Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

A high-yielding serum-free, suspension cell culture process to manufacture recombinant adenoviral vectors for gene therapy

We have developed an efficient, reproducible, and scaleable cell culture process for a recombinant adenoviral vector expressing therapeutic transgenes for clinical trials. HEK 293 cells – which support the propagation of E1 deficient adenovirus – were first adapted to serum free media and suspension growth. Subsequent studies focused on the infection, virus production and harvest from suspension culture bioreactors. Future studies are planned to address the kinetics of adenovirus production in HEK 293 as well as in other cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

A model for beta-galactosidase production with a recombinant yeast Saccharomyces cerevisiae in fed-batch culture.

An unstructured model is developed to describe the growth and product formation behavior of a recombinant yeast Saccharomyces cerevisiae using beta-galactosidase as a model protein. The model shows good agreement with the experimental data over a range of conditions. It also accurately predicts the effect of growth rate on yield coefficient and gene expression. The simplicity and accuracy of the model make it suitable for designing and implementing control and optimization strategies for the production of recombinant proteins at large scale.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l⁻¹). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l⁻¹ representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.     

Insect cell culture for industrial production of recombinant proteins

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.     

重组单克隆抗体药物的工程细胞培养工艺研究

单克隆抗体(monoclonal antibody, mAb)是一类重要的基因工程生物技术衍生药物,其药物适应证包括自身免疫性疾病、移植后并发症、心血管疾病、感染性疾病和各种类型的癌症。此类适应证药物通常使用时间长、使用剂量大,因此需具备大批量生产的能力。由于mAb药物的生产成本以及产品质量控制的复杂性,要求有效且经济地提供批间一致性较好的高质量药物。中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞是生物技术产业中用于生产治疗性糖蛋白最常用的真核表达宿主,也是生产治疗性抗体药物最常使用的细胞。在细胞培养过程中,工程细胞株、培养基和工艺条件是mAb药物生产中影响其质量的三个重要因素。现分别从培养基、培养参数和培养模式等对生产mAb药物的细胞培养技术作一概述。     

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.     

Controlling trisulfide modification in recombinant monoclonal antibody produced in fed-batch cell culture

Molecular heterogeneity was detected in a recombinant monoclonal antibody (IgG1 mAb) due to the presence of a trisulfide linkage generated by the post‐translational insertion of a sulfur atom into disulfide bonds at the heavy–heavy and heavy–light junctions. This molecular heterogeneity had no observable effect on antibody function. Nevertheless, to minimize the heterogeneity of the IgG1 mAb from run‐to‐run, an understanding of the impact of cell culture process conditions on trisulfide versus disulfide linkage formation was desirable. To investigate variables that might impact trisulfide formation, cell culture parameters were varied in bench‐scale bioreactor studies. Trisulfide analysis of the samples from these runs revealed that the trisulfide content in the bond between heavy and light chains varied considerably from <1% to 39%. Optimizing the culture duration and feeding strategy resulted in more consistent trisulfide levels. Cysteine concentration in the feed medium had a direct correlation with the trisulfide level in the product. Systematic studies revealed that cysteine in the feed and the bioreactor media was contributing hydrogen sulfide which reacted with the IgG1 mAb in the supernatant leading to the insertion of sulfur atom and formation of a trisulfide bond. Cysteine feed strategies were developed to control the trisulfide modification in the recombinant monoclonal antibody. Biotechnol. Bioeng. 2012; 109: 2523–2532. © 2012 Wiley Periodicals, Inc.     

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.     

High level production of human proapo A-I by fed-batch culture of recombinant Escherichia coli

Three Escherichia coli strains, two recA⁻ strains (DH1 and YK537) and one recA⁺ strain (KS476) harboring human proapo A-I expression plasmid pUS(pAI), were cultivated in fed-batch mode using a synthetic medium and the amounts of human proapo A-I accumulation were compared under various cultivation conditions. In the expression plasmid, nine proapo A-I genes were tandemly ligated downstream of the tac promoter. Experimental results indicated that selection of the host strain and cultivation temperature was important. Among the three E. coli strains checked, strain DH1 yielded the most effective production of human proapo A-I at 30°C.     

A T-flask based screening platform for evaluating and identifying plant hydrolysates for a fed-batch cell culture process

This paper presents a T-flask based screening platform for evaluating and identifying plant hydrolysates for cell culture processes. The development of this platform was driven by an urgent need of replacing a soy hydrolysate that was no longer available for the fed-batch process of recombinant Sp2/0 cell culture expressing a humanized antibody. Series of small-scale experiments in T-flasks and 3-l bioreactors were designed to gain an insight on how this soy hydrolysate benefits the culture. A comprehensive, function-oriented screening platform then was developed, consisting of three T-flask tests, namely the protection test, the growth promotion test, and the growth inhibition test. The cell growth in these three T-flask tests enabled a good prediction of the cell growth in the fed-batch bioreactor process. Fourteen plant hydrolysate candidates were quickly evaluated by this platform for their ability to exert strong protection, high cell growth promotion, and low cell growth inhibition to the culture. One soy hydrolysate was successfully identified to support the comparable cell growth as the discontinued soy hydrolysate. Because of the advantage of using small-scale batch culture to guide bioreactor fed-batch culture, this proposed platform approach has the potential for other applications, such as the medium and feeding optimization, and the mechanism study of plant hydrolysates, in a high throughput format.     

Fault detection and diagnosis in an industrial fed-batch cell culture process

A flexible process monitoring method was applied to industrial pilot plant cell culture data for the purpose of fault detection and diagnosis. Data from 23 batches, 20 normal operating conditions (NOC) and three abnormal, were available. A principal component analysis (PCA) model was constructed from 19 NOC batches, and the remaining NOC batch was used for model validation. Subsequently, the model was used to successfully detect (both offline and online) abnormal process conditions and to diagnose the root causes. This research demonstrates that data from a relatively small number of batches (approximately 20) can still be used to monitor for a wide range of process faults.     

Development of a simple fed-batch process for the high-yield production of recombinant Japanese encephalitis virus protein

Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy affecting children and adolescents in the tropics. Optimization of media was carried out for enhanced production of recombinant JE virus envelope domain III (EDIII) protein in Escherichia coli. Furthermore, batch and fed-batch cultivation process in E. coli was also developed in optimized medium. Expression of this protein in E. coli was induced with 1 mM isopropyl-β-thiogalactoside and yielded an insoluble protein aggregating to form inclusion bodies. The inclusion bodies were solubilized in 8 M urea, and the protein was purified under denaturing conditions using Ni-NTA affinity chromatography. After fed-batch cultivation, the recombinant E. coli resulted in cell dry weight and purified protein about 36.45 g l⁻¹ and 720 mg l⁻¹ of culture, respectively. The purity of the recombinant JE virus EDIII protein was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, and reactivity of this protein was determined by Western blotting and ELISA with JE virus-infected human serum samples. These results establish the application of this protein to be used for the diagnosis of JE virus infection or for further studies in vaccine development. This process may also be suitable for the high-yield production of other recombinant viral proteins.     

Experimental optimization of fed-batch culture for poly-beta-hydroxybutyric acid production

The optimal feed rate profiles of glucose and ammonium hydroxide were calculated using a proposed model, and implemented for the production of poly-beta-hydroxybutyric acid (PHB) by Alcaligenes eutrophus. By implementing these optimal feed rates with a high glucose feed concentration of 700 g/L and an ammonium hydroxide concentration of 7%(w/w), it was possible to achieve a high final cell concentration of 141 g/L and a high PHB concentration of 105 g/L in 40 h of fed-batch operation. The PHB productivity was as high as 2.63 g/(L hr). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 697-705, 1997.     

Enhanced alpha-amylase production in recombinant Bacillus brevis by fed-batch culture with amino acid control

Fed-batch culture with controlled L-amino acid composition was performed to improve production of a recombinant gene product in Bacillus brevis. The maximum recombinant protein (alpha-amylase) level and specific activity increased from 5.14 kU/mL and 0.77 kU/mg dry cell in conventional fed-batch culture to 12.01 kU/mL and 2.64 kU/mg dry cell, respectively, when L-amino acid concentration was controlled at 5 mM using an asparagine (Asn)- and isoleucine (Ile)-enriched nitrogen source. The L-amino acid concentration in the culture was monitored by an automatic biotech analyzer and controlled at 2-20 mM using a mixture of polypeptone and yeast extract. Although L-amino acid concentrations were controlled at low levels, the alpha-amylase activity increased only 1.3 times compared to an uncontrolled batch culture; accumulation of ammonium ion was not reduced. When L-amino acid was controlled at the high level, more cell mass and less recombinant gene product were produced than in those with low control level. To overcome ammonium ion inhibition, the specific amino acids Asn and Ile were substituted to improve the production of gene product. Addition of these amino acids to a flask culture led to an improvement in the enzyme production level and specific activity to 2.9 and 5.1 times, respectively, as high as that without them. Both the control of amino acids at low concentrations and the enrichment of Asn and Ile were effective for the improvement of recombinant protein production from recombinant B. brevis cells. (c) 1996 John Wiley & Sons, Inc.     

A fully defined,fed‐batch,recombinant NS0 culture process for monoclonal antibody production

To manufacture a glycoprotein, mammalian cells expressing the desired protein are often grown in fed‐batch mode. Feeding an undefined, nonanimal hydrolysate helps the cells receive sufficient nutrition, but makes systems difficult to optimize. Even different lots of the same hydrolysate may have significant variability; furthermore, individual components may actually be detrimental to the cells. Switching to fully defined feeds could eliminate these issues. For monoclonal antibody (mAb) production by fed‐batch NS0 cells, this article describes the replacement of a hydrolysate‐based feed with a fully defined, animal‐component‐free feed system. The defined feed initially had 67 components, but additional experiments allowed a reduction to 25 components. The mAb titer is approximately 20% higher than in the undefined system, and the feed volume is circa 20% lower. The two systems generated antibodies with similar glycosylation profiles. Other benefits of the defined feed system include lower raw material costs, the ability to optimize key nutrient concentrations, greater confidence in raw material quality, and the elimination of potential, hydrolysate‐associated endotoxin issues. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli

Secretory production of human granulocyte colony-stimulating factor fusion protein (hG-CSF) by fed-batch culture of Escherichia coli was investigated in both 2.5-L and 30-L fermentors. To develop a fed-batch culture condition that allows efficient production of hG-CSF, different feeding strategies including pH-stat, exponential and constant feeding were examined. Among these, the constant feeding strategy (0.228 g glucose2min^-1) and the exponential feeding that supports a low specific growth rate (µ=0.116 h^-1) resulted in the best hG-CSF production. Under these conditions, 4.4 g2L^-1 of hG-CSF was produced. The effect of induction time on the protein production was also investigated. For the fed-batch cultures carried out with the pH-stat and exponential feeding strategies, induction at higher cell density (late-exponential phase) resulted in more hG-CSF production compared with induction at lower cell density (early to mid-exponential phase). The constant feeding strategy that supported best hG-CSF production was applied to the scale-up production of hG-CSF in 30 L of fermentor. The maximum dry cell weight and hG-CSF concentration of 51.7 and 4.2 g2L^-1, respectively, was obtained.     

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellularviral components, and the heterologous product titers. The model describes the whole processes of viral infection and theeffect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave goodagreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infectionin batch and fed-batch culture were studied using the model.