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.     

Ammonia inhibition of hybridomas propagated in batch, fed-batch, and continuous culture

The nature and temporal development of ammonia inhbition were investigated in batch, fed-batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 --> 3.7 --> 4.0 --> 4.9 --> 7.7 --> 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady-state specific growth kinetics or specific antibody productivity. (c) 1994 John Wiley & Sons, Inc.     

Vancomycin production in batch and continuous culture

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific production (Y(p/x)) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylalanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h(-1)), specific vancomycin production rate (q(vancomycin)) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, q(vancomycin) was a function of specific growth rate; the maximum value was observed at D = 0.087 h(-1) (52% of the maximum specific growth rate). Under phosphate limited growth conditions, q(vancomycin) was threefold higher (0.37 mg/g dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). (c) 1996 John Wiley & Sons, Inc.     

Fermentative hydrogen production from fresh leachate in batch and continuous bioreactors

This research for the first time investigated hydrogen production from the fresh leachate originated from municipal solid wastes. We found that fermentation of the leachate generated H₂ and was very much enhanced in the presence of extra phosphate in the batch reactor. The continuous expanded granular sludge bed (EGSB) reactor started to generate H₂ at day 20 and continued to 176 days with 120 mg/l of extra phosphate present. The highest chemical oxygen demand (COD) removal efficiency (66.9%) was achieved at liquid up-flow velocity of 3.7 m/h and hydraulic retention time of 12 h. Under proposed optimal operation conditions, the mean H₂ production rate reached up to 2155 ml/(l day). We also found that over 80% liquid metabolites were acetic acid and ethanol, suggesting the ethanol-type fermentation was dominant in the bioreactor. These findings indicate that the fresh leachate can be used as the source for continuous hydrogen production.     

Specificity of anti-polynucleotide monoclonal antibodies from human-human hybridomas

Summary Nine human-human hybridoma clones, secreting monoclonal antibodies reactive with nucleic acids, were generated by fusing with lymphocytes of lung cancer or systemic lupus erythematosus patients. These hybridoma antibodies were classified into 5 types, in terms of reactivities with DNA, RNA, various synthetic nucleic acids and cardiolipin. Hybridoma clone SU-1 secreted antibody reacting with dsDNA, ssDNA and RNA (type I). Clone HL-321 did not react with these, but with poly (dT), poly (I) and poly (G) (type II). Clone HL-349 was reactive with almost all nucleic acids tested and also with cardiolipin (type III). Clones HF-4, HF-7, HB-7 and HL-259 reacted with ssDNA, poly(A), poly(G) and cardiolipin, but not with RNA (type IV). HB-5 and SH-9 antibodies were reactive only with poly (dT) (type V). Editor’s Statement This paper describes isolation and characterization of human-human hybridoma clones producing antibodies to nucleic acids. Isolation of such hybridomas from lymphocytes of cancer patients and the similarity of some isolates to those obtained from mice exhibiting autoimmune disease represent interesting observations that may lead to future insights.     

Growth and product inhibition kinetics of T-cell hybridomas producing lymphokines in batch and continuous culture.

The kinetics of growth and lymphokine formation by T-cell hybridomas were investigated in batch and continuous culture. Product inhibitions by ammonia and lactic acid were considered in the proposed model and experimental data were used to determine the kinetic and inhibition constants. The effect of dilution rate on specific substrate consumption and product formation rates was investigated.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

An unstructured kinetic model of macromolecular metabolism in batch and fed-batch cultures of hybridoma cells producing monoclonal antibody

Growth profiles of the batch and fed-batch culture of hybridoma cells producing monoclonal antibody were simulated using an unstructured model. The model describes the production of cellular macromolecules and monoclonal antibody, the metabolism of glucose and glutamine with the production of lactate and ammonia, and the profiles of cell growth in batch and fed-batch culture. Equations describing the cells arrested in G1 phase [T.I. Linardos, N. Kalogerakis, L.A. Behie, Biotechnol. Bioeng. 40 (1992) 359–368; E. Suzuki, D.F. Ollis, Biotechnol. Bioeng. 34 (1989) 1398–1402] were included in this model to describe the increase of the specific antibody productivity in the near-zero specific growth rate, which was observed in the recent experiments in fed-batch cultures of this study and the semi-continuous culture of hybridoma cells [S. Reuveny, D. Velez, L. Miller, J.D. Macmillan, J. Immnol. Methods 86 (1986) 61–69]. This model predicted the increase of specific antibody production rate and the decline of the specific production rate of cellular macromolecules such as DNA, RNA, protein, and polysaccharide in the late exponential and decline phase of batch culture and at lower specific growth rates in the fed-batch culture.     

An integration of cultivation and purification in membrane bioreactors: production of monoclonal antibodies and lymphokines by microencapsulated hybridomas

Engineering and physiologic aspects of growth and production processes associated with encapsulated cells, mostly of anchorage-independent type, are reviewed. They include membrane diffusion characterization, mechanical properties of microcapsules, process limiting phenomena, physiological state of cells, process modeling and product purity and concentration. Finally, application areas are spelled out with an outlook discussion.     

Enhancing effects of retinoic acid on monoclonal antibody production of human-human hybridomas

The effects of retinoids on the production of monoclonal antibody of human-human hybridomas were examined. IgG antibody secretion of a hybridoma CLNH11 was enhanced up to about two- to fourfold by retinoic acid (RA) at concentrations ranging from 10(-9) to 10(-5) M, where RA had little effect on the growth rate and saturation density of the cell. Among other retinoids, retinol magnified the antibody production as well as RA. Retinal and retinyl acetate had weak effects. Retinyl palmitate showed no effect. RA also enhanced the production of monoclonal antibodies from other human-human hybridomas: SLNF10, IgG-producing; CoLNE10, IgA-producing; TOS/H8, IgM-producing. RA and human hybridomas provide a defined system to study the effects of retinoids on immune responses at a molecular level.     

Protease production by Clostridium perfringens in batch and continuous culture

A single covalently closed circular plasmid isolated from Selenomonas ruminantium HD4 migrated at 3–5 kilobase pairs (kb). A second band migrating at 23 kb could not be confirmed as plasmid DNA. The plasmid was digested by HindIII. Extraction of plasmid DNA from S. ruminantium HD4 was facilitated by the use of a carbonate buffer wash during cell harvest that allowed for rapid and complete lysis by lysozyme and markedly improved the release of DNA.     

Ethanol production in multistage continuous, single stage continuous, Lactobacillus-contaminated continuous, and batch fermentations

Saccharomyces cerevisiae-based ethanol fermentations were conducted in batch culture, in a single stage continuous stirred tank reactor (CSTR), a multistage CSTR, and in a fermentor contaminated with Lactobacillus that corresponded to the first fermentor of the multistage CSTR system. Using a glucose concentration of 260 g l^–1 in the medium, the highest ethanol concentration reached was in batch (116gl^–1), followed by the multistage CSTR (106gl^–1), and the single stage CSTR continuous production system (60gl^–1). The highest ethanol productivity at this sugar concentration was achieved in the multistage CSTR system where a productivity of 12.7gl^–1h^–1 was seen. The other fermentation systems in comparison did not exceed an ethanol productivity of 3gl^–1h^–1. By performing a continuous ethanol fermentation in multiple stages (having a total equivalent working volume of the tested single stage), a 4-fold higher ethanol productivity was achieved as compared to either the single stage CSTR, or the batch fermentation.     

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.     

A structured kinetic modeling framework for the dynamics of hybridoma growth and monoclonal antibody production in continuous suspension cultures

A structured kinetic model is developed to describe the dynamics of hybridoma growth and the production of monoclonal antibodies and metabolic waste products in suspension culture. The crucial details of known metabolic processes in hybridoma cells are incorporated by dividing the cell mass into four intracellular metabolic pools. The model framework and structure allow the dynamic calculation of the instantaneous specific growth rate of a hybridoma culture. The steady state and dynamic simulations of the model equations exhibit excellent agreement with experimentally observed trends in substrate utilization and product formation. The model represents the first to include any degree of metabolic detail and structure in describing a hybridoma culture. In so doing, it provides the basic modeling framework for incorporating further details of metabolism and can be a useful tool to study various strategies for enhancing hybridoma growth as well as viability and the production of monoclonal antibodies in suspension cultures.     

Immobilized chloroplast membranes: kinetic aspects of their continuous use in batch and chemostat

To study the biophotolytíc processes involved in the production of hydrogen gas the photosynthetic material of isolated chloroplast membranes was first stabilized. This was achieved by immobilization in a serum albumin-glutaraldehyde matrix. Some kinetic data of the photochemical reactions carried out in closed and continuous flow reactor systems have been investigated. The use of a CSTR chemostat enabled the functional stability of the immobilized photosystems to be examined.     

Advantages of continuous over batch reactors for the kinetic analysis of enzymes inhibited by an unknown substrate impurity

A new experimental technique, employing a continuous stirred-tank reactor, for studying enzyme kinetics in the presence of inhibitor-contaminated substrate is described. The proposed method is simulated mathematically for competitive, uncompetitive, and mixed-type noncompetitive inhibition. The step-by-step experimental procedure is described, as is the necessary data analysis for determining the kinetic parameters. Differences in system response for enzyme inhibition by excess substrate and by an impurity are illustrated, and a stability analysis of the system is performed.     

The production of monoclonal antibody in growth-arrested hybridomas cultivated in suspension and immobilized modes.

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.     

Optimized 1,3-propanediol production from crude glycerol using mixed cultures in batch and continuous reactors

Bioprocess and Biosystems Engineering - The production of 1,3-propanediol from crude glycerol and mixed anaerobic sludge was investigated in batch experiments and continuous reactors. Using a 23...     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara