Bradley A. Plantz

Modeling pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.

Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

Fed-batch fermentation of a methanol utilization plus (Mut+) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut+ Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (νgly, νMeOH) were established. A mixed feeding strategy with desired μgly/μMeOH =1, 2, 3, 4 (desired μMeOH set at 0.015xa0h−1) was employed to study growth interactions and their effect on production. The results show that the optimal desired μgly/μMeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3xa0mg/g wet cells.

Improved production of recombinant ovine interferon-τ by Mut+ strain of Pichia pastoris using an optimized methanol feed profile

Recombinant ovine interferon‐τ (r‐oIFN‐τ) production by Pichia pastoris was studied using methanol as the sole carbon source during induction. The cells were grown on glycerol up to a certain cell density before induction of the AOX1 promoter by methanol for expression of the recombinant protein. Cell growth on methanol has been modeled using a substrate‐feed equation, which served as the basis for an effective computer control of the process. The r‐oIFN‐τ concentration in the culture began to decline despite continued cell growth after 50 (± 6) h of induction, which was associated with an increase in proteolytic activity of the fermentation broth. A specific growth rate of 0.025 h‐1 was found to be optimal for r‐oIFN‐τ production. No significant improvement in r‐oIFN‐τ production was observed when the specific growth rate was stepped up before the critical point when r‐oIFN‐τ concentration started decreasing during fermentation. However, best results were obtained when the specific growth rate was stepped down from 0.025 to 0.02 h‐1 at 38 h of induction, whereby the active production period was prolonged until 70 h of induction and the broth protease activity was correspondingly reduced. The corresponding maximum protein yield was 391.7 mg·L‐1 after 70 h of fermentation. The proteolytic activity could be reduced by performing fermentations at specific growth rates of 0.025 h‐1 or below. The recombinant protein production can be performed at an optimal yield by directly controlling the methanol feed rate by a computer‐controlled model. The production profile of r‐oIFN‐τ was found to be significantly different from other secreted and intracellular recombinant protein processes, which is an indication that recombinant protein production in Pichia pastoris needs to be optimized as individual processes following established principles.

Design of methanol feed control in Pichia pastoris fermentations based upon a growth model

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut+ (methanol utilization plus) strain. Pichia fermentation is conducted in a fed‐batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed‐batch phase (induction phase) in the fermentation. A simple “on‐off” control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed‐loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters Kc (controller gain), θI (controller integral time constant), and θD (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed‐batch phase. Fermentations were conducted using four Pichia strains , each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

A growth-associated model was applied to the production of recombinant ovine interferon-τ (rOvIFN-τ) with Pichia pastoris for the purpose of manufacturing preclinical and clinical active material. This model predicts that product yields will be the greatest when the specific growth of the culture is maintained at a steady and optimal rate. However, rOvIFN-τ yields did not meet the expected linear model but most closely corresponded to a polynomial relationship. After transitioning from glycerol to methanol, product accumulated for 31–45xa0h, and then the yield decreased. This production shift, which has been termed decoupling, was clearly related to time on methanol and not culture density. It was determined that a correlation exists between the decoupling point and a drop in energy state of the cell when expressing β-galactosidase. By assigning decoupling as a constraint that limits productivity and by reformulating the growth medium, the time prior to decoupling increased to 46.8±2.4xa0h, product yield improved for rOvIFN-τ from 203 to 337xa0mg l−1, and the coefficient of variation for yield decreased from 67.9 to 23.3%. A robust and stable fermentation process was realized, resulting in a 210% improvement in total yield from 557±357 to 1,172±388xa0mg.

Application of synchrotron FTIR microspectroscopy for determination of spatial distribution of methylene blue conjugated onto a SAM via"click" chemistry.

We report, for the first time, the application of synchrotron FTIR microspectroscopy to determine the spatial distribution of methylene blue conjugated onto a self-assembled monolayer surface via Sharpless click chemistry.

Evaluation of metals in a defined medium for Pichia pastoris expressing recombinant β-galactosidase

Culture growth and recombinant protein yield of the Pichia pastoris GS115 methanol utilization positive system were studied in response to the types and levels of metals present in the growth medium and the supplemental salts typically used for these fermentations. Magnesium and zinc were both required to support cell growth but at significantly reduced levels compared to the control. However, supplementation with calcium, cobalt, iron, manganese, iodine, boron, and molybdenum were not required to sustain cell mass. When the medium was reformulated with only zinc and magnesium, the cells grew to 12–15 generations, which are expected for high cell density fed‐batch fermentations. Product yields of the recombinant protein β‐galactosidase were significantly influenced by the trace metal concentrations. By using response surface and full factorial designs, maximum protein yield occurred when the concentration of zinc salt was limited to the level necessary only to support cell mass while protein yield positively correlated to increasing levels of the remaining trace metal salts. These studies are the first to show that excess trace metals must be optimized when developing P. pastoris based fed‐batch fermentations.

Detection of non-host viable contaminants in Pichia pastoris cultures and fermentation broths

The ability to detect viable contaminants in cultures propagated from the original host-expression system ensures that the integrity and purity of seed banks, fermentation broths, and ultimately the final product are continually controlled and maintained. The method developed to detect such agents must be selective for a broad spectrum of microbes, which may be present at very low levels, while discriminating from the host organisms. Although Pichia pastoris strains are frequently used as cell lines for the expression of heterologous proteins, a method that is specific for monitoring culture purity has yet to be reported for this type of organism. An assay that is capable of recovering contaminating bacteria, fungi, and closely related yeast from cultures of P. pastoris at parts per million detection limits is described here.