Methanol induction optimization for scFv antibody fragment production in Pichia pastoris

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.     

Oxygen-limited control of methanol uptake for improved production of a single-chain antibody fragment with recombinant Pichia pastoris

The yeast Pichia pastoris is a suitable production system for recombinant proteins due to its strong methanol-inducible AOX1 promoter. A key parameter of the production process is the specific methanol uptake rate. To control the methanol uptake and simultaneously maintain a constant methanol concentration during the production phase, two strategies were developed to generate purposeful oxygen limitation and to feed-forward control the specific methanol uptake rate into the optimum range. First, the cell density at induction was adjusted by prolonged preinduction glycerol feeding. Alternatively, the airflow rate was restricted and increased in parallel with the biomass. While the product accumulation started 20 h earlier with the first approach, the specific production rate of a single-chain antibody fragment was three times higher in the latter case. After 70 h of production, both schemes yielded product concentrations in the gram-per-liter range. Moreover, they release the requirement for dosage of pure oxygen and thereby can facilitate the scale-up of the production process. The different production profiles indicate that the impact of specific methanol uptake rate on protein production by recombinant P. pastoris depends on the control mode.     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

Extended operation of a pressurized 75-L bioreactor for shLkn-1 production by Pichia pastoris using dissolved oxygen profile control

In this study, a dissolved oxygen (DO)-stat fed-batch process was conducted in a pressurized 75-L bioreactor, resulting in the production of the short version of human leukotactin-1 (shLkn-1) using Pichia pastoris as the host, with control of the DO-stat profile and an extension of the recombinant shLkn-1 production phase. By regulation of the exhaust-gas valve, we were able to maintain the vessel pressure at up to 120 kPa, in order to overcome DO limitations associated with the use of the DO-stat. The lowest DO value was adjusted by varying the feed pump speed, allowing us to control the DO-stat profile. This principle was successfully applied to both glycerol feeding during the growth phase and methanol feeding for the induction of shLkn-1. The extension of the methanol induction phase to a total of 192 h of culture time resulted in a shLkn-1 concentration of 2.5 g/L, and a total of 102 g of cumulative production. During this extended induction period, the C-terminal residue of shLkn-1 was truncated and this was confirmed by both reverse-phase HPLC and mass spectrometry.     

毕赤酵母发酵产S-腺苷蛋氨酸的甘油-甲醇混合流加策略

在毕赤酵母发酵生产S-腺苷蛋氨酸(SAM)的诱导阶段,以不同甘油-甲醇比例的甘油-甲醇混合培养基进行诱导培养,结果表明以10%(w/v)甘油含量的甘油-甲醇混合培养基进行诱导培养时最有利于SAM的表达,SAM产量达6.09 g/L,比0%甘油含量条件下的SAM产量提高了20.4%。对诱导方式进行优化,先以100%甲醇诱导24 h,然后再连续流加10%(w/v)甘油含量的甘油-甲醇混合培养基,SAM产量可达7.94 g/L,在此基础上,进一步改进诱导方式,SAM产量得到进一步的提高,达到9.80 g/L。     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

Dissolved-oxygen-stat controlling two variables for methanol induction of rGuamerin in Pichia pastoris and its application to repeated fed-batch

A DO-stat control strategy for two variables was introduced to the rGuamerin production process in Pichia pastoris and applied to repeated fed-batch culture. Two interrelated variables, namely the ratio of partial pressure of pure O2 in the inlet air-stream and the methanol feed rate, were controlled simultaneously. By using this control strategy, methanol feeding for induction could be controlled automatically while efficiently controlling the dissolved oxygen level. As a result, the cell concentration reached more than 140 g l(-1) and rGuamerin expression level 450 iu l(-1). rGuamerin was secreted into the culture medium and reached a level that was 40% higher than achieved in a fed-batch process using manual control of the methanol feeding rate. Repeated rGuamerin induction was achieved by repeating the methanol feeding and withdrawing the culture broth during extended production. During more than 250 h of culture, expression of rGuamerin was maintained at an average of about 430 iu l(-1 )(473 mg l(-1)), without causing the cell density to decrease. In addition to the rGuamerin production process, the proposed control system might be applied to cultivation of other methylotrophic yeasts in the production of therapeutic proteins.     

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Optimization of protein production from methanol‐induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol‐feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut⁺ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5‐fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014     

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.     

Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris

A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.     

酵母表达肌肉生长抑制素前肽发酵工艺的优化

突变型肌肉生长抑制素前肽（MMP）在治疗肌肉萎缩症和培育多肌肉牲畜上有着广泛的应用前景。以重组表达MMP的毕赤酵母工程菌为模式,对该工程菌在30L发酵灌中培养与诱导备件进行优化,建立该表达系统大规模发酵的最佳生产条件,以期荻得最短的发酵时间和最低的生产损耗。毕赤酵母发酵过程通常分为3个阶段：分批培养（基础培养）阶段、分批补料培养阶段、诱导阶段。对发酵过程的第2与第3阶段进行了优化。通过分步提高甘油流加速度：以20mL／L·h^-1的速度流加5h、以30mL／L·h^-1的速度流加5h、以50mL／L·h^-1速度流加14h,并通入纯氧气,维持60％的溶氧度,使甘油流加阶段的时间从常规的48h缩短至24h,即只需24h即可达到常规方法48h才能达到的菌体密度。在诱导表达阶段,通过甘油与甲醇的交替流加,同时对发酵液中甲醇含量的实时监测,保持甲醇的浓度不超过0．5％的高限,使诱导的时间从常规的72h缩短至36h,而且表达量提高了约1倍。优化后,整个发酵周期从120～148h缩短至72～80h,显著提高了MMP蛋白的生产效率。     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度,在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明,以下条件：初始甘油浓度40g／L、初始甲醇浓度3.1g甲醇／gDCW、每24h添加0.51g甲醇／gDCW、诱导表达周期72h、250mL三角瓶诱导培养基装液量30mL、初始pH6,0,最适于菌体生长与产物表达。在此基础上,7L罐上通过恒速流加甘油进一步提高细胞密度,诱导阶段甲醇采取前期恒速流加和后期DO-stat,发酵结束菌体干重达80g／L,酶活为217U／mL,比摇瓶结果提高了66.2％。     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度, 在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明, 以下条件：初始甘油浓度40 g/L、初始甲醇浓度3.1 g甲醇/g DCW、每24 h添加0.51 g甲醇/g DCW、诱导表达周期72 h、250 mL三角瓶诱导培养基装液量30 mL、初始pH 6.0, 最适于菌体生长与产物表达。在此基础上, 7 L罐上通过恒速流加甘油进一步提高细胞密度, 诱导阶段甲醇采取前期恒速流加和后期DO-stat, 发酵结束菌体干重达80 g/L, 酶活为217 U/mL, 比摇瓶结果提高了66.2%。     

Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris

Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing.     

Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is a powerful system for production of recombinant proteins, showing high ability to secrete properly folded proteins. A major plus is the strong AOX1 promoter highly induced by methanol. During growth on methanol, however, oxygen readily becomes limiting. In oxygen-limited cultivations of recombinant Pichia pastoris, the methanol concentration had a strong impact on the production of a single-chain antibody fragment (scFv). High methanol concentrations were required to compensate the lack of oxygen and fully induce recombinant protein production, at the same time reducing gratuitous biomass formation due to a lower biomass yield. Product concentrations of 60, 150, and 350 mg/L were obtained with methanol concentrations of 0.3, 1, and 3% (v/v). Moreover, accumulation of a putative product fragment that cannot be removed during affinity purification was prevented at high methanol concentrations. Cell vitality after 100 h was maintained above 98% and 96% of the culture with 0.3% and 3% methanol, respectively. In cultivations supplemented with oxygen, in contrast, methanol concentration between 0.3% and 3% did not influence the product yield of 300-400 mg/L. Thus, efficient recombinant protein production under oxygen-limitation seems to require high methanol concentrations, enabling product concentration as high as otherwise obtained only with expensive supply of pure oxygen.     

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.     

Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.     

Enhancement of alkaline polygalacturonate lyase production in recombinant Pichia pastoris according to the ratio of methanol to cell concentration

Polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. It was found that the ratio of methanol to cell concentration had a significant influence on PGL production. In this study, an advanced glycerol exponential feeding strategy was developed for biomass accumulation in cell growth phase, by which cell concentration reached 140 g L(-1) after 19 h glycerol feeding. In subsequent production phase, a methanol feeding profile was proposed according to the optimal ratio of methanol to cell concentration at a range of 0.163-0.171 g g(-1), and PGL activity and productivity reached 430 U mL(-1) and 4.34 U mL(-1)h(-1), respectively. The strategy for enhancing PGL production by controlling the optimal ratio may provide an alternative approach to enhance heterologous protein production with P. pastoris.     

Improvement of specific growth rate of <Emphasis Type="BoldItalic">Pichia pastoris</Emphasis> for effective porcine interferon-α production with an on-line model-based glycerol feeding strategy

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model-based glycerol feeding strategy improved the cell density to 120 g DCW/L, while the specific growth rate decreased from 0.15 to 0.18 to 0.03–0.08 h⁻¹ during the last 10 h of the glycerol feeding stage leading to a variation of the porcine interferon-α production, as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model-based feeding strategy to maintain the specific growth rate above 0.11 h⁻¹. With this feeding strategy, the pIFN-α concentration reached a level of 1.43 g/L, more than 1.5-fold higher than that obtained with the previously adopted feeding strategy. Our results showed that increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective porcine interferon-α production have been achieved by our novel glycerol feeding strategy.     

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.