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Optimization of humanized IgGs in glycoengineered Pichia pastoris

Abstract

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class1. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells2,3,4,5,6. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.

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Figure 1: Characterization of yeast-derived antibody.
Figure 2: N-glycan analysis of antibodies.
Figure 3: Receptor binding assays of rituximab glycovariants.
Figure 4: B-cell depletion assays of rituximab/Rituxan, GS WT, GS3.0 and GS4.0.

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Acknowledgements

We wish to acknowledge Ron Hitzman and Max Vasquez for their continued input on improving antibody expression and quality. Erin Giaccone, Sujatha Gomathinayagam, Heather Lynaugh, Teresa Mitchell and Alissa Thompson are acknowledged for excellent technical assistance and Jim Posada is acknowledged for his insistence on developing a yeast-based antibody expression platform.

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Correspondence to Tillman U Gerngross.

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All authors, with the exception of T.U.G., J.P.H. and R.S., who hold faculty positions at Dartmouth College, are employess of GlycoFi. All authors, excluding J.P.H. and R.S., have a financial interest in the company.

Supplementary information

Supplementary Fig. 1

Plasmid construct pDX478 used for the expression of rituximab in P. pastoris. (PDF 211 kb)

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Li, H., Sethuraman, N., Stadheim, T. et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol 24, 210–215 (2006). https://doi.org/10.1038/nbt1178

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