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 class. 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 cells. 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.

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

“…P. pastoris yAS309 [24] was used for generation of rhLF producing strains. Protein expression studies were done at room temperature in a 96-well plate format with buffered glycerol-complex medium (BMGY) consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast nitrogen base, 4×10 −5 % biotin, and 1% glycerol as a growth medium.…”

“…When an OD600 of 20±5 was reached, the seed culture was transferred to the production fermenter. The systems was controlled by Applikon 1030 Biocontroller with closed loop control of pH, temperature, dissolved oxygen concentration and foam control as described earlier [24]. The pH was maintained at 6.0 throughout the fermentation.…”

“…Thus, each enzyme must be properly targeted and must function at high efficiency in its respective location in the secretory pathway. The application of a combinatorial library approach has been essential to generate P. pastoris strains harboring combinations of mannosidases, glycosyltransferases, GlcNAc/Gal transporters, and Gal epimerase [24]. Over the past years we have created a library of glycoengineered P. pastoris strains each capable of displaying defined N-linked glycans at high uniformity [5,8,12,17,24].…”

Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption

“…The light and heavy chains of the human monoclonal anti-CD20 IgG (Li et al 2006) were genetically fused to the secretion signal comprising amino acids 1-17 of the endogenous yeast acid phosphatase (PHO5 gene).…”

“…The advantages of this host as an expression system include a long history of scientific research leading to a vast amount of data from single component to cellular level. With the advent of glycoengineered yeasts, the production of therapeutic glycoproteins with yeast is now tangible (Li et al 2006;Parsaie Nasab et al 2013;Piirainen et al 2014). However, it is known that bottlenecks in the secretion pathway lead to suboptimal and variable production levels.…”