Natarajan Sethuraman

Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.

Optimization of humanized IgGs in glycoengineered Pichia pastoris

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.

Optimization of erythropoietin production with controlled glycosylation-PEGylated erythropoietin produced in glycoengineered Pichia pastoris

Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris.

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.

Inactivation of a GAL4-Like Transcription Factor Improves Cell Fitness and Product Yield in Glycoengineered Pichia pastoris Strains

ABSTRACT With a completely reengineered and humanized glycosylation pathway, glycoengineered Pichia pastoris has emerged as a promising production host for the manufacture of therapeutic glycoproteins. However, the extensive genetic modifications have also negatively affected the overall fitness levels of the glycoengineered host cells. To make glycoengineered Pichia strains more compatible with a scalable industrial fermentation process, we sought to identify genetic solutions to broadly improve cell robustness during fermentation. In this study, we report that mutations within the Pichia pastoris ATT1 (PpATT1) gene (a homolog of the Saccharomyces cerevisiae GAL4 [ScGAL4] transcriptional activator) dramatically increased the cellular fitness levels of glycoengineered Pichia strains. We demonstrate that deletion of the PpATT1 gene enabled glycoengineered Pichia strains to improve their thermal tolerance levels, reduce their cell lysis defects, and greatly improve fermentation robustness. The extension of the duration of fermentation enabled the PpATT1-modified glycoengineered Pichia strains to increase their product yields significantly without any sacrifice in product quality. Because the ATT1 gene could be deleted from any Pichia strains, including empty hosts and protein-expressing production strains alike, we suggest that the findings described in this study are broadly applicable to any Pichia strains used for the production of therapeutic proteins, including monoclonal antibodies, Fc fusions, peptides, hormones, and growth factors.

Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.

Erratum to: Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Unfortunately, in the original article, the name of the author Meehl Meehl was given incorrectly. The correct name is Michael Meehl for which we correctly presented above.