Fed-batch sorbose fermentation using pulse and multiple feeding strategies for productivity improvement

Microbial oxidation of D-sorbitol tol-sorbose byAcetobacter suboxydans is of commercial importance since it is the only biochemical process in vitamin C synthesis. The main bottleneck in the batch oxidation of sorbitol to sorbose is that the process is severely inhibited by sorbitol. Suitable fed-batch fermentation designs can eliminate the inherent substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted by using two nutrient feeding strategies. For fed-batch fermentation with pulse feeding highly concentrated sorbitol (600 g/L) along with other nutrients were fed intermittently in four pulses of 0.5 liter in response to the increased DO signal. The fed-batch fermentation was over in 24 h with a sorbose productivity of 13.40 g/L/h and a final sorbose concentration of 320.48 g/L. On the other hand, in fed-batch fermentation with multiple feeds, two pulse feeds of 0.5 liter nutrient medium containing 600 g/L sorbitol was followed by the addition of 1.5 liter nutrient medium containing 600 g/L sorbitol at a constant feed rate of 0.36 L/h till the full working capacity of the reactor. The fermentation was completed in 24 h with an enhanced sorbose productivity of 15.09 g/L/h and a sorbose concentration of 332.60 g/L. The sorbose concentration and productivity obtained by multiple feeding of nutrients was found to be higher than that obtained by pulse feeding and was therefore a better strategy for fed-batch sorbose fermentation.     

Fed-batch cultivation of Acetobacter suboxydans for the microbial oxidation of D-sorbitol to L-sorbose

Microbial oxidation of D-sorbitol to L-sorbose is commercially important since it is the only biochemical process in Vitamin-C manufacture. The main bottleneck in the batch oxidation of D-sorbitol is that the process is severely inhibited by sorbitol. By conducting fed-batch fermentation, the inherent substrate inhibition present in batch fermentation can be eliminated. Batch fermentation with an initial sorbitol concentration of 200 g l^у featured a productivity of 14.2 g l^у h^у and a final sorbose concentration of 200 g l^у. Fed-batch fermentation conducted by feeding nutrients containing 600 g l^у of sorbitol at a constant feed rate of 0.36 l h^у yielded a productivity of 17.7 g l^у h^у and a final sorbose concentration of 320 g l^у.     

Productivity enhancement in l-sorbose fermentation using oxygen vector

Sorbose, an intermediate of Vitamin-C is produced by biological oxidation of sorbitol by Acetobacter suboxydans. The utility of oxygen vector (N-hexadecane) in enhancing the sorbose accumulation and its productivity was examined for sorbitol to sorbose bioconversion process. Shake flask fermentations were conducted using 1% to 6% (v/v) N-hexadecane. Higher sorbose production was observed in shake flask containing 1-4% N-hexadecane as compared to shake flasks without N-hexadecane. A maximum of 82.12 kgm(-)(3) sorbose accumulated in 24 h by the addition of 4% N-hexadecane as against 64.83 kgm(-)(3) sorbose without the addition of N-hexadecane. However, the concentration of sorbose produced in the same time decreased when 5% and 6% N-hexadecane were used. Batch sorbose fermentation conducted using 4% N-hexadecane demonstrated decrease in the process time from 14 h to 12 h. The productivity increased from 14.3 kgm(-)(3) h(-)(1) to 16.7 kgm(-)(3) h(-)(1) when 4% N-hexadecane was used. Fed-batch fermentation using 4% N-hexadecane was over in 20 h with a productivity of 15.9 kgm(-)(3) h(-)(1), and a sorbose accumulation of 311.68 kgm(-)(3) was obtained. The same fed-batch fermentation finished in 25 h when no N-hexadecane was used. A productivity of 12.6 kgm(-)(3) h(-)(1) and a final sorbose accumulation of 316 kgm(-)(3) were obtained. The increase in productivity and lower process time were achieved by the addition of N-hexadecane to the fermentation medium.     

Novel spiking methods developed for anion exchange chromatography operating in a continuous process

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a nonhomogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in-line spiking. This is challenging to be implemented at the contract research organization performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow-through anion exchange chromatography step in a monoclonal antibody (mAb) manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of anion exchange chromatography unit operating in continuous processing.     

Fed-batch sorbitol to sorbose fermentation by A. suboxydans

Batch kinetics for sorbitol to sorbose bioconversion was studied at 20% sorbitol concentration. The culture featured 90% conversion of sorbitol to sorbose in 20 hours. Increasing the initial substrate concentration in the bioreactor decreased the culture specific growth rate. At 40% initial sorbitol concentration no culture growth was observed. The batch kinetics and substrate inhibition studies were used to develop the Mathematical Model of the system. The model parameters were identified using the original batch kinetic data (S _o =20%). The developed mathematical model was adopted to fed-batch cultivation with the exponential nutrient feeding. The fed-batch model was simulated and implemented experimentally. No substrate inhibition was observed in the fed-batch mode and it provided an overall productivity of 12.6?g/l-h. The fed-batch model suitably described the experimentally observed results. The model is ready for further optimization studies.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Influence of Product Addition on the Oxidation of D-Sorbitol to L-Sorbose by the Strain Acetobacter suboxydans

The kinetics of the D-sorbitol to L-sorbose biotransformation catalysed by the strain Acetobacter suboxydans is studied. The product inhibits the bacterial growth but the transformation is an autocatalytic process. However, higher initial concentrations of sorbose lead to a considerable decrease of the rate constant of the reaction, although the autocatalytic process takes place too. The addition of sorbose in the exponential phase of bacterial growth, or in the stationary phase, leads to a considerable shortening of the process duration, compared to the traditional fermentation.

The rate constants calculated from the kinetic curves are dependent on the initial dry substances concentration and there is a correlation between these levels and the biomass concentration in the stationary phase.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Lower fat deposition and energy utilization of growing rats fed diets containing sorbose

1. Growing rats were fed diets containing graded levels (0, 100, 200 and 300 g/kg diet) of sorbose for 6 weeks. Protein, fat and energy deposition were determined by carcass analysis. 2. The values for growth, serum insulin level, digestible energy (DE), metabolizable energy (ME) and fat and energy deposition declined with the increment of dietary sorbose. 3. The efficiency of protein utilization (protein retained/protein consumed) was hardly affected by dietary sorbose. The DE and ME of sorbose per se was calculated as 14.09 and 12.35 kJ/g respectively. The efficiency of energy utilization (energy retained/ME intake) decreased with the increase of dietary sorbose, although sorbose had an ME. 4. The relative weights of gastro-intestinal tract and liver were positively associated with dietary sorbose level, although the reverse was true for the amount of stomach content, being heavier with higher dietary sorbose. 5. It is suggested that dietary sorbose, as a sweetener as well as a bulky agent, seems to be a suitable sugar for the obese and diabetic with special reference to lower body fat and energy deposition without reducing protein utilization.     

新组合菌系SCB329-SCB933利用L-山梨糖发酵产生维生素C前体2-酮基-L-古龙酸的研究Ⅲ.发酵过程的特征和条件控制

在分析了新组合菌系ＳＣＢ３２９－ＳＣＢ９３３发酵过程特征的基础上,对流加发酵工艺中的种子培养、ｐＨ、溶氧的控制,以及发酵液初始培养基中的Ｌ－山梨糖浓度和流加起始点进行了优化,获得了比分批发酵更为满意的结果：发酵最终总糖达１３％（ｗ／ｖ）左右,发酵周期４０～５０ｈ,产２－酮基－Ｌ－古龙酸达１１５－１３０ｍｇ／ｍｌ,克分子转化率达８８ｍｏｌ％左右。     

Life cycle assessment of vitamin D3 synthesis: from batch to photo-high p,T

Purpose

Novel process windows allow the development of faster, flexible, and greener processes. Therefore, novel process windows were applied to develop a greener process for the synthesis of vitamin D₃. In this study the environmental impacts of several batch pathways to obtain vitamin D₃ are benchmarked against the continuous microflow process, where novel process windows such as high temperature and pressure were applied. To evaluate the environmental impact of these processes, life cycle assessments were conducted.

Methods

A new process concept was developed to optimize and simplify the synthesis of crystalline vitamin D₃. This process was conducted in microflow by combining UV photoirradiation and high-p,T (photo-high-p,T) processing. Microreactors allow a high photon flux and enable the harsh conditions, respectively. The process was coupled with an integrated continuous crystallization, and its feasibility has been proven and reported before. The potential environmental impacts were assessed from a cradle-to-gate perspective. Both processes, continuous and batch, were modeled in Aspen Plus using foreground data from the experimental continuous setup, and background data from different patents. The assessment was performed in the software Umberto NXL LCA using the ReCiPe Midpoint 2008 method.

Results and discussion

The continuous process has a significantly lower environmental impact than the batch processes. This lower impact is largely due to the fact that fewer amounts of material, particularly solvents, are used. Moreover, the continuous process is faster and has fewer steps, i.e., process-simplified. Among the industrial processes, the synthesis conducted in isopropanol has the lowest environmental impact, although, even in this case, the impact is between 20 and 30 times higher—depending on the conditions—compared with the continuous process. When the batch process is conducted in benzene, the worst environmental impact is obtained. Finally, recycle of the solvent for the best batch case was assessed. This improved the batch process to make it comparable with the continuous process.

Conclusions

The continuous production of vitamin D₃ leads to an interesting alternative to the industrial process. Continuous manufacturing of vitamin D₃ is faster, requires fewer steps, and uses less solvents compared with the industrial synthesis. However, although the environmental impact of this continuous process is already lower than that of the batch processes, the continuous process can still benefit from further optimization, particularly the introduction of a recycle loops for the solvents methyl tert-butyl ether and acetonitrile.

     

Comparison of specific rates of hybridoma growth and metabolism in batch and continuous cultures

For the mouse hybridoma cell line VO 208, kinetics of growth, consumption of glucose and glutamine, and production of lactate, ammonia and antibodies were compared in batch and continuous cultures. At a given specific growth rate, different metabolic activities were observed: a 40% lower glucose and glutamine consumption rate, but a 70% higher antibody production rate in continuous than in batch culture. Much higher metabolic rates were also measured during the initial lag phase of the batch culture. When representing the variation of the specific antibody production rate as a function of the specific growth rate, there was a positive association between growth and antibody production in the batch culture, but a negative association during the transient phase of the continuous culture. The kinetic differences between cellular metabolism in batch and continuous cultures may be result of modifications in the physiology and metabolism of cells which, in continuous cultures, were extensively exposed to glucose limitations.Institut National Polytechnique de Lorraine, ENSAIA BP 172, 2 avenue de la forêt de Haye, 54505, Vandoeuvre Cedex France     

维生素C二步发酵中巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产酸的影响

通过测定氧化葡萄糖酸杆菌转化L－山梨糖中成ZKGA的细胞酶活性、摇瓶发酵及中长变化,研究了Vc:步发酵中巨大茅孢杆菌对氧化葡萄糖酸杆菌生长和产酸作用的影响。结果显示：巨大芽孢杆菌胞外液和胞内液均可促进氧化葡萄糖酸杆菌的增殖,主要表现为缩短其中长周期中的延迟期;巨大芽孢杆菌通过所产生的部分生物活性物质增强氧化葡萄糖酸杆菌产酸的细胞酶活性,促进氧化葡萄糖酸杆菌转化L一山梨糖生成2KGA.     

Continuous fractionation of human plasma proteins by precipitation from the suspension of the recycling stream

The conventional cold-ethanol batch fractionation method of human plasma is converted to an automatically controlled continuous fractionation process. The selected protein fractions are precipitated by mixing in the recycled product stream of the suspension. Compared to the batch process, the continuous fractionation process generates less coprecipitation and less spontaneous nucleation, allowing efficient centrifugation of precipitates, and the yield and purity of albumin in the final fraction is significantly increased.     

Process modelling and simulation of a transketolase mediated reaction: Analysis of alternative modes of operation

A previously proposed model of a transketolase catalysed carbon–carbon bond formation reaction condensing β-hydroxypyruvate and glycolaldehyde to synthesise l-erythrulose has been extended to describe various modes of operation as an alternative to a batch process. The alternative continuous and fed-batch operations, with each substrate being fed separately and together have been analysed using the extended model. The analysis was carried out simulating the product concentration after a given time under defined process conditions. Comparison of product concentration and yield on catalyst as two process metrics were used to identify promising cases for further process development.     

Economic assessment of continuous processing for manufacturing of biotherapeutics

Continuous processing offers a promising approach to revolutionize biotherapeutics manufacturing as reflected in recent years. The current study offers a comparative economic assessment of batch and continuous processing for the production of biotherapeutic products. Granulocyte-colony stimulating factor (GCSF), a protein expressed in E. coli, and an IgG1 monoclonal antibody, were chosen as representatives of microbial and mammalian derived products for this assessment. Economic indicators—cost of goods (COGs), net present value (NPV), and payback time have been estimated for the assessment. For the case of GCSF, conversion from batch to integrated continuous manufacturing induced a $COGs/g reduction of 83% and 73% at clinical and commercial scales, respectively. For the case of mAb therapeutic, a 68% and 35% reduction in $COGs/g on translation from batch to continuous process was projected for clinical and commercial scales, respectively. Upstream mAb titer was also found to have a significant impact on the process economics. With increasing mAb titer, the $COG/g decreases in both operating modes. With titer increasing from 2 to 8 g/L, the $COG/g of batch process was reduced by 53%, and that of the continuous process was reduced by 63%. Cost savings in both the cases were attributed to increased productivity, efficient equipment and facility utilization, smaller facility footprint, and reduction in utilization of consumables like resin media and buffers actualized by the continuous processing platform. The current study quantifies the economic benefits associated with continuous processing and highlights its potential in reducing the manufacturing cost of biotherapeutics.     

Fermentation Kinetics and Continuous Process of L-Asparaginase Production

For the purpose of obtaining L-asparaginase in quantities from Erwinia aroideae, cell growth and enzyme formation were investigated in both batch and continuous fermentation. Using yeast extract as a growth-limiting substrate, the relationship between specific growth rate and substrate concentration was found to fit the Monod equation. The optimum temperature for enzyme production was 24 C, although cell growth was higher at 28 C. The enzyme yield reached its maximum of 4 IU/ml during the negative acceleration growth phase which occurs just prior to stationary growth. Compared to batch fermentations, the continuous fermentation process gave a lower enzyme yield except when the fermentation was conducted at a dilution rate of 0.1 hr(-1). The graphical method frequently used for prediction of continuous fermentation does not apply to L-asparaginase production by E. aroideae. The optimum temperature for enzyme production in continuous process was 24 C, which was the same as in batch process. Increasing the temperature from 24 to 28 C resulted in a 20% loss of enzyme yield.     

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo‐enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single‐phase concept was applied in a fed‐batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single‐phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed‐batch process. Employing the fed‐batch process reaction rates up to 36 mmol h^?1 per gram_cat, and a maximum yield of 96 % was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long‐term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55 h without any loss of activity and with a space‐time yield of 154 mmol L^?1 h^?1, which is three times higher than in the fed‐batch process. The higher catalytic productivity compared to the fed‐batch process (34 vs. 18 g_Prod g^?1_cat) shows the increased enzyme stability in the continuous process.     

Regulatory function of sorbose-resistance gene C in sugar transport of Neurospora

Summary A sorbose-resistant double mutant sor ^r A-10/sor ^r C-17 produces larger colonies in sorbose containing test-medium than the respective single mutants; wildtype colonies remain very small. Resistance of the single mutants was shown to be connected with a decreased rate of sorbose-uptake into their conidia; however, sorbose uptake of the double mutant had not been measured. To check, whether the improved performance of the double mutant on test medium is correlated with a further decrease of sorbose uptake in this strain, studies on the uptake of fructose, sorbose and deoxyglucose by ungerminated conidia of the two single mutants, the double mutant and the wildtype were conducted, using C¹⁴-marked sugars, the millipore filter technique, and conidia either untreated or pretreated with 1% sorbose for 4 hours.If sorbose uptake is referred to that of fructose as basis of calculations, as in the earlier studies, the sorbose uptake by cells of the double mutant is smaller than that of both single mutants for conidia not pretreated with sorbose (Fig. 7a). However, for conidia pretreated with sorbose, this correlation does not hold. Rather, cells of the double mutant take up less sorbose than those of the C-mutant, but as much or slightly more than those of the A-mutant (Fig. 7b). If sorbose uptake is referred to that of deoxyglucose for an independent point of reference, cells of the double mutant take up less sorbose than those of the C-mutant, but much more than those of the A-mutant. This holds for untreated and sorbose pretreated cells (Fig. 5 a and b). These data rule out a correlation between colony size and transport defect for at least one of the strains used here, i.e. the C-mutant.The following data suggest a new interpretation: In contrast to the earlier findings with germinated conidia, ungerminated untreated cells of the C-mutant take up much more fructose and sorbose than those of the wildtype (Fig. 3 a and 1a). The uptake of fructose by cells of the C-mutant can not be improved by sorbose pretreatement (Fig. 3 b), but in both wildtype and A-mutant it is increased (Figs. 1b and 2b). Uptake of deoxyglucose was nearly equal for all three strains either untreated or pretreated. Untreated cells of the A-mutant take up as much sorbose as those of the wildtype (Figs. 2 a and 1 a). On pretreatment their sorbose uptake remains nearly constant (Figs. 2b), in contrast to wildtype cells, where it increases drastically and without an increase of fructose uptake by an equivalent amount (Fig. 1b).The new interpretation suggests that gene C is of the regulator type. Mutation of it in the C-strain used here has lead to the simultaneous de-repression of a system for fructose and sorbose uptake. Deoxyglucose uptake is not served by this system. Gene A is a structural gene, harbouring the information for the inducible synthesis of a carrier or permease specifically engaged in sorbose uptake. It is not under the controll of gene C.This interpretation is supported by results on untreated cells of the double mutant. However, fructose uptake of such cells is roughly equal to that of C-mutant cells (Fig. 6a) and sorbose uptake is less (Fig. 5a). Hence, a secondary effect of the A-gene, i.e. on the amount of de-repression of sorbose uptake by mutation in gene C, is indicated.     

Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism

In Kluyveromyces marxianus grown on a glucose-containing synthetic medium four different sugar transporters have been identified. In cells, harvested during the exponential phase, only the constitutive glucose/fructose carrier, probed with 6-deoxy-D-glucose or sorbose, appeared to be active. In cells from the stationary phase three proton symporters can be active, recognizing 6-deoxyglucose (a glucose/galactose carrier), sorbose (a fructose carrier) and galactosides (lactose carrier), respectively. These symporters appeared to be sensitive to catabolite inactivation. This process is induced by incubating cells in the presence of glucose, fructose or mannose. Catabolite inactivation was not influenced by the inhibitor of protein synthesis, anisomycin. Derepression of the proton/sorbose and the proton/galactoside symporters proceeded readily when cells were incubated in a medium without glucose. Activation of the proton/galactose symporter needed, in addition, the presence of specific molecules (inducers) in the medium. The activation of each of these active transport systems was inhibited by anisomycin, showing the involvement of protein synthesis.