Transient growth- and product-formation kinetics of acetic acid bacteria

Rates of growth and product formation under non-stationary conditions were measured in fermentations of industrial acetic acid bacteria. A repeated-batch process, where conditions change rapidly, and a slower shift experiment in CSTR culture were examined. Significant deviations from the steady-state kinetics determined in continuous fermentations were found for cell growth as well as for the formation of acetic acid. Algebraic functions of the inhibiting acid concentration were identified to describe the rates of reaction under stationary conditions. Transient kinetics are modeled by phenomenological differential equations. The data from both the repeated-batch experiments and the CSTR shift is consistently reproduced. Measurements and simulation results are presented in phase diagrams of the reaction rates over the concentration of acetic acid. Due to the dynamic effects, which enhance the transient rates of both growth and product formation, the repeated-batch process is superior to a continuous fermentation in terms of total volumetric productivity and final acid concentration.     

Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes

The advantages of nanofiltration membranes coupled with a CSTR were demonstrated for the semicontinuous production of lactic acid from whey permeate. Lactic acid was removed from the growth medium while lactose was kept in the bioreactor with the bacterial cells; moreover, Mg²⁺ ions were also recycled in the bioreactor at 96% and the nanofiltrate color was greatly reduced. The highest volumetric productivity achieved with this device was 7.1 g l⁻¹ h⁻¹ and the lactate concentration was 55 g l⁻¹. The specific productivity was 3.54 h⁻¹. More than 99% of the membrane fouling after 44 h of fermentation was reversible. The initial permeate flux was restored easily by a water rinse. The performance of this type of membrane bioreactor was discussed.     

Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process

Controlled feeding of nutrient supplements to a cell culture to enhance monoclonal antibody productivity has been practiced widely in high-yield, fed-batch processes. In this study, a similar feeding concept has been applied to a perfused culture and evaluated for the effects on bioreactor productivity and product quality. Our experimental results show that, by using such a "controlled-fed perfusion" approach, the volumetric antibody productivity (antibody per liter per day) was significantly increased by nearly twofold over the perfusion process, and surpassed fed-batch and batch processes by almost tenfold. The substantial boost in the overall productivity is attributable primarily to the combined effects of increased cell density as well as reduced product dilution. Both were achieved through careful nutrient supplementation in conjunction with metabolite minimization. As the manufacturing process evolved from roller bottles to the controlled-fed perfusion bioreactor system, the immunoreactivity and the cDNA sequences of the antibody were well preserved. However, the product glycosylation distribution patterns did alter. The controlled-feed perfusion process demonstrated a unique encompassment of the advantages of fed-batch and perfusion methods; that is, high product concentration with high volume throughput. Therefore, it may be very suitable for large-scale production of monoclonal antibodies.     

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

High efficiency ethanol fermentation by entrapment of Zymomonas mobilis into LentiKats

AIMS: To examine the potential of Zymomonas mobilis entrapped into polyvinylalcohol (PVA) lens-shaped immobilizates in batch and continuous ethanol production. METHODS AND RESULTS: Cells, free or immobilized in PVA hydrogel-based lens-shaped immobilizates - LentiKats, were cultivated on glucose medium in a 1 l bioreactor. In comparison with free cell cultivation, volumetric productivity of immobilized batch culture was nine times higher (43.6 g l(-1) h(-1)). The continuously operated system did not improve the efficiency (volumetric productivity of the immobilized cells 30.7 g l(-1) h(-1)). CONCLUSIONS: We demonstrated Z. mobilis capability, entrapped into LentiKats, in the cost-efficient batch system of ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported here emphasize the potential of bacteria in combination with suitable fermentation technology in industrial scale. The innovation compared with traditional systems is characterized by excellent long-term stability, high volumetric productivity and other technological advantages.     

A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity

A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.     

Scale-up of virus-like particles production: effects of sparging, agitation and bioreactor scale on cell growth, infection kinetics and productivity

The baculovirus-insect cells expression system was used for the production of self-forming Porcine parvovirus (PPV) like particles (virus-like particles, VLPs) in serum-free medium. At 2l bioreactor scale an efficient production was achieved by infecting the culture at a concentration of 1.5 x 10(6)cells/ml using a low multiplicity of infection of 0.05 pfu per cell. In a continuous bioreactor, it was shown that the uninfected insect cells were not sensitive to local shear stress values up to 2.25 N/m2 at high Reynolds numbers (1.5 x 10(4)) in sparging conditions. Uninfected insect cells can be grown at scaled-up bioreactor at high agitation and sparging rates as long as vortex formation is avoided and bubble entrapment is minimized. An efficient process scale-up to 25 l bioreactor was made using constant shear stress criteria for scale-up. The kinetics of baculovirus infection at low multiplicity of infection, either at different cell concentration or at different scales, are very reproducible, despite the different turbulence conditions present in the bioreactor milieu. The results suggest that the infection kinetics is controlled by the rate of baculovirus-cell receptor attachment and is independent of the bioreactor hydrodynamic conditions. Furthermore, the achieved specific and volumetric productivities were higher at the 25 l scale when compared to the smaller scale bioreactor. Different rates of cell lysis after infection were observed and seem to fully explain both the shift in optimal harvest time and the increase in cell specific productivity. The results emphasize the importance of integrated strategies and engineering concepts in process development at bioreactor stage with the baculovirus insect cell system.     

Effect of specific growth rates on productivity in continuous open and partial cell retention animal cell bioreactors.

A clonal derivative of a transfectant of the SP2/0 myeloma cell line producing a chimeric monoclonal antibody was cultivated in both continuous open and continuous partially-closed bioreactors. Using an open system for the determination of kinetic parameters, we showed that the production of this chimeric mAb was growth associated. As such, the volumetric productivity increased linearly with increasing dilution rate up to the maximum dilution rate. Three continuous cultivations employing partial cell retention were conducted. In agreement with mathematical predictions, the product titer and volumetric productivity were independent of the degree of cell retention when the total dilution was held constant. When cells were maintained at a low specific growth rate, the product titer was independent of dilution rate and the volumetric productivity increased with increasing dilution rate, again in agreement with mathematical predictions. Since the partially-closed bioreactor could be operated at dilution rates in excess of the maximum specific cellular growth rate, volumetric productivities were greater than those achievable in the open bioreactor. However, when cells were maintained at a high specific growth rate, cell accumulation was limited and product titers decreased at high dilution rates. Therefore, the volumetric productivity in this latter case did not increase at higher dilution rates.     

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.     

Cell growth and protein formation on various microcarriers

A large number of microcarriers are commercially available. The capability of cells to successfully proliferate on microcarriers varies with cell lines and media. Choosing the right microcarrier for a particular cell line is more than a choice of a microcarrier. It is part of an integrated process design. A detailed picture of cell growth and product formation will not only be essential in identifying the kind of microcarrier, but also in determining other parts of the process, such as operation mode and media. Our initial screening on thirteen microcarriers showed that cultures on some microcarriers reached a low cell density but high cell-specific productivity, and high density microcarrier cultures have a low specific productivity. The result is a similar product output per unit volume and time for these two types of cultures. An ideal culture system shall have increased volumetric productivity at elevated cell density. This requires the process goal to be incorporated as early as cell line construction and screening. A high output process can then be realized through high density culture. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.     

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.     

A continuous process for the production of baculovirus using insect-cell cultures

Summary A continuous culture of insect cells (Spodoptera frugiperda) was used for continuous production of baculovirus (nuclear polyhedrosis virus fromAutographa californica). The system consisted of a cascade of two continuous stirred tank reactors (CSTRs). In CSTR I the insect cells were grown in suspension. This suspension was fed continuously to CSTR II where the virus infection occurred. For a period of about 25 days the average volumetric productivity was about 10⁷ polyhedra (virus particles occluded in protein capsules) and 10⁸ infectious NOVs (non-occluded virus particles) per cm³ effluent. This is equivalent to 25 polyhedra and 250 NOVs per infected cell, respectively. In one case, the percentage of infected cells was 65%, which is close to the theoretical value of 68%. After a run-time of 32 days a decrease of process productivity was observed, probably due to the so-called passage effect, a degeneration of the virus DNA.     

Cultural and physiological factors affecting expression of recombinant proteins

The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physicochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR).Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture. The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode. Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate.The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line. The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min^–1) and dilution rates (1 d^–1) and glutamine concentration (2.5 mmol l^–1), plus the lowest values for bead size (2 mm) and inoculum density (10⁷ ml^–1). Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system.Abbreviations FIBR fluidised bed bioreactor - FBR fixed bed reactor - STR stirred tank reactor - SEAP secreted alkaline phosphatase - rAb recombinant antibody     

Oxygen supply for CHO cells immobilized on a packed-bed of Fibra-Cel disks

Packed-bed bioreactors (PBR) have proven to be efficient systems to culture mammalian cells at very high cell density in perfusion mode, thus leading to very high volumetric productivity. However, the immobilized cells must be continuously supplied with all nutrients in sufficient quantities to remain viable and productive over the full duration of the perfusion culture. Among all nutrients, oxygen is the most critical since it is present at very low concentration due to its low solubility in cell culture medium. This work presents the development of a model for oxygenation in a packed-bed bioreactor system. The experimental system used to develop the model was a packed-bed of Fibra-Cel disk carriers used to cultivate Chinese Hamster Ovary cells at high density ( approximately 6.1 x 10(7) cell/mL) in perfusion mode. With the help of this model, it was possible to identify if a PBR system is operated in optimal or sub-optimal conditions. Using the model, two options were proposed, which could improve the performance of the basal system by about twofold, that is, by increasing the density of immobilized cells per carrier volume from 6.1 x 10(7) to 1.2 x 10(8) cell/mL, or by increasing the packed-bed height from 0.2 to 0.4 m. Both strategies would be rather simple to test and implement in the packed-bed bioreactor system used for this study. As a result, it would be possible to achieve a substantial improvement of about twofold higher productivity as compared with the basal conditions.     

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of ∼5 × 10⁶ cells mL^-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell^-1 day^-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized (∼40 mg L^-1) at 0.2 nL cell^-1 day^-1. The volumetric protein productivity (∼60 mg L^-1 day^-1 was maximized above 0.3 nL cell^-1 day^-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

Perfusion seed cultures improve biopharmaceutical fed‐batch production capacity and product quality

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof‐of‐concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high‐seed fed‐batch production cultures. First, we optimized the perfusion N‐1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N‐1 duration, reaching >40 × 10⁶ vc/mL at the end of the perfusion N‐1 stage. The cultures were subsequently split into high‐seed (10 × 10⁶ vc/mL) fed‐batch production cultures. This strategy significantly shortened the culture duration. The high‐seed fed‐batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low‐seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N‐1 and high‐seed fed‐batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low‐seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:616–625, 2014     

Packed bed bioreactor with porous ceramic beads for animal cell culture

A packed bed bioreactor was investigated as means for the cultivation of mammalian cells. The packed bed is comprised of porous ceramic particles with pores sufficiently large for cell immobilization as well as for intraparticle convective flow. In this way, the transport of limiting nutrients such as oxygen can be significantly enhanced, allowing maintenance of cell viability and productivity in an environment protective of adverse shear effects. The extent of intraparticle convective medium flow was experimentally quantified relative to the reactor operating conditions, and was found to be the dominant mechanism of nutrient transport to cells immobilized in the particle interior. An approximate linear relationship was obtained between overall reactor productivity and the extent of intraparticle convection. As the latter can be controlled at the single-particle level through total flow rate control, this relationship is a useful scale-up tool for the design of bioreactors. The high cell densities and the high volumetric productivities achieved by using small lab-scale reactors underline the potential of this simple bioreactor configuration for large-scale cell culture applications. (c) 1993 John Wiley & Sons, Inc.