A Single Nutrient Feed Supports Both Chemically Defined NS0 and CHO

A Single Nutrient Feed Supports Both Chemically Defined NS0and CHO Fed-Batch Processes:Improved Productivity and Lactate Metabolism Ningning Ma,JoAnn Ellet,Centy Okediadi,Paul Hermes,Ellen McCormick,and Susan Casnocha Bioprocess R&D,Global Biologics,Pfizer Inc,Chesterfield,MO63017DOI10.1002/btpr.238Published online July27,2009in Wiley InterScience().A chemically defined nutrient feed(CDF)coupled with basal medium preloading wasdeveloped to replace a hydrolysate-containing feed(HCF)for a fed-batch NS0process.TheCDF not only enabled a completely chemically defined process but also increased recombi-nant monoclonal antibody titer by115%.Subsequent tests of CDF in a CHO process indi-cated that it could also replace the hydrolysate-containing nutrient feed in this expressionsystem as well as providing an80%increase in product titer.In both CDF NS0and CHOprocesses,the peak lactate concentrations were lower and,more interestingly,lactate metab-olism shifted markedly from net production to net consumption when cells transitioned fromexponential to stationary growth phase.Subsequent investigations of the lactate metabolicshift in the CHO CDF process were carried out to identify the cause(s)of the metabolicshift.These investigations revealed several metabolic features of the CHO cell line that westudied.First,glucose consumption and lactate consumption are strictly complementary toeach other.The combined cell specific glucose and lactate consumption rate was a constantacross exponential and stationary growth phases.Second,Lactate dehydrogenase(LDH)ac-tivityfluctuated during the fed-batch process.LDH activity was at the lowest when lactateconcentration started to decrease.Third,a steep cross plasma membrane glucose gradientexists.Intracellular glucose concentration was more than two orders of magnitude lowerthan that in the medium.Fourth,a large quantity of citrate was diverted out of mitochondriato the medium,suggesting a partially truncated tricarboxylic acid(TCA)cycle in CHO cells.Finally,other intermediates in or linked to the glycolytic pathway and the TCA cycle,whichinclude alanine,citrate,isocitrate,and succinate,demonstrated a metabolic shift similar tothat of lactate.Interestingly,all these metabolites are either in or linked to the pathwaydownstream of pyruvate,but upstream of fumarate in glucose metabolism.Although the spe-cific mechanisms for the metabolic shift of lactate and other metabolites remain to be eluci-dated,the increased understanding of the metabolism of CHO cultures could lead to futureimprovements in medium and process development.V C2009American Institute of ChemicalEngineers Biotechnol.Prog.,25:1353–1363,2009Keywords:CHO,NS0,chemically defined process,lactate,metabolic shift,metabonomics,metabolomicsIntroductionHydrolysates,especially those of nonanimal origin,are widely used as serum replacements in therapeutic protein production processes using mammalian cells.Hydrolysates can improve cell growth and protein yield in serum-free processes(Burteau et al.,2003;Heidemann et al.,2000; Sung et al.,2004;Xie et al.,1997).However,there are a few limitations that complicate hydrolysates’utilization.First of all,lot-to-lot variation exists for hydrolysates(Luo and Chen,2007;Zhang et al.,2003).Prescreening using small-scale performance testing is typically required to eliminate underperforming lots.Second,the nutrient composition in hydrolysates is not balanced for cell consumption.Various components,such as ash and salts,are either not required by the cells or are required at much lower levels than supplied. Third,the chemically undefined nature of hydrolysates hin-ders the scientists’understanding process of cell metabolism. For instance,it is not well understood how mammalian cells metabolize amino acids in peptides,so that amino acid con-sumption analysis based on free amino acids measurements may or may not be accurate(Nyberg et al.,1999). Development of various chemically defined media for NS0cells and CHO cells has been reported(Chen et al., 2000;Hata et al.,1992;Huang et al.,2007;Zhang and Rob-inson,2005),and commercial chemically defined media are readily available from all major mammalian cell culture me-dium manufacturers.However,the development of chemi-cally defined fed-batch processes that support rapid cell proliferation,high cell density,and high production rate has been challenging.Chemically defined fed-batch processes have been reported recently.Gong et al.(2006)developed a chemically defined fed-batch process for hybridoma cells.Correspondence concerning this article should be addressed to N.Maat ningning.ma@pfi.V C2009American Institute of Chemical Engineers1353Peak viable cell density reached about8Â106cells/mL,but viable cells dropped sharply after a5-day culture.Burky et al.(2006)developed a generic chemically defined fed-batch process for NS0cells.The process supported peak via-ble cell density around1Â107cells/mL and product titer of 2.64g/L on Day13.Lactic acid,or lactate,has long been recognized as a major by-product in cell culture processes(Hu et al.,1987; Miller et al.,1989;Ozturk and Palsson,1992).Base addition to neutralize lactic acid elevates medium osmolarity,which, in turn,inhibits cell growth and causes viability to drop. Reducing lactic acid production had been shown to increase peak cell density,extend process duration,and increase recombinant protein yield.Four strategies had been reported in the literature to reduce lactate production.Thefirst one is to reduce medium glucose concentration to limit glucose supply(Cruz et al.,1999;Zhou et al.,1995,1997b).Using an online dynamic feeding protocol,Zhou et al.(1995, 1997b)successfully maintained glucose at a low level of about0.5mM.As a result,lactate production was reduced and higher peak cell densities and higher viability were achieved.A second strategy is to use alternative,slow-con-suming carbon sources(Altamirano et al.,2004).Altamirano et al.(2004)used a feeding strategy in which glucose and galactose feeds were alternated.This strategy forced CHO cells to consume more residual lactate than the culture fed with glucose only.As a result,harvest viability was mod-estly improved.The third strategy is to downregulate lactate dehydrogenase-A(LDH-A)(the LDH subunit that effectively catalyzes pyruvate to lactate conversion)using a wide vari-ety of techniques including homologous recombination (Chen et al.,2001),antisense mRNA(Jeong et al.,2001; Paredes et al.,1999),and small interfering RNA(Kim and Lee,2007a).Chen et al.(2001)compared a LDH-A downre-gulated hybridoma clone to a control clone.In the culture utilizing the LDH-A downregulated cell line,lactate concen-tration was about35%lower and monoclonal antibody titer was about200%higher.However,although Kim and Lee (2007a)reduced lactate production rate even further(by45–79%),the recombinant thrombopoietin product rates of three LDH-A downregulated clones were not statistically better than the control clone.A fourth strategy is to enhance the flow of glucose carbon into tricarboxylic acid(TCA)cycle.A focus area has been the overexpression of pyruvate car-boxylase of yeast(Irani et al.,1999,2002)or human origin (Kim and Lee,2007b).In a perfusion culture,Irani et al. (2002)demonstrated that pyruvate carboxylase-transfected BHK-21cells produced half of the lactate when compared with the control cells.Cell density was about50%higher and recombinant erythropoietin production rate was about 100%higher.More recently,it was reported that the expres-sion of antiapoptosis genes,E1B-19K and Aven(EA167), could reduce lactate production of CHO cells(Dorai et al., in press).In batch cultures with or without glucose limita-tion,a CHO clone transfected with antiapoptotic genes gave lower lactate concentration compared with the control cell line at corresponding conditions.In this study,we developed a chemically defined nutrient feed(CDF)with companion production medium preloading to replace a hydrolysate-containing feed(HCF)for a fed-batch NS0process.The CDF was later investigated in a CHO process.In both NS0and CHO processes,CDF resulted in higher product titer and lower peak lactate con-centration.The CDF processes also exhibited a dramatic lac-tate metabolic shift from net production to net consumption. At the end of the process,lactate concentration in the CDF processes was almost undetectable.Subsequent investigations were carried out to better understand this metabolic shift in the CDF CHO process.Materials and MethodsCellsNS0and CHO cells producing test recombinant monoclo-nal antibodies were used in this study.Both cell types used glutamine synthatase as the transfection selection marker. ProcessNS0and CHO cells were routinely subcultured in250mL shakeflasks(Corning,Acton,MA)in chemically defined hy-bridoma and CHO media,respectively.Both hybridoma and CHO media are commercially available.The CHO medium was supplemented with25mM methionine sulfoximine(MSX). Two-liter Applikon bioreactors(Applikon,Foster city, CA)with a1-L working volume were used in this study.In these bioreactors,two pitched blade impellers provided mixing and gas bubble dispersion.Temperature was controlled at 36.5 C using a heating blanket.pH was controlled at7.0by carbon dioxide sparging through a ring sparger and by adding either0.5N sodium hydroxide or7.5%sodium bicarbonate. Dissolved oxygen(DO)was controlled at30%air saturation using air overlay and oxygen sparging.The fed-batch process for NS0and CHO cells was very similar.Cells are seeded at 2.5Â105viable cells/mL in corresponding NS0or CHO pro-duction media.Starting from Day3,a nutrient feed was added into the bioreactors at28mL/L/day and16mL/L/day for NS0 and CHO cells,respectively,till the end of the process.Glucose was fed separately to control glucose concentration around2g/ L.Bioreactors were sampled daily for offline measurements. Routine bioreactors offline measurementsCell density and viability were measured using a Cedex cell counter(Innovatis,Bielefeld,Germany).Offline DO, pH,glucose concentration,and lactate concentration were measured with a BioProfile chemical analyzer(Nova Bio-medical,Waltham,MA).A portion of the broth from each daily sampling wasfiltered through a0.2l m polyethersul-fonefilter(Pall,Ease Hills,NY)for product titer and amino acid measurements.Monoclonal antibody titer was measured using an Agilent HPLC(Hewlett Packard,Waldbronn,Germany)with a POROS protein-A affinity column(Applied Biosystems,Fos-ter City,CA).The column was calibrated using correspond-ing reference standards before titer quantification. Intracellular LDH activity measurementAbout2–4mL of broth was taken out of bioreactors and transferred to a centrifuge tube chilled on ice.The cells were washed twice with cold phosphate buffered saline(PBS).In each wash,the cells were pelletted in a refrigerated centri-fuge at150g for5min,the supernatant was discarded,and the cell pellet was gently resuspended in4mL of cold PBS. After the second wash,cells were diluted to5Â105viable cells/mL and lysed using freeze-thaw.In the freeze-thaw procedure,cells werefirstflash frozen in liquid nitrogen andthen thawed overnight at2–8 C.LDH activity was quantified using a colorimetric LDH assay kit(CytoTox96nonradioac-tive cytotoxicity assay,Promega,Madison,WI)and a spec-trophotometer(Molecular Devices,Sunnyvale,CA).Sample preparation and measurement followed manufacturers’instructions.The LDH assay kit quantifies LDH activity based on the conversion rate of lactate and NADþto pyru-vate and NADH.Sample collection for metabonomics assayBoth cell and spent medium samples were collected for metabonomics analysis.To collect cell samples,cell culture broth containing2Â107viable cells was removed from the bioreactor and immediately chilled in an ice bucket.The cells were pelleted at1,000g for3min in a refrigerated cen-trifuge(Bechman Coulter,Fullerton,CA)and then rinsed twice using cold PBS.After two washes,the cell pellet was flash frozen in liquid nitrogen and stored atÀ80 C.A2mL spent medium sample was collected where cell broth was first centrifuged at1,000g for3min and then the supernatant wasfiltered through a0.2l m polyethersulfonefilter.Spent medium samples were also stored atÀ80 C.After all sam-ples were collected,they were sent on dry ice to the analysis provider.Sample preparation and metabonomics assaySample preparation and metabonomics analysis were con-ducted at Metabolon(Durham,NC).The metabonomics methodology is detailed elsewhere(Lawton et al.,2008)but briefly,the samples were thawed,small molecules were extracted,and the reconstituted extracts were split for analy-sis by GC and LC mass spectrometry.Chromatographic sep-aration of all ions in each sample was followed by library matching of these ions to Metabolon’s reference library of standards.The identity of metabolites was determined by matching the combination of chromatographic retention index and mass spectra signatures compared with the refer-ence library entries.Results and DiscussionDevelopment of a chemically defined nutrientfeed for NS0processA CDF was developed to replace a hydrolysate-containingnutrient feed(HCF)that was previously used for a fed-batch NS0process.In the hydrolysate-containing fed-batch pro-cess,the basal medium is chemically defined,but the nutri-ent feed contains a nonanimal-derived hydrolysate.The limitations of hydrolysates,such as lot-to-lot variation, prompted the development of the CDF to achieve a fully chemically defined process.The development took a top-down approach,as illustrated in Figure1.There were three major development steps:(1) develop an over-rich nutrient feed,(2)simplify formula by removing unnecessary components and reduce the concentra-tion of overfeed components,and(3)polishing and formula-tion.The plan for the over-rich solution was that it should contain all nutrients that NS0cells require and all required nutrients should be at sufficiently high concentrations.To ensure that all necessary components were included in the over-rich solution,over90components were incorporated. These components came from the formula of a Pfizer propri-etary chemically defined NS0medium and a previously developed CDF.To ensure that all necessary components were in ample supply,most components were added at a high concentration.In the next step,nutrients in the over-rich solution were divided into eight rough groups to balance their ratio.Grouping was based on nutritional category(trace elements,nucleotides,etc.)or by solubility(low pH,ethanol, etc.).In the subsequent formula simplification steps,compo-nents were screened in groups for two cycles,first in rough groups and then in refined groups.In thefirst cycle,two of eight rough groups were found unnecessary where their exclusion rendered no detectable difference in process per-formance(cell density,viability,and product titer).Compo-nents in these two subgroups were subsequently removed from the formula.The remaining components were divided into13refined groups again based on nutritional category and solubility.Most groups contained less thanfive compo-nents.In the second cycle of screening,five refinedgroups Figure1.The top-down approach used in the chemically defined nutrient feed development.The development included three major steps:(I)obtain an overrich nutrient feed,(II)formula simplification,and(III)polish-ing and formulation.were found to offer no detectable beneficial (or detrimental)effect on the process performance and were removed.In the final step,the components in the simplified formula,with less than 40components,were balanced for optimization.Amino acids were optimized based on spent medium analy-sis,whereas the other components were optimized relied on refined group titration study.Finally,all components were formulated into a single pH neutral nutrient feed.For com-ponents with low solubility in the feed,the portion over the solubility limit was preloaded into the basal medium.All de-velopment studies were conducted in fed-batch shake flasks.At each milestone,the performance was confirmed in 2L bench-scale bioreactors.The whole development took $9months,with each of three major steps for about 3months.A comparison of the CDF process and HCF process in 2L bioreactors is presented in Figure 2,where results are the average of three bioreactor runs and error bars indicate 1SD.Cell growth phase was extended in the CDF process where the peak viable cell density reached 15Â106cells/mL,$50%higher than that in the HCF process.Viability was modestly improved in the CDF process,the harvest via-bility was about 10%higher in the CDF process.Product ti-ter was much higher in the CDF process after Day 5.At harvest on Day 11,product titer in the CDF process was 115%higher than that in the HCF process.This 115%increase was from a 33%increase in the integral viable cellconcentration and a 62%increase in cell specific productiv-ity (Q p ).These results demonstrate that CDF not only allows for removing hydrolysate from the process but also delivers better cell growth and productivity.Another benefit of the CDF process is the lower lactate concentration.As shown in Figure 2b,lactate in the CDF process was lower throughout the whole process,and the peak concentration was only half of that in the HCF process.In both processes,cells switched from net lactate production to net consumption around Day 5.In the CDF process,residual lactate was consumed in 2days and remained lower than the detection limit until har-vest.In the HCF process,the lactate concentration was gen-erally higher and net lactate production resumed,although at a very slow rate,after Day 8.The benefit of the top-down approach to nutrient feed de-velopment is that it eliminates nutrient limitation up front,thereby allowing the development to focus on nutrient bal-ancing and simplification.The risk of this strategy is the inclusion of unnecessary components and overfeeding,which could be toxic or inhibitory to cell growth or production.The successful development of a CDF demonstrates that the risk is manageable.During the development,many compo-nents,such as vitamins and minerals,were found to be non-toxic even at a concentration one or two orders of magnitude higher than what the cells needed.This observation agrees with Xie and Wang (1994),who found that a high concentra-tion of vitamins was not harmful to animal cells.Amino acids have the potential to be inhibitory to cell growth at high concentrations.Fortunately,amino acid overfeeding can be avoided using spent medium analysis.Evaluation of the CDF in CHO processesThe CDF developed for NS0cells was later tested on CHO cells and compared with the CHO HCF process.The medium used in both processes is a commercially available chemically defined CHO medium.As shown in Figure 3,the CDF process offered clear advantages over the HCF process.Results shown in Figure 3were the average of two duplicate bioreactors.Peak viable cell density reached $18Â106cells/mL in both processes.However,viability was main-tained better in the CDF process.When the processes were terminated on Day 15,viability for the CDF process was around 80%,whereas that for the HCF process had declined to 50%.Product titer was similar in both processes till Day 10,after which titer started to level off in the HCF process,whereas that in the CDF process kept rising linearly till har-vest.By Day 15,product titer in the CDF process was 82%higher than that in the HCF ctate concentration was much lower in the CDF process after Day 7.By Day 10,lactate concentration in the CDF process became close to undetectable,whereas that in the HCF process stayed at 1.8–2.6g/L.Lactate metabolic shift in the CDF processesA major metabolic feature of the CDF processes is the dramatic shift of lactate metabolism from net production to net consumption.On one hand,the metabolic shift is benefi-cial for the process as it significantly reduced the concentra-tion of a major cellular metabolic by-product.At the end of the process,lactate concentration was almost undetectable.On the other hand,the shift could be due to a glucose carbon limitation,which may have a detrimental effect on thecellFigure parison of the CDF (solid lines)and HCF (dot-ted lines)NS0processes.(a)Viable cell density and viability.(b)Product titer and lac-tate concentration.The results shown are the average of three replicate batches.Error bar indicates 1SD.growth and productivity.Although glucose supply was not limited in both CDF NS0and CDF CHO processes (glucose concentration [1g/L),possible limitations could still exist in the glucose transportation step or the flux through the gly-colytic pathway.To better understand and to identify the possible mechanism(s)for the lactate metabolic shift,we examined carbon metabolisms in the CDF CHO process.The lactate metabolic shift in fed-batch processes has been previ-ously reported with different cell expression systems,includ-ing DHFR-CHO (Tsao et al.,2005),GS-NS0(Zhou et al.,1997a),and a non-GS-NS0(Burkey et al.,2006).However,no thorough investigation of this phenomenon has been pub-lished.We focused our investigation on CHO cells,where the cell line used was a subclone of the CHO cell line used in the initial CDF evaluation (Figure 3).The CDF fed-batch process was kept the same.As indicated in Figure 4a,the lactate metabolic shift took place in the same period when cells were transitioning from exponential to stationary growth phase.The same timing cor-relation between the lactate metabolic shift and cell growth phase transition exists in previous publications using differ-ent mammalian cell types (Burkey et al.,2006;Tsao et al.,2005;Zhou et al.,1997a).Whether there is a cause-effect relationship between lactate metabolic shift and cell growth slowdown is unknown.One possibility is that the metabolic shift results from the cell growth phase transition.High lac-tate production is a hallmark of transformed cells,a phenom-enon established more than 70years ago (Warburg,1930).High lactate production has been attributed in the literature to the upregulation of various enzymes in the glycolytic pathway (Board et al.,1990;Dang and Semenza,1999;Kondoh et al.,2005).As cells exit the rapid proliferating phase,they may concurrently cease high lactate production.Alternatively,the lactate metabolism shift could be a result of glucose carbon limitation because of restricted flux through the glycolytic pathway.Analysis of glucose consumption revealed a strong com-plementary relationship between glucose and lactate con-sumption.Cumulative glucose utilization,lactate production,and net glucose consumption (defined as combined glucose and lactate consumption)from the two duplicate bioreactors referred to in Figure 4a are illustrated in Figure 4b.The slope of the curves in Figure 4b represents either cell-spe-cific substrate consumption (glucose or net glucose)or pro-duction (lactate)rate.The glucose consumption and lactate production rates fluctuated during the process.However,net glucose consumption rate was steady throughout the whole process.This observation inferred that (1)cell-specificFigure parison of the CDF (solid lines)and HCF (dot-ted lines)CHO processes.(a)Viable cell density and viability.(b)Product titer and lac-tate concentration.The results shown are the average of two duplicatebatches.Figure 4.Cell growth and glucose and lactate metabolism ofCHO cells in the CDF process in two duplicate bioreactors.Bioreactor 1,solid symbols;Bioreactor 2,hollow symbols.(a)Cell growth and lactate profile.The arrow indicates the correla-tion between shift of lactate metabolism and cell growth phase transition.(b)Weight cumulative glucose consumption,lactate production,and net glucose consumption.The net glucose con-sumption is defined as glucose consumption minus lactate pro-duction.The glucose and lactate metabolism can be divided into three phases:(I)high glucose consumption and net lactate production,(II)low glucose consumption and net lactate consumption,and (III)high glucose consumption and very low residual lactate concentration.pyruvate consumption rate in TCA cycle was a constant throughout the whole process and (2)glucose and lactate consumption rate was tightly controlled so that the flux from glucose to pyruvate and the flux from lactate to pyruvate are strictly complementary to each other.NADH homeostasis,as previously being suggested (e.g.,Zhou et al.,1997a),could be the cause of the tight control of glucose and lactate consumption.The three major path-ways related to NADH homeostasis are illustrated in Figure 5.The conversion of pyruvate to lactate recycles NADH pro-duced in glycolysis back to NAD þ.When the conversion is reversed,it competes with glycolysis for NAD þ.The reduced availability of NAD þcould slow down the glycoly-sis rate.NADH is also recycled back to NAD þinside mito-chondria through the aspartate–malate shuttle (Figure 5)or similar shuttle systems.We need to assume that the shuttle rate was a constant for NADH homeostasis to exert the com-plementation effect on glucose and lactate consumption.It should be noted that in Stage II and III of Figure 4b,all NADH needed to be recycled in mitochondria as no NADH was recycled through pyruvate to lactate conversion.LDH activity changes during the processCell-specific LDH activity was monitored in two bioreactor runs using the same CHO subclone and CDF process.As shown in Figure 6,cell-specific LDH activity oscillated in both runs.During the exponential growth phase,LDH activ-ity first increased and then decreased.It further decreased and reached the lowest level during the growth phase transi-tion from exponential to stationary phase.With the decrease of cell-specific LDH activity,lactate metabolism shifted from net production to net consumption.After the lactate meta-bolic shift,LDH activity gradually increased and reached its maximum at the end of the process.Cell size was consistent during the fed-batch process (data not shown),so that the LDH activity variation was not due to a cell volume change.Although there seems to be a linkage between LDH activity and lactate metabolism,the results were hard to interpret.First,in the LDH assay that we used,LDH activity is quanti-fied by the rate of lactate to pyruvate conversion,hence it is counterintuitive to find that the lowest activity measured off-line correlated to the highest lactate to pyruvate conversion rate in the process.Second,although decreased LDH activity was correlated with net lactate consumption,subsequent increase in LDH activity did not reverse the metabolism backto lactate production.More studies are needed to fully under-stand the role of LDH activity variation in lactate metabolic shift,such as the ratio between the two major LDH subunits,LDH-A and LDH-B,which favor the pyruvate–lactate con-version in opposite directions,and the ratio between the other two substrates in the pyruvate–lactate conversion reaction,NAD þand NADH,where a high NAD þ/NADH ratio favors lactate to pyruvate conversion.Shift of amino acid metabolismIn addition to lactate metabolic shift,several amino acids also demonstrated shifts of metabolism during the fed-batch process,as shown in Figure 7.The results were from a sin-gle bioreactor run.The alanine shift is of particular interest as similar to lactate,alanine can be derived from pyruvate.Figure 7showed that the pattern of alanine metabolic shift was similar to that of lactate,although the alanine production and consumption rate was much lower than that of lactate.Asparagine net consumption rate was initially very high,but decreased rapidly midway through the process.The decrease was due to asparagine supply limitation.As most asparagine is likely utilized in the TCA cycle through aspartate and then oxaloacetate,under asparagine limitation,net aspartate consumption rate would increased,as observed in Figure7.Figure 5.Major NAD 1and NADH conversionpathways.Figure 6.Variation of LDH activity in the CHO CDF process.Results were from two similar bioreactor runs using the same clone.Bioreactor 1,solid symbols;Bioreactor 2,hollow symbols.。