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.     

脲对棒状链霉菌棒酸合成的影响

【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂,其合成过程中产生副产物脲,旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明:低浓度脲降低棒酸产量,当添加脲浓度达到20 mmol/L时,完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子,导致铵离子浓度及pH提高,因此,通过阻断棒状链霉菌脲酶活性,可以更准确地反映脲对棒酸合成的影响。结果发现,脲酶敲除株发酵液中脲大量积累,浓度高达10 mmol/L,但棒酸产量没有明显降低,说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量,可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用;另外,pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用,证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因,为进一步阐明棒酸合成调控机制提供了根据。     

Effective and stable porcine interferon-α production by Pichia pastoris fed-batch cultivation with multi-variables clustering and analysis

Efficient porcine interferon-α (pIFN-α) expression in high density recombinant Pichia pastoris cultivation was achieved in a 5 l bench-scaled bioreactor. The results indicated that a high and stable oxygen uptake rate (OUR) during induction phase was closely related with pIFN-α production efficiency. The multi-variables clustering and analysis results showed that the achievement of a high and stable OUR relied on a higher glycerol consumption rate during fed-batch culture phase and a moderate methanol level (around 10 g/l) during induction phase. In the high and stable OUR environments (200–300 mmol/l/h), the highest pIFN-α antiviral activity could reach a level of 6.7 × 10⁶ IU/ml, which was more than 10–300-folds higher than those obtained at lower OUR (80–200 mmol/l/h) using the same bioreactor and those obtained in shaking flasks. Clustering and analysis of the specific growth and glycerol consumption rates data during culture phase could detect the ill fermentation state at early stage, potentially provided a simple and effective fault alarming/diagnosis method for the achievement of stable pIFN-α production.     

Simultaneous production and decomposition of clavulanic acid during Streptomyces clavuligerus cultivations

 Clavulanic acid (CA) was produced by Streptomyces clavuligerus in medium containing glycerol and soy meal or soy meal extract. With regard to growth and CA productivity, the microorganism showed significant differences if solid soy meal as such or its extract were applied as the major nitrogen source. If the extract is used, growth and CA production take place simultaneously and in the stationary phase the CA concentration is stagnant or reduces. If soy meal is used, growth is threefold faster and CA is only generated in the stationary phase. In the case of using the soy meal extract, the decrease of the CA concentration is mainly due to decomposition or re-metabolisation of CA in the presence of the microorganism. This conclusion is supported by in vivo and in vitro data on CA decomposition. Received: 17 July 1995 / Received revision: 7 September 1995 / Accepted: 13 September 1995     

Oxygen supply controls the onset of pristinamycins production by Streptomyces pristinaespiralis in shaking flasks

Antibiotics are secondary metabolites, generally produced during stationary phase of growth under different nutritional and hydrodynamic stresses. However, the exact mechanisms of the induction of antibiotics production are still not clearly established. In a previous study, the induction of pristinamycins production by Streptomyces pristinaespiralis as well as product concentrations were correlated with power dissipation per unit of volume (P/V) in shaking flasks. In this study, detailed kinetics of growth, substrate consumption, oxygen transfer rate and pristinamycins production under varying P/V conditions have been obtained and analyzed. Our results showed that higher P/V resulted in a higher concentration of biomass and promoted an earlier nutrient limitation and ultimately an earlier induction of pristinamycins production. The maximal specific growth rate, specific oxygen consumption rate and specific consumption rate of glutamate increased with P/V while influence was less marked with specific consumption rate of glucose, arginine, ammonium ions and phosphate. When oxygen uptake rate (OUR) was limited by free-surface oxygen transfer, pristinamycins production was not detected despite the occurrence of nitrogen and/or phosphate sources limitation. The threshold value for OUR observed was around 25 mmol L(-1) h(-1). This suggested that a limitation in nitrogen and/or phosphate alone was not sufficient to induce pristinamycins production by S. pristinaespiralis pr11. To induce this production, the oxygen transfer had to be non-limiting.     

Clavulanic acid degradation in Streptomyces clavuligerus fed-batch cultivations

Clavulanic acid (CA) is an important antibiotic that is produced by Streptomyces clavuligerus. CA is unstable and product degradation has turned out to have a major impact on product titers in fed-batch cultivations. Three different types of experiments have been used to elucidate CA degradation under fed-batch cultivation conditions. First, the influence of individual medium compounds was examined. Second, degradation was monitored during the exponential growth phase in batch cultivations. Third, CA degradation was studied in the supernatant of samples taken during a fed-batch. In addition, data from six fed-batch cultivations were studied to derive information about CA degradation during the production phase. These cultivations were based on a mineral medium, containing glycerol, glutamate, ammonium, and phosphate as the main nutrients. The ammonium concentration had a large influence on the degradation rate constant. In addition, either changes in the substrate availability or high concentrations of ammonium or glycerol cause a major increase in the degradation rate constant. Finally, a linear and a fuzzy logic model were made to predict CA degradation rates in these fed-batches.     

Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale

Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures. Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags. Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature. For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum. Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L. In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium. It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium. Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages. Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

A new method for on-line measurement of the volumetric oxygen uptake rate in membrane aerated animal cell cultures

Oxygen is a key substrate in animal cell metabolism and its consumption is thus a parameter of great interest for bioprocess monitoring and control. A system for measuring it based on an oxygen balance on the liquid phase was developed. The use of a gas-permeable membrane offered the possibility to provide the required quantity of oxygen into the culture, while avoiding problems of foaming or shear stress generally linked to sparging. This aeration system allowed moreover to keep a known and constant k(L)a value through cultures up to 400 h. Oxygen uptake rate (OUR) was measured on-line with a very good accuracy of +/-5%, and the specific OUR for a CHO cell line was determined during batch (growth phase) and continuous culture as, respectively, equal to 2. 85x10(-13) and 2.54x10(-13) mol O(2) cell(-1) h(-1). It was also shown that OUR continuous monitoring gives actually more information about the metabolic state of the culture than the cell concentration itself, especially during transition phases like the end of the growth phase in a batch culture.     

Oxygen uptake rate regulation during cell growth phase for improving avermectin B<Subscript>1a</Subscript> batch fermentation on a pilot scale (2 m<Superscript>3</Superscript>)

Avermectin B_1a batch fermentation of Streptomyces avermitilis in a 2 m³ fermentor was investigated by oxygen uptake rate (OUR) regulation during cell growth phase. OUR was controlled by adjusting of aeration and agitation. Result showed that OUR strongly affected cell growth and antibiotics production. Avermectin B_1a biosynthesis could be effectively enhanced when OUR was stably regulated at an appropriate level in batch fermentation of S. avermitilis. Avermectin B_1a yield reached 5568 ± 111 mg/l by controlling maximal OUR between 15 and 20 mmol/l/h during cell growth phase, which was increased by 21.8% compared with the control (maximal OUR above 20 mmol/l/h). The stimulation effect on avermectin B_1a production could be attributed to the improved supply of propionic acid and acetic acid, the precursors of avermectin B_1a, in the cells. Hence, this OUR control method during cell growth phase may be a simple and applicable way to improve industrial production of avermectin.     

Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.     

Improving an on-line feeding strategy for fed-batch cultures of hybridoma cells by dialysis and `Nutrient-Split'-feeding

The concept of the feeding strategy was to minimise the formation of inhibiting metabolites and to increase the yield of monoclonal antibodies in fed-batch cultures of hybridoma cells by a balanced supply of substrates. A process control system based on fieldbus technology was used for monitoring and control. External program routines were implemented to control dissolved oxygen (DO) and to calculate the oxygen uptake rate (OUR) and cumulative oxygen consumption (COC) simultaneously. A concentrated feed solution was supplied according to the off-line estimated stoichiometric ratio between oxygen and glucose consumption (GC). Feeding was initiated automatically when the OUR decreased due to substrate limitation. The antibody concentration increased three-fold compared to the conventional batch culture by applying this strategy. But it was not possible to avoid inhibition by ammonia during the fed-batch phase. This was accomplished by the use of a dialysis membrane. Dialysis fed-batch cultures were performed in a membrane dialysis reactor with a `nutrient-split' feeding strategy, where concentrated medium is fed to the cells and toxic metabolites are removed into a buffer solution. This resulted in a ten-fold increase of the antibody concentration compared to the batch. Amino acid concentrations were analysed to identify limiting conditions during the cultivation and to analyse the performance of the nutrient supply in the fed-batch and dialysis fed-batch.     

A comparison of the clavam biosynthetic gene clusters in Streptomyces antibioticus Tü1718 and Streptomyces clavuligerus

The production of clavam metabolites has been studied previously in Streptomyces clavuligerus , a species that produces clavulanic acid as well as 4 other clavam compounds, but the late steps of the pathway leading to the specific end products are unclear. The present study compared the clavam biosynthetic gene cluster in Streptomyces antibioticus , chosen because it produces only 2 clavam metabolites and no clavulanic acid, with that of S.?clavuligerus. A cosmid library of S.?antibioticus genomic DNA was screened with a clavaminate synthase-specific probe based on the corresponding genes from S. clavuligerus, and 1 of the hybridizing cosmids was sequenced in full. A clavam gene cluster was identified that shows similarities to that of S.?clavuligerus but also contains a number of novel genes. Knock-out mutation of the clavaminate synthase gene abolished clavam production in S.?antibioticus, confirming the identity of the gene cluster. Knock-out mutation of a novel gene encoding an apparent oxidoreductase also abolished clavam production. A potential clavam biosynthetic pathway consistent with the genes in the cluster and the metabolites produced by S. antibioticus, and correspondingly different from that of S.?clavuligerus, is proposed.     

Computer-aided on-line monitoring of physiological variables in suspended cell cultures of Perilla frutescens in a bioreactor

A computer-aided on-line real-time monitoring system for plant cell bioprocesses was established and applied to the cultivation of Perilla frutescens plant cells in a bioreactor. This system calculated several informative process variables which were useful for the identification of the physiological states of the plant cells during cultivation. Some variables, such as the respiratory quotient (RQ), pH, and specific carbon dioxide evolution rate (SCER), could be used for the identification of the growing phase of cell cultures. The results also suggest that the oxygen uptake rate (OUR) and the specific OUR (SOUR) may depend on the accumulation of anthocyanin (a secondary metabolite) in P. frutescens cell cultures.     

Biosynthesis of citric and isocitric acids from ethanol by mutant Yarrowia lipolytica N 1 under continuous cultivation

The effect of ethanol, zinc, and iron (Fe²⁺ and Fe³⁺) concentration and of oxygen supply on cell growth and the production of citric acid (CA) and isocitric acid (ICA) from ethanol by mutant Yarrowia lipolytica N 1 was studied under continuous cultivation. The following peculiarities of Y. lipolytica metabolism were found: (1) intensive CA production occurred under yeast growth limitation by nitrogen; (2) inhibition of yeast growth by ethanol was accompanied by significant alterations in fatty acid composition of lipids; (3) the production of CA and ICA from ethanol required high concentrations of zinc and iron ions; (4) the intracellular iron concentration determined whether CA or ICA was predominantly formed; (5) the cell's requirement for oxygen depended on the intracellular iron concentration. The events taking place in the production of CA and ICA were evaluated through the activities of enzyme systems involved in the metabolism of ethanol and CA in this strain. Electronic Publication     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures

Respiration rates in Spodoptera frugiperda (Sf-9) cell bioreactor cultures were successfully measured on-line using two methods: The O(2) uptake rate (OUR) was determined using gas phase pO(2) values imposed by a dissolved oxygen controller and the CO(2) evolution rate (CER) was measured using an infrared detector. The measurement methods were accurate, reliable, and relatively inexpensive. The CER was routinely determined in bioreactor cultures used for the production of several recombinant proteins. Simple linear relationships between viable cell densities and both OUR and CER in exponentially growing cultures were used to predict viable cell density. Respiration measurements were also used to follow the progress of baculoviral infections in Sf-9 cultures. Infection led to increases in volumetric and per-cell respiration rates. The relationships between respiration and several other culture parameters, including viable cell density, cell protein, cell volume, glucose consumption, lactate production, viral titer, and recombinant beta-galactosidase accumulation, were examined. The extent of the increase in CER following infection and the time postinfection at which maximum CER was attained were negatively correlated with the multiplicity of infection (MOI) at multiplicities below the level required to infect all the cells in a culture. Delays in the respiration peak related to the MOI employed were correlated with delays in the peak in recombinant protein accumulation. DO levels in the range 5-100% did not exert any major effects on viable cell densities, CER, or product titer in cultures infected with a baculovirus expressing recombinant beta-galactosidase. (c) 1996 John Wiley & Sons, Inc.     

A comparison of different methods to determine the end of exponential growth in CHO cell cultures for optimization of scale-up

Maximizing cell growth rate and cell yield are among the most important features of a successful mammalian cell culture production process. To minimize time and resources needed to scale up cell mass it is important to maintain the cultures in exponential growth at every scale. Here we report results comparing viable cell counts, packed cell volume, intracellular nucleotide ratios, cell cycle analysis, and on-line oxygen uptake rates (OUR) and optical density for the determination of the end of exponential growth to optimize transfer times during scale-up of CHO cell cultures. Viable cell concentration, packed cell volume, and relative abundance of cells in S-phase were not very reliable at determining the end of exponential growth during the process. In contrast, on-line determination of OUR and off-line determination of intracellular nucleotide ratios (U-ratio) were very sensitive to changes in growth rate, enabling clear determination of the end of exponential growth within a short time. Although on-line OUR was found to be the most convenient and fastest method, it is restricted to instrumented and continuously monitored cultures. In contrast the nucleotide method can be applied with any culture scale and condition but needs the availability of an operator running an HPLC system and takes about an hour from sampling to result. Optical density showed an inflection along with OUR and U-ratio but was less sensitive in determining the end of exponential growth.     

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures

Shake-flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10-fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.