Stephen R. Hamilton

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.

Production of sialylated O-linked glycans in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.

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

ABSTRACTPurposeP. 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.MethodsTNFR2: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.ResultsThe 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.ConclusionsSialic acid content is a critical attribute for modulating the pharmacokinetics of recombinant TNFR2:Fc produced in glycoengineered P. pastoris.

Elimination of β-mannose glycan structures in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing β-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the β-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of β-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of β-Man-associated glycans and their related antigenicity. Taken together, the elimination of β-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.

Characterization of the Pichia pastoris Protein- O -mannosyltransferase Gene Family

The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, either N-linked or O-linked, can elicit an immune response or enable the expressed protein to bind to mannose receptors, thus reducing their efficacy. Previously we have reported the elimination of β-linked glycans in this organism. In the current report we have focused on reducing the O-linked mannose content of proteins produced in P . pastoris , thereby reducing the potential to bind to mannose receptors. The initial step in the synthesis of O-linked glycans in P . pastoris is the transfer of mannose from dolichol-phosphomannose to a target protein in the yeast secretory pathway by members of the protein-O-mannosyltransferase (PMT) family. In this report we identify and characterize the members of the P . pastoris PMT family. Like Candida albicans, P . pastoris has five PMT genes. Based on sequence homology, these PMTs can be grouped into three sub-families, with both PMT1 and PMT2 sub-families possessing two members each (PMT1 and PMT5, and PMT2 and PMT6, respectively). The remaining sub-family, PMT4, has only one member (PMT4). Through gene knockouts we show that PMT1 and PMT2 each play a significant role in O-glycosylation. Both, by gene knockouts and the use of Pmt inhibitors we were able to significantly reduce not only the degree of O-mannosylation, but also the chain-length of these glycans. Taken together, this reduction of O-glycosylation represents an important step forward in developing the P . pastoris platform as a suitable system for the production of therapeutic glycoproteins.

Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris.

Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins

The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.

The Impact of Sialic Acids on the Pharmacokinetics of a PEGylated Erythropoietin

Erythropoietin (EPO) is an important molecule in the erythropoiesis and various forms of EPO have been marketed in managing anemia in humans. Long acting EPOs for less frequent dosing have been generated either by increasing the number of glycosylation sites of the EPO molecule or by linking it to a polyethylene glycol (PEG). We have generated recombinant human EPO (rhEPO) using glycoengineered Pichia pastoris strains and evaluated the pharmacokinetics (PK) in rats of this molecule linked to a 40 kDa PEG (PEGylated rhEPO), in relation to its glycosylation patterns. As expected, the PEGylated rhEPO exhibited a significant improvement in half-life of serum when compared with the non-PEGylated version. Interestingly, the PK properties of the PEGylated rhEPO molecule were also significantly influenced by the glycosylation profile. Specifically, PEGylated rhEPO with a significantly higher sialic acid content in the biantennary structure (high A2) exhibited lower systemic clearance and higher systemic exposure than those with a lower sialic acid content (low A2) following either intravenous or subcutaneous administrations. These results suggest that A2 content may be one of the important criteria for release in manufacturing PEGylated rhEPO to ensure consistent PK.

Progress in Yeast Glycosylation Engineering.

While yeast are lower eukaryotic organisms, they share many common features and biological processes with higher eukaryotes. As such, yeasts have been used as model organisms to facilitate our understanding of such features and processes. To this end, a large number of powerful genetic tools have been developed to investigate and manipulate these organisms. Going hand-in-hand with these genetic tools is the ability to efficiently scale up the fermentation of these organisms, thus making them attractive hosts for the production of recombinant proteins. A key feature of producing recombinant proteins in yeast is that these proteins can be readily secreted into the culture supernatant, simplifying any downstream processing. A consequence of this secretion is that the proteins typically pass through the secretory pathway, during which they may be exposed to various posttranslational modifications. The addition of glycans is one such modification. Unfortunately, while certain aspects of glycosylation are shared between lower and higher eukaryotes, significant differences exist. Over the last two decades much research has focused on engineering the glycosylation pathways of yeast to more closely resemble those of higher eukaryotes, particularly those of humans for the production of therapeutic proteins. In the current review we shall highlight some of the key achievements in yeast glyco-engineering which have led to humanization of both the N- and O-linked glycosylation pathways.

Selection of Pichia pastoris strains expressing recombinant immunoglobulin G by cell surface labeling

A simple cell labeling method for sorting yeast Pichia pastoris antibody expressing strains is described. A small portion of secreted recombinant antibody retained on the cell surface was labeled with fluorescence detection antibody. The signal intensity of the labeled cell was correlated with the cells antibody productivity. Using this labeling technique to sort a mixture model induced in the same fermenter where the cells of high producing strain were spiked into a population of a low producing strain at the frequency of 1:100,000, one round of sorting achieved a approximately 5000-fold enrichment of the high producing strain. A variety of P.pastoris strains expressing antibody sorted based on the signal intensity on the cell surface yielded titer improvements by 30% to 300%. Our data demonstrate that Pichia cell surface labeling is a simple, effective and reliable method for sorting Pichia antibody expressing strains for productivity improvement.