Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.     

Patterns of RNA synthesis in batch and cyclic fed-batch cultures of tylosin-producingStreptomyces fradiae

Ribonucleic acid (RNA) synthesis was studied in batch and cyclic fed-batch cultures of tylosin-producingStreptomyces fradiae. In batch culture, net RNA synthesis increased with increasing growth rate then decreased as the fermentation progressed. A cyclic response pattern of net RNA synthesis was observed when glucose and glutamate solutions were cyclically fed to the batch culture; RNA synthesis rate increased sharply to a maximum level following the feeding cycles then decreased to lower values between the maxima. Data obtained from an extended cyclic fed-batch culture showed that cyclic feeding of the key nutrients allowed RNA and tylosin syntheses to continue during a period when such activities had ceased in the control batch culture.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Efficient conversion of lactic acid to butanol with pH-stat continuous lactic acid and glucose feeding method by Clostridium saccharoperbutylacetonicum

In order to achieve high butanol production by Clostridium saccharoperbutylacetonicum N1-4, the effect of lactic acid on acetone–butanol–ethanol fermentation and several fed-batch cultures in which lactic acid is fed have been investigated. When a medium containing 20 g/l glucose was supplemented with 5 g/l of closely racemic lactic acid, both the concentration and yield of butanol increased; however, supplementation with more than 10 g/l lactic acid did not increase the butanol concentration. It was found that when fed a mixture of lactic acid and glucose, the final concentration of butanol produced by a fed-batch culture was greater than that produced by a batch culture. In addition, a pH-controlled fed-batch culture resulted in not only acceleration of lactic acid consumption but also a further increase in butanol production. Finally, we obtained 15.5 g/l butanol at a production rate of 1.76 g/l/h using a fed-batch culture with a pH-stat continuous lactic acid and glucose feeding method. To confirm whether lactic acid was converted to butanol by the N1-4 strain, we performed gas chromatography–mass spectroscopy (GC-MS) analysis of butanol produced by a batch culture during fermentation in a medium containing [1,2,3-¹³C₃] lactic acid as the initial substrate. The results of the GC-MS analysis confirmed the bioconversion of lactic acid to butanol.     

Optimization of erythritol production by Candida magnoliae in fed-batch culture

A two-stage fed-batch process was designed to enhance erythritol productivity by the mutant strain of Candida magnoliae. The first stage (or growth stage) was performed in the fed-batch mode where the growth medium was fed when the pH of the culture broth dropped below 4.5. The second stage (or production stage) was started with addition of glucose powder into the culture broth when the cell mass reached about 75 g dry cell weight l⁻¹. When the initial glucose concentration was adjusted to 400 g l⁻¹ in the production stage, 2.8 g l⁻¹ h⁻¹ of overall erythritol productivity and 41% of erythritol conversion yield were achieved, which represented a fivefold increase in erythritol productivity compared with the simple batch fermentation process. A high glucose concentration in the production phase resulted in formation of organic acids including citrate and butyrate. An increase in dissolved oxygen level caused formation of gluconic acid instead of citric acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 100–103. Received 25 February 2000/ Accepted in revised form 08 June 2000     

Increased CHO cell fed-batch monoclonal antibody production using the autophagy inhibitor 3-MA or gradually increasing osmolality

Modulating autophagy provides a new method to increase CHO cell protein production. A fed-batch protocol using the autophagy inhibitor 3-methyl adenine (3-MA), developed for a tissue-plasminogen activator (t-PA) expressing DHFR based CHO cell line, was successfully adapted to a monoclonal antibody (MAb) expressing CHOK1-SV based CHO cell line. By optimizing the timing and dose of 3-MA treatment, the cell-specific productivity was increased 4-fold, resulting in 2-fold increased total MAb production. The positive effect of the 3-MA treatment appeared to be reduced when the amino acid feed concentration was increased 5-fold. Further investigation revealed that by slowly increasing osmolality up to ∼450 mOsm/kg, both the cell-specific productivity and the total MAb almost doubled. This effect was replicated with a DUXB-based CHO cell line expressing a human–llama chimeric antibody. The positive effect of gradually increasing osmolality was then combined with the positive effects of the 3-MA treatment, however their combined effect were not additive. Thus, either increased osmolality or 3-MA treatment were equally effective in increasing MAb-CHO cell fed-batch production on the cell lines tested. Analysis of protein glycosylation showed that both of these fed-batch modifications did not substantially influence the overall glycan profiles of the MAb product.     

Production of tissue plasminogen activator (t-PA) in Aspergillus niger

A protease-deficient strain of Aspergillus niger has been used as a host for the production of human tissue plasminogen activator (t-PA). In defined medium, up to 0.07 mg t-PA (g biomass)(-1) was produced in batch and fed-batch cultures and production was increased two- to threefold in two-phase batch cultures in which additional glucose was provided as a single pulse at the end of the first batch growth phase. Production was increased [up to 1.9 mg t-PA (g biomass)(-1)] by the addition of soy peptone to the defined medium. The rate of t-PA production in batch cultures supplemented with soy peptone (0.2 to 0.6 mg t-PA L(-1) h(-1)) was comparable to rates observed previously in high-producing mammalian or insect cell cultures. In glucose-limited chemostat culture supplemented with soy peptone, t-PA was produced at a rate of 0.7 mg t-PA L(-1) h(-1). Expression of t-PA in A. niger resulted in increased expression of genes (bipA, pdiA, and cypB) involved in the unfolded protein response (UPR). However, when cypB was overexpressed in a t-PA-producing strain, t-PA production was not increased. The t-PA produced in A. niger was cleaved into two chains of similar molecular weight to two-chain human melanoma t-PA. The two chains appeared to be stable for at least 16 h in culture supernatant of the host strain. However, in general, <1% of the t-PA produced in A. niger was active, and active t-PA disappeared from the culture supernatant during the stationary phase of batch cultures, suggesting that the two-chain t-PA may have been incorrectly processed or that initial proteolytic cleavage occurred within the proteolytic domain of the protein. Total t-PA (detected by enzyme-linked immunoassay) also eventually disappeared from culture supernatants, confirming significant extracellular proteolytic activity, even though the host strain was protease-deficient.     

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 10⁷ cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.     

Heuristic optimization of antibody production by Chinese hamster ovary cells

Large-scale production of monoclonal antibodies necessitates the development of a commercially viable process using the appropriate bioreactors, culture medium, and optimal feeding strategies. In the development of feeding strategies for higher antibody titers it is critical to assess the effects of limiting substrates on cell culture longevity and antibody production. In this study, glucose and L-glutamine were identified as limiting substrates and their effects on culture longevity and antibody production were evaluated in small-scale experiments. The results suggested that an optimal feeding strategy should account for the osmolality profile of the culture. The heuristic approach taken to optimize the antibody production showed that the fed-batch cultivation is superior to batch culture and maintaining low osmolality during growth phase increases cumulative viable cell density and thus leads to higher final antibody titer.     

Application of hypoosmolar medium to fed-batch culture of hybridoma cells for improvement of culture longevity

Cell culture longevity in fed-batch culture of hybridomas is often limited by elevated medium osmolality caused by repeated nutrient feeding. Shotwise feeding of 10x Dulbecco's modified Eagle's medium (DMEM) concentrates elevated the osmolality of medium up to 540 mOsm/kg at the end of fed-batch culture of S3H5/gamma2bA2 hybridoma which is known to be lethal to most hybridomas. S3H5/gamma2bA2 hybridoma has been shown to grow without significant growth depression at 219 mOsm/kg in DMEM supplemented with 10% fetal bovine serum. To improve culture longevity in fed-batch cultures of S3H5/gamma2bA2 hybridoma, a hypoosmolar medium (223 mOsm/kg) was used as an initial basal medium. The use of hypoosmolar medium delayed the onset of severe cell death resulting from elevated osmolality and allowed one more addition of 10x DMEM concentrates to the culture. As a result, a final antibody concentration obtained was 121.5 microg/mL which is approximately 1.5-fold higher compared to fed-batch culture using a standard medium (335 mOsm/kg). When compared to batch culture, a more than 5-fold increase in the final antibody concentration was achieved. Taken together, the use of hypoosmolar medium as an initial medium in fed-batch culture improved culture longevity of S3H5/gamma2bA2 hybridoma, resulting in a substantial increase in the final antibody concentration.     

批次和流加培养中国仓鼠卵巢工程细胞的细胞周期相关基因转录谱分析

以表达人重组尿激酶原中国仓鼠卵巢 (CHO) 工程细胞系11G-S为研究对象,运用基因芯片技术比较了CHO工程细胞在批次及流加培养不同生长阶段基因表达水平的差异,在此基础上采用Genmapp软件,同时结合已知的细胞周期信号通路图,着重分析了批次及流加培养CHO工程细胞的细胞周期调控基因转录谱差异。在基因芯片涉及的19 191个目标基因中,批次和流加培养不同生长阶段CHO工程细胞的下调表达的基因数量多于上调表达基因数目;两种培养模式下的基因差异表达有着明显的不同,尤其是在细胞生长的衰退期,流加培养CHO工程细胞中下调表达的基因数量明显多于批次培养。有关调控细胞周期关键基因的转录谱分析表明,CHO工程细胞主要是通过下调表达CDKs、Cyclin及CKI家族中的Cdk6、Cdk2、Cdc2a、Ccne1、Ccne2基因及上调表达Smad4基因,来达到调控细胞增殖及维持自身活力的目的。     

Bacteriocin production with Lactobacillus amylovorus DCE 471 is improved and stabilized by fed-batch fermentation

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.     

Feed development for fed‐batch CHO production process by semisteady state analysis

Semisteady state cultures are useful for studying cell physiology and facilitating media development. Two semisteady states with a viable cell density of 5.5 million cells/mL were obtained in CHO cell cultures and compared with a fed‐batch mode control. In the first semisteady state, the culture was maintained at 5 mM glucose and 0.5 mM glutamine. The second condition had threefold higher concentrations of both nutrients, which led to a 10% increase in lactate production, a 78% increase in ammonia production, and a 30% reduction in cell growth rate. The differences between the two semisteady states indicate that maintaining relatively low levels of glucose and glutamine can reduce the production of lactate and ammonia. Specific amino acid production and consumption indicated further metabolic differences between the two semisteady states and fed‐batch mode. The results from this experiment shed light in the feeding strategy for a fed‐batch process and feed medium enhancement. The fed‐batch process utilizes a feeding strategy whereby the feed added was based on glucose levels in the bioreactor. To evaluate if a fixed feed strategy would improve robustness and process consistency, two alternative feeding strategies were implemented. A constant volume feed of 30% or 40% of the initial culture volume fed over the course of cell culture was evaluated. The results indicate that a constant volumetric‐based feed can be more beneficial than a glucose‐based feeding strategy. This study demonstrated the applicability of analyzing CHO cultures in semisteady state for feed enhancement and continuous process improvement. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures

The influence of Bcl-2 expression on the suppression of apoptosis during the cultivation of an NS0 cell line expressing a chimeric antibody was investigated. Following selection of transfectants in medium containing G418, Western analysis revealed evidence of some up-regulation of endogenous Bcl-2 expression even in the control vector transfectants. Cultivation of the two cell lines in suspension batch cultures clearly demonstrated the enhanced robustness of the bcl-2 vector transfected cells. Suppression of apoptosis resulted in an approximately 20% increase in maximum viable cell number, and a doubling in culture duration compared to the control transfected cells. However, despite the significant affect on viability, Bcl-2 expression did not result in an increase in final antibody titre in comparison with the control cell line. Exposure of cells to various nutrient limited conditions further emphasised the influence of Bcl-2 on cell survival. After 3 days of exposure to serum, glucose, glutamate and asparagine deprivation, the viable cell number and viability were significantly higher in the bcl-2 transfected cell line. When control cells were deprived of all amino acids, there was a complete loss of viability and viable cell number within 3 days. By contrast, the bcl-2 transfected cell line retained greater than 75% of the initial viable cell number and about 70% viability. In response to exposure to 8 mM thymidine (a cytostatic agent) the control cell line underwent complete loss of viability and viable cell number after 6 days. This compared with 18 days for complete loss of viability in the bcl-2 transfected cell line. As under batch culture conditions, there was no difference between the two cell lines in final antibody titre, which indicated that MAb synthesis is limited by nutrient availability during the latter stages of culture in both cases. When fed batch cultures were carried out using a concentrated essential amino acid feed, the bcl-2 cell line exhibited a 60% increase in maximum viable cell number and a 50% increase in culture duration, when compared to the control cell line. Moreover, the bcl-2 cell line exhibited a greater than 40% increase in maximum antibody titre.     

Fed-batch fermentation for production of nitrile hydratase byRhodococcus rhodochrous M33

To enhance the productivity and activity of nitrile hydratase inRhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.     

The effect of amino acid supplementation in an industrial Chinese Hamster Ovary process

The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.     

Defined protein and animal component-free NS0 fed-batch culture

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

Fed-batch culture optimization of a growth-associated hybridoma cell line in chemically defined protein-free media

An investigation was made to study the processes of fed-batch cultures of a hybridoma cell line in chemically defined protein-free media. First of all, a strong growth-associated pattern was correlated between the production of MAb and growth of cells through the kinetic studies of batch cultures, suggesting the potential effectiveness of extending the duration of exponential growth in the improvement of MAb titers. Second, compositions of amino acids in the feeding solution were balanced stepwisely according to their stoichiometrical correlations with glucose uptake in batch and fed-batch cultures. Moreover, a limiting factor screening revealed the constitutive nature of Ca²⁺ and Mg²⁺ for cell growth, and the importance of their feeding in fed-batch cultures. Finally, a fed-batch process was executed with a glucose uptake coupled feeding of balanced amino acids together with groups of nutrients and a feeding of CaCl₂ and MgCl₂ concentrate. The duration of exponential cell growth was extended from 70 h in batch culture and 98 h in fed-batch culture without Ca²⁺/Mg²⁺ feeding to 117 h with Ca²⁺/Mg²⁺ feeding. As a result of the prolonged exponential cell growth, the viable and total cell densities reached 7.04 × 10⁶ and 9.12 × 10⁶ cells ml⁻¹, respectively. The maximal MAb concentration achieved was increased to approximately eight times of that in serum supplemented batch culture.     

Optimisation of L-lysine production byCorynebacterium sp in fed-batch cultures

Summary The influence of initial concentration of glucose from 60 to 233 g/l on the production of L-lysine byCorynebacterium sp was studied first in batch culture. The maximum conversion rate into L-lysine was obtained at 165 g/l and the best specific production rate for L-lysine was observed at 65 g/l of glucose. In fed-batch fermentations, better conversion and the specific production rates were obtained. Maintaining of a high glucose concentration in the fed-batch technique allowed a 54% increase of the L-lysine production compared to the batch culture.