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A high-resolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns

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Abstract

N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese hamster ovary (CHO) cells are typically glycosylated during biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donors and impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on the use of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed.

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Abbreviations

ADP:

Adenosine diphosphate

AMP:

Adenosine monophosphate

ATP:

Adenosine triphosphate

CDP:

Cytidine diphosphate

CMP:

Cytidine monophosphate

CTP:

Cytidine triphosphate

CMP-SA:

Cytidine monophosphate-sialic acid

GDP:

Guanosine diphosphate

GMP:

Guanosine monophosphate

GTP:

Guanosine triphosphate

GDP-Fuc:

Guanosine diphosphate fucose

GDP-Man:

Guanosine diphosphate mannose

IP-RP:

Ion-pair reverse phase

Trp:

Tryptophan

UDP:

Uridine diphosphate

UMP:

Uridine monophosphate

UTP:

Uridine triphosphate

UDP-Gal:

Uridine diphosphate galactose

UDP-Glc:

Uridine diphosphate glucose

UDP-Hex:

Uridine diphosphate hexosamine

UDP-GlcNAc:

Uridine diphosphate N-acetylglucosamine

UDP-HexNAc:

Uridine diphosphate N-acetylhexosamine

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Acknowledgments

The authors gratefully thank Dr. Maurizio Cattaneo for providing the cell line and Dr. Kurt Brorson for the support of this study.

Funding

The study was supported by grants from NSF (1706731) and NSF/IUCRC AMBIC (1624718).

Author information

Authors and Affiliations

  1. Biomedical Engineering and Biotechnology, University of Massachusetts Lowell, Lowell, MA, 01854, USA

    Sha Sha & Seongkyu Yoon

  2. Biomedical & Nutritional Sciences, University of Massachusetts Lowell, Lowell, MA, 01854, USA

    Garry Handelman

  3. U.S. FDA, CDER/OBP/Division of Biotechnology Review and Research II, Silver Spring, MD, 20993, USA

    Cyrus Agarabi

  4. Chemical Engineering, University of Massachusetts Lowell, Lowell, MA, 01854, USA

    Seongkyu Yoon

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Correspondence to Seongkyu Yoon.

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Sha, S., Handelman, G., Agarabi, C. et al. A high-resolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns. Anal Bioanal Chem 412, 3683–3693 (2020). https://doi.org/10.1007/s00216-020-02608-6

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  • DOI: https://doi.org/10.1007/s00216-020-02608-6

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