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Development of Scale-Down Techniques for Investigation of Recombinant Escherichia coli Fermentations: Acid Metabolites in Shake Flasks and Stirred Bioreactors Mary Ellen Dahlgren,’ Alan L. Powell, Randolph L. Greasham, and Hugh A. George Department of Bioprocess Research and Development, Merck Research Laboratories, Rahway, New Jersey 07065
We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGFa-PE40, by recombinant Escherichia coli JM109 in stirred bioreactors. When a nutrient-rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations. Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask. The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of “scale-down” conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium. Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation. For the complex MlOl medium, optimal shake-flask performance in 250-mL, nonbaffled flasks a t 37 O C occurred with 0.3X medium strength, supplementation with0.3 M HEPES buffer (pH 7.51, and 10 mL of medium per flask. Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements a t 600 nm) and maintained a pH near neutrality. Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGFa-PE40 was comparable to stirred bioreactors induced for expression a t various biomass levels. When culture parameters were controlled within these limits, similar results were also observed in the defined FM medium.
Introduction Large-scale culture of recombinant Escherichia coli strains is used commerciallyin the manufacture of several polypeptide products (Khosrovi and Gray, 1985). As with natural products of microbial origin, desirable strains are commonly identified in shake-flask cultures before a production process is developed and scaled up to large bioreactors. Optimal culture conditionsfor manufacture, including medium composition and growth stage at induction, vary widely and often are not predictable from host genotype or plasmid construction. Rapid evaluation of these influences in flask cultures will reduce development times in stirred bioreactors, provided that the limitations of growth and expression in flask cultures are well documented. In contrast to many natural product fermentations, shake-flaskculture models have not proven reliable for the evaluation of scale-up parameters for high cell density recombinant fermentations. A principal reason for this is the large composition differencesbetween media practical for large-scale cultures and those used to identify producing recombinant strains. The most economical production scheme would employ both a high cell concentration in the fermentor and a high level of product expression. Production media used for E. coli fermentations typically either contain high initial concentrations of glucose or are supplemented by glucose feeding during culture, since this convenient and relatively inexpensive carbon source supports growth to high cell
* Author to whom correspondence should be addressed. 8756-7938/93/3009-0580$04.00/0
densities (Rinas et al., 1989; Luli and Strohl, 1990). In contrast, the culture media usually used for screening E. coli, such as LB (Sambrook et al., 19891, contain no carbohydrate and support low biomass growth. In this study, we have explored approaches to studying glucosecontaining media for growth and expressionof recombinant E . coli in shake-flask cultures. A complication of E . coli growth on glucose under aerobic conditions is incomplete glucose oxidation, resulting in the accumulation of metabolic acids, particularly acetate; this is sometimes referred to as the bacterial “Crabtree effect”(Sambrooket al., 1989;Tempest and Neijssel, 1987; Zabriskie e t al., 1987). Acetate accumulation leads to both reduced culture growth and reduced recombinant protein expression (Zabriskie et al., 1987;Bauer et al., 1990;BechJensen and Carlsen, 1990; MacDonald and Neway, 1990; Rinas et al., 1989; Luli and Strohl, 1990). The potential complicationof acetate accumulation imposes restrictions upon three interrelated factors in E . coli shake-flask fermentations: the initial carbohydratelevel, the buffering capacity of the test medium, and the cell densities at the time of induction. Additionally, as a facultativeanaerobe,E . coli is capable of producing metabolites other than acetate; these include pyruvate, lactate, and ethanol. Cultures grown in stirred bioreactors or in shake flasks may accumulate these metabolites whenever cell densities and oxygen demand exceed aeration capacity. Oxygen limitation in shakeflask cultures is usually reached at lower cell densities than in stirred bioreactors, since the strategies available
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to maintain aeration in stirred bioreactors, viz., increased agitation, air flow, and back pressure or oxygen enrichment, generally do not apply to flask cultivation. We have previously reported that expression levels, respiratory profiles, and biomass levels in stirred bioreactor cultivation of E. coli JM109 expressing recombinant TGFa-PE40 are dependent upon the cell densities present a t the time of induction (George et al., 1992). Moreover, acid metabolite profiles, even under adequate aeration, were altered by the induction of expression. Pyruvate excretion accompanied expression in both complex and defined media; however, pyruvate was also excreted in uninduced cultures when dissolved oxygen levels fell to less than 10%of saturation, a condition sometimes referred to as "microaerobic". These data indicated that carbon flow to acid metabolites was altered by the induction of expression in a manner similar to the alterations observed during oxygen limitation. In the present study, we sought to develop shake-flask screening conditions that are relevant to and predictive of TGFa-PE40 expression in stirred bioreactors. Acid metabolites and ethanol were used as general indicators of both carbon flow during expression and oxygen limitation. Variations in medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of scale-down conditions for expression in a general medium background similar to that used in stirred bioreactors.
Materials and Methods Bacterial Strains and Fermentation Conditions. The plasmid construction used for TGFa-PE40 expression has been described previously (Heimbrook et al., 1990). The host strain transformed with the complete expression plasmid is termed JM109 (pTAC-TGF57-PE40). Expansions of the master seed bank, prepared by liquid passage in selective medium and stored in 0.5-mL aliquots at -70 "C,were supplied to us by that laboratory. One frozen seed vial was thawed for each inoculum. The shake-flask cultures, with the exception of inoculum development, were conducted in 250-mL, nonbaffled Erlenmeyer flasks containing the indicated volumes (between 10 and 50 mL) of the two medium formulations, MlOl and FM. The flasks were incubated at 220 rpm (1-in. throw) at 37 "C for the indicated times. Culture pH determinations were performed within 60 s of removal of the flasks from the shaking incubator. All shake-flask experiments employed multiple replicate flasks. Individual flasks removed at various times for pH determination and sampling were not returned to the shaking incubator. Stirred bioreactor fermentations were performed in 22-L vessels (Chemap, Basel, Switzerland) using a 15-L working volume. In stirred bioreactor fermentations, the pH was controlled by the automatic addition of the base solution specified with each medium formulation; dissolved oxygen was maintained a t 50% saturation by increasing the agitation, air flow, and back pressure. At the times indicated, those cultures which were induced for the expression of TGFaPE40 received 1.0 mM (final concentration) isopropyl8-Dthiogalactopyranoside (IPTG). Inoculum Development. Inocula were grown in M9 medium (Sambrook et al., 1989) adjusted with NaOH to pH 7.3 before sterilization and supplemented with separately sterilized components to the following final concentrations: glucose, 2 g/L; neomycin sulfate 75 mg/L (equivalent to 50 mg/L neomycin base); CaC12, 0.01 M; MgS0~7H20,0.5g/L; and thiamine hydrochloride, 2 mg/ L. The contents of one frozen seed vial were used to inoculate a 2-L, baffled Erlenmeyer flask containing 250
mL of supplemented M9 medium. The flask was incubated at 200 rpm (1-in. throw) for 15-17 h at 37 OC. Transfers of inocula were timed at or near the time of glucose depletion. Shake-flask cultures in complex medium were inoculated with 5% volume, and those with defined medium were inoculated with 10-20% volume. Stirred bioreactor cultures in either complex or chemically defined medium employed a two-stage inoculum development regime. For the second stage, the entire contents of a flask grown as described above were transferred to 14.8 L of M9 medium, supplemented as above, in a 22-L stirred bioreactor. After an additional 6-7 h of incubation (37 "C, 0.5 wm, 400 rpm, 0.3 bar gauge pressure), 750-mL portions were transferred to the production stage. As with the inoculum transfer from the flask, this transfer occurred at or near the time of glucose depletion. Medium Formulations. MlOl medium contained the following (in grams per liter final concentrations): KH2Pod, 3.5; K2HP04, 3.5; (NH4)2S04, 3.0; yeast extract, 10; hydrolyzed soy peptone, 50. The medium was adjusted to pH 7.2 with 50% (w/v) NaOH prior to sterilization (25 minat 122 OC). Glucose, MgS04-7H20,neomycin sulfate, and thiamine hydrochloride were sterilized separately and added to final concentrations of 30 g/L, 2 g/L, 75 mg/L, and 2 mg/L, respectively. In stirred bioreactors, the culture pH was maintained a t 7.2 by the automatic addition of 20% (w/v) NaOH; no pH control was employed in the shake flasks. Chemically defined FM medium, adapted from Fass et al. (19891, contained the following (in grams per liter final concentrations): KH2P04,lO; K2HPO4,14; (NH4)2HP04,3;NaH@OpH20,4. Glucose, MgS04.7H20, neomycin sulfate, and thiamine hydrochloride were sterilized separately and added to final concentrations of 10 g/L, 0.45 g/L, 75 mg/L, and 50 mg/L, respectively. A concentrated trace element mixture containing (in grams per liter of 1.2 N HC1) 27 FeClr6H20,3 ZnS04*7H20,2 CoCL6H20,2 NaMoO4-2H20,lCaCL2H20,l CuC12, and 0.8 Na2B40~10H20was added to the final formulation at a level of 1mL/L. Cultures in stirred bioreactors were maintained at pH 7.2 by the automatic addition of NH4OH [15% (v/v) NH31, which also activated the addition of an equivalent volume of a glucose and MgS04.7HzO feed solution from a separate reservoir. The feed solution contained 480 g/Lglucose and 8.5 g/L MgS04-7H20,which amounts to a ratio of 8 g of glucose/g of NHs. The pH control and feeding strategy was not employed in shake
flasks. Analytical Methods for Fermentation Substrates and Metabolites. The analytical methods for TGFaPE40 quantification, biomass estimation, and measwement of carbohydrate, alcohol, and acidic product levels have already been described in detail (Georgeet al., 1992). Briefly, TGFa-PE40 concentrations in guanidine hydrochloride extracts of cell pellet samples were estimated by reverse-phase binary gradient HPLC using a Glycotech (Hamden, CT) Hytach C-18column. Total cellular protein concentrations were determined in the same extracts by the Pierce (Rockford, IL) BCA method. The specific expression of TGFa-PE40 in a sample was calculated as the micrograms of TGFa-PE40 protein divided by the milligrams of total cellular protein. Biomasswas estimated by measuring light scattering a t 600 nm. One optical density unit corresponded to 0.62 g of dry cell weight/L. Carbohydrate, alcohol, and acidic product levels were measured in acidified culture supematants by HPLC using a Bio-Rad (Richmond, CA) Aminex HPX87H column eluted isocratically with 0.3 N HzS04. Residual glucose was also determined using a Beckman Glucose Analyzer 2 and the manufacturer's reagents and protocol.
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Reagent-grade chemicals except for IPTG were obtained from Sigma Chemical Company (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Dioxane-free IPTG was obtained from Chem-Impex International (Wood Dale, IL). Yeast extract was supplied by Difco (Detroit, MI) and soy peptone by Sheffield (Norwich, NY).
Results Growth and Expression in MlOl Medium in Shake Flasks. The initial series of experiments examined uninduced E. coli JM109 (pTAC-TGF57-PE40) cultures in MlOl complex medium in stirred bioreactors and shake flasks. The medium composition, percent inocula, and temperature were identical for all of the culture vessels, and the agitation conditions were the same for all of the flasks. Thus, the major differences among the vessels are physical parameters, particularly the inability to control pH and dissolved oxygen in the flasks. In the shake flasks, variable medium volumes (10, 25, and 50 mL) were employed to test the hypothesis that a higher medium volume would result in poorer aeration of the culture. Growth and Metabolic Profile of Uninduced Cultures in Flasks. E. coli JM109 (pTAC-TGF57-PE40) cultures grown on high-glucose medium M101, either in shake flasksor in stirred bioreactors, achieved substantially higher cell densities than were observed with glucose-free LB medium. LB medium supported growth to an A m of only 2.1 (datanotshown). Medium MlOl results are shown in Figure 1,which compares the Am, glucose consumption, and pH profiles during growth in a stirred bioreactor and in the shake flasks. In the stirred bioreactor, growth continued until the biomass had accumulated to an A m of 30. However, growth ceased when the A m measured between 6 and 11in the shake flasks, and the greater the medium volume, the lower the biomass level at which growth stopped (Figure 1A). During growth in the stirred bioreactor, all of the glucose, originally 30 g/L, was exhausted, while approximately 20 g/L glucose remained in the flasks when growth ceased (Figure 1B). The pH profile was also influenced by the volume of medium: the greater the volume, the more rapidly the pH dropped, even though lower growth occurred at the higher volumes (Figure 1C). Metabolite Accumulation in Uninduced Cultures. Figure 2 summarizes the differences observed in the pattern of accumulation of glucose metabolites in shakeflask and stirred bioreactor cultures. In shake flasks, the pattern was related to medium volume. The differences in growth rate noted previously (Figure 1A) complicate the interpretation of metabolic profiles when the comparison is made on the basis of culture age. Accordingly, Figure 2 summarizes the glucose metabolites for the four vessels at a constant biomass level, as estimated by an A m of 6. Since growth was most rapid in the stirred bioreactor and the flask with the 10-mL medium volume, this biomass level was achieved earlier in those vessels than in the flasks with higher volumes. At a constant biomass level, both the stirred bioreactor and the various volume shake flasks exhibited similar patterns of acetate accumulation. The acetate concentration in the 10-mL flask was comparable to that in the stirred bioreactor, and the concentration increase was progressive with increasing medium volume. In contrast, the pyruvate accumulation was qualitatively different in the stirred bioreactors and the flasks. Pyruvate accumulation was not observed in the stirred bioreactor, but was observed in all of the shake flasks and was only modestly higher with the 50-mL medium volume than with the 10 mL. We had previously observed (George et
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Figure 1. Culture growth (A), glucose consumption (B),and pH profile (C) of E. coli JM109 (pTAC-TGF57-PE40) in stirred bioreactors and in shake flasks containing 10, 25, or 50 mL of complex MlOl medium (30g/L glucose). Culture growth was estimated by measuring absorbance at 600 nm. Specificgrowth rates were calculated for the initial 6 h in each vessel. Those rates and the correlation coefficients (r2values) for the correspondence of the growth curve to a theoretical exponential curve are as follows: stirred bioreactor, 0.68 (r2= 0.99); 10-mL flask, 0.69 (0.98); 25-mL flask, 0.60 (0.96); 50-mL flask,0.56 (0.93). All data points shown are the means of determinations that were made on duplicate flasks or medium samples.
al., 1992)that pyruvate accumulated in stirred bioreactors when the dissolved oxygen fell to less than 10% of saturation. This had led us to expect an increase in pyruvate concentration at higher medium volumes similar to the increase observedwith acetate. Instead, we observed that lactate and ethanol, metabolites associated with anaerobic fermentation which were not detectable in the stirred bioreactor and the low medium volume flasks, both accumulated in the flasks with higher medium volumes. These results indicate that serve oxygen limitation occurred in MlOl medium in shake flasks at biomass levels well below those achieved in stirred bioreactors, especially at higher medium volumes.
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Acetate Pyruvate [PPI Lactate
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Culture vessel Figure 2. Medium concentrations of glucose metabolites measured at a constant biomass level in stirred bioreactors and in shake flasks containing 10, 25, or 50 mL of complex medium M101. The biomass level was estimated by measurement of absorbance at 600 nm. In order to compare results at a constant biomass level, measurements were made at different postinoculationtimes in each type of culture vessel (seegrowth pattern in Figure 1). Table I. Effect of Dilution and Buffering of Complex Medium MlOl on Growth, pH, and Specific Glucose Consumption by E. coli JMlO9 (pTAC-TGF57-PE40) Grown in Shake Flasks. buffer medium concentration concentration (M) 0.1x 0.3X 0.5X 5.6 5.5 5.5 0 PH A600
SPGC 0.1
PH A600
SPGC 0.3
PH A600
0.5
SPGC PH A600
1.1 0.67
5.1 0.83
6.7 2.8 0.89
6.2 8.0 0.95
10.2 0.72
7.2 2.8 0.96
7.0 7.3 1.07
6.9 9.1 0.75
1.3 2.2 1.19
7.3 5.3 0.99
7.3
1
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0.46 5.8
3ca 6.0
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0.38 SPGC All flasks contained 10 mL of medium volume. The data shown were measured at 6 h after inoculation. SPGC is the specificglucose consumption, expressed as g/L glucose consumed per Am unit.
The pH and metabolite patterns demonstrate that a shake-flask model should employ the smallest feasible medium volume in order to maximize aeration, but that minimum volume by iteelf cannot overcome all of the limitations of the shake-flask model. Subsequent recombinant protein expression studies employed 10 mL of medium in 250-mL Erlenmeyer flasks. Modificationof Bioreactor Medium for the ShakeFlask Scale-Down Model. Growth, pH, and Metabolites in Dilute, Buffered Medium. The limitations observed in the shake flasks led us to rebalance the glucose concentration and buffering capacity of MlOl medium. We anticipated that a reduction in the glucose available for metabolism might limit the accumulation of undesirable acid metabolites. The medium strength, including carbohydrate concentration, was varied by diluting the complete medium, and various levels of HEPES buffer were incorporated into the medium. Our objective was to identify a combination of buffering and medium strengh which would allow culture growth to continue at neutral pH until the medium glucose was exhausted, without the accumulation of either pyruvate or the anaerobic markers lactate and ethanol. Table I summarizes the A m and pH
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Culture age, hours Figure 3. Comparison of the culture growth (A), glucose consumption(B),and pH profile (C) in unmodified complexMlO1 medium in the stirred bioreactor and in 10 mL of modified (0.3X dilution, buffered with 0.3 M HEPES at pH 7.5) medium in a shake flask.
values measured after 6 h of culture growth a t several medium strengths and buffer concentrations. We also calculated the glucose consumed, in grams per liter, per Am unit. This is abbreviated in the table as SPGC (for specific glucose consumption). The lowest HEPES buffer concentration which maintained a neutral pH until the cultures reached A m = 6 was 0.3 M. HEPES buffer had a modest adverse effect upon cell growth a t 0.3 M and a marked negative effect at 0.5 M. In the presence of 0.3 M buffer, 0.3X concentrated medium was sufficient to achieve an A m of 6. The specific glucose consumption was highest with the combination of 0.3 M HEPES buffer and 0.3X concentrated medium. The time course of growth of JM109 (pTAC-TGF67PE40) in shake flasks in 0.3X medium with 0.3 M HEPES buffer is compared to the full-strength medium in a stirred bioreactor in Figure 3A. In contrast to full-strength complex medium in shake flasks, growth in the dilute, buffered medium in flasks continued until the medium
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Table 11. Induction of TGFa-PE40 Expression in Original and Modified Complex MlOl Medium in Stirred Bioreactors and Shake Flasks.
stirred bioreactor A000
PH pyruvate (g/L)
2.0 7.2 0.1
early induction original modified medium medium Parameters at Induction 2.0 1.8 6.9 7.3 0.0
0.0
stirred bioreactor 6.0 7.2 0.0
late induction original medium
modified medium
6.0 6.6 0.3
5.8 6.9 0.0
Final Parameters 4.5 4.3 14.3 9.7 7.5 6.4 7.1 7.2 5.7 6.9 1.1 1.1 1.0 3.1 1.2 1.9 Protein Expression 186 207 182 118 73 136 TGFa-PE40 (pglmg of protein) 112 98 % bioreactor 62 115 Both early and late inductions were investigated in the stirred bioreactor and in the shake-flask model. As described in the text, biomass was estimated by Am; an absorbance of 2.0 was chosen as the model of early induction and 6.0 was the model for late induction. Culture pH and medium pyruvate concentrations were measured both at the time of IPTG addition and 4 h later. Specific protein expression is shown as pg of TGFa-PEPO per mg of total cell protein. The ratio of specific protein expression in the shake flask to expression in the comparable stirred bioreactor is shown as % bioreactor. The differences in final biomass level, as reflected by the absorbance at 600nm, reflect the growth differences as shown in Figure 3A. All shake flasks contained IO-mL medium volumes. 7.7 7.2
glucose (originally9 g/L) was exhausted (Figure 3B). When the growth reached an Am equivalent to 6 after 6 h of growth, the acetate concentration in the medium was 1.9 g/L, which was comparable to the 2.0 g/L measured in a stirred bioreactor at the same cell density. At that time, pyruvate, lactate, and ethanol were undetectable (data not shown), and the pH was 6.9 (Figure 3C). Thus, this combination of buffering and dilution met the criteria for investigating induction in complex medium in the shakeflask model. Growth and glucose consumption continued a t rates comparable to those in the stirred bioreactor until the medium glucosewas exhausted, the pH was maintained near neutrality, and there was no evidence of oxygen limitation. Recombinant Protein Expression in Full-Strength and ModifiedM101 Medium. IPTG-induced expression of recombinant TGFa-PE40 was compared in a stirred bioreactor, in shake flasks with full-strength medium, and in flasks with the dilute, buffered medium characterized above. Replicate cultures of each type were induced for expression at cell densities selected to model early and late logarithmic growth in stirred bioreactors: an A m of 2 was used to model the early logarithmic phase, and an Am equal to 6, while still within the logarithmic phase in stirred bioreactors, was used for the late logarithmic phase because of the limitations we had observed on physiological behavior in shake-flask cultures a t higher cell densities. In both cases, the specific TGFa-PE40 concentration was measured 4 h after induction for expression. The results are summarized in Table 11. Early induction of the shake flask with unmodified full-strength medium occurred when the pH was 6.9 and pyruvate and other metabolites characteristic of oxygen-limited growth were not yet detectable. Under these circumstances, specificexpression was comparable in the flask and the stirred bioreactor: 207 mg of TGFa-PE40lg of protein in the shake flask and 186 mg/g in the stirred bioreactor. With early induction, the specific expression level in the dilute buffered medium, 182 mg/g, was also comparable. Both conditions also duplicated our previous observation in the stirred bioreactor (George et al., 1992) that pyruvate accumulation accompanied the expression of TGFa-PE40. However, the situation is very different for later induction. As previously reported for the stirred bioreactor, late induction in the shake flasks led to a lower level of specific protein expression than did early induction. In the unmodified shake flask, by the time the A6w had reached 6, the pH had droppedto 6.6 and oxygen limitation
was indicated by the accumulation of pyruvate to a concentration of 0.3 g/L. IPTG induction of the unmodified shake flask at that time resulted in expression at only 62 % of the level reached in the stirred bioreactor. In contrast, the dilute buffered medium shake flask exhibited a pH of 6.9 at the time of late induction, pyruvate was undetectable in the medium, and induction resulted in specific protein expression comparable to late induction in the bioreactor. The culture growth after IPTG induction was less in all cases in the shake flasks than in the comparable stirred bioreactors; this did not affect the specific protein expression level. We attribute the failure of the unmodified medium to reflect the situation in the bioreactor to the poor physiological state of the shake-flask culture a t this age: its low pH and its compromised aeration as reflected in the accumulation of metabolites that are markers of poor oxygenation. The culture in the unmodified medium had already accumulated over 0.3 g/L pyruvate at the time of IPTG induction. In contrast, control of pH and metabolite accumulation in the dilute, buffered flask resulted in induced expression levels which were very similar to those in the stirred bioreactor. On the basis of this demonstration, we postulate that a shake-flask model for recombinant protein expression must meet several criteria to predict behavior in a stirred bioreactor. The buffering capacity of the medium must be sufficient to maintain the pH within physiological limits throughout the growth of the culture. The smallest possible medium volume should be employed to maximize aeration. In this investigation, 10 mL was the lowest practical volume which provided samples adequate for metabolite assays. Aeration should be monitored by measuring the accumulation of metabolites that are markers of oxygen-limited growth, and conclusions about expression levels should be based upon experiments where these metabolites are no higher than in stirred bioreactors a t comparable cell densities. For strain JM109 (pTAC-TGF57-PE40)in0.3X MlOl buffered with 0.3 M HEPES, pyruvate, lactate, and ethanol are undetectable and acetate is present at concentrations comparable to those in stirred bioreactors until an upper limit for A m of about 6. Application of the Scale-Down Model to Defined Medium Development. A similar evaluation was then conducted in a chemically defined medium. Growth, pH, and Metabolite Profiles in Defined Medium. Figure 4 summarizes the growth, glucose consumption, and pH profile of JM109 (pTAC-TGF57-
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Culture vessel Figure 5. Medium concentrations of glucose metabolites measured at a constant biomass level in stirred bioreactors and in shake flaskswith 10 or 50 mL of chemicallydefined FM medium. Table 111. Induction of TOFU-PE40Expreaaion in Chemically Defined FM Medium in Stirred Bioreactora and Shake Flasks. early induction late induction bioreactor flask bioreactor flask Parameters at Induction 1.9 1.5 6.8 5.6 Am 7.2 6.8 7.2 6.7 PH 0.0 0.0 pyruvate W L ) 0.0 0.0 Final Parameters &OO 2.6 2.9 9.8 5.7 PH 7.2 6.7 7.2 6.5 pyruvate (g/L) 1.4 1.0 3.8 2.4 Protein Expression TGFa-PEN (pg/mg 108 96 111 104 of protein) 89 % bioreactor 94 ~
6.0
Except for the medium composition, the experimental design was identical to that for Table 11. The late-induced flask exhausted the medium glucose without increasing ita biomass level after induction. The stirred bioreactor, which was glucose-fed, demonstrated a modest increase in biomass level after induction, but lea than was observed in complex medium (Table 11).
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C . b 0 2 4 6 8 10
Culture age, hours Figure 4. Culturegrowth (A),glucose consumption (B),and pH profiie (C) in stirred bioreactors and in shake flash with chemically defined FM medium containing 10 g/L glucose.
PE40) in fed-batch stirred bioreactors and in shake flasks with either 10or 50 mL of synthetic FM medium. Growth in both medium volumes continued until the available glucose (originally 10 g/L) was exhausted, but as in the complex medium, the final biomass level was higher in the flasks with the lower medium volume. In the stirred bioreactor, the early portion of the growth curve was comparable to the 10-mL flask; glucose feeding allowed biomass to continue to accumulate up to an A m of 24. In the shake flasks,the phosphate buffer in the FM medium as formulated was adequate to maintain a nearly neutral PH. Figure 5 summarizes accumulated glucose metabolites at a constant biomass in synthetic medium. As with complex medium, the constant biomass selected was indicated by A m 5-6. Better oxygenation, as indicated by the absence of accumulation of pyruvate and lactate, was achieved with a lower shake-flask medium volume. Acetate accumulation in FM medium was lower than in the MlOl medium, and much lower in the bioreactor and
the 10-mLflasks than in the 50-mL flasks. Both pyruvate and lactate were detectable in the cultures with the 50mL volume, but not in the 10-mL cultures or bioreactors. No ethanol was detected in any of the cultures. The culture of JM109 (pTAC-TGF57-PE40) in 10 mL of FM medium in a shake flask meeta the requirements previously established with complex medium for a model system for evaluation of recombinant protein expression: culture growth to full exhaustion of the available glucose while maintaining the pH at a physiological level without accumulation of the metabolites associated with oxygenlimited growth. Recombinant Protein Expression. Recombinant TGFa-PE40 protein expression in FM medium in flasks and stirred bioreactors is shown in Table 111. As in complex medium, both early and late inductions were evaluated by adding IPTG at biomass levels corresponding to Am = 2 and A m = 6. In synthetic FM medium, only modest increases in biomass levels were measured following IPTG addition, although more culture growth occurred in the glucose-fed, stirred bioreactor. At both induction times, specific recombinant TGFa-PE40 protein expression in the shake-flask model paralleled the behavior in the comparable stirred bioreactor. Additionally, pyruvate accumulated in the medium in response to induction, as it does in complex medium.
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Discussion The present study has examined several interrelated factors that complicate the direct utilization of high cell density production media in shake-flask studies and has defined physiological parameters which can be measured to predict the validity of a shake-flask model of expression in a stirred bioreador. Thishas allowed us to devise shakeflask culture conditions for JM109 (pTAC-TGF57-PE40) in both complex and defined media, so that induction for expression of TGFa-PE40 produces specific expression levels comparable to those in stirred bioreactors. The physiological parameters monitored in shake flasks were culture pH, acetate accumulation, and aeration as indicated by metabolites associated with oxygen-limited growth. In unbuffered, high-glucose complex medium, the specific expression was comparable to that of stirred bioreactors only when induction occurred at very low biomass levels. Specific expression was lower when induction occurred after metabolites indicative of compromised aeration had appeared in the medium. In contrast, when the medium was diluted and buffered and adequate oxygenation was demonstrated at the time of inducer addition, specific expression in flasks and stirred bioreactorswas comparable over a wider range of induction times. Similar results were obtained in a chemically defined medium when these same factors were controlled. As already noted, recombinant protein expression is known to be sensitive to acetate concentration and, therefore, to initial glucose concentration (Zabriskie et al., 1987; Bauer et al., 1990; Bech-Jensen and Carlsen, 1990; MacDonald and Neway, 1990; Rinas et al., 1989; Luli and Strohl, 1990). This study has demonstrated that monitoring the aeration of shake-flask cultures by measurement of the concentrations of metabolites associated with oxygen-limitedgrowth can be used to define the limits of culture growth in a shake flask that are models of growth and function in a stirred bioreactor. While there is little documentation of the influence of 0 2 limitation on expression levels in E. coli except in the special case of 02-regulated promoters (Oxer et al., 1991; Khosla et al., 1990),it is possible that shifting to anaerobic metabolism could influence gene expression by affecting protein synthesis or other cellular processes. The use of flasks withdifferent geometries (Tunac, 1989) or higher agitation may extend the period of oxygen sufficiency. The defined medium shake-flask model has been documented here to correspond well to the stirred bioreactor. This provides a convenient means for investigating the effects of medium supplementation and modification on TGFa-PE40 expression in defined medium. The objective is to develop a defined medium in which expression levels are comparable to those achieved in the complex medium. For example, since the product of interest is a protein, amino acids are the most likely candidates for nutritional supplementation. The effect of supplementation on both growth and expression could be investigated by making amino acid additions either before or after IPTG induction. Such a series of experiments would be technically overwhelming, even in smallscale stirred bioreactors, but feasible in shake flasks. In such experiments, it would be important to continue to monitor pH and metabolite accumulation during growth to confirm that the model still corresponds to the pH-
controlled, aeration-controlledsituation which exists in a stirred bioreactor.
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Abstract publishedinAdvance ACSAbstracts, August 15,1993.
