At-line process analytical technology (PAT) for more efficient scale up of biopharmaceutical microfiltration unit operations.

Tangential flow microfiltration (MF) is a cost-effective and robust bioprocess separation technique, but successful full scale implementation is hindered by the empirical, trial-and-error nature of scale-up. We present an integrated approach leveraging at-line process analytical technology (PAT) and mass balance based modeling to de-risk MF scale-up. Chromatography-based PAT was employed to improve the consistency of an MF step that had been a bottleneck in the process used to manufacture a therapeutic protein. A 10-min reverse phase ultra high performance liquid chromatography (RP-UPLC) assay was developed to provide at-line monitoring of protein concentration. The method was successfully validated and method performance was comparable to previously validated methods. The PAT tool revealed areas of divergence from a mass balance-based model, highlighting specific opportunities for process improvement. Adjustment of appropriate process controls led to improved operability and significantly increased yield, providing a successful example of PAT deployment in the downstream purification of a therapeutic protein. The general approach presented here should be broadly applicable to reduce risk during scale-up of filtration processes and should be suitable for feed-forward and feed-back process control.

Characterization of the Pichia pastoris protein-O-mannosyltransferase gene family.

The methylotrophic yeast, Pichiapastoris, 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 ß-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.

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.

Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production.

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast.

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9gL(-1) vs. 1.2gL(-1)), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.

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.

High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies.

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.

Antibody expression kinetics in glycoengineered Pichia pastoris.

Growth of the antibody market has fueled the development of alternative expression systems such as glycoengineered yeast. Although intact antibody expression levels in excess of 1 g L(-1) have been demonstrated in glycoengineered yeast, this is still significantly below the titers reported for antibody fragments in fungal expression systems. This study presents a simplified approach to estimate antibody secretion kinetics and oxygen uptake rate requirements as a function of growth-rate controlled by a limiting methanol feed rate in glycoengineered Pichia pastoris. The yield of biomass from methanol and the specific oxygen requirements predicted in this study compare well with values reported in the literature for wild-type P. pastoris, indicating the intrinsic nature of these yields independent of glycoengineering or the heterologous protein expressed. Specific productivity was found to be a non-linear function of specific growth rate. Based on comparison with relationships between specific growth rate and specific productivity reported in the literature this correlation seems empirical in nature and cannot be established a priori. These correlations were then used in a simple mass balance based model to predict the cultivation performance of carbon limited cultivations under oxygen transfer limited conditions to indicate the usefulness of this approach to predict large scale performance and aid in process development.

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 cell's 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.

Production of monoclonal antibodies by glycoengineered Pichia pastoris.

The growing antibody market and the pressure to improve productivity as well as reduce cost of production have fueled the development of alternative expression systems. The therapeutic function of many antibodies is influenced by N-linked glycosylation, which is affected by a combination of the expression host and culture conditions. This paper reports the generation of a glycoengineered Pichia pastoris strain capable of producing more than 1 g l(-1) of a functional monoclonal antibody in a robust, scalable and portable cultivation process with uniform N-linked glycans of the type Man(5)GlcNAc(2). N-linked glycan uniformity and volumetric productivity have been maintained across a range of cultivation process conditions including pH (5.5-7.5), temperature (16-24 degrees C), dissolved oxygen concentration (0.85-3.40 mg l(-1)) and specific methanol feed rate (9-19 mg g(-1) h(-1)) as well as across different cultivation scales (0.5, 3.0, 15 and 40 l). Compared to a marketed CHO-produced therapeutic antibody, the glycoengineered yeast-produced antibody has similar motilities on SDS-PAGE, comparable size exclusion chromatograms (SEC) and antigen binding affinities. This paper provides proof of concept that glycoengineered yeast can be used to produce functional full-length monoclonal antibodies at commercially viable productivities.

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: production of complex humanized glycoproteins with terminal galactose.

A significant percentage of eukaryotic proteins contain posttranslational modifications, including glycosylation, which are required for biological function. However, the understanding of the structure-function relationships of N-glycans has lagged significantly due to the microheterogeneity of glycosylation in mammalian produced proteins. Recently we reported on the cellular engineering of yeast to replicate human N-glycosylation for the production of glycoproteins. Here we report the engineering of an artificial glycosylation pathway in Pichia pastoris blocked in dolichol oligosaccharide assembly. The PpALG3 gene encoding Dol-P-Man:Man(5)GlcNAc(2)-PP-Dol mannosyltransferase was deleted in a strain that was previously engineered to produce hybrid GlcNAcMan(5)GlcNAc(2) human N-glycans. Employing this approach, combined with the use of combinatorial genetic libraries, we engineered P. pastoris strains that synthesize complex GlcNAc(2)Man(3)GlcNAc(2) N-glycans with striking homogeneity. Furthermore, through expression of a Golgi-localized fusion protein comprising UDP-glucose 4-epimerase and beta-1,4-galactosyl transferase activities we demonstrate that this structure is a substrate for highly efficient in vivo galactose addition. Taken together, these data demonstrate that the artificial in vivo glycoengineering of yeast represents a major advance in the production of glycoproteins and will emerge as a practical tool to systematically elucidate the structure-function relationship of N-glycans.