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Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response

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Abstract

Traditional production of therapeutic glycoproteins relies on mammalian cell culture technology. Glycoproteins produced by mammalian cells invariably display N-glycan heterogeneity resulting in a mixture of glycoforms the composition of which varies from production batch to production batch. However, extent and type of N-glycosylation has a profound impact on the therapeutic properties of many commercially relevant therapeutic proteins making control of N-glycosylation an emerging field of high importance. We have employed a combinatorial library approach to generate glycoengineered Pichia pastoris strains capable of displaying defined human-like N-linked glycans at high uniformity. The availability of these strains allows us to elucidate the relationship between specific N-linked glycans and the function of glycoproteins. The aim of this study was to utilize this novel technology platform and produce two human-like N-linked glycoforms of recombinant human lactoferrin (rhLF), sialylated and non-sialylated, and to evaluate the effects of terminal N-glycan structures on in vitro secondary humoral immune responses. Lactoferrin is considered an important first line defense protein involved in protection against various microbial infections. Here, it is established that glycoengineered P. pastoris strains are bioprocess compatible. Analytical protein and glycan data are presented to demonstrate the capability of glycoengineered P. pastoris to produce fully humanized, active and immunologically compatible rhLF. In addition, the biological activity of the rhLF glycoforms produced was tested in vitro revealing the importance of N-acetylneuraminic (sialic) acid as a terminal sugar in propagation of proper immune responses.

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Abbreviations

Man:

mannose

Gal:

galactose

GlcNAc:

N-acetylglucosamine

Sia:

sialic acid

hLF:

human lactoferrin

rhLF:

recombinant human lactoferrin

anti-hLF:

anti-human LF antibody

anti-HCP:

anti-host cell protein antibody

CV:

column volume

AFC:

antibody forming colonies

MALDI-TOF:

matrix-assisted laser desorption/ionization time of flight

MS/MS:

tandem mass spectrometry

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Acknowledgments

This research was supported by the National Institutes of Health: R42AI051050-02 and R41GM079810-01. We thank Teresa Mitchell for the technical assistant and Bing Gong for the critical reading of the manuscript.

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Authors and Affiliations

  1. GlycoFi, Inc., a wholly owned subsidiary of Merck & Co., Inc., Lebanon, NH, USA

    Byung-Kwon Choi, Sandra Rios, Marc d’Anjou, Terrance A. Stadheim, Shannon Warburton, Erin Giaccone, Michael Cukan, Huijuan Li, Angela Kull, Nathan Sharkey & Stefan Wildt

  2. The University of Texas Health Science Center at Houston, Houston, TX, USA

    Jeffrey K. Actor

  3. Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland

    Maja Kocięba, Jolanta Artym & Michal Zimecki

  4. University at Buffalo, The State University of New York, Buffalo, NY, USA

    Paul Gollnick

  5. PharmaReview Corporation, 7505 Fannin Street, Suite #313, Houston, TX, 77054, USA

    Marian L. Kruzel

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  1. Byung-Kwon Choi
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  2. Jeffrey K. Actor
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  3. Sandra Rios
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  9. Huijuan Li
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  14. Jolanta Artym
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  15. Michal Zimecki
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  16. Marian L. Kruzel
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Correspondence to Marian L. Kruzel.

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Choi, BK., Actor, J.K., Rios, S. et al. Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response. Glycoconj J 25, 581–593 (2008). https://doi.org/10.1007/s10719-008-9123-y

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