Comparison of growth and recombinant protein expression in two different insect cell lines in attached and suspension culture

Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPLBeta-Sf21-AE) and Trichoplusia ni (Tn 5Beta-1-4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150-160 rpm were necessary for maximum growth of suspended Tn 5Beta-1-4 cells compared to 125-150 rpm for Sf-21 cells. An inoculum size of 5 x 10(5) cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for beta-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5Beta-1-4 cells are 2-4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of beta-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5Beta-1-4 cells produced 2.6-4.4 and 2.7-3 times more beta-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the beta-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of beta-galactosidase by Tn 5Beta-1-4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production.     

The effect of dissolved oxygen (DO) concentration on the glycosylation of recombinant protein produced by the insect cell-baculovirus expression system.

The effect of dissolved oxygen concentration on human secreted alkaline phosphatase (SEAP) glycosylation by the insect cell-baculovirus expression system was investigated in a well-controlled bioreactor. Oligomannose-type N-linked glycans (i.e., Man2 to Man6 and Man3F) were present in SEAP produced by Spodoptera frusiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines. The relative amounts of the most highly processed glycans (i.e., Man3F and Man2 in the SEAP from Sf-9 and Tn-5B1-4 cells, respectively) were significantly higher at 50% of air saturation than at either 10% or 190% of air saturation. That is, glycan processing was inhibited at both low and high dissolved oxygen concentrations.     

The response of virally infected insect cells to dissolved oxygen concentration: recombinant protein production and oxidative damage

The effects of dissolved oxygen (DO) concentration on virally infected insect cells were investigated in 3-L bioreactor culture. Specifically, cultures of Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) were infected with Autographa californica multiple nucleopolyhedrovirus expressing secreted alkaline phosphatase (SEAP). Following infection at a DO concentration of 50% air saturation, the DO concentration was adjusted to a final value of either 190%, 50%, or 10% air saturation. Recombinant SEAP production, cell viability, protein carbonyl content, and thiobarbituric acid reactive substances (TBARS) content were monitored. The increases in protein carbonyl and TBARS contents are taken to be indicators of protein oxidation and lipid oxidation, respectively. DO concentration was found to have no noticeable effect on SEAP production or cell viability decline in the Sf-9 cell line. In the Tn-5B1-4 cell line, cells displayed an increased peak SEAP production rate for 190% air saturation and displayed an increased rate of viability decline at increased DO concentration. Protein carbonyl content showed no significant increase in the Sf-9 cell line by 72 h postinfection (pi) at any DO concentration but showed a twofold increase at 10% and 50% DO concentration and a threefold increase at 190% DO concentration by 72 h pi in Tn-5B1-4 cells. TBARS content was found to increase by approximately 50% in Sf-9 cells and by approximately twofold in Tn-5B1-4 cells by 72 h pi with no clear relationship to DO concentration. It is hypothesized that oxygen uptake changes due to the viral infection process may bear a relation to the observed increases in protein and lipid oxidation and that lipid oxidation may play an important role in the death of virally infected insect cells.     

Inducing single-cell suspension of BTI-TN5B1-4 insect cells: I. The use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production

Sulfated polyanions have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell line which has a high intrinsic capacity for recombinant protein production but clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. The efficacy of inducing single-cell suspension correlated positively with the increase in sulfation of the added polyanion. Unsulfated polyanions, neutral polymers, polycations, disaccharides, and monosaccharides were ineffective in inducing single-cell suspension.Elimination of clumping in suspension culture by adding a dispersing agent can lead to enhanced recombinant protein production. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in a four-fold increase in volumetric yield of the recombinant glycosylated protein, human secreted alkaline phosphatase, and a two-fold increase in volumetric yield of the recombinant cytoplasmic protein, beta-galactosidase. High yields of 82 U/ml (ca. 110 mg/L) for alkaline phosphatase, and 705 U/mL (ca. 2.3 g/L) for beta-galactosidase under elevated oxygen have been obtained. The optimum volumetric yield of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen but unsupplemented medium is 6 to 11-fold higher than attached cultures, and 3-fold higher than the best yield obtained for SF21 cells in suspension at elevated oxygen and with nutrient supplementation. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:191-205, 1997.     

Use of mannosamine for inducing the addition of outer arm N-acetylglucosamine onto N-linked oligosaccharides of recombinant proteins in insect cells

Glycosylation is a cellular process accomplished by a series of sequential enzymatic processing steps through the endoplasmic reticulum and Golgi apparatus with vesicle transport between the membranous organelles. The capacity for complex glycosylation is considered to be conferred by cell genetics, while the roles of nongenetic factors in protein processing are often ignored. It was hypothesized that the glycosyltransferase reactions in the insect cell-baculovirus system were limited by the small supply of sugar donor cosubstrates. By adding mannosamine, the glycosylation of a human secreted alkaline phosphatase in Spodoptera frugiperda (Sf-21) cells was extended to include terminal N-acetylglucosamine structures which were not seen in control cultures, and in Trichoplusic ni (BTI-Tn5B1-4) cells the amount of terminal N-acetylglucosamine structures was increased.     

High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: comparison to a batch Sf-9-BEV system

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.     

Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Fivetrade mark cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Fivetrade mark cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Fivetrade mark cell cultures, but not in Sf-9 cell cultures. In addition, High Fivetrade mark cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Fivetrade mark cell cultures. It was also found that the medium had a significant effect on High Fivetrade mark cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.     

Transient responses of hybridoma metabolism to changes in the oxygen supply rate in continuous culture

Oxygen is an important nutrient that may limit the productivity of commercial cell culture reactors. The transient responses of hybridoma growth and metabolism to step changes in the oxygen supply rate have been examined for dissolved oxygen concentrations (DO) ranging from 0.1% to 10% of air saturation in continuous culture. Metabolic quotients are reported for glucose, lactate, ammonia, oxygen, glutamine, alanine and other amino acids. A majority of the estimated ATP production was due to oxidative phosphorylation under all conditions tested. Decreases in the oxygen supply rate below the value required to maintain 0.5% DO caused the viable cell concentration to decrease. Glycolysis was enhanced at the lower oxygen concentrations, and after an initial decrease, the specific glutamine consumption rate was also higher. High residual glutamine concentrations occurred below 0.5% DO. Oxidation of other amino acids and production of serine were also inhibited. The cells subsequently adapted to low oxygen concentrations. The increase in cell concentration following the return to 10% DO was preceded by increased biosynthetic activity, as evidenced by transiently reduced yields of lactate from glucose, and alanine and ammonia from glutamine.     

Design of an efficient medium for insect cell growth and recombinant protein production

We report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1 x 10(6) cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3-4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2 x 10(6) cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 lU/ml (58 mg/L) and 28 lU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.     

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 °C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.     

An actively mixed mini-bioreactor for protein production from suspended animal cells

Biopharmaceutical production would benefit from rapid methods to optimize production of therapeutic proteins by screening host cell line/vector combination, culture media, and operational parameters such as timing of induction. Miniaturized bioreactors are an emerging research area aiming at improving the development speed. In this work, a 3 mm thick mini-bioreactor including two 12 mm wide culture chambers connected by a 5 mm wide channel is described. Active mixing is achieved by pressure shuttling between the two chambers. Gas-liquid phase exchange for oxygen and carbon dioxide is realized by molecular diffusion through 50 microm thick polymethylpentene membranes. With this unique design, a velocity difference between the middle area and the side areas at the interfaces of the culture chambers and the connecting channel is created, which enhances the mixing efficiency. The observed mixing time is on the order of 100 s. The combination of high permeability toward oxygen of polymethylpentene membranes and fluid movement during active pressure shuttling enables higher volumetric oxygen transfer coefficients, 5.7 +/- 0.4-14.8 +/- 0.6 h(-1), to be obtained in the mini-bioreactors than the values found in traditional 50 mL spinner flasks, 2.0-2.5 h(-1). Meanwhile, the calculated volume averaged shear stress, in the range of 10(-2)-10(-1) N/m(2), is within the typical tolerable range of animal cells. To demonstrate the applicability of this mini-bioreactor to culture suspended animal cells, the insect cell, Spodoptera frugiperda, is cultured in mini-bioreactors operated under a K(L)a value of 14.8 +/- 0.6 h(-1) and compared to the same cells cultured in 50 mL spinner flasks operated under a K(L)a value of 2.2 h(-1). Sf-21 cells cultured in the mini-bioreactors present comparable length of lag phases and growth rates to their counterparts cultured in 50 mL spinner flasks, but achieve a higher maximum cell density of 5.3 +/- 0.9 x 10(6) cell/mL than the value of 3.4 +/- 0.4 x 10(6) cell/mL obtained by cells cultured in 50 mL spinner flasks. Sf-21 cells infected with SEAP-baculovirus produce a maximum SEAP concentration of 11.3 +/- 0.7 U/mL when cultured in the mini-bioreactor. In contrast, infected Sf-21 cells cultured in 50 mL spinner flasks produce a maximum SEAP concentration of 7.4 +/- 0.9 U/mL and onset of production is delayed from 18 h in minibioreactor to 40 h in spinner flasks.     

Glutamine is not an essential amino acid for Sf-9 insect cells

Summary Spodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of -galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.     

Extracellular phosphatases of Chlamydomonas reinhardi and their regulation.

Chlamydomonas reinhardi, cultured under normal growth conditions, secreted significant amounts of protein and carbohydrates but not lipids or nucleic acids. A fivefold increase in light intensity led to a tenfold increase in secreted protein and carbohydrate. Among the proteins secreted was acid phosphatase with a pH optimum at 4.8 like the enzyme in the cells. Phosphorus depleted algae grown on minimal orthophosphate contained and secreted both acid and alkaline phosphatase. The pH optimum of the intracellular alkaline phosphatase was 9.2. When phosphorus-depleted cells were grown with increasing orthophosphate, intra- and extracellular alkaline phosphatase was almost completely repressed and intra- and extracellular acid phosphatase was partially repressed. Extracellular acid and alkaline phosphatase increased with the age of the culture. Electrophoresis indicated only one acid and one alkaline phosphatase in phosphorus-satisfied and phosphorus-depleted cells. Chlamydomonas cells suspended in an inorganic salt solution secreted only acid phosphatase; the absence of any extr-cellular cytoplasmic marker enzyme indicated that there was little, if any, autolysis to account for the extracellular acid enzyme. Phosphorus-depleted cells were able to grow on organic phosphates as the sole source of orthophosphate. Ribose-5-phosphate was the best for cell multiplication, and its utility was shown to be due to the cell's ability to use the ribose as well as the orthophosphatase for cell multiplication.     

Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production

The influence of ammonia and lactate on cell growth, metabolic, and antibody production rates was investigated for murine hybridoma cell line 163.4G5.3 during batch culture. The specific growth rate was reduced by one-half in the presence of an initial ammonia concentration of 4 mM. Increasing ammonia levels accelerated glucose and glutamine consumption, decreased ammonia yield from glutamine, and increased alanine yield from glutamine. Although the amount of antibody produced decreased with increasing ammonia concentration, the specific antibody productivity remained relatively constant around a value of 0.22 pg/cell-h. The specific growth rate was reduced by one-half at an initial lactate concentration of 55 mM. Although specific glucose and glutamine uptake rates were increased at high lacatate concentration, they showed a decrease after making corrections for medium osmolarity. The yield coefficient of lactate from glucose decreased at high lactate concentrations. A similar decrease was observed for the ammonia yield coefficient from glutamine. At elevated lactate concentrations, specific antibody productivities increased, possibly due to the increase in medium osmolarity. The specific oxygen uptake rate was insensitive to ammonia and lactate concentrations. Addition of ammonia and lactate increased the calculated metabolic energy production of the cells. At high ammonia and lactate, the contribution of glycolysis to total energy production increased. Decreasing external pH and increasing ammonia concentrations caused cytoplasmic acidification. Effect of lactate on intracellular pH was insignificant, whereas increasing osmolarity caused cytoplasmic alkalinization.     

Localization of alkaline phosphatase in cells of Bacillus intermedius

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.     

Kinetic studies on hybridoma cells immobilized in fixed bed reactors

Cultures with immobilized hybridoma cells were performed in fixed bed systems. "Steady state" values for volume-specific substrate uptake and metabolite production rates were determined at various perfusion rates and superficial flow velocities of the medium within the carrier matrix. Data from fixed bed volumes between 50 and 600 ml did not show any difference. The volume-specific glutamine and glucose uptake rate turned out to be independent of the superficial flow velocity, but decreased with decreasing glutamine and glucose concentration. The volume-specific oxygen uptake rate increased with increasing superficial flow velocity and substrate concentration, respectively. A similar behavior was observed for the ratio between oxygen and glucose uptake rate. The production rate for monoclonal antibodies was neither affected by the substrate concentration nor by the superficial flow velocity. The metabolic parameters of the immobilized cells were put into kinetic equations and compared to those of suspended cells. It could be concluded that the metabolism of the immobilized cells is determined by the oxygen supply within the macroporous carriers. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 535-541, 1997.     

Induction of a metabolic switch in insect cells by substrate-limited fed batch cultures

Insect cell metabolism was studied in substrate-limited fed batch cultures of Spodoptera frugiperda (Sf-9) cells. Results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine and a batch culture were compared. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Although the metabolism was influenced by substrate limitation, the specific growth rate was similar in all cultures. Alanine formation must involve incorporation of free ammonium, if ammonium formation is mediated by glutaminase and glutamate dehydrogenase, as our data suggest. On the basis of the results, two possible pathways for the formation of alanine in the intermediary metabolism in insect cells are suggested. The cellular yield on glucose was increased 6.6 times during glucose limitation, independently of the cellular yield on glutamine, which was increased 50–100 times during glutamine limitation. The results indicate that alanine overflow metabolism is energetically wasteful and that glutamine is a dispensable amino acid for cultured Sf-9 cells. Preliminary data confirm that glutamine can be synthesised by the cells themselves in amounts sufficient to support growth.     

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".