosylation site was determined based on reporter signal intensities of the TMT labeling reagents. This is the first report of site-specific simultaneous qualitative and quantitative comparison of glycoforms for Cerezyme® and VPRIV. The results demonstrate that this method could be utilized for biosimilarity determination and counterfeit identification of glycoproteins.

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Acknowledgments

The authors gratefully acknowledge receipt of materials from Genzyme Corporation (Cerezyme®) and Shire Human Genetic Therapies, Inc. (VPRIV) which were used in this work. The work performed in the study was supported in part by the FDA Critical Path grant program awarded to H. Ye.

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The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy.

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Authors and Affiliations

  1. Division of Pharmaceutical Analysis, CDER, US Food and Drug Administration, 645 South Newstead Avenue, St. Louis, Missouri, 63110, USA

    Hongping Ye, Ashley C. Gucinski, Michael T. Boyne II & Lucinda F. Buhse

  2. 1921 Cheyenne Dr., Carrollton, Texas, 75010, USA

    John Hill

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  1. Hongping Ye
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Correspondence to Hongping Ye.

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Ye, H., Hill, J., Gucinski, A.C. et al. Direct Site-Specific Glycoform Identification and Quantitative Comparison of Glycoprotein Therapeutics: Imiglucerase and Velaglucerase Alfa. AAPS J 17, 405–415 (2015). https://doi.org/10.1208/s12248-014-9706-4

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