Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.     

基因工程抗体在巴斯德毕赤酵母中表达的研究进展

简述了运用巴斯德毕赤酵母系统表达基因工程抗体从构建载体到表达的一般过程,及如何改善和提高抗体的表达。侧重介绍运用该系统获得抗体高表达、高产量的研究近况。     

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.     

Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations.

In the last few years the Pichia pastoris expression system has been gaining more and more interest for the expression of recombinant proteins. Many groups have employed fermentation technology in their investigations because the system is fairly easy to scale up and suitable for the production in the milligram to gram range. A large number of heterologous proteins from different sources has been expressed, but the fermentation process technology has been investigated to a lesser extent. A large number of fermentations are carried out in standard bioreactors that may be insufficiently equipped to meet the demands of high-cell-density fermentations of methylotrophic yeasts. In particular, the lack of on-line methanol analysis leads to fermentation protocols that may impair the optimal expression of the desired products. We have used a commercially available methanol sensor to investigate in detail the effects of supplementary glycerol feeding while maintaining a constant methanol concentration during the induction of a Mut(+) strain of Pichia pastoris. Specific glycerol feed rates in the range of 38-4.2 mg. g(-1). h(-1) (mg glycerol per gram fresh weight per hour) were investigated. Expression of the recombinant scFv antibody fragment was only observed at specific feed rates below 6 mg. g(-1). h(-1). At low specific feed rates, growth was even lower than with methanol as the sole carbon source and the harvest expression level of the scFv was only half of that found in the control fermentation. These results show that glycerol inhibits expression driven by the AOX1 promoter even at extremely limited availability and demonstrate the benefits of on-line methanol control in Pichia fermentation research.     

High-level secretory expression of immunologically active intact antibody from the yeast Pichia pastoris

We have produced a functional murine antibody to dioxin in the culture medium of the methylotrophic yeast Pichia pastoris. Complementary DNA copies encoding the light () and heavy () chains of the dioxin monoclonal antibody, DD1, were each placed under the control of P.pastoris alcohol oxidase (AOX1) promoter and Saccharomyces cerevisiae -mating factor secretion signal sequence. The resulting expression cassettes were assembled into a single plasmid (pPICZDD1) to permit co-expression of both light and heavy chains of the antibody molecule. P.pastoris SMD1168 (pep4, his4) transformed with pPICZDD1 was able to secrete intact antibody into the culture medium. As high as 36 mg l^–1 of the antibody was produced in shake-flask cultures after 96-h induction with methanol. Functional analysis using immunoassay confirmed murine nature of the recombinant antibody and its ability to bind dioxin.     

Production of Humanized Fab Fragment against Human High Affinity IgE Receptor in Pichia pastoris

Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10-40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.     

人源抗HBsAg单链抗体在巴氏毕赤酵母中的表达

在P.pastoris中分泌表达非融合抗HBsAg单链抗体（HBscFv）。设计引物从pGEMHBscFv上扩增目的基因,亚克隆至P.pastoris表达载体pPICZαA中,线性化后转化P. pastorisGS115;转化子经菌落PCR、高浓度Zeocin抗性筛选鉴定后,甲醇诱导目的蛋白表达。结果发现,重组HBscFv可以在α因子的引导下,分泌至培养基中,产量为80mg/L;分泌至培养基中的HBscFv具有结合HBsAg活性,活性总量在诱导培养72h后达最高峰,在诱导培养后期,HBscFv活性下降;PAS糖显色结果表明,酵母表达HBscFv是一种低糖基化蛋白或非糖基化蛋白。     

Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding

Secretory production of a single-chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth-limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol-feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol-feeding phase was extended to 18 h. Interestingly, despite a significantly lower cell density in the cultivation with extended glycerol feeding, the same amount of functional product of 300 mg/L was obtained about 30 h after the start of glycerol feeding with both methods. mRNA analysis revealed that the higher and faster production of the product was related to longer lasting induction of the scFv mRNA. Additional effects of a better adaptation of the secretion machinery may be suggested by higher expression of unfolded protein response-related genes KAR2 and PDI. A clear benefit of the longer glycerol-feeding phase was a 75% reduction of the consumption of both pure oxygen and methanol, and a significantly lower cell density, which would be beneficial for down-stream purification of the product.     

发酵重组Pichia pastoris生产腺苷甲硫氨酸的研究

在5L发酵罐中对高产S腺苷甲硫氨酸的重组Pichia pastoris发酵进行了研究。重组菌在pH5．0生长,然后调为pH6．0积累腺苷甲硫氨酸,在30℃、溶氧5％及流加甲硫氨酸和尿素的条件下培养82h后,产量达4．3g／L。     

人源性抗HBsAg抗体Fab段在酵母中的表达

通过分步整合的方式,将人源性抗乙肝表面抗原（HBsAg）抗体Fab的轻、重链基因分步整合到巴斯德毕赤（Pichia pastoris）酵母GS115菌株的染色体上,经甲醇诱导,成功地分泌表达出抗HBsAg抗体的Fab片段,表达量达50～80mg/L。ELISA结果显示重组酵母分泌表达出的Fab具有较强的结合HBsAg的能力。通过抗Fab的抗体柱亲和层析,纯化出了纯度较高的Fab产品。     

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris

Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.     

Genome‐scale analysis of library sorting (GALibSo): Isolation of secretion enhancing factors for recombinant protein production in Pichia pastoris

A method combining fluorescence activated cell sorting (FACS) and DNA microarray assisted clone identification was developed and termed Genome‐Scale Analysis of Library Sorting (GALibSo). Genes enhancing the production of secreted heterologous proteins in Pichia pastoris were identified out of a cDNA library by cell surface display and FACS. The trends of gene enrichment during consecutive FACS rounds were monitored by DNA microarrays. In a case study a P. pastoris cDNA library was co‐expressed in a strain secreting the Fab fragment of a monoclonal antibody against human immunodeficiency virus type 1 as a model protein. Three genes were identified, increasing the relative expression level of the surface‐displayed model protein up to 45%. While one of these genes had a positive effect on three out of four tested proteins, the product specific effect of the other two suggested that the effects of the co‐expressed secretion enhancing factors are partly dependent on the protein to be produced. The microarray based monitoring of the enrichment of genes causing enhanced protein secretory capacity led to novel insights into the limitation of protein secretion. Biotechnol. Bioeng. 2010; 105: 543–555. © 2009 Wiley Periodicals, Inc.     

一种简化的巴斯德毕赤酵母基本培养基

通过逐步减少巴斯德毕赤酵母基本培养基中生长因子和微量元素的组成种类, 最终确认维持巴斯德毕赤酵母在平板上的生长, 除生物素外其它生长因子和微量元素不必额外添加。据此提出了一种简化的巴斯德毕赤酵母基本培养基SPMD, 其成分为6种无机盐、生物素和葡萄糖。除葡萄糖单独灭菌外, 配制时可以高温高压灭菌。使用该培养基用于转化子的筛选实验中, 获得了与使用常规MD培养基基本相当的转化效率。     

Production process for recombinant human angiostatin in Pichia pastoris

A pilot-scale production method of recombinant human angiostatin, a 38-kD fragment of plasminogen which has been reported to have antiangiogenic activity, has been successfully established by expressing the protein in the methylotrophic yeast Pichia pastoris. The secreted protein inhibited cultured endothelial cell proliferation in vitro and Lewis lung carcinoma growth in mice. The fermentation process was carried out using an on-line methanol controller, administering methanol to the growing culture and keeping its concentration under 2 g L⁻¹. The fermentation lasted 90 h, of which 70 h were growth on methanol. During growth on methanol the culture volume increased 64%, from 7 L to 11.5 L, producing 200 mg angiostatin and 5 kg of biomass. Journal of Industrial Microbiology & Biotechnology (2000) 24, 31–35. Received 12 May 1999/ Accepted in revised form 06 September 1999     

毕赤酵母高密度发酵研究进展

巴斯德毕赤酵母表达系统是近年发展起来的一种优秀的真核表达系统.本从培养基、温度、pH值、溶氧、甲醇的流加等角度对国内外毕赤酵母高密度发酵的研究进展做了较详细的综述,并提出了目前毕赤酵母高密度发酵过程中存在的问题.     

Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures

Recently, we engineered a Pichia pastoris Mut⁺ strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced. To further improve the product yield, in this work, we evaluated L. vannamei trypsinogen production in P. pastoris using a bioreactor and two recombinant P. pastoris strains with different methanol utilization (Mut) phenotypes. The effect of pH and temperature during the induction step on the trypsinogen production was also evaluated. The results indicate that temperature, pH, and Mut phenotypes influence the production of the recombinant protein, with almost no observed effect on cell growth. All cultures with the Mut⁺ strain had significant operational difficulties, such as in lowering the induction temperature, maintaining dissolved oxygen (DO) above 20%, and maintaining the methanol concentration at a constant value, and showed a decrease in metabolic activity due to trypsinogen toxicity to the cell host. In the culture with the Mut^s strain, however, the temperature, methanol concentration, and DO could be more easily controlled, the temperature could be easily decreased, and the trypsinogen caused the lowest toxicity to the host cells. After 96 h of Mut^s strain induction (pH 6 and 25°C), about 250 mg/L recombinant trypsinogen was detected in the culture medium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

抗H5亚型禽流感病毒单链抗体在毕赤酵母中的分泌型表达和生物活性分析

在本实验室研制出的多株针对H5N1亚型禽流感血凝素单抗中,13D4单抗对所有H5亚型病毒均有血凝抑制和中和活性,具有特异性高、反应性强和识别广的特点,且在小鼠实验中显示了对各种代表株禽流感的感染和发病均具有良好的治疗效果。在此研究基础上,本实验通过基因工程构建含有13D4单链抗体(scFv)基因的毕赤酵母表达载体,实现目的蛋白的分泌性表达和纯化。经过竞争法和血凝抑制检测其活性,表明获得的单链抗体具有与原始鼠源抗体相近的反应活性和相同的识别表位。H5N1广谱中和单抗13D4的单链抗体的成功构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础。     

响应面分析优化产木聚糖酶毕赤酵母基因工程菌培养条件

研究不同碳源、氮源和无机盐对毕赤酵母AX181菌株产木聚糖酶的影响。实验表明,分别采用葡萄糖和玉米浆干粉为碳源和氮源可以明显提高木聚糖酶的产量。无机盐单因子优化实验显示添加适量的（NH4）2SO4、KH2PO4、MnSO4·H2O、FeSO4·7H2O也可以部分提高木聚糖酶产量。在此基础上利用响应面法优化毕赤酵母产木聚糖酶培养基,利用12次实验的Plackett—Burman设计实验筛选出影响产木聚糖酶的3个主要因素,即玉米浆干粉、MnSO4·H2O和FeSO4·7H20。并进一步通过最陡爬坡路径逼近最大响应区域,采用中心组合实验设计确定最佳条件。优化后的产木聚糖酶培养基组分为（g/L）：葡萄糖40．00,玉米浆干粉80．84,（NH4）2SO46．25,KH2PO41．25、MnSO4·H2O0．35,FeS04-7H2O1．31。培养基优化后,实际产酶2883．86u／mL,是优化前YPD培养基产酶的2．51倍。     

毕赤酵母高密度发酵表达血管紧张素转化酶C-结构域

血管紧张素转化酶（ACE, EC3.4.15.1）在调节血压方面具有重要作用。研究证实,ACE的C结构域（ACE-C）是使血管紧张素I (AngI)分解的主要活性位点。在5 L 发酵罐中, 对重组毕赤酵母表达ACE C-结构域的发酵工艺进行优化,探讨温度、pH、甲醇浓度等主要因素对重组蛋白表达量和酶活力的影响。结果表明,当工业培养基添加2%蛋白胨为氮源时, ACE C-结构域的降解现象得到了有效控制;采用诱导温度为26℃,pH5.5,甲醇含量为1.5%的表达条件,ACE C-结构域表达量和酶活力分别达到446 mg/L和38.2U/ml,比活力达到86U/mg,是Sigma公司ACE标准品比活力的2倍,为大规模制备ACE C-结构域蛋白,筛选专一性更强的ACE C-结构域抑制剂奠定了基础。