Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115

A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7-380 mL columns and the process was performed at the pilot scale using 0.5-3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01g of rBoNTE(Hc)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50's of BoNT/E.     

Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris

A recombinant vaccine candidate was developed that protected mice against botulinum neurotoxin serotype F (BoNTF) intoxication. A synthetic gene encoding BoNTF fragment C (rBoNTF(H(c))) was designed, constructed, and inserted into a plasmid for expression in the yeast Pichia pastoris. A total cell protein content of 2.9 g was obtained per liter of fermentation broth. Recombinant rBoNTF(H(c)) was purified from the soluble yeast extract in two chromatographic steps. The process employed Mono S cation exchange chemistry followed by Alkyl-Superose hydrophobic interaction chromatography, producing material judged to be greater than 98% pure by SDS-PAGE. The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste. rBoNTF(H(c)) was also purified from the insoluble fraction of the yeast cell lysate. Because the fragment C in the pellet was 35% of the total insoluble protein, only a Mono S cation exchange chromatography step was necessary to achieve a purity greater than 98%. Mice that received three injections of 0.2 microgram of purified soluble rBoNTF(H(c)) were completely protected when challenged with 1000 mouse ip LD(50) of BoNTF toxin. Similarly, three doses of 1 microgram of purified resolubilized rBoNTF(H(c)) completely protected mice from a challenge of 5000 mouse ip LD(50) of BoNTF toxin. Individual serum antibody ELISA titers of mice injected with soluble rBoNTF(H(c)) correlated with survival as all 34 mice with ELISA titers of 100 or greater survived toxin challenge. The work presented here demonstrates that purified rBoNTF(H(c)) is able to protect against a high challenge dose of neurotoxin.     

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.     

Production and purification of the heavy chain fragment C of botulinum neurotoxin, serotype A, expressed in the methylotrophic yeast Pichia pastoris

A recombinant H(C) fragment of botulinum neurotoxin, serotype A (rBoNTA(H(C))), has been successfully expressed in a Mut(+) strain of the methylotrophic yeast Pichia pastoris for use as an antigen in a proposed human vaccine. Fermentation employed glycerol batch, glycerol-fed batch, and methanol-fed batch phases to achieve high cell density. Induction times were short to maximize rBoNTA(H(C)) production while minimizing proteolytic degradation. Concentration of rBoNTA(H(C)) in yeast cell lysates was generally 1-2% of the total protein based on ELISA analysis. The H(C) fragment was purified from cell lysates using a multistep ion-exchange (IEC) chromatographic process, including SP, Q, and HS resins. The zwitterionic detergent Chaps was included in the buffer system to combat possible interactions, such as protein-protein or protein-DNA interactions. Following IEC was a hydrophobic interaction chromatography (HIC) polishing step, using phenyl resin. The H(C) fragment was purified to >95% purity with yields up to 450 mg/kg cells based on ELISA and Bradford protein assay. The purified H(C) fragment of serotype A was stable, elicited an immune response in mice, and was protected upon challenge with native botulinum type A neurotoxin.     

应用流式细胞术检测毕赤酵母的细胞活性

选取两种细胞活性染色试剂二乙酸荧光素(fluoresceindiacetate,FDA)和碘化丙锭(propidiumiodide,PI),应用流式细胞术(flowcytometry,FCM)检测毕赤酵母细胞活性。比较FDA/PI双染色与PI单染色的FCM图谱,后者能够很好地将死活细胞区分开来并得到正确的比例。利用PI单染色检测发酵过程细胞活性的变化,甘油补料阶段几乎没有细胞死亡,进入甲醇补料阶段后,随着细胞密度的增加,细胞的活性不断降低,发酵88h时细胞活性仅为73.8%。     

Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(H(c))] expressed in Pichia pastoris

A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H_c)], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H_c) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H_c) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel?), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5–100 mL columns and the process was performed at the pilot scale using 0.6–1.6 L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H_c)/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H_c)/kg WCW at the pilot scale. The purified rBoNTC(H_c) was stable for at least 3 months at 5 and ?80 °C as determined by reverse phase-HPLC and SDS–PAGE and was stable for 24 months at ?80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H_c) was intact.     

流式细胞术检测毕赤酵母发酵过程中胞内活性氧水平

以2′,7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)和碘化丙锭(PI)为标记探针,通过DCFH-DA/PI双染色与PI单染色的对照,检测毕赤酵母胞内活性氧(reactive oxygen species,ROS)的水平及其影响。研究发现发酵过程细胞活性下降与胞内ROS积累相关。在甘油生长期,细胞几乎没有ROS积累,细胞活性接近100%。在甲醇诱导初期,部分细胞积累少量的ROS,细胞活性仍然很高,死亡细胞所占比例只有1.5%。在甲醇诱导后期,94.0%的细胞积累了大量的ROS,高含量的ROS造成细胞损伤,引起部分细胞丧失了活性,在总共29.1%的死亡细胞中,高ROS积累的死亡细胞占了25.4%。     

High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris expression system

A continuous fermentation process has been developed in Pichia pastoris (P. pastoris) with the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in order to produce large quantities of recombinant human chitinase (rh-chitinase) for preclinical studies as a potential high-dose antifungal drug. Expression levels of about 200 to 400 mg/L have been demonstrated in fed-batch fermentations using strains with either the traditional methanol-inducible or the constitutive GAP promoter. Proteolytic degradation of the enzyme was typically seen in fed-batch fermentations. Continuous production of the enzyme by P. pastoris with the GAP promoter was demonstrated in a 1.5-L working volume fermentor using either glucose or glycerol as the carbon source. The fermentation could be extended for >1 month with a steady-state protein concentration of approximately 300 mg/L. Cell densities were >400 g/L wet cell weight (WCW) (approximately 100 g/L dry cell weight [DCW]) at a dilution rate (D) of 0.83 day(-1) or 1.2 volume exchanges per day (VVD). No proteolytic degradation of the enzyme was seen in the continuous fermentation mode.     

表达重组咖啡豆α-半乳糖苷酶的酵母工程菌的高密度发酵

用5 L发酵罐优化了重组咖啡豆α-半乳糖苷酶酵母工程菌pPIC9K-Gal/GS115(本室构建)的高密度发酵工艺.通过对发酵条件的优化,包括甘油补充量及补充时机、甲醇诱导量及诱导时机、溶氧控制、诱导时间等,重组咖啡豆α-半乳糖苷酶在毕赤酵母中得到了高效表达.利用所确定的最适条件进行发酵,菌体密度最终达到368 g/L以上,每批发酵液离心后可获得3.5 L的发酵上清,上清中的蛋白含量达到3 g/L以上,目的蛋白占上清总蛋白的50%以上,含量约为1.5 g/L,上清中α-半乳糖苷酶的活性维持在80 U/ml左右.确立工艺后又进行了3次发酵试验,证明了工艺的可行性和稳定性.为重组咖啡豆α-半乳糖苷酶在B→O血型改造和酶解大豆低聚糖方面的应用奠定了基础.     

Production and Purification of the Heavy-Chain Fragment C of Botulinum Neurotoxin, Serotype B, Expressed in the Methylotrophic YeastPichia pastoris

A recombinant H_cfragment of botulinum neurotoxin, serotype B (rBoNTB(H_c)), has been successfully expressed in a Mut⁺strain of the methylotrophic yeastPichia pastorisfor use as an antigen in a proposed human vaccine. The fermentation process consisted of batch phase on glycerol, followed by glycerol and methanol fed-batch phases yielding a final cell mass of 60 g/L (dcw) and was easily scaled-up to 60 L. A multistep ion-exchange chromatographic purification process was employed to produce 99% pure H_cfragment. The final yield of the purified antigen was 390 mg per kilogram of wet cell mass. The purified H_cfragment of serotype B was stable, elicited an immune response in mice, and protected upon challenge with native botulin.     

重组毕赤酵母发酵牛肉风味肽的中试研究

研究分泌表达16拷贝牛肉风味肽（beefy meaty peptide, BMP）毕赤酵母工程菌株(Pichia pastoris GS115-16B2)的500L发酵罐中试发酵工艺。在500L发酵罐中采用三步发酵法进行了3批次中试发酵实验,设定菌体培养阶段温度为30℃,pH5.0,诱导表达阶段温度为28℃,pH3.0。在发酵程中通过调节搅拌速度、通气量和罐压等措施来使DO维持在20%~35%左右,总共发酵126h（诱导时间为96h）,当诱导88h后（发酵118h）,600nm波长下的光密度值(OD600)达到184.5,菌体湿重达到318.7g/L,目的产物BMP的表达量达到最高为172 mg/L,实现了BMP的高效表达。通过中试发酵初步建立了工程菌P. pastoris GS115-16B2的中试发酵工艺,为BMP的进一步研究开发奠定了良好基础。     

中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统的建立及活性检测

中性粒细胞蛋白酶抑制剂elafin是中性粒细胞弹性蛋白酶特异性抑制剂。为建立中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统,通过逆转录PCR扩增获取elafin cDNA,双酶切后定向克隆至穿梭质粒pPIC9K上。SalI酶切线性化后,电穿孔法转至毕赤酵母GS115菌株中,PCR筛选出阳性克隆,接种至最小甘油缓冲培养基中,甲醇诱导表达后,取上清阳离子交换层析纯化靶标蛋白。分别以western-blot、弹力纤维蛋白-聚丙烯酰胺凝胶电泳检测重组elafin免疫原性、抗蛋白酶活性。重组elafin在毕赤酵母系统中呈分泌性表达,产量及活性高,分离纯化简便。此表达系统的建立为其后续研究及临床应用奠定了基础。     

不同甲醇流加策略对重组毕赤醇母高密度发酵生产水蛭素的影响

考察了不同甲醇流加策略对毕赤酵母高密度发酵生产水蛭素的影响。溶氧控制法不能有效地防止甲醇的过量流加。气相色谱离线检测法虽然防止了甲醇流加过量 ,但甲醇浓度的波动较大。利用甲醇传感器在线检测控制甲醇的流加可维持较恒定的甲醇浓度。在流加甲醇的同时 ,以限制性速率流加甘油可以增加表达期间的能量供应 ,提高产物的表达量。经优化后 ,采用甲醇甘油混合流加时细胞干重达到 16 2g L ,水蛭素活性达到 2 4×10 4ATU mL ,即 1 7g L。     

A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris

A novel feeding strategy for enhanced protein production of hepatitis B virus surface antigen (HBsAg) in fed-batch fermentation, recombinant Pichia pastoris, has been developed. A minimal salt medium was used to grow cells in the initial batch fermentation, followed by a glycerol+salts fed-batch phase. At the end of the fed-batch phase a dry cell weight of 130 g l⁻¹ was achieved. In the absence of basal salts, the same amount of glycerol feed resulted in only 90 g l⁻¹ cell dry weight. When a limited amount of casamino acids were also included every 24 h during methanol induction, there was a two-fold increase in expression levels of HBsAg. After 192 h of induction, the expression levels of HBsAg (soluble and insoluble) reached >1 g l⁻¹ using the Mut⁻ strain. Thus, the use of basal salts in the glycerol feed, along with the addition of limited amounts of casamino acids with the methanol feed, resulted in an increased expression of total HBsAg.     

Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization

A cell permeabilization method for the release of intracellular proteins from microbial cells was developed. The method was applied to the recovery of recombinant botulinum neurotoxin fragments, expressed intracellularly in the yeast Pichia pastoris, by suspending the cells in an aqueous solution containing N,N-dimethyltetradecylamine. For the botulinum neurotoxin serotype B C-terminal heavy chain fragment, 1.8 mg g(-1) biomass were recovered. For the botulinum neurotoxin serotype A C-terminal heavy chain fragment, 3.7 mg g(-1) biomass were recovered. The concentration of recombinant protein in the cell extracts remained stable for up to 48 and 24 h for the serotype B and serotype A fragments, respectively. The permeabilization method was compared with high-pressure homogenization; the permeabilization method proved to be both more selective and more efficient.     

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.     

An ultra scale-down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges

Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra‐scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot‐scale disk‐type machines, was used in this work. Two recombinant P. pastoris strains, namely a X‐33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot‐ and a large‐scale disk‐type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X‐33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot‐scale runs during early stages of process development for therapeutic products. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1029–1036, 2012     

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600mg/l while residual methanol level was maintained below 2g/l. Clarification of culture supernatant through a 0.1μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5×10(8)IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.     

Two-stage glycerol feeding for enhancement of recombinant hG-CSF production in a fed-batch culture of Pichia pastoris

Recombinant hG-CSF was expressed in Pichia pastoris under the control of the AOX1 promoter. In this study, the glycerol feeding rate was adjusted to achieve the maximum attainable specific growth rate before induction. Using a two-stage glycerol feeding method, the specific growth rate was changed from a maximum value of 0.21 h⁻¹ (at the beginning of feeding) to 0.15 h⁻¹ prior to induction. With this approach, the final dry cell wt and rhG-CSF yield achieved was close to 120 g l⁻¹ and 320 mg l⁻¹, respectively. Our study found that the two-stage feeding method allowed the overall productivity of rhG-CSF to increase 2.9 times that of the conventional fed-batch method.     

Production, characterization and crystallization of the Plasmodium falciparum aquaporin

The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.