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.

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 β-linked glycans in this organism.

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.

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.

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.

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.

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.

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.

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).

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.