High-End pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO...

ARTICLE

High-End pH-Controlled Delivery of GlucoseEffectively Suppresses Lactate Accumulation inCHO Fed-Batch Cultures

Matthew Gagnon,1 Gregory Hiller,1 Yen-Tung Luan,1 Amy Kittredge,2 Jordy DeFelice,1

Denis Drapeau1

1Pfizer, Inc., 1 Burtt Road, Andover, Massachusetts; telephone: 978-247-3173;

fax: 978-247-2602; e-mail: [email protected], Inc., Billerica, Massachusetts

Received 7 October 2010; revision received 23 December 2010; accepted 18 January 2011

Published online 15 February 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23072

ABSTRACT: A simple method for control of lactate accu-mulation in suspension cultures of Chinese hamster ovary(CHO) cells based on the culture’s pH was developed. Whenglucose levels in culture reach a low level (generally below1mM) cells begin to take up lactic acid from the culturemedium resulting in a rise in pH. A nutrient feeding methodhas been optimized which delivers a concentrated glucosesolution triggered by rising pH. We have shown that thishigh-end pH-controlled delivery of glucose can dramaticallyreduce or eliminate the accumulation of lactate during thegrowth phase of a fed-batch CHO cell culture at both benchscale and large scale (2,500 L). This method has provenapplicable to the majority of CHO cell lines producingmonoclonal antibodies and other therapeutic proteins.Using this technology to enhance a 12-day fed-batch processthat already incorporated very high initial cell densities andhighly concentrated medium and feeds resulted in anapproximate doubling of the final titers for eight cell lines.The increase in titer was due to additional cell growth andhigher cell specific productivity.

Biotechnol. Bioeng. 2011;108: 1328–1337.

� 2011 Wiley Periodicals, Inc.

KEYWORDS: CHO cell culture; mammalian cell culture;fed-batch; glucose limitation; lactate; monoclonal antibodyproduction

Introduction

For decades of mammalian cell culture, lactic acid secretionduring cell growth and production of protein therapeuticshas been problematic due to the resulting increases of lactateconcentration and osmolality, both of which can adverselyaffect cell growth and productivity (Hassell et al., 1991; Yoonet al., 2006). Even in the presence of sufficient levels of

dissolved oxygen, transformed mammalian cells tend to useglucose inefficiently, converting much of it to lactic acid(Miller et al., 1987, 1988).

Relative to lab scale experiments, reduced processperformance of Chinese hamster ovary (CHO) fed-batchprocesses in large scale (>1,000 L) bioreactors has oftenbeen associated with variable and inconsistent rates of lacticacid formation during the growth phase, and unreliable ratesof lactic acid uptake during the production phase (Zhu et al.,2005).

Limiting the glucose concentration has long beenrecognized as a way to reduce lactic acid secretion(Glacken et al., 1986). However, the methodologiesemployed to keep glucose concentrations low in a batchor fed-batch production bioreactor can reduce the rate ofgrowth and productivity, and tend to be difficult toeffectively implement in a controllable and reproduciblemanner at large scale (Fleischaker and Sinskey, 1981; Huet al., 1987; Zhou et al., 1995).

The use of alternative six-carbon sugars such as galactose,fructose, or mannose to substitute for, or in combinationwith, glucose can also help to control lactate accumulation inmammalian cell cultures (Altamirano et al., 2000; Andersonet al., 1994; Reitzer et al., 1979;Wagner et al., 1991; Luan et al.,US Patent 7,429,491 B2 2008). Unfortunately, the growthrates and peak cell densities tend to be lower in the presence ofthese alternate primary metabolites.

With the goal of lactic acid control, suppression of six-carbon sugar membrane transporters using chemicalinhibitors, and genetic engineering techniques includingthe amplification, suppression, addition, or silencing ofcertain enzymes or substrate membrane transporters,have also been attempted (Chen et al., 2001; Irani et al.,2002; Wlaschin and Hu, 2007). Unfortunately, while insome cases lactate accumulation was reduced, usuallythe cells have eventually reverted to their original wild-type metabolism.Correspondence to: G. Hiller

1328 Biotechnology and Bioengineering, Vol. 108, No. 6, June, 2011 � 2011 Wiley Periodicals, Inc.

In the present work, we describe a method for very effectivesuppression of lactate accumulation during the growth phaseof fed-batch cultures of CHO cells. The method is simple,scalable, requires no significant capital outlay at the largescale, and can result in dramatic increases in peak andsustained cell densities, cell viabilities, and overall bioreactorproductivities. The method has been given the convenientacronym HIPDOG (HI-end pH-controlled Delivery OfGlucose), which will be described in detail later in the text.

Materials and Methods

Cells Lines

All experiments used CHO-K1 cells, pre-adapted to serum-free suspension growth prior to transfection. Cells werestably transfected with proprietary DNA vectors encodingrecombinant proteins and the selectable marker dihydro-folate reductase.

Medium and Feeds

All experiments utilized highly concentrated proprietarymedia and feed solutions developed at Pfizer.

Equipment

Cells were grown in 1 or 2-L working volume Applikonbioreactors utilizing Applikon 1010 or 1030 controllers(Applikon, Inc., Schiedam, Netherlands). All bioreactorsmaintained continuous control of pH near 7.0 using either1M sodium/potassium carbonate, or a slow addition of anutrient feed containing concentrated glucose. At the labscale during the growth phase, the high-end pH control wasperformed with syringe pumps, model NE1000X (New EraPump Systems, Framingdale, NY). Dissolved oxygen wascontrolled at 25–40% of air saturation by micro-bubblesparging of a mixture of 7% CO2/air and a continuousdelivery of the same mixture to the bioreactor headspace.Single or dual Rushton impellors operating at approxi-mately 275 RPM provided agitation at a calculated powerper unit volume of approximately 90W/m2. Temperaturewas controlled at 37 or 318C with electric heating blankets.

During fed-batch experiments, bioreactors were bolus fedvarying quantities (2–3% of reactor volume per day startingon day 4) of a concentrated nutrient solution dependingupon the cell density and the previous day’s consumptionpattern. The consumption patterns were determined bymeasuring the changes in glucose and lactate levels, and onchanges in osmotic strength of the culture.

The production bioreactor inocula were expanded in pH-adjusted shake flasks or in controlled bioreactors. Flaskswere continuously shaken at approximately 60 RPM.Incubators were humidified and controlled at 7% carbondioxide and 378C.

Analytical Methods

Cell counts were performed using CEDEX model AS20(Roche Innovatis AG, Bielefeld, Germany) or VI-CELL(Beckman-Coulter, Inc., Brea, CA) automated cell countingdevices using trypan blue exclusion. Glucose and lactatelevels in the culture medium were determined using aNOVA 400 BioProfile automated analyzer (NovaBiomedical, Waltham, MA). A model 3900 freezing-pointosmometer (Advanced Instruments, Inc., Norwood, MA)was used for osmolality determinations. Offline gas and pHmeasurements were performed with a model 248 CIBA-Corning blood gas analyzer (Bayer AG, Leverhusen,Germany). Titer analysis was performed by a ForteBioOctet model analyzer (ForteBio, Inc., Menlo Park, CA) orprotein A HPLC (model 1100 HPLC, Agilent Technologies,Inc., Santa Clara, CA, protein A column model 2-1001-00,Applied Biosystems, Foster City, CA).

The N-linked oligosaccaride profiling methodologyinvolves enzymatic removal of the oligosaccharides fromthe protein, followed by derivatization of the oligosacchar-ides with the fluorophore 8-aminopyrene-1,3,6-trisulfonicacid (APTS). The derivatized oligosaccarides are thenseparated by capillary electrophoresis using laser-inducedfluorescence (LIF) detection. The separation of oligosac-charides is based on the mass-to-charge ratio of the labeledoligosaccharide. The APTS serves as a fluorophore thatattaches to the reducing end of an oligosaccharide, allowingfor detection. Additionally the APTS contains threenegatively charged sulfate groups that provide charge toneutral carbohydrates, allowing for migration through thecapillary to occur.

Results and Discussion

Shake Flask Experiments With Sodium LactateAdditions

Figure 1a shows the significant and dose-dependentsuppression of integrated viable cell density that occurswhen sodium lactate is added to a culture. CHO cellsproducing a monoclonal antibody were removed from a 1-Lbioreactor on day 2 after inoculation. The culture wasseparated into 50-mL aliquots and distributed into multiple250-mL shake flasks with additions of sodium lactate from 0to 6.0 g/L (�54mM or 4.8 g/L lactate ion). pH wasmaintained near 7.0 for an additional 12 days by dailysampling and additions of 1M sodium bicarbonate.Figure 1b shows that the specific cellular productivity andday 14 titer were also depressed in a dose-dependent mannerwith increasing lactate levels. The experiment utilized twoosmotic strength controls. The osmotic strength controlsused additions of a concentrated sodium chloride solutionto mimic the increase in osmotic strength that would occursimply due to the sodium lactate additions (54 and108mOsm). From Figure 1 it is clear that the increase in

Gagnon et al.: pH-Controlled Lactate Suppression in CHO Cultures 1329

Biotechnology and Bioengineering

osmotic strength accounts for some, but not all of thegrowth and productivity inhibition that results from theadditions of sodium lactate. The lactate ion itself is clearlyinhibitory to both growth and productivity.

Encounter With a Cell Line with an Unusual Propensityfor Lactic Acid Consumption

CHO fed-batch processes tend to include a growth phaseduring which viable cell density rises, and a productionphase during which viable cell density remains constant ordeclines. Commonly the switch from growth phase to

production phase is triggered by changing the pH and/ortemperature, or by adding chemicals that induce highercellular productivity (Jiang and Sharfstein, 2008; Mooreet al., 1997; Yoon et al., 2006). The switch to the productionphase is generally accompanied by a decrease in the rate oflactic acid secretion, and this is often followed by ametabolicshift to lactic acid consumption, which results in a risein pH. Lactic acid consumption also results in a decrease ofosmolality, which is beneficial, because nutrient feedsolutions can then be added without driving the osmolalityupward to an inhibitory level.

During the initial screening of CHO clones producing atherapeutic glycoprotein of�115 kDa, an unusual clone was

Effect of Lactate on Integrated Viable Cell Density

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Figure 1. The effect of additions of sodium lactate to pH-controlled shake flask cultures of a CHO cell line producing a monoclonal antibody. Included in the experiment were

two osmolality controls consisting of flasks with only sodium chloride additions to mimic increases of lactate ion and the additional sodium that would result in the neutralization of

the lactic acid (2.4 and 4.8 g/L). a: Effect on integrated viable cell density. b: Effect on final (day 14) titer and accumulated cellular (specific or per cell) productivity. [Color figure can

be seen in the online version of this article, available at http://wileyonlinelibrary.com/bit]

1330 Biotechnology and Bioengineering, Vol. 108, No. 6, June, 2011

encountered. This clone, when inoculated at a very high celldensity, began consuming lactic acid while still in the growthphase and without any external intervention (e.g., notemperature shift) after an initial short period of lactic acidsecretion. Figure 2 shows the growth curve and lactateconcentration profile of a pair of fed-batch bioreactorsutilizing this clone operating under identical conditions,maintained continuously at 378C. Under these conditions,this clone grew to much higher cell densities than underconditions where lactic acid was not consumed, and alsogrew to much higher cell densities than other cell linesproducing the same molecule that did not consume lacticacid until after a temperature shift (data not shown).

Development of the HIPDOG (HI-End pH-ControlledDelivery of Glucose) Technology and PotentialMetabolic Mechanisms Involved

It has been reported in the literature that in some casesmammalian cells growing on six-carbon substrates otherthan glucose may have significant reductions in lactateproduction (Anderson et al., 1994; Marquis et al., 1996;Reitzer et al., 1979; Wagner et al., 1991). In preliminaryexperiments in our laboratories with CHO fed-batchcultures, when the alternative carbon substrate galactosewas used as a substitute for glucose, cellular growth wasgreatly slowed. When galactose was used in combinationwith glucose the uptake rate of glucose and the productionrate of lactate were significantly reduced, but again, cellulargrowth was also slowed appreciably when compared tocultures with freely available glucose. The membrane sugartransporter GLUT1, which can transport both glucose andgalactose, is active in CHO cells (Harrison et al., 1991;Mueckler et al., 1985). It is possible that in our experimentsin the presence of glucose, galactose might be competing foravailable GLUT1 membrane transporters and potentiallyslowing the transport of glucose across the cell membrane,though additional experimentation was not performed toconfirm this supposition.

The above experimental results led to the hypothesis thatlimiting lactic acid secretion during a fed-batch culture,potentially by restricting the availability of glucose, mightlead to improved growth and cellular productivity. Theresults also suggested that spontaneously rising pH is asurrogate indicator of glucose limitation that is more readilymeasurable than glucose concentration itself, and could beused for instant and continuous feedback control for glucosedelivery (Luan et al., US Patent 7,429,491 B2 2008). The pHof a culture could be controlled on the high end by thesimple, slow addition of a solution containing a highconcentration of glucose whenever the pH rose to the set-point.

Schematically, the potential sequence of metabolic eventsduring this control loop is elucidated in Figure 3. The

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Figure 2. Time course of a fed-batch experiment of a metabolically unusual cell

line producing a protein therapeutic. When cultured under appropriate conditions the

cell line consumed lactic acid from the surrounding culture medium without any

external manipulations. The consumption of lactic acid improved culture health and

allowed the cells to grow to unusually high cell densities. The data in the figure

represent two bioreactors that used identical culture conditions and were run at

the same time from the same inoculum source. Main figure shows time course of

viable cell density, inset shows time course of culture lactate ion concentrations.

[Color figure can be seen in the online version of this article, available at http://

wileyonlinelibrary.com/bit]

Lactic acid Produced

pH Rises Above Setpoint

Glucose Pump Turns On (slow rate)

pH Drops Below Setpoint

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Lactate and glucose carryover from N-1 (seed reactor)

Lactic acidConsumed

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Figure 3. Pictorial representation of the hypothesized sequence of events occurring in the bulk cell culture fluid during the growth phase of a bioreactor utilizing the HIPDOG

control scheme for limiting lactic acid accumulation.



glucose level of the initial production bioreactor medium iszero or low. Once glucose carried over from the N-1 (seedreactor) is consumed down to a very low level, lactic acid willstart to be taken up from the surrounding culture fluid. Theselective uptake of lactic acid will increase bulk pH,eventually triggering the slow addition of glucose whenthe upper set-point is reached. When glucose increases tosome critical concentration, cells will again convert some ofthe pyruvate generated from glucose catabolism to formlactic acid, lowering the bulk fluid pH as the acid istransported out of the cells, and triggering the cessation ofglucose delivery to the bioreactor. Once the glucose againdrops to a low concentration due to cellular uptake andmetabolism, the entire cycle begins again at the top andcontinues in this manner for the duration of the growthphase of the fed-batch. The net result of the control schemeis very effective control of lactate at a low level throughoutthe course of the growth phase. The technology is called HI-end pH-controlled delivery of glucose, and has been giventhe convenient acronym HIPDOG. From a practical standpoint, during the entire time that the HIPDOG scheme isfunctioning during a production bioreactor experiment, theglucose levels are undetectable using the standard laboratorymethods described in the Materials and Methods Section.Similarly, the transient changes in lactate levels are too smallto be accurately measured.

Direct Comparison of HIPDOG Technology toConventional Fed-Batch for Eight Clones Producing aMAb

The HIPDOG technology was applied to eight differentclones, all producing the same humanized monoclonal IgGantibody, in fed-batch cultures in 2-L bioreactors. Astandard fed-batch for each clone, using the same medium(except with higher initial levels of glucose [15 g/L] andreduced levels of sodium chloride so that the initialosmolality of the medium was nearly identical), was usedas a control. All cultures utilized a temperature shift from 37to 318C to trigger a production phase. Other than during thegrowth phase for the cultures using the HIPDOGtechnology, the glucose levels in all bioreactors weremaintained well above 1mM by regular bolus additionsof glucose as necessary.

Figure 4a shows the time course of the lactateconcentration for the control cultures. The eight cloneshad variable rates of lactic acid secretion during the growthphase, and variable rates of lactic acid secretion orconsumption after the temperature shift. In dramaticcontrast, the HIPDOG cultures maintained comparativelyvery low levels of lactate, all below 3 g/L (34mM)throughout both the growth phase and the productionphase (Fig. 4b). Due to the suppression of lactic acidsecretion, the bioreactors using the HIPDOG technologycould be maintained at 378C for an additional day of growth(temperature shift performed on day 5 rather than on day 4,as for the controls). Experiments with other cell lines when

developing the HIPDOG technology have suggested thatcontinuing the HIPDOG phase (and by default in this casethe time that the culture is maintained at 378C) beyond acertain point may lead to diminishing returns with respectto additional cell growth, and sometimes can lead to fasterlosses in cell viability.

Figure 5 shows the time course of viable cell density forthe two sets of cultures. Likely due to the low lactate levels inthe bioreactors using the HIPDOG technology, some clones(e.g., clones 674 and 179) had much more growth after thetemperature shift in the HIPDOG conditions. Also of note,during the initial 4 days of the culture, the growth rate foreach clone was identical or only slightly lower in theHIPDOG relative to the control culture.

Clone 674, one of the highest lactate producers underconventional conditions (Fig. 4a), dropped to a cell viabilityof 75% on the final day (day 11) in the conventional fed-batch culture (data not shown). All other clones maintainedviability above 86% as measured by trypan blue exclusion

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Figure 4. Production bioreactor clone screening experiment. Time course of

lactate accumulation. When two lactate values are shown for single time point

the second value was calculated based on known dilutions after a nutrient feed.

a: Conventional fed-batch bioreactor, temperature shifted from 37 to 318C on day 4.

b: Bioreactor utilizing the HIPDOG control strategy, temperature shifted from 37 to 318Con day 5. [Color figure can be seen in the online version of this article, available at

http://wileyonlinelibrary.com/bit]


and automated cell counting for the duration of theexperiment under either the conventional fed-batch, orHIPDOG fed-batch conditions (day 10 viabilities; Table I).

Other than during the growth phase for the bioreactorsusing the HIPDOG technology, the residual glucose levels inall bioreactors were maintained in excess, well above 1mM,by regular bolus additions of glucose as necessary (data notshown). It has been our experience that in fed-batchprocesses with most CHO cell lines, the glucose consump-tion rates and lactate production rates do not varyappreciably on a per cell basis over a very wide range ofextracellular glucose concentrations (1–75mM).

When compared to the control cultures, the day 10 titersof the HIPDOG cultures were significantly higher for all ofthe clones (Fig. 6). The clones that had high rates of lacticacid formation in the control culture (Fig. 4a) benefitedmost from the application of the HIPDOG technique, withclones 179, 674, and 701 achieving 5.9-, 2.3-, and 2.5-foldday 10 titer increases, respectively (Table I).

While the HIPDOG technology allowed most clones togrow to significantly higher cell densities (Fig. 5), theincrease in titer realized in these reactors was also due in partto significant increases in the cellular productivity (Fig. 7and Table I). Clones 179 and 701 seemed to benefit the mostfrom the HIPDOG technology, with increases in cellularproductivity of 4.1- and 2.6-fold over those in the controlcultures.

While it is clear that the HIPDOG technology significantlysuppresses lactic acid formation rates for most cell lines,there is a concomitant increase in the secretion ofammonium. Table I shows that for all clones, the peakammonium levels were higher in the HIPDOG cultures, andfor all but one, the day 10 ammonium levels were alsoslightly higher. While ammonium is inhibitory, any adverseeffects on growth or cellular productivity of the higherammonium levels were clearly dwarfed by the beneficialeffects of the lower lactate levels.

With two of these cell lines from the above clone screeningexperiment, the HIPDOG approach was modified by raisinginitial cell density and increasing the volume of nutrientfeeds. In these partially optimized fed-batch productionbioreactor experiments for clone 701, a titer of 7.8 g/L was

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viable cell density. a: Conventional fed-batch bioreactor, temperature shifted from 37

to 318C on day 4. b: Bioreactor utilizing the HIPDOG control strategy, temperature

shifted from 37 to 318C on day 5. [Color figure can be seen in the online version of this

article, available at http://wileyonlinelibrary.com/bit]

Table I. Metabolic parameters from an experiment involving eight CHO cell lines producing the same monoclonal antibody.

Parameter Mode of bioreactor operation

Clone number

12 48 179 400 601 674 701 896

Day 10 titer (g/L) Conventional fed-batch 1.2 2.3 0.9 3.4 1.8 1.0 1.9 1.5

HIPDOG culture 2.3 3.0 5.4 4.9 3.8 2.4 4.7 2.2

Fold increase in day 10 titer (HIPDOG/conventional) 1.9 1.3 5.9 1.5 2.2 2.3 2.5 1.5

Day 10 percent viability Conventional fed-batch 97 95 93 94 94 82 96 95

HIPDOG culture 96 87 95 92 96 88 97 97

Day 10 integrated viable cell density (e6 cell day/mL) Conventional fed-batch 113 131 114 127 148 145 161 99

HIPDOG culture 164 142 165 158 167 201 149 118

Day 10 cumulative cell specific productivity (pg/cell/day) Conventional fed-batch 11 18 8 26 12 7 12 15

HIPDOG culture 14 21 33 31 23 12 31 19

Peak ammonium concentration (day 4 or 5, mM) Conventional fed-batch 4.0 3.7 4.4 3.6 3.6 4.7 3.8 3.2

HIPDOG culture 6.3 6.9 6.3 5.4 6.9 8.2 6.5 6.4

Day 10 ammonium concentration (mM) Conventional fed-batch 2.5 1.3 4.5 1.4 2.2 2.3 0.8 1.8

HIPDOG culture 4.0 2.9 4.8 4.0 2.0 3.4 3.1 3.2

Comparison of the two bioreactor operational modes, conventional fed-batch, and fed-batch using the HIPDOG nutrient control scheme.



attained at day 12 (integrated viable cell density of 286e6 cell day/mL, and cellular-specific productivity of 27 pg/cell/day) and for clone 48, a titer of 8.3 g/L was attained atday 17 (integrated viable cell density of 365 e6 cell day/mL,and cellular specific productivity of 23 pg/cell/day).

N-1 or Seed Bioreactor Use of HIPDOG Technology

The HIPDOG technology can also be applied to the N-1(seed) bioreactor to reach higher cell densities while stillserving as a healthy inoculum source.

In an experiment designed to test the utility of theHIPDOG technology in an N-1 bioreactor, clone 701(referred to above), was grown in N-1 bioreactors with orwithout the HIPDOG technology. Figure 8 inset shows thecell densities reached in these bioreactors; the viabilities at alltimes were above 95% (viabilities not shown). Each of theN-1 bioreactors was used to seed two production bioreactorsat 5� 106 viable cells/mL, one operating with, and onewithout the HIPDOG technology. Temperature shifts from

37 to 318C were performed on all production cultures onday 4. The cells coming out of a HIPDOG N-1 bioreactorexperienced reduced lag phases in the production bioreactorwhen compared with cells coming from an N-1 bioreactorthat had been pushed to equally high cell densities withoutusing the HIPDOG technology (Fig. 8). Additionally, theinoculum from a HIPDOG N-1 bioreactor will carrysignificantly lower levels of lactate and osmotic strengthinto the production reactor. Cells coming from theHIPDOG N-1 and using the HIPDOG technology in theproduction bioreactor grew to the highest cell densities,reaching 3.8� 107 viable cells/mL by day 5 of culture, andmaintaining a viability of nearly 95% out to day 12 (Fig. 8).The calculated final integrated viable cell densities, and titersand specific cellular productivities relative to the conven-tional N-1 seeding a conventional fed-batch condition forthis experiment are shown in Table II. The culture using aninoculum from an N-1 bioreactor run as a HIPDOG culture,seeding a production bioreactor also run as a HIPDOGculture, produced the highest titer, 3.9-fold higher than the

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product concentration. a: Conventional fed-batch bioreactor, temperature shifted from

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article, available at http://wileyonlinelibrary.com/bit]

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cellular (cell specific, or per cell) productivity. a: Conventional fed-batch bioreactor,

temperature shifted from 37 to 318C on day 4. b: Bioreactor utilizing the HIPDOG control

strategy, temperature shifted from 37 to 318C on day 5. [Color figure can be seen in the

online version of this article, available at http://wileyonlinelibrary.com/bit]


conventional batch N-1 seeding the conventional fed-batchproduction bioreactor. No glycosylation pattern analysiswas performed on protein produced in bioreactors whichutilized the HIPDOG technology in both the N-1 (seed) andproduction bioreactors.

Application of HIPDOG Technology to LegacyProcesses

The HIPDOG technology was evaluated for its potential toimprove productivities for several processes previously usedto produce protein for clinical studies (legacy processes).Additionally, clones that had previously been rejectedduring initial clone screening experiments due either totheir tendency to secrete high amounts of lactic acid or totheir lack of propensity to consume lactic acid duringthe production phase were tested using the HIPDOGcontrol scheme. Significant improvements in overall processproductivity were achieved for the majority of cell lines/processes tested. Figure 9 shows titers for the originalprocesses, for processes with higher cell densities and a more

concentrated medium and feeding scheme (enhancedplatform), and finally for HIPDOG processes (which alsoincorporated the features of the enhanced platform). Forprojects MAb-26, MAb-63, and MAb-54, the high titer ofthe HIPDOG process was in part due to the use of a moreproductive clone than in the original process. In each ofthese cases, the clone that was used for the HIPDOG processhad been rejected during the original clone screeningbecause of its high lactic acid secretion rate.

Scale Up of CHO Fed-Batch Processes Utilizing theHIPDOG Technology

Three processes using the HIPDOG technology havebeen scaled up to the 180, 500, and/or 2,500-L productionreactor scales. With appropriately sized pumps to feed theconcentrated glucose solution and proper adjustment ofcontrol loops, the process performance improvements withthe HIPDOG technology translated well to the large scale.The HIPDOG control scheme was used only during thegrowth phase of the cultures. Nearly equivalent performanceto the bench scale was realized upon scale up. For oneprocess, used for a clinical resupply of a non-glycosylatedtherapeutic protein, the HIPDOG process increased theday 12 titer by 76% (N¼ 6 batches for each process at 2,500-L scale, std. dev. 30%, P¼ 0.0005). For another such process,producing a glycosylated therapeutic protein, the day 12titer increase was 3.2-fold (N¼ 4 batches original process,N¼ 3 batches HIPDOG process, 2,500-L scale, std. dev. 0.3-fold, P¼ 0.0001).

Additional Potential Advantages of the HIPDOGTechnology

An additional advantage of the HIPDOG approach is that itcan reduce the need for adding base to the culture during thegrowth phase and for adding carbon dioxide or other acidsduring the production phase. A conventional fed-batchprocess has an early phase in which lactic acid issecreted from the cells and a later phase in which lacticacid may be reabsorbed and metabolized. To maintain aneutral pH during the early phase, sodium carbonate (orother base) is typically added to the culture to neutralizethe secreted lactic acid. The resulting increase in osmolalitycan be detrimental to cell growth and productivity. Later

Viable Cell Density and Viability

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12Time (days)

Viab

le C

ells

(e6

cells

/mL)

30%

40%

50%

60%

70%

80%

90%

100%

Perc

ent V

iabi

lity

HIPDOG N-1 to HIPDOG ProductionHIPDOG N-1 to Conv. F-Batch

Conv. Batch N-1 to HIPDOG ProductionConv. Batch N-1 to Conv. F-Batch

0

5

10

15

20

25

0 1 2 3 4Time (days)

Viab

le C

ells

(e6

cell/

ml)

Conv. BatchHIPDOG N-1

Figure 8. HIPDOG technology applied to N-1 bioreactors. Viable cell density and

viability. Inset shows viable cell density for N-1 (seed) bioreactors operated as batch

cultures with and without the HIPDOG technology. Main figure shows viable cell

density and viability in the production bioreactors. Cultures from N-1 using HIPDOG

technology (circles), cultures from N-1 using conventional batch (squares). Solid

symbols for production reactors using HIPDOG technology, open symbols for those

using conventional fed-batch. All cultures were temperature shifted from 37 to 318C onday 4. [Color figure can be seen in the online version of this article, available at http://

wileyonlinelibrary.com/bit]

Table II. Day 12 parameters from an experiment investigating the use of HIPDOG technology in seed bioreactors.

N-1 (seed bioreactor)

operational mode

Production reactor

operational mode

Integrated viable cell

density (e6 cell day/mL) Relative titer

Relative cumulative

cell-specific productivity

HIPDOG culture HIPDOG culture 352 3.9 2.1

HIPDOG culture Conventional fed-batch 275 1.5 1.0

Conventional batch HIPDOG culture 300 2.9 1.9

Conventional batch Conventional fed-batch 192 1.0 1.0

Day 12 integrated viable cell density, and titer and cumulative cell specific productivity relative to the conventional batch seed bioreactor inoculating aconventional fed-batch bioreactor.



when the cells consume lactic acid, the pH will rise, and ifno form of high-end pH control is employed, the pH canreach 7.5. This is high enough to affect cell viability, and forsome molecules, to affect product quality. Since theHIPDOG technology very effectively suppresses lactic acidsecretion during the growth phase, there is very littlerequirement for base to control pH on the low end, and isvery little pH rise during the production phase when whatlittle lactic acid that was produced is absorbed andmetabolized.

Product Quality Differences for HIPDOG Fed-BatchProduced Proteins

While only a limited number of proteins have yet beenproduced utilizing the HIPDOG technology, to date therehave been no significant differences in levels of high orlow molecular weight species, acidic species, or N-linkedglycosylation patterns for those proteins that containglycosylation sites, when compared with a conventional

fed-batch process. For the process described earlier in thetext in which three 2,500-L production bioreactor batcheswere completed using the HIPDOG process, the N-linkedglycosylation results are compared to the original processrun at the same scale (Table III). One potential reason for nosignificant changes in product quality characteristics couldbe due to the fact that the HIPDOG control scheme isoperating only in the growth phase. Figure 6b shows thatlittle as twenty percent of the total protein might beproduced during this growth phase. It is also possible thatwhile cells are glucose limited in a HIPDOG fed-batch, theymight never be glucose starved, as any minor increaseof pH resulting from a small consumption of lactic acidresults in an immediate addition of concentrated glucosesolution. CHO cells may regulate their intracellular levels ofglycosylation-related precursor molecules sufficiently thatglycosylation is not significantly affected by transient lowextracellular glucose levels. Comparisons of the glycosyla-tion of protein produced only during the HIPDOG growthphase, to that produced later in culture have to date not beenperformed.

Comparison of Process PerformanceDay 12 Titer (unless otherwise noted)

0

1

2

3

4

5

6

7

8

9

10

MAb-94SPT-03MAb-54MAb-63SPT-87MAb-26

Molecule / Project Name

Tit

er (

gra

ms/

L)

Original Phase I Platform

High Seed / Higher Nutrient Medium and Feeds

HIPDOG Technology Applied

HIPDOG Technology Applied - Day 15

Figure 9. A comparison of the process performance of the original phase I platform process to an enhanced platform process (higher seed densities and more enriched

medium and feeds), and the enhanced process with the HIPDOG technology applied. For projects MAb-26 and MAb-63, a different clone was used for the HIPDOG experiments. For

project MAb-54, a different clone was used for the enhanced platform and HIPDOG experiments as explained in the text. SPT¼ small protein therapeutic. [Color figure can be seen

in the online version of this article, available at http://wileyonlinelibrary.com/bit]

Table III. Glycosylation pattern for the original standard fed-batch process (process I), and process II utilizing the HIPDOG technology for a glycosylated

protein produced at the 2,500-L production reactor scale.

Process G0-core fucosylated (%) G1-core fucosylated (%) G2-core fucosylated (%)

Original fed-batch (N¼ 4) 74.2� 2.2 9.4� 1.0 2.5� 0.2

HIPDOG fed-batch (N¼ 3) 69.4� 1.7 10.7� 0.4 2.4� 0.3

Average and standard deviation values are listed.


Conclusions

CHO fed-batch processes utilizing the HIPDOG controlscheme have shown dramatic increases in process perfor-mance, and more consistent process performance batchto batch over conventional fed-batch conditions due toextremely effective suppression of the inhibitor lactic acid.For the majority cell lines, the HIPDOG control schemeenables CHO cell production bioreactors to be inoculated athigher cell densities, reach higher peak cell densities, andmaintain higher cell-specific productivities than conven-tional fed-batch cultures. The technology has been tested atthe 2,500-L scale and shown to perform equivalently to thebench scale. While adding a level of complexity to a fed-batch process, the HIPDOG control scheme need only beoperational during the growth phase of a culture, andrequires no significant capital outlay to be utilized at largescale.

We thank Wenge Wang for help with medium formulations. Special

thanks for help implementing the technology at large scale, Amy

Walsh, Meredith Kolpak, Carlos Cabrera, and Penninah Wanjeri.

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