Expression of functional single-chain variable domain fragment (scFv) antibody against Metolcarb in Pichia pastoris

A Pichia pastoris system was used to express a single-chain variable fragment (scFv) antibody targeted against Metolcarb. The specific scFv gene was amplified from the phage-display scFv library and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C-scFv, was linearized and transformed into P. pastoris strain X-33. A transformant named X-33-Pp-SMW-12-6, which showed strong expression of antibodies, was isolated, and the culture conditions, including methanol induction concentrations, inoculum densities, and pH, were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of the scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against Metolcarb are comparable to those of scFv antibodies produced in E. coli. Recoveries of Metolcarb demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of Metolcarb residue in environmental and agricultural samples using a competitive inhibition enzyme-linked immunosorbent assay. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against Metolcarb for downstream applications.     

Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv)

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75?h, typically between 140 and 160?g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5?% of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0?% boundary for longer periods of time when compared to a traditional proportional–integral–derivative algorithm, which tends to destabilize with increasing cell density.     

Model‐based optimization and pO2 control of fed‐batch Escherichia coli and Saccharomyces cerevisiae cultivation processes

A model‐based approach for optimization and cascade control of dissolved oxygen partial pressure (pO₂) and maximization of biomass in fed‐batch cultivations is presented. The procedure is based on the off‐line model‐based optimization of the optimal feeding rate profiles and the subsequent automatic pO₂ control using a proposed cascade control technique. During the model‐based optimization of the process, feeding rate profiles are optimized with respect to the imposed technological constraints (initial and maximal cultivation volume, cultivation time, feeding rate range, maximal oxygen transfer rate and pO₂ level). The cascade pO₂ control is implemented using activation of cascades for agitation, oxygen enrichment, and correction of the preoptimized feeding rate profiles. The proposed approach is investigated in two typical fed‐batch processes with Escherichia coli and Saccharomyces cerevisiae. The obtained results show that it was possible to achieve sufficiently high biomass levels with respect to the given technological constraints and to improve controllability of the investigated processes.     

Two‐compartment processing as a tool to boost recombinant protein production

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.     

Multiparameter optimization method and enhanced production of secreted recombinant single-chain variable fragment against the HIV-1 P17 protein from Escherichia coli by fed-batch fermentation

The single-chain fragment variable (scFv) was used to produce a completely functional antigen-binding fragment in bacterial systems. The advancements in antibody engineering have simplified the method of producing Fv fragments and made it more efficient and generally relevant. In a previous study, the scFv anti HIV-1 P17 protein was produced by a batch production system, optimized by the sequential simplex optimization method. This study continued that work in order to enhance secreted scFv production by fed-batch cultivation, which supported high volumetric productivity and provided a large amount of scFvs for diagnostic and therapeutic research. The developments in cell culture media and process parameter settings were required to realize the maximum production of cells. This study investigated the combined optimization methods, Plackett–Burman design (PBD) and sequential simplex optimization, with the aim of optimize feed medium. Fed-batch cultivation with an optimal feeding rate was determined. The result demonstrated that a 20-mL/hr feeding rate of the optimized medium can increase cell growth, total protein production, and scFv anti-p17 activity by 4.43, 1.48, and 6.5 times more than batch cultivation, respectively. The combined optimization method demonstrated novel power tools for the optimization strategy of multiparameter experiments.     

Batch and fed-batch growth of Pichia pastoris under increased air pressure

Pichia pastoris CBS 2612 behavior under air pressures of 1, 3 and 5 bar in culture media of glycerol (pure and crude) and methanol was studied. Generally, the increase in oxygen transfer rate due to the increase of total pressure improved cellular growth for all carbon sources and for batch and fed-batch processes with different feeding rate strategies. In batch cultures, 1.4-, 1.2-, and 1.5-fold improvement in biomass production was obtained with the increase of air pressure up to 5 bar, using methanol, pure glycerol, and crude glycerol, respectively. The increase of air pressure to 5 bar using exponential feeding rate led to 1.4-fold improvement in biomass yield per glycerol mass consumed, for crude and pure glycerol. The current low cost of crude glycerol from the biodiesel production together with the present results shows the possibility of improving cell mass production of P. pastoris using increased air pressure.     

Effects of environmental conditions and methanol feeding strategy on lipase-mediated biodiesel production using soybean oil

Methanol is a commonly used acyl acceptor for lipase-driven biodiesel production, but a high concentration of methanol is detrimental for lipase activity. To overcome this drawback, a simple fed-batch process was developed by optimization of the methanol feeding strategy and reaction conditions. For the feeding strategy, an equal volume of pure methanol was fed twice with specified time intervals into a reactor initially containing a 1:1 molar ratio of soybean oil to methanol in order to adjust the net molar ratio of the oil to methanol to 1:3. In contrast with the batch reaction, a higher agitation speed in the fed-batch process elevated the conversion yield of soybean oil to biodiesel. An agitation speed of 600 rpm and a reaction temperature of 70°C were chosen as the optimal environmental conditions. Residual lipase activities for the fed-batch operation at 40 ∼ 70°C and 600 rpm were 7.1 ± 1.4 times higher than that of the batch method at 40°C with the same agitation speed, indicating that methanol feeding can prevent significant deactivation of lipase. Finally, two times feeding methanol at 2 and 6 hr resulted in a biodiesel productivity of 10.7%/h and 94.9% final conversion yield under the optimal conditions.     

Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris

Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60–70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.     

Optimal Bioreactor Operational Policies for the Enzymatic Hydrolysis of Sugarcane Bagasse

The consolidation of the industrial production of second-generation (2G) bioethanol relies on the improvement of the economics of the process. Within this general scope, this paper addresses one aspect that impacts the costs of the biochemical route for producing 2G bioethanol: defining optimal operational policies for the reactor running the enzymatic hydrolysis of the C6 biomass fraction. The use of fed-batch reactors is one common choice for this process, aiming at maximum yields and productivities. The optimization problem for fed-batch reactors usually consists in determining substrate feeding profiles, in order to maximize some performance index. In the present control problem, the performance index and the system dynamics are both linear with respect to the control variable (the trajectory of substrate feed flow). Simple Michaelis–Menten pseudo-homogeneous kinetic models with product inhibition were used in the dynamic modeling of a fed-bath reactor, and two feeding policies were implemented and validated in bench-scale reactors processing pre-treated sugarcane bagasse. The first approach applied classical optimal control theory. The second policy was defined with the purpose of sustaining high rates of glucose production, adding enzyme (Accellerase® 1500) and substrate simultaneously during the reaction course. A methodology is described, which used economical criteria for comparing the performance of the reactor operating in successive batches and in fed-batch modes. Fed-batch mode was less sensitive to enzyme prices than successive batches. Process intensification in the fed-batch reactor led to glucose final concentrations around 200 g/L.     

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut^S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.     

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Production of salmosin, a snake venom-derived disintegrin, in recombinant Pichia pastoris using high cell density fed-batch fermentation

Salmosin, a snake venom-derived disintegrin, was successfully expressed in the methylotrophic yeast Pichia pastoris and secreted into the culture supernatant, as a 6 kDa protein. High-cell density fermentation of recombinant P. pastoris was optimized for the mass production of salmosin. In a 5 L jar fermentor, recombinant P. pastoris was fermented in growth medium containing 5% (w/v) glycerol at the controlled pH of 5.0. After culturing for 21 h, glycerol feeding medium was fed at one time into the culture broth. After 7 h (a total of 28 h), induction medium that contained methanol was increasingly added until the culture time totaled 75 h. Finally, these optimized culture conditions produced a high cell density of recombinant P. pastoris (dry cell weight of 113.38 g/L) and led to the mass production of salmosin (a total protein concentration of 369.2 mg/L). The culture supernatant containing salmosin inhibited platelet aggregation, resulting in a platelet aggregation of 9% compared to that of 94% in the control experiment, without culture supernatant. These results demonstrate that recombinant salmosin in culture supernatant from high cell density fed-batch fermentation can serve as a platelet aggregation inhibitor.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.     

Model-based optimization of antibody galactosylation in CHO cell culture

Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality. In this work, we present a modeling platform that quantifies the impact of glycosylation precursor feeding – specifically that of galactose and uridine – on cellular growth, metabolism as well as antibody productivity and glycoform distribution. The platform has been parameterized using an initial training data set yielding an accuracy of ±5% with respect to glycoform distribution. It was then used to design an optimized feeding strategy that enhances the final concentration of galactosylated antibody in the supernatant by over 90% compared with the control without compromising the integral of viable cell density or final antibody titer. This work supports the implementation of Quality by Design towards higher-performing bioprocesses.     

Construction,production, and characterization of recombinant scFv antibodies against methamidophos expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris was used to express a recombinant scFv antibody against methamidophos derived from a recombinant phage-display library. The specific scFv gene was amplified from a positive clone and then subcloned into the expression vector pPICZα C. The resulting plasmid, pPICZα C–scFv, was linearized and transformed into P. pastoris (X-33). A transformant named X-33-Pp-Met-28D4, which showed strong expression of antibodies, was isolated, and the culture conditions were optimized. Under optimal conditions, P. pastoris cultures yielded much higher levels of scFv product than the Escherichia coli expression system. Immunochemical characterization of the scFv antibodies produced in P. pastoris indicated that the affinity and specificity of scFv against methamidophos are comparable to those of scFv antibodies produced in E. coli. Recoveries of methamidophos-fortified samples demonstrated that the P. pastoris-derived scFv antibodies can be used to determine the content of methamidophos residue in environmental and agricultural samples. For our purposes, expression in Pichia proved to be an efficient and economical method for the large-scale production of functional scFv antibodies against methamidophos for downstream applications.     

Constitutive expression of alkaline β-mannanase in recombinant Pichia pastoris

Alkaline β-mannanase has important applications for specific industrial processes like pulp bleaching and the detergent industry. The low yield of alkaline β-mannanase produced from native microbes such as alkaliphilic Bacillus limits its applications. Pichia pastoris is the most efficient heterologous system to produce alkaline mannanase. However, the previous use of the AOX system required large amount of methanol and sophisticated operation strategy, which are undesirable in large scale production. In this study, we established a safe and simple constitutive expression process for mannanase production in P. pastoris. The mannanase gene was successfully expressed under the control of GAP promoter. Sequential optimization of the constructed strains was also performed including the copy number optimization and co-expression of chaperone genes. A two-stage feeding strategy was then applied for the finally optimized strain. After 96 h fermentation, a production level of 2980 U/mL was finally reached, illustrating the potential of the GAP constitutive expression system for industrial scale preparation of alkaline β-mannanase.     

Metabolic engineering of Pichia pastoris for malic acid production from methanol

The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP‐CM301 could produce 8.55 g/L malic acid from glucose, which showed a 3.45‐fold increase compared to the parent strain. To improve the efficiency of site‐directed gene knockout, NHEJ‐related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by‐product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose‐6‐phosphate isomerase was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for the metabolic engineering of P. pastoris to produce value‐added chemicals from methanol.