Osmolality as a lever to modulate the N-glycolylneuraminicacid (Neu5Gc) level of a recombinant glycoprotein produced in Chinese hamster ovary cells.

Glycoproteins could be highly sialylated, and controlling the sialic acid levels for some therapeutic proteins is critical to ensure product consistency and efficacy. N-acetylneuraminic acid (Neu5Ac, or NANA) and N-glycolylneuraminic acid (Neu5Gc, or NGNA) are the two most common forms of sialic acids produced in mammalian cells. As Neu5Gc is not produced in humans and can elicit immune responses, minimizing Neu5Gc formation is important in controlling this quality attribute for complex glycoproteins. In this study, a sialylated glycoprotein was used as the model molecule to study the effect of culture osmolality on Neu5Gc. A 14-day fed-batch process with osmolality maintained at physiological levels produced high levels of Neu5Gc. Increase of culture osmolality reduced the Neu5Gc level up to 70-80%, and the effect was proportional to the osmolality level. Through evaluating different osmolality conditions (300-450 mOsm/kg) under low or high pCO2 , we demonstrated that osmolality could be an effective process lever to modulate the Neu5Gc level. Potential mechanism of osmolality impact on Neu5Gc is discussed and is hypothesized to be cytosol NADH availability related. Compared with cell line engineering efforts, this simple process lever provides the opportunity to readily modulate the Neu5Gc level in a cell culture environment.

Investigation of Air-Liquid Interface Rings in Buffer Preparation Vessels: the Role of Slip Agents.

Air-liquid interface rings were observed on the side walls of stainless steel buffer vessels after certain downstream buffer preparations. Those rings were resistant to regular cleaning-in-place procedures but could be removed by manual means. To investigate the root cause of this issue, multiple analytical techniques, including liquid chromatography with tandem mass spectrometry detection (LC-MS/MS), high-resolution accurate mass liquid chromatography with mass spectrometry, nuclear magnetic resonance, Fourier transform infrared spectroscopy, and scanning electron microscopy with energy-dispersive X-ray spectroscopy have been employed to characterize the chemical composition of air-liquid interface rings. The main component of air-liquid interface rings was determined to be slip agents, and the origin of the slip agents can be traced back to their presence on raw material packaging liners. Slip agents are commonly used in plastic industry as additives to reduce the coefficient of friction during the manufacturing process of thin films. To mitigate this air-liquid interface ring issue, an alternate liner with low slip agent was identified and implemented with minimal additional cost. We have also proactively tested the packaging liners of other raw materials currently used in our downstream buffer preparation to ensure slip agent levels are appropriate. LAY ABSTRACT: Air-liquid interface rings were observed on the side walls of stainless steel buffer vessels after certain downstream buffer preparations. To investigate the root cause of this issue, multiple analytical techniques have been employed to characterize the chemical composition of air-liquid interface rings. The main components of air-liquid interface rings were determined to be slip agents, which are common additives used in the manufacturing process of thin films. The origin of the slip agents can be traced back to their presence on certain raw material packaging liners. To mitigate this air-liquid interface ring issue, an alternate liner with low slip agent was identified and implemented.

Evaluation of Mut+ and MutS Pichia pastoris phenotypes for high level extracellular scFv expression under feedback control of the methanol concentration.

Extracellular secretion of over 4 g x L(-1) of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut+ and MutS phenotypes were compared in a bioreactor under closed-loop PID control of the methanol level. As expected, the MutS phenotype has a growth rate lower than that of the Mut+ (0.37 vs 1.05 d(-1)) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut+ (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g x L(-1) for the MutS also has a positive effect on the final product concentration. Both Mut+ and MutS phenotypes reach a maximum biomass density around 450 g x L(-1) wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOX promoters, without prior knowledge of the culture growth parameters.

An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris.

The expression of a humanized single-chain variable domain fragment antibody (A33scFv) was optimized for Pichia pastoris with yields exceeding 4 g L(-1). A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for immunotherapy of colon cancer. P. pastoris with a MutS phenotype was selected to express A33scFv, which was cloned under regulation of the methanol-inducible AOX1 promoter. We report the optimization of A33scFv production by examining methanol concentrations using fermentation technology with an on-line methanol control in fed-batch fermentation of P. pastoris. In addition, we examined the effect of pH on A33scFv production and biomass accumulation during the methanol induction phase. A33scFv production was found to increase with higher methanol concentrations, reaching 4.3 g L(-1) after 72 h induction with 0.5% (v/v) methanol. Protein production was also greatly affected by pH, resulting in higher yields (e.g., 4.88 g L(-1)) at lower pH values. Biomass accumulation did not seem to vary when cells were induced at different pH values, but was greatly affected by lower concentration of methanol. Purification of A33scFv from clarified medium was done using a two-step chromatographic procedure using anion-exchange and hydrophobic interaction chromatography, resulting in 25% recovery and >90% purity. Pure A33scFv was tested for functionality using surface plasmon resonance and showed activity against immobilized A33 antigen. Our results demonstrate that functional A33scFv can be produced in sufficient quantities using P. pastoris for use in further functionality studies and diagnostic applications.