Defining process design space for monoclonal antibody cell culture

The concept of design space has been taking root as a foundation of in‐process control strategies for biopharmaceutical manufacturing processes. During mapping of the process design space, the multidimensional combination of operational variables is studied to quantify the impact on process performance in terms of productivity and product quality. An efficient methodology to map the design space for a monoclonal antibody cell culture process is described. A failure modes and effects analysis (FMEA) was used as the basis for the process characterization exercise. This was followed by an integrated study of the inoculum stage of the process which includes progressive shake flask and seed bioreactor steps. The operating conditions for the seed bioreactor were studied in an integrated fashion with the production bioreactor using a two stage design of experiments (DOE) methodology to enable optimization of operating conditions. A two level Resolution IV design was followed by a central composite design (CCD). These experiments enabled identification of the edge of failure and classification of the operational parameters as non‐key, key or critical. In addition, the models generated from the data provide further insight into balancing productivity of the cell culture process with product quality considerations. Finally, process and product‐related impurity clearance was evaluated by studies linking the upstream process with downstream purification. Production bioreactor parameters that directly influence antibody charge variants and glycosylation in CHO systems were identified. Biotechnol. Bioeng. 2010;106: 894–905. © 2010 Wiley Periodicals, Inc.     

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.     

Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production

Pichia pastoris is extensively used to produce various heterologous proteins. Amounts of biopharmaceutical drugs and industrial enzymes have been successfully produced by fed-batch high-cell-density fermentation (HCDF) of this cell factory. High levels of cell mass in defined media can be easily achieved and therefore large quantities of recombinant proteins with enhanced activities and lower costs can be obtained through HCDF technology. A robust HCDF process makes a successful transition to commercial production. Recently, efforts have been made to increase the heterologous protein production and activity by the HCDF of P. pastoris. However, challenges around selecting a suitable HCDF strategy exist. The high-level expression of a specific protein in P. pastoris is still, at least in part, limited by optimizing the methanol feeding strategy. Here, we review the progress in developments and applications of P. pastoris HCDF strategies for enhanced expression of recombinant proteins. We focus on the methanol induction strategies for efficient fed-batch HCDF in bioreactors, mainly focusing on various stat-induction strategies, co-feeding, and the limited induction strategy. These processes control strategies have opened the door for expressing foreign proteins in P. pastoris and are expected to enhance the production of recombinant proteins.     

Production of recombinant protein in Pichia pastoris by fermentation

This protocol is applicable to recombinant protein expression by small-scale fermentation using the Pichia pastoris expression system. P. pastoris has the capacity to produce large quantities of protein with eukaryotic processing. Expression is controlled by a methanol-inducible promoter, which allows a biomass-generation phase before protein production is initiated. The target protein is secreted directly into a protein-free mineral salt medium, and is relatively easy to purify. The protocol is readily interfaced with expanded bed adsorption for immediate capture and purification of recombinant protein. The setting up of the bioreactor plus the fermentation itself takes 1 wk. Making the master and user seed lots takes approximately 2 wk for each individual clone.     

A model-based feeding strategy for fed-batch fermentation of recombinant Pichia pastoris

A two stage, exponential feeding strategy with mixed glycerol/methanol substrate was used in a fed-batch recombinant Pichia pastorisfermentation. The feeding strategy was developed using a simple model based on mass balances, Monod-type growth kinetics, and constant specific heterologous protein production rate. The model accurately predicted cell growth, and demonstrated the usefulness of a rational, model-based approach for improving the productivity of recombinant P. pastoris fermentation.     

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     

海参i-型溶菌酶在毕赤酵母基因工程菌中优化表达及纯化

将实验室已构建的毕赤酵母基因工程茵(pPIC9K-SjLys/GS115)作为海参i-型溶茵酶生产菌株,本研究分别从甲醇浓度、培养基pH、温度和诱导时间对其产酶发酵条件进行优化.实验得出甲醇诱导浓度为1.0％,发酵培养基初始pH 6.0,温度30℃,培养96 h为最佳目的蛋白表达条件,其发酵液中海参i-型溶菌酶含量达10.63 mg/L.将发酵液经离心和超滤浓缩后得到上清液,再经离子交换和凝胶过滤层析纯化获得海参i-型溶菌酶产品,其酶活力达826.44 U/mg.经测定该酶对革兰氏阳性菌溶壁微球菌和革兰氏阴性菌副溶血弧菌均具有明显的抑菌作用.     

酶法测定甲醇酵母发酵液中甲醇浓度

用醇氧化酶－过氧化氢酶联合的酶促反应法,测定甲醇酵母发酵诱导阶段变化的发酵液中甲醇浓度,建立一种能够快速测定发酵液中甲醇浓度的方法,测定的最佳浓度范围是0．5－5％。     

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 31–45 h, 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±2.4 h, product yield improved for rOvIFN-τ from 203 to 337 mg 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±388 mg.     

Response surface optimization of fermentation conditions for xylanase production by recombinant Pichia pastoris

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、KH2 PO4、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、MrSO4 ·H2O和FeSO4·7H2O.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)2SO4 6.25,KH2PO4 1.25、MnSO4·H2O 0.35,FeSO4 ·7H2O 1.31.培养基优化后,实际产酶2 883.86 U/mL,是优化前YPD培养基产酶的2.51倍.     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997     

Optimization of fermentation conditions of xylanase produced by recombinant Pichia pastoris

采用单因素实验确定重组毕赤酵母产木聚糖酶生长相的最适条件,然后利用Plackett-Burman实验设计对诱导相培养基成分和培养条件的10个因素进行筛选,方差分析结果表明,影响木聚糖酶表达的主要因子为酵母膏、诱导pH和摇床转速;在此基础上,用Box-Behnken的响应面方法对3个因素进行进一步优化,当酵母膏为11.13 g/L,pH为6.38,摇床转速为228 r/min时酶活有最大值,为262.77 U/mL,较优化前提高了175.44％.优化后的摇瓶发酵条件应用于7L发酵罐并连续诱导培养120 h,发现诱导72 h后的木聚糖酶酶活最高,为2054.89U/mL.     

Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris

Glycerol/methanol and sorbitol/methanol mixed-feed fermentation strategies for the production of recombinant proteins by Pichia pastoris were compared in order to examine sorbitol's potential as a carbon source. Although P. pastoris does have a lower cell yield on sorbitol than on glycerol, the specific rate of product formation is higher (60 g protein g^–1 dry wth for sorbitol/methanol, vs 45 g protein g^–1 dry wth for glycerol/methanol), resulting in comparable final recombinant expression levels. Importantly, the presence of residual sorbitol in the growth medium appears to be less repressive to the alcohol oxidase promoter in this organism, providing a more forgiving means of operating mixed-feed fed-batch recombinant P. pastoris fermentations.     

Enhancing the production of Fc fusion protein in fed-batch fermentation of Pichia pastoris by design of experiments

This study focuses on the feasibility of producing a therapeutic Fc fusion protein in Pichia pastoris (P. pastoris) and presents an optimization design of experiment (DOE) strategy in a well-defined experimental space. The parameters examined in this study include pH, temperature, salt supplementation, and batch glycerol concentration. The effects of these process conditions were captured by statistical analysis focusing on growth rate and titer responses. Batch medium and fermentation conditions were also investigated prior to the DOE study in order to provide a favorable condition to enable the production of this Fc fusion protein. The results showed that approximately 373 mg/L of the Fc fusion protein could be produced. The pH was found to be particularly critical for the production of this Fc fusion protein. It was significantly higher than the conventional, recommended pH for P. pastoris fermentation. The development of this process shows that protein production in P. pastoris is protein specific, and there is not a set of pre-defined conditions that can work well for all types of proteins. Thorough process development would need to be performed for every type of protein in order for large-scale production in P. pastoris to be feasible.     

Glycerophosphate as a phosphorus source in a defined medium for Pichia pastoris fermentation

Pichia pastoris has emerged as a commercially important yeast for the production of a vast majority of recombinant therapeutic proteins and vaccines. The organism can be grown to very high cell densities using a defined basal salts media (BSM). However, BSM contains bi-cation or tri-cation phosphate, which precipitates out of the medium at pH above 5.5, although the optimal fermentation pH of most recombinant protein fermentation varies between 5.5 and 7.0. In this article, the application of glycerophosphates was investigated as a substitute phosphate source in an effort to eliminate precipitation. The solubility of BSM containing sodium or potassium glycerophosphates was examined before and after autoclaving at various pHs. Sodium glycerophosphate was found stable at autoclave temperature but formed complexes with coexisting magnesium and calcium ions that were insoluble above pH 7.0. Medium where sodium glycerophosphate was autoclaved separately and then added to the growth medium did not produce any precipitate up to pH 10.5. The performance of P. pastoris fermentations expressing α-galactosidase and ovine interferon-τ using a glycerolphosphate-based medium was found to be comparable to a conventional BSM. The results from this work demonstrate that sodium glycerophosphate can be assimilated by the P. pastoris strains and can be employed as a reliable phosphorus source for both cell growth and recombinant protein production.     

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood β-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q _O/q _O,max), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90–95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.     

An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation

Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ～ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.     

Design of a novel automated methanol feed system for pilot-scale fermentation of Pichia pastoris

Large-scale fermentation of Pichia pastoris requires a large volume of methanol feed during the induction phase. However, a large volume of methanol feed is difficult to use in the processing suite because of the inconvenience of constant monitoring, manual manipulation steps, and fire and explosion hazards. To optimize and improve safety of the methanol feed process, a novel automated methanol feed system has been designed and implemented for industrial fermentation of P. pastoris. Details of the design of the methanol feed system are described. The main goals of the design were to automate the methanol feed process and to minimize the hazardous risks associated with storing and handling large quantities of methanol in the processing area. The methanol feed system is composed of two main components: a bulk feed (BF) system and up to three portable process feed (PF) systems. The BF system automatically delivers methanol from a central location to the portable PF system. The PF system provides precise flow control of linear, step, or exponential feed of methanol to the fermenter. Pilot-scale fermentations with linear and exponential methanol feeds were conducted using two Mut(+) (methanol utilization plus) strains, one expressing a recombinant therapeutic protein and the other a monoclonal antibody. Results show that the methanol feed system is accurate, safe, and efficient. The feed rates for both linear and exponential feed methods were within ± 5% of the set points, and the total amount of methanol fed was within 1% of the targeted volume.     

Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

     

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.