Heuristic Optimization of Antibody Production by CHO

NOTES

Heuristic Optimization of Antibody Production by Chinese Hamster Ovary Cells

Sandeepa Sandadi,Semsi Ensari,*and Brian Kearns

Biotechnology Development,Schering-Plough Research Institute,1011Morris Avenue,Union,New Jersey07083 Large-scale production of monoclonal antibodies necessitates the development of a

commercially viable process using the appropriate bioreactors,culture medium,and

optimal feeding strategies.In the development of feeding strategies for higher antibody

titers it is critical to assess the effects of limiting substrates on cell culture longevity

and antibody production.In this study,glucose and L-glutamine were identified as

limiting substrates and their effects on culture longevity and antibody production were

evaluated in small-scale experiments.The results suggested that an optimal feeding

strategy should account for the osmolality profile of the culture.The heuristic approach

taken to optimize the antibody production showed that the fed-batch cultivation is

superior to batch culture and maintaining low osmolality during growth phase

increases cumulative viable cell density and thus leads to higher final antibody titer.

Introduction

Cultured mammalian cells are widely used for the production of potential therapeutics.The use of optimal feeding strategies and culture medium,in addition to appropriate bioreactors,enhances the yield of the anti-body product.A typical fed-batch operation involves feeding the nutrients necessary for cell growth,the precursors for product formation,or the inducers.

For most animal cells glucose and L-glutamine are the major sources of carbon and nitrogen,respectively.The metabolism of both glucose and L-glutamine is inter-related,and the proportion of each nutrient consumed by the different cellular pathways depends on the meta-bolic state of the cells(1).Therefore,their effect on culture longevity and antibody production needs to be studied when developing feeding strategies.The signifi-cance of glucose and L-glutamine for growth and main-tenance of Chinese hamster ovary(CHO)cells has been extensively studied.In a recent study,Sun et al.(2) showed that L-glutamine cannot support recombinant CHO cell growth and maintenance in the absence of glucose.Subsequently,the metabolism of CHO cells at low glucose concentrations was investigated by determin-ing intracellular metabolites.It was observed that the shortage of energy from reduced glucose metabolism resulted in cellular growth limitation,and amino acid catabolism could not replenish the energy deficit(3). Alternatively,a large glucose addition leads to significant changes in the osmolality of the culture.This is particu-larly true in bioreactors,where the osmolality of the culture increases as a result of two reasons,base addi-tions to control the pH and glucose additions to supply necessary energy for the culture.Past studies emphasized the importance of osmolality on cell culture and antibody production.Among various methods to increase antibody production,hyperosmolality has been recognized as an economical solution(4-9).Moreover,in a fed-batch culture,repeated feeding of medium concentrates leads to a substantial increase in medium osmolality(10).In addition to antibody production,elevated osmolality can also affect growth rate,duration of exponential growth, and cell death rate.However,within a range of osmolality (300-400mOsm/kg),an increased specific antibody production rate can compensate for a decreased growth rate and cell concentration to yield a higher final antibody concentration(11).Previously,a few culture strategies were developed to optimize antibody production using the osmolality dependence of the cultures(6,9).In contrast, Zhu et al.(12)observed that increased specific productiv-ity in hyperosmolal batch culture did not result in a significant improvement in final titer because of sup-pression of cell growth.Therefore,the effects of osmolality on the culture should also be considered when interpret-ing the effects of glucose in the development of feeding strategies for the bioreactors.

With the availability of a limited number of bioreactors, there have been intensive efforts to develop microbio-reactor systems to diminish scale-up problems.Addition-ally,recent studies showed that shake flasks can be modified to mimic conventional stirred tank systems(13, 14).Previous studies also investigated the feasibility of using shake flasks to culture animal cells and verified their scalability to larger volumes(15,16).Although bioreactors offer a controlled environment for process parameters,it is very difficult to maintain constant levels of pH and dissolved oxygen in shake flasks.Therefore, the coupled effects of pH control and osmolality in the bioreactors should be considered when interpreting the results of shake flask experiments.

*To whom correspondence should be addressed.Ph:(908)820-6354.Fax:(908)820-6995.E-mail:semsi.ensari@https://www.360docs.net/doc/993164718.html,.1537

Biotechnol.Prog.2005,21,1537?1542

10.1021/bp0501266CCC:$30.25?2005American Chemical Society and American Institute of Chemical Engineers

Published on Web07/27/2005

In shake flasks,statistically designed experiments

provide effective and efficient ways to investigate the

effects of factors on response variables.The sequential

and predictive nature of these designs allows greater

understanding of the effects of these factors on culture

performance when compared to the classical one-factor-

at-a-time approach and can detect potential interactions

among the factors.When experimenting with known

factors,their effects and interactions have to be first

estimated using factorial designs,followed by response

surface methods to optimize the response(17).On the

other hand,when only a few factors are under investiga-

tion,an alternative approach is to skip factorial designs.

The response surface graph generated from the effect of

two or more quantitative factors may provide useful

information to develop experimental strategies.In the

past,response surface methods were used for rational

optimization of culture media and/or operating conditions

of both microbial and cell cultures.In contrast,the use

of response surfaces to develop heuristic strategies for

bioreactor optimization has not been reported.In this

study,the effects of limiting nutrients and osmolality on

culture longevity and antibody production were investi-

gated in shake flasks using response surface methods.

Because the shake flask experiments do not allow for

rational optimization of cultivations in bioreactors,the

results from the response surface for antibody production

were interpreted to develop and test heuristic osmolality

profiles to improve final antibody titer in bioreactors.

Materials and Methods

Cell Line and Culture Conditions.CHO dihydro-

folate reductase(DHFR)-deficient mutant cell line DXB-

11expressing a proprietary recombinant protein was

propagated in shake flasks at37°C with7.5%CO2

overlay at100rpm using protein-free,animal-component-

free CHO medium(Sigma C5467,St.Louis,MO)con-

taining4g/L glucose and supplemented with4mM L-glutamine.Cellbags(Wave Biotech LLC,Bridgewater, NJ)for inoculum were cultured at37°C with7.5%CO2

overlay,and the platform rocker was set at10rocks per

minute with an angle of8°.Cultures were passaged in

their exponential phase of growth using split ratios of

1:3,1:4,or1:5to achieve seeding densities around0.3×

106cells/mL.

Offline Measurements.Samples were analyzed for

viable and total cell counts by Cedex System(Innovatis

AG,Bielefeld,Germany),glucose/lactate and L-glutamine/ L-glutamate by YSI2700Select(YSI Inc.,Yellow Springs, OH),pH/pO2/pCO2by ABL5(Radiometer America Inc., Westlake,OH),NH4+by Nova BioProfile100Plus(Nova Biomedical Corp.,Waltham,MA),and osmolality by Advanced Micro-Osmometer(Advanced Instruments, Norwood,MA).The antibody content of the culture supernatant samples was quantified by a reverse-phase chromatography assay that was based upon a reported technique(18).The liquid chromatography system(Agi-lent1100system,Palo Alto,CA)was equipped with an R2/10Poros column(30mm×2.1mm,Applied Biosys-tems,Cambridge,MA).The column temperature was maintained at70°C.The flow rate was initially set at 0.1mL/min until the column temperature stabilized at 70°C,and it was set to2mL/min during all gradient segments.Solution A was0.2%trifluoroacetic acid in water,and Solution B was0.2%acetic acid in aqueous acetonitrile(90:10).The gradient program started with 0.5min at32%Solution B followed by a5-min gradient from32%to60%Solution B.Consequently,a1-min gradient to100%Solution B was followed by a1-min gradient back to32%Solution B.The program was

finished by a2-min equilibration at32%Solution B.The

detection was carried out at280nm by either a photo-

diode array detector or a multiwavelength detector using

5-100μL injections depending on the titer of the culture.

The linear range for the assay is from0.2to2.5μg of

antibody and the assay variability is3%.

Response Surface Methods.Response surface graphs

visualize the effects of two factors very effectively.Box

and Wilson(19)introduced central composite design

(CCD)as an alternative to3n factorials to estimate

quadratic response surface equations where n is the

number of https://www.360docs.net/doc/993164718.html,D consists of2n factorial points with

2n additional treatment combinations called axial points

and m replications at the center of the design.The

designs used in this study were rotatable and contained

eight factorial points,four axial points,and four center

points(Design-Expert,Version6.0.10,Stat-Ease,Inc.,

MN).In rotatable designs the variance of estimated

values is constant at points equally distant from the

center point(uniform precision).Two sets of response

surface designs were used to observe the effects of

glucose,L-glutamine,and osmolality on the culture

longevity and antibody production.First,the effects of

glucose and L-glutamine were investigated,using a range

of glucose concentrations between1and15g/L and L-glutamine concentrations between0.5and14.5mM. Second,the effect of osmolality was studied using NaCl

and glucose,with NaCl concentrations between0and83

mM and glucose concentrations between4and16g/L.

No KCl was supplied along with NaCl in order to mimic

base control in the bioreactors,which is accomplished

with sodium-containing bases only.All experiments were

carried out in250-mL shake flasks containing72(5mL

culture.The cultures were incubated at37°C with7.5%

CO2overlay at100rpm and sampled on days0,1,2,3,

4,5,10,and15.The response variables were final

antibody titer and the cumulative viable cell density

(CVC)calculated using the trapezoidal rule for the

integral given by

where t f is the final time.The values are reported as

percent of maximum.

Bioreactor Operating Conditions.Celligen Plus

bioreactors(3L working volume,H/D) 1.2:1,New

Brunswick Scientific Co.,Inc.,Edison,NJ)were run at

37°C,with a constant gas sparging rate of0.03vvm,and

agitated by marine blade impellers at250rpm.The

bioreactors were equipped with a condenser to minimize

any evaporative losses due to off-gas.The dissolved

oxygen was maintained by controlling the composition

of the inlet gas mixture consisting of air,nitrogen,

oxygen,and carbon dioxide.The pH was controlled at6.8

by carbon dioxide gas sparging and1M sodium hydroxide

addition.All cultures were inoculated with0.75L of seed

culture and filled up to a working volume of3L with L-glutamine and glucose-enriched protein-free,animal-component-free CHO medium(Sigma C5467,St.Louis, MO).Initial L-glutamine concentration was11.6mM.The initial glucose concentration in the batch culture was12 g/L,and glucose additions for the fed-batch cultures were done according to the heuristic approach described below. The working volume in fed-batch cultures remained constant as a result of the use of highly concentrated feeds.

Heuristic Optimization.To maximize the yield of the

cell culture,a heuristic approach was proposed by

CVC)∫0t f X d t(1)

manipulating initial glucose concentration and feeding profile.Mainly,two heuristics were developed using the response surface graph for the final antibody titer gener-ated from shake flask studies.In both heuristics,the glucose concentration was maintained between8and10 g/L by adding15mL of glucose solution(450g/L)when the concentration was less than8g/L.For the first heuristic(Heuristic1),initial glucose concentration was chosen at the point of maximum yield observed in the response surface for the final antibody titer(9g/L).The second heuristic(Heuristic2)was set up at the basal glucose concentration(4g/L).To maintain low osmolality profile during the growth phase,three gradually increas-ing bolus feeds were charged on days2,3,and4.Each bolus feed was calculated using the following equation to attain a glucose concentration of9g/L on day4: where V(t)is the amount of glucose to be added to the bioreactor on a given day t,V is the working volume(3 L),S F and S(t)are the glucose concentration in the feed (450g/L)and bioreactor,respectively.

Results and Discussion

The effects of glucose and L-glutamine were studied in shake flasks using response surface methods.To compare the results of the batch culture experiments,the two parameters considered were the final titer and CVC calculated by eq1.The values are reported as percent of maximum.

Response Surface Study1.The response surface graphs of the final antibody titer and CVC are depicted in Figure1as a function of initial L-glutamine and glucose concentration.In the top panel,the final antibody titer increased with increase in L-glutamine and glucose concentrations,suggesting that the two nutrients were limiting for the given system.On the other hand,the CVC was maximum at the center where the concentra-tions of L-glutamine and glucose were7.5mM and8g/L, respectively(see bottom panel).As indicated in Figure 1,when glucose and L-glutamine were increased from4 g/L glucose and4mM glutamine to8g/L glucose and 7.5mM glutamine,both final antibody titer and CVC increased by55%and12%,respectively.Although an additional increase in the initial concentrations of glucose and L-glutamine to12g/L and11.6mM,respectively, improved the final antibody titer further by23%,there

was an6%decrease in CVC.The specific productivity, defined as the ratio of final antibody titer to CVC, increased with increasing L-glutamine and glucose con-centrations.The enhanced specific productivity could also be due to increase in osmolality of the culture.Hence,it was vital to distinguish the effect of initial osmolality and glucose concentration,along with their interaction on the cell culture.

Response Surface Study2.To facilitate the under-standing of the effect of initial osmolality and glucose concentration on the cell culture,it was imperative to ensure that there was no L-glutamine limitation.The results from the first study indicated that L-glutamine at an initial concentration above11.6mM was not depleted during the15-day cultivation.Thus the initial L-glutamine concentration in the second study was chosen as11.6mM,and the range for initial glucose concentration was between4and16g/L.The results of the second response surface study are depicted in Figure 2as a function of initial NaCl and glucose concentrations. The highest final antibody titer was observed between8 and10g/L glucose concentration and40to44mM NaCl, which corresponds to400to420mOsm/kg osmolality(see top panel).Although the final titer showed a nonmono-tonic dependence on the osmolality,CVC decreased monotonically with increasing osmolality(see bottom panel).At the initial glucose concentration of10g/L,the final antibody titer improved by24%and the CVC decreased by30%as the osmolality of the culture increased from360to420mOsm/kg.The specific pro-ductivity in this case improved by77%,confirming that the specific productivity increases with increase in os-molality.Hence,the elevated titers observed in the shake flasks,at glucose concentrations greater than10g/L, were due to the effects of increased osmolality and not the effects of glucose,per se.It should however be noted

V(t))V(9-S(t))

S

F

(5-t)

(2)

Figure1.Response surface graph for the effect of glucose and

L-glutamine on the final antibody titer(top panel)and cumula-

tive viable cell density(bottom panel).The numbers of replicates

are depicted along with the design points as solid circles and

the final antibody titer and CVC values(%of maximum)are

shown by contour lines.

that this increase in titer was attained at the expense of CVC.Thus in a bioreactor,where the osmolality of the culture inherently increases with base and glucose ad-ditions,it would not be feasible to start at 420mOsm/kg osmolality as suggested from the shake flask experi-ments.In other words,both batch and fed-batch mode of operations in the bioreactor would be subjected to additional increase in osmolality due to base additions.This may in turn lead to a decrease in final titer as the increased specific productivity cannot compensate for the drastic decrease in CVC.However,the dependence of specific productivity on osmolality suggests that an appropriate osmolality profile may improve the titer in fed-batch operation of the bioreactors.

Heuristic Optimization.To maximize the final an-tibody titer,an optimal solution would maximize both CVC and specific productivity.Previously,biphasic cul-

ture strategies based on the osmotic stress improved the titers in batch cultures significantly (6,9).In this study,a heuristic approach was proposed to determine the “best”feasible solution by interpreting the results ob-tained from the response surface studies.The idea was to first maximize the cell growth by maintaining low osmolality and then maximize the specific productivity by gradually increasing the osmolality.Two heuristics were developed using the response surface graphs for the final titer generated from the shake flask studies.The details of the two heuristics are described in Materials and Methods.

The first response surface study in shake flasks indicated that the “best”culture condition for a batch bioreactor was 11.6mM L -glutamine and 12g/L glucose.This condition was selected as the Control culture for the bioreactors.The second response surface showed that the highest final antibody titer was between 8and 10g/L glucose concentration (see Figure 2)and 400to 420mOsm/kg osmolality.Hence,Heuristic 1started with 9g/L glucose concentration and was maintained between 8and 10g/L to gradually increase the osmolality during the cultivation.In addition,the second response surface study showed that whereas CVC was maximal at the basal osmolality,the specific productivity increased with increasing osmolality.The dependence of specific pro-ductivity and CVC on osmolality suggested that the final titer could be improved further,by first maximizing the cell growth at low osmolality and then increasing the osmolality gradually to maximize the final titer.Hence,Heuristic 2started at 4g/L basal glucose concentration and was charged with the three bolus feeds of glucose on days 2,3,and 4,to increase the osmolality gradually during the exponential phase.Thereafter Heuristic 2was also maintained between 8and 10g/L glucose concentra-tion,similar to Heuristic 1.

The glucose,osmolality,CVC,and titer profiles for three bioreactor runs are depicted in Figure 3.As intended,the glucose concentration for Heuristic 1was maintained between 8and 10g/L,whereas in Heuristic 2,glucose was fed on days 2,3,and 4to increase the osmolality gradually (see panel A).At the end of the cultivation Heuristics 1and 2had 26.5and 28.1g/L total glucose addition,respectively,whereas only 12g/L of glucose was added to the batch culture.It should be noted that the additional glucose was only used for increasing the osmolality of the culture and hence was not com-pletely utilized as an energy source.In accord with the initial glucose concentrations,all three cultures started at three different osmolalities.While the osmolality profile for the batch culture increased slightly,the profiles for both heuristics were similar after day 5(see panel B).This increase in osmolality in batch culture is due to lactate production and base addition for pH control.As depicted in panel C,CVC profile for Heuristic 2was higher than the other two cultures.As expected,the results are in accord with the low osmolality profile that Heuristic 2attained during growth phase.Similarly,Heuristic 2reached the highest titer among three cul-tures (see panel D).Overall,Heuristics 1and 2,respec-tively,reached approximately 10%lower and 20%higher values for both CVC and titer when compared to the Control culture.Therefore,overall specific productivities were similar for all the three cultures.While both Heuristic 1and 2attained osmolalities higher than that of the batch culture,all three cultures averaged similar osmolalities and specific productivities.Additionally,the Control culture in the bioreactor attained 25%higher specific productivity when compared to the shake

flask

Figure 2.Response surface graph for the effect of glucose and sodium chloride on the final antibody titer (top panel)and cumulative viable cell density (bottom panel).The numbers of replicates are depicted along with the design points as solid circles and the final antibody titer and CVC values (%of maximum)are shown by contour lines.

culture with the same initial conditions.The improved specific productivity could be mainly due to increase in osmolality as a result of base additions and partly due to strict dissolved oxygen and pH control in the bioreac-tor.The specific productivities of the Control culture remained almost constant for the duration of the cultiva-tion and the values increased slightly for Heuristic 1.On the other hand,in Heuristic 2,the specific productivity at the end of the cultivation was 50%higher than the specific productivity at the beginning.While there were significant differences in the CVC and titer,L -glutamine,lactate,and ammonium concentrations were similar in all the three cultures (data not shown).Consequently,the specific rates of L -glutamine consumption and lactate and ammonium production were lowest in the culture with gradual increase in osmolality (Heuristic 2).In summary,Heuristic 2,which experienced a gradual increase in the osmolality from the basal condition,improved the yield by approximately 20%as compared to the culture with quasi-steady osmolality profile.The results of the bioreactor runs were process significant since the typical batch-to-batch variability is within 4%.

Conclusions

The effects of glucose and L -glutamine on the culture were studied in shake flasks using response surface methods.The shake flask studies indicated that the best culture condition for a batch bioreactor was 11.6mM L -glutamine and 12g/L glucose.Increased specific pro-ductivity was observed with an increase in the concentra-tions of glucose and L -glutamine.However,to distinguish between the effects of increased glucose and osmolality

on the culture,a second response surface study was implemented by using a constant initial L -glutamine concentration of 11.6mM and varying the initial glucose and salt concentrations.Due to the strong dependence of specific productivity and CVC on the osmolality of the culture,a heuristic approach was adopted to maximize the final antibody titer by maximizing both CVC and specific productivity.The idea was first to maximize the viable cell density and then gradually increase the osmolality to maximize the antibody production.The heuristic profile improved the yield by approximately 20%as compared to the culture with quasi-steady osmolality profile.The results suggest that for biological systems,which display nonmonotonic behavior with respect to osmolality,the final titers can be further improved by maintaining low osmolality during the growth phase and then gradually increasing the osmolality with glucose/base additions during the late growth phase.

Acknowledgment

We thank Dennis Rendeiro and two anonymous reviewers for their comments.We also acknowledge Jack Cardoso,Mike Cuzzola,Carmine Giannetta,James Jimenez,Debbie Lutz,and Rich Stout for assistance with experiments and Steve Blaisdell,Brittany Larkin,and Ed Previte for performing reverse-phase analysis.

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