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The Impact of Glycosylation on the Pharmacokinetics of a TNFR2:Fc Fusion Protein Expressed in Glycoengineered Pichia Pastoris

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ABSTRACT

Purpose

P. pastoris has previously been genetically engineered to generate strains that are capable of producing mammalian-like glycoforms. Our objective was to investigate the correlation between sialic acid content and pharmacokinetic properties of recombinant TNFR2:Fc fusion proteins generated in glycoengineered P. pastoris strains.

Methods

TNFR2:Fc fusion proteins were generated with varying degrees of sialic acid content. The pharmacokinetic properties of these proteins were assessed by intravenous and subcutaneous routes of administration in rats. The binding of these variants to FcRn were also evaluated for possible correlations between in vitro binding and in vivo PK.

Results

The pharmacokinetic profiles of recombinant TNFR2:Fc produced in P. pastoris demonstrated a direct positive correlation between the extent of glycoprotein sialylation and in vivo pharmacokinetic properties. Furthermore, recombinant TNFR2:Fc produced in glycoengineered Pichia, with a similar sialic acid content to CHO-produced etanercept, demonstrated similar in vivo pharmacokinetic properties to the commercial material. In vitro surface plasmon resonance FcRn binding at pH6.0 showed an inverse relationship between sialic acid content and receptor binding affinity, with the higher affinity binders having poorer in vivo PK profiles.

Conclusions

Sialic acid content is a critical attribute for modulating the pharmacokinetics of recombinant TNFR2:Fc produced in glycoengineered P. pastoris.

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Abbreviations

ASGPR:

asialoglycoprotein receptor

AUC:

area under the curve

BR3-Fc:

B cell-activating factor receptor 3 (BR3) Fc fusion protein

CHO:

Chinese hamster ovary

Fc:

crystallizable fragment of antibody

FcRn:

neonatal Fc receptor

GlcNAc:

n-acetylglucosamine

IgG1:

immunoglobulin G subclass 1

IMID:

immune-mediated inflammatory disease

IV:

intravenous

LFA3TIP:

lymphocyte function-associated molecule 3 Fc fusion protein

PK:

pharmacokinetics

rhEPO:

recombinant human erythropoietin

SC:

subcutaneous

TNF:

tumor necrosis factor

TNFR2:

tumor necrosis factor receptor 2

TSA:

total sialic acid content

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank members of the Strain Development, Fermentation, Purification and Analytical groups of Biologics Discovery (GlycoFi, Merck), that supported this study. We would also like to thank Tamara Pittman and Scott Faulty for their in vivo support, and Dr Paul Peloso for reviewing this manuscript. All funding for this project was provided by Merck & Co., Inc.

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Authors and Affiliations

  1. Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism Merck Research Laboratories, West Point, Pennsylvania, 19486, USA

    Liming Liu, Lora Hamuro, Thomayant Prueksaritanont & Weirong Wang

  2. Biologics Discovery (GlycoFi, Merck), Merck Research Laboratories, 21 Lafayette Street (Suite 200), Lebanon, New Hampshire, 03766, USA

    Sujatha Gomathinayagam, Terrance A. Stadheim & Stephen R. Hamilton

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  1. Liming Liu
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  2. Sujatha Gomathinayagam
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  3. Lora Hamuro
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  4. Thomayant Prueksaritanont
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  6. Terrance A. Stadheim
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  7. Stephen R. Hamilton
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Correspondence to Stephen R. Hamilton.

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Liu, L., Gomathinayagam, S., Hamuro, L. et al. The Impact of Glycosylation on the Pharmacokinetics of a TNFR2:Fc Fusion Protein Expressed in Glycoengineered Pichia Pastoris . Pharm Res 30, 803–812 (2013). https://doi.org/10.1007/s11095-012-0921-3

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  • DOI: https://doi.org/10.1007/s11095-012-0921-3

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