A Review of Advanced Small

A Review of Advanced Small-Scale Parallel Bioreactor Technology for

Accelerated Process Development: Current State and Future Need

Rachel Bareither and David PollardBiologics New & Enabling Technologies, Biologics Development, Merck Research Laboratories, Merck & Co. Inc., Rahway, NJ 07065

DOI 10.1002/btpr.522Published online November 9, 2010 in Wiley Online Library (wileyonlinelibrary.com).

The pharmaceutical and biotech industries face continued pressure to reduce developmentcosts and accelerate process development. This challenge occurs alongside the need forincreased upstream experimentation to support quality by design initiatives and the pursuitof predictive models from systems biology. A small scale system enabling multiple reactionsin parallel (n � 20), with automated sampling and integrated to purification, would providesignificant improvement (four to fivefold) to development timelines. State of the art attemptsto pursue high throughput process development include shake flasks, microfluidic reactors,microtiter plates and small-scale stirred reactors. The limitations of these systems are com-pared to desired criteria to mimic large scale commercial processes. The comparison showsthat significant technological improvement is still required to provide automated solutionsthat can speed upstream process development. VVC 2010 American Institute of ChemicalEngineers Biotechnol. Prog., 27: 2–14, 2011Keywords: bioreactor, process development, integrated, automation

Introduction

Importance of bioprocesses and the need tocontrol development costs

The application of biotechnology for the commercializa-tion of pharmaceuticals continues to increase in importanceas biologics research becomes [50% of pharma’s pipelineportfolio. Over 600 biologics are under development eachyear with the majority comprising of monoclonal antibodies(mAbs) providing the fastest growth of new therapeuticswith a global expectation of around �$49 B by 2013.1 Thisincreasing influence of biologics development comes at atime of continued pressure on the pharma industry from thechallenges of cost pressures from healthcare providers, reve-nue reduction from patent expiration, pipeline concerns andincreasing generics competition.1,2 The focus of cost controlapplies not only to manufacturing but also to developmentwhere costs have risen exponentially during the last decade.2

The increasing size and complexity of clinical trials contrib-utes to these costs, but equally there is a need to lower drugsupply development costs, and reduce the time from discov-ery to market.3 Companies recognize the need to pursue newtechnological methods to gain development efficiency andpotential competitive advantage.

Accelerating process development by highthroughput bioreactor technology

The high throughput methods implemented for strain de-velopment, using 96 well plate screens with integrated ana-lytical automation, has shifted the rate limiting step toward

strain evaluation and process development. Figure 1 demon-strates the intensive upstream experimentation required forprocess development of a biologic. This is a significantresource burden, requiring integration with purification, toovercome the issues of process influence to product qualityand obtaining sufficient process understanding for robustnessand scalability. The remainder of this introduction providesfurther clarity to the justification for technology implementa-tion to accelerate development.

Improving Process Efficiency: Cost of Goods andDevelopment Timelines. The currently approved mAbs areprimarily produced in mammalian cell lines, typically eitherChinese Hamster Overy (CHO) or Murine myeloma cells(NS0, SP2/0).4 In 2006, the average production cost for amAb was determined in the range of $300 to $3000 pergram.5 In the late 1990s the upstream bioreactor productiontiter was the main driver for process economics with 600 mgL�1 as an average titer.5 Titers greater than 2 g L�1 are nowcommon place while titers in the 5 to 10 g L�1 range areachievable.6,7 At these titers the process economics shift to-ward the purification costs and deter the need for futureupstream titer improvement.6 This puts increasing emphasison improving product development timelines and achievingdesired product quality attributes.8 The use of faster growingmicrobial systems offers the ability to reduce developmenttimelines and production costs compared to slower growingmammalian cell lines. The replacement of mammalian cellculture with glycoengineered Pichia pastoris libraries, thatreplicate the most essential glycosylation pathways found inmammals, is now becoming a commercial reality.8–10 mAbtiters of �1 g L�1 have routinely been expressed in Pichiawith highly uniform human N-linked glycans and productiv-ities of [50 mg h L�1 are likely to be achievable.8 Thiscapability adds to the existing microbial expression host

Correspondence concerning this article should be addressed toD. Pollard at [email protected].

2 VVC 2010 American Institute of Chemical Engineers

toolbox11 which includes inclusion body production using

E. coli,12 virus like particles expression by Saccharomyces,13

and periplasmic expression with a Pseudomonas host.14 This

resurgence of the use of microbial expression systems is also

driven from the expanding research and development efforts

for alternative fuels and fine chemicals.15 This assists to in-

vigorate the demand for improved technology to speed pro-

cess development.16

Maintaining Product Quality and Process Reproducibilitythroughout the Development Phases. The therapeutic effi-ciency of many antibodies is influenced from the N-linkedglycosylation which modulate the interaction with specificFc receptors, and determine antibody dependent cytotoxicity(ADCC) function.17 These complex glycoproteins are sus-ceptible to heterogeneity and modifications, such as variantsof N terminal pyroglutamate and C-terminal lysine, methio-nine oxidation, disulphide bond scrambling, aggregation, andfragmentation. It is well-established that the combination ofthe upstream process (expression host and culture conditions)and the interaction with the primary recovery and purifica-tion processes will influence the product quality. It is under-stood that multifactorial statistical experimentation is essentialto investigate the complex relationships, especially as opti-mum conditions for titer often compete with the conditionsfor desired quality attributes. Using this approach has led tomany examples of improved product quality, such as theimpact of media composition process conditions (dO2, dCO2,pH, substrate feed rates) and use of additives to influence theglycosylation during expression.18–20 This experimental bur-

den requires high intensity resources, especially for long dura-tions with cell culture (10–21 days processes) and wouldclearly benefit from high throughput automated technology.

Increased Experimentation to Support Cellular FunctionModels. In the coming years the acceleration of process de-velopment will be assisted from the use of predictive modelsof complex cellular behavior.21 Over expression is often ratelimited by an enzyme of a complex multicompartment pro-cess, where the kinetics are poorly understood. Establishingthe interconnection between genomic sequences to phenotypefunction leads to identifying rate the limiting steps. Signifi-cant effort has been made to advance the technical methodsrequired for systems biology, such as the cost reduction ofmicroarrays22 combined with improved methods for proteo-mics,23 metabolic flux analysis24 metabolite analysis25 andstoichiometric network analysis.

These advances have recently enabled the genome wideanalysis of cell physiology to become routinely applied toprocess development. To ultimately build effective cellularfunction models demands the systems biology tools to beapplied to industrially relevant manufacturing conditions tounderstand the impact of feed rates, physical and chemicalstresses. The requirement of large data sets dictates the ma-jority of the work to be carried out at lab scale with scaledown technology that mimics the industrial fed batch highcell density processes. This significant upstream experimen-tal burden is only achievable with effective automated reac-tor technology that allows parallel cultures (n � 20). Thedevelopment of such automated small-scale fermentation

Figure 1. The upstream workflow to development a microbial manufacturing process.

This example is for protein/mAb expression from Pichia process taking up to 1.5–3 years of development time.

Biotechnol. Prog., 2011, Vol. 27, No. 1 3

technology tools to assist this work lags behind the advance-ment of the systems biology technology.

Scale Up Evaluation and Late Stage Process Characteri-zation. Optimization begins with the baseline lab scale pro-cess using operating constraints that mimic the limitationsexpected at large scale. This will find key stress factors andparameters that influence cell growth, yield, and productquality and identify areas for further optimization. Thedevelopment process must include pilot scale runs to verifythe scalability and validate the lab scale down process (Fig-ure 1). During late stage development, process validationincludes critical process characterization and robustnessassessment. Extensive statistical DOE’s determine the ac-ceptable ranges for critical operational parameters and definethe edge of failure (Figure 1). This design space definitionforms a key part of the new quality by design paradigm.26 Ittypically requires at least 50–60 experiments for fermenta-tion characterization of a process taking up to 6–8 months tocomplete. An effective scaledown model is required for thiswork and is currently performed at the 1 to 30 L scale wherefermentation kinetics, product titer, and quality are represen-tative of a large scale batch (Figure 2).

Integrating Upstream with Downstream Purification. Fi-nally, to streamline development for the increasing ex-perimentation demand, the linking of high throughputfermentation to automated purification would clearly providebenefits. A few examples have shown this advantage withmicroscale purification steps operated on standard liquid han-dling decks. Methods have included low volume, high pres-sure cell disruption with a microfluidizer or adaptive focusedacoustics along with microcentrifuges and micropipette chro-matography columns (\100 lL) (PhyTips, PhyNexus).27–30

The microcolumns were operated in a simple step gradientmode with load, wash, and elution steps that enabled a com-parable performance to lab scale.27 These methods wereused to rapidly understand the upstream impact on the qual-ity attributes, aggregation, conformation, and purity, for theproduction of virus-like particles.27 Similar micromethodswere implemented for the centrifugation and periplasmicrelease of Fab fragments from E. coli, where post inductionfermentation feed rate influenced fragment antibody leakagefrom the periplasmic space.30

In summary this review goes beyond previous work (Ref.29, 31, 32) by providing not only a detailed update on thecurrent state of the art but also, the definition of an idealautomated system using process criteria from large scaleexamples. Industrial quantitative and qualitative criteria foran automated small-scale parallel bioreactor for both mam-malian and microbial cell culture are presented (Table 1).This is compared to the current state of the art technology(Table 2–4) which establishes the technological deficienciesneeded to be overcome to reach the desired future state.

Criteria for Automated Small-Scale ParallelBioreactor System

Upstream process development work is usually carried outat the lab scales of 1 to 30 L with 6–8 reactors available perproject. A typical approach is iterative rounds of statisticaldesign of experiments with each round evaluating 5 to 10parameters requiring 20–30 experiments. This would usuallyrequire up to 4 weeks to complete for a microbial processand up to 8–12 weeks for a 10 day mammalian cell process.The application of an automated small-scale parallel reactorsystem enabling up to 20–24 reactors, to be operated at anyone time by a single researcher, would clearly have a signifi-cant impact to the process development timeline. The systemwould need to be capable of mimicking large scale processesof high cell densities and ideally be integrated with purifica-tion to enable an overall process development capability ofthe total process.

The desired criteria for such an automated reactor system,with dual capability for microbial and mammalian cell cul-ture, are outlined in Table 1 and reflect the significant tech-nology challenge. The diversity of the operating differencesbetween mammalian and microbial culture, such as shearsensitivity and oxygen demand are clearly well-established.A common approach across the expression systems is theuse of fed batch feeding regimes to maximize productivityby building high cell densities, typically upto three timeshigher than batch phase. In general the feeding regime strat-egy can vary from simple bolus additions, to fixed continu-ous feeds, or more advanced exponential feed control. Acontinuous feed of concentrated growth limiting substratecan minimize catabolic regulation, oxygen limitation, andheat generation that can often occur during unlimited batchprocesses with excess carbon. The feed rate of a slowlymetabolized carbon source, such as glucose, glycerol ormethanol, often below the maximum uptake rate, is used tooptimize the specific growth rate. This in turn builds biomassto high cell densities and maximizes product formation.Examples include the controlled glucose feeding to preventprevention of acetate accumulation for E. coli,52 ethanol in-hibition of S. cerivisae and methanol inhibition of the AOX1 promoter for Pichia.8 Often product secretion is growthassociated so that specific productivity is a function of

Figure 2. Fermentation scalability for monoclonal antibodyexpression by Pichia pastoris process.

3L ~, 30L l, 1200L n. Comparison of biomass level, oxygenuptake rate and product titer. Product quality with respect to N-glycans, O-glycans, integrity and purity were also found to besimilar (data not shown).

4 Biotechnol. Prog., 2011, Vol. 27, No. 1

specific growth rate. This requires careful optimization oflimited substrate feed to achieve the correct balance ofgrowth and production rates to maximize productivity.53 Analternative strategy to carbon limited feeding is to controlthe growth rate by limited dissolved oxygen, usually set tozero, to control the balance of growth rate to specific produc-tivity.14 In this case the carbon usually remains in residualconcentration while the operating conditions (agitation, airflowrate) control the oxygen uptake rate (OUR) which ultimatelycontrols the balance of growth rate to specific productivity.Feed on demand systems have also been implemented such asdissolved oxygen stat54 or pH stat55 approaches which avoidaccumulation of toxic residual carbon levels.

Using a reactor configuration with geometric similarity tolarge scale would enable a number of key assumptions to bemaintained during scale up. This includes equal aspect ratioallowing the prediction of hydrostatic pressure and oxygensolubility. Similar principles for achieving oxygen transferand mixing can be maintained including the calculation ofpower input. It also enables similar fluid dynamics and flow

properties that influence mass transfer and mixing. This is akey factor as a number of small-scale reactor research groupshave shown that different methods of agitation can producedifferent flow regimes, which in turn can impact the abilityto reproduce large scale cultivation conditions. For example,using reactor systems with orbital shaking are more prone tosurface aeration influences. For this reason it is recom-mended to evaluate clone selection using the same reactortechnology as used at large scale.

For accelerated process development short set up times(\2 h) between experiments is desired and this could beaided by using presterilized disposable reactors. This com-pares to the current practice of requiring 1–2 days to com-plete clean up, setup, sterilization, and maintain a set ofautoclavable benchtop reactors (n of 6). Automated samplingprocedures speed process development by providing highthroughput sample collection and release time for researchersto carry out other development activities. The improved sam-pling frequency, such as overnight data collection, increases theunderstanding of rate kinetics and accelerates the knowledge

Table 1. Criteria for an Ideal Small Scale Automated Parallel Reactor System Based Upon Parameter Ranges Achieved

for Current Industrial Processes

Process Characteristic Cell Culture Yeast E. coli Comments

Growth rate (/h) 0.041–0.075 0.5–4 0.1–4Doubling time (h) 15–24 h 0.5–5 h 0.1–4 hCell density 106–107 cells/mL 200–500 g/L wet

cell weight200–500 g/L wet

cell weightOxygen update rate (OUR)

(mmol/Lh)\5 mmol/Lh Upto 300 mmol/Lh Upto 300 mmol/Lh Require vent gas

analysis for onlineprocess measurement

Power per unit vol (Pg/V) 0.8 to 2 W/L 2 to 10 W/L 2 to 10 W/LkLa (/h) 1–15/h 200–400/h 200–400/hDissolved oxygen

(%air saturation)[20% [20% [20%

Agitation (rpm) 50–150 rpm 100–3000 rpm 100–3000 rpm Desired agitationexpected at 100 mLworking volume

Dissolved CO2 35–80 mmHg (possibletoxicity[110 mmHg)

\5% \5%

Temp (�C) 32 to 38 �C 18 to 30�C 18 to 37�CpH 6.8 to 7.15 4 to 8 6–7.5pH control mechanism Caustic or CO2 addition Acid or caustic additionFed batch capability 20–90 pg/cell/day 0.5 to 9 g/LhAdvanced automation

required for feed controlFeeding strategies (linear ramp, exponential, constant, bolus additions)

and nutrient additions by event triggers or feedingvia pH stat or DO stat

Example of MeOHfeed initiationtriggered uponOUR drop afterglycerol depletion.

Geometric similarity tostirred tank

Equal HL/Di, DT/Di, impeller spacing, baffles : number & spacing

Cycle time Duration of 20 days Duration upto 7 days Duration\ 4 days Acceptable to allowdaily manual removalof accumulated samples

Sufficient working volumeto enable parallelprocessing andautomated sampling

1–2 mL for primary screening Enable sufficientvolume for productquality analysisafter 1st chrom step

50–150 mL for development

At least 24 reactors runin parallel withfast set up time

[20 reactors allows single partial factorial DOE with 4–6 parameters tobe run by 1 scientist

Set up time \2h to setup using disposable reactors and feed manifoldsIntegrated with automated

small scale purificationSample processing to enable purificiation sufficient to analyze product quality. Apply microscale

purification methodsof centrifugation,filtration andchromatography

The system is defined as �24 reactors of dual capability for mammalian culture or microbial culture at 100 mL working volume with automated sam-pling. The system needs to mimic the growth and product kinetics of an upstream commercial process.


build of the process, which ultimately improves the successof meeting the desired process targets.

Sufficient broth volume is needed to enable sampling forprocess characterization including product titer and qualityanalysis. Therapeutic proteins or monoclonal antibodiesaround 3–5 mg of purified product is needed for full charac-terization which includes assays for O glycan, N glycan, LCMS, sialic acids, potency, aggregates, residual DNA, and pu-rity from SDS PAGE gels. The purification usually includesthe first stage of chromatography required, such as protein Afor a mAb, to provide sufficient purity for the quality assays.The automation of these methods onto standard liquid han-dling formats would provide significant speed improvementto process development.

Current State of the Art for Small-ScaleParallel Reactors

This section summarizes the current state of the art for re-actor applications as compared to the desired system criteriaoutlined in the previous section (Table 1). It will becomeapparent that no single device has solved all the challengesof miniaturization to mimic large scale process conditionswhile maintaining the full functionality of conventional bio-reactors. The applications cover a range of systems fromshakeflasks, microfluidic reactors, microtiter plates andsmall-scale stirred tank reactors. Their key specifications andperformance are outlined in Table 2–4.

Shakeflasks

Shake flasks continue to be the common tool of choice ofmost microbial manufacturing processes for growing suffi-cient culture inoculum between the frozen vial and the firstseed stage reactor or directly into the production vessel.They are convenient and simple to use with the availabilityof disposable flasks with vented caps from the 10 mL to 3 Lformat. The reliance on surface aeration and orbital shakingfor agitation makes reduced oxygen transfer a major limita-tion when compared to stirred tank reactors. Oxygen transfercan be maximized by the use of baffled flasks with high agi-tation and minimizing the working volume.33 For example, a20 mL working volume in a 250 mL flask with the standard5 cm orbital throw enabled kLa values upto 150 (h�1)34 (Ta-ble 2). At Merck optical densities values up to 45 have beenachieved for Pichia inoculum seed train expansion. This typ-ically uses 400 mL volume for a 3L flask (250 rpm, 5 cmthrow) with growth eventually becoming pH limited. Theapplication of shake flasks to process development has beenlimited by not only the oxygen transfer issue but also diffi-culty to maintain feed capability, control pH, DO, and thelack of similarity to stirred tanks. Shake flask developmentwork has been shown for slow growing cultures, such asstrain selection and media design of fungal natural prod-ucts,56 yeasts, or animal cell culture.57 The use of stackableincubators enables statistical Plackett Burman type screeningexperiments with 50 to 100 flasks with sufficient volume fortime course profiling.58

The application of fluorescence dye patches has madesome improvement to pH and DO monitoring. Dynamic lu-minescence quenching of the specific fluorophore, such asfrom ruthenium oxide, by oxygen or protons concentrationsis described by the Stern-Volmer equation. The dye is incor-porated into a patch which is adhered to the base of the flaskT

able

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150/h

140–160

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Agitationtype

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withstirredtankgeometry

Orbital

shaking

150–300rpm

Orbital

shaking

300rpm

Orbital

shaking

800–1300rpm

(3mm

throw)

Orbital

shaking

500rpm

(3mm

throw)

Orbital

shaking800–1300

(3mm

throw)

20rpm

circular

rotation

Disposable

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or coated onto the tip of a fiber optic linked probe.34 Pre-sterilized shake flasks containing patches for DO and pHmeasurement are now commercially available (Sensoluxfrom Sartorius stedim biotech and SFR from PreSens). Theflasks are integrated with a shaker tray which houses the op-tical fiber sources for reading the patch sensors. The pHmonitoring is currently limited to a range of 5.5 to 8.5. Thispatch technology is now becoming common place for dispos-able mammalian cell culture reactor systems.4,57 Offgas anal-ysis for oxygen uptake and carbon dioxide evolution rates isalso available.59 Some rudimentary shakeflask feeding sys-tems have been applied to E. coli high producer screeningstrategies.60 Alternative approaches of controlling nutrientlevels include the application of feed beads for the slowrelease of glucose61 and enzyme based gel storage systemsfor the slow diffusion of starch which is enzymatically cata-lyzed to glucose.62 Despite these attempts the shakeflaskremains primarily an inoculum expansion tool and screeningtool at the 10 to 20 mL scale. Where possible the majorityof screening work has transferred to the micro titer plateformat.

Microtiter plates

Over the last decade the microtiter plate has become anestablished screening tool alternative to shakeflasks. Thetechnology has been easily integrated into standard lab auto-mation liquid handling decks and the measurements for pH,OD, and DO are routinely possible via the incorporation offluorescence patch sensors into the base of each well. Forbioprocess strain selection the 96 well plates are routinelyused for primary screening (Figure 1). The agitation by shak-ing applied to microtiter plates generates a centrifugalinduced rotational movement similar to that of the shake-flasks whereby the gas transfer is solely by surface aeration.The gas liquid exchange area is the most limiting factor anddevelopment efforts have focused on improvements throughagitation.35 Surface tension is another important factor inthis regime, which counteracts the flow and movement ofthe liquid g-forces arising from orbital shaking and isimpacted by the culture media components. The combinationof issues complicates the modeling of the process specificoxygen transfer and fluid mixing regime. These factors haveshown that a satisfactory oxygen transfer rate can beachieved despite a poor degree of mixing within the bulk ofthe liquid.35

For conventional 96 well plates (100 lL volume) the kLavalues are limited to those for shakeflasks (140–160 h�1:300 rpm, 5 cm orbital throw) (Table 2).35 Higher agitationresults in spilling and splashing of the contents due to theshallow well design. The use of deep well (2 mL) 96 wellplates with 300 to 500lL working volume has become thestandard for microbial screening which is sufficient volumefor analytical analysis of the desired titer attribute (Figure1). High agitation rates (800 to 1400 rpm) are used in com-bination with a short orbital throw of 3mm, generating kLavalues equivalent to stirred tanks (kLa 500–800 h�1, OTRs100–200 mmol O2 L

�1h�1) (Table 2). These conditions weredeveloped from the knowledge gained from a number ofgroups.35,36,63 including work at Merck, that confirmed theOTR to be proportional to shaking amplitude and frequency.It is also known that the OTR is inversely proportional tothe height/ fill volume at the high shaking speeds 800–1000rpm and short orbital throws (3 mm).35,63 Minimizing the

working volume of the well expands the available headspace to maximize the agitated liquid height. Such effortshave shown to gain a 10-fold improvement in available sur-face area for gas liquid transfer.34 In addition, the square-well plates provide 30 to 50% higher oxygen transfer (kLa828 h�1) compared to round wells at 800 rpm.63 The cornersof the well were found to act as baffles limiting the vortexand increasing turbulence. The high surface to volume ratiocan lead to evaporation so gas permeable sealing membranesare routinely applied.

A number of the studies demonstrated predictive scale upfrom 96 deep well plate (2 mL) scale to lab scale stirredtank (3–10 L scale).35,36,63,64 Micheletti et al. 2006 showedmicroscale reproducibility of transketolase over expressionfrom E. coli from 2 mL to 1.4 L scale based upon constantkLa. The microwell generated maximum kLa values of 288h�1 at 1000 rpm with biomass levels limited to less than 7 gL�1 dry cell weight63 (Table 2). Similarly, mammalian cul-tures of CHO cells were successfully scaled from 800 lL to100 mL shakeflask using mean energy dissipation rates.63

Improved biomass levels were achieved using the 24 deepwell plate system (micro 24) supplied by Applikon. Theround well baffless plate has a total well-volume of 10 mLwith a 3 to 5 mL working volume using orbital short throwagitation. The single reactor plate is placed inside a reactorunit that controls temperature, DO, and pH across the plate.Each well has a sintered sparger point at the base of the wellincluding fluorescence patch sensors for pH, DO, and ODmeasurements. The technology has become established in anumber of industrial screening processes as the next step af-ter the primary 96 well deep plate screening38,65 (Figure 1).Chen et al. 2009 implemented the technology for screeninghigh producing CHO cells at Genentech.38 They demon-strated similar performance as a 2-L stirred tank reactor withrespect to kLa ([30 h�1), doubling time (1.5 to 1.7 days),metabolite production rate (lactate : 2.52–2.86 pmol per cellday), antibody production (1.5 to 1.6 g L�1), and qualityattributes. Surface aeration was relied upon as direct sparg-ing produced foaming issues. The manual addition of car-bonate was required as the mechanism for stripping CO2 asN2 gassing was not sufficient to reach the desired pH set-point. The dual control of both pH and DO control was notpossible. They also combined multiple reactor units with theproprietary robotics technology into a single biosafety cabi-net.38 The automated system enabled the simultaneous load-ing, sampling and feeding of cells for 24 independentreactions. A number of industrial examples used the micro24 system for microbial cultures of, E. coli, Pichia, and Sac-charomyces with automated pH and DO control.37,64,65 Per-formance was found to match lab scale (up to 20 L scale)for nutrient consumption rates and biomass formation. Isettet al. 2007 showed well to well reproducibility across theplate for DO and pH control, using ammonia.39 Air spargekLa values of 30 h�1 supported biomass levels of OD 10(dry cell weight \10 g L�1). KLa values were improved to150–160 h�1 using 100% oxygen. The feeding regime waslimited to manual bolus addition of MeOH feed to all wellsregardless of demand. The combined use of oxygen blendingand manual MeOH additions produced Pichia wet cellweight up to �250 g L�1. Other changes included the useof gas permeable membranes to replace the well filter caps,which became blocked from foaming. The resulting Pichiaprocesses showed reproducibility with titer and glycan qual-ity for Mab production from 3 mL to 2000 L.65


An important challenge is how to control the pH of asmall volume reactor and particularly how to deliver a smallvolume addition of acid or base. Buchenauer et al. 200937

addressed this issue with a 48 well microtiter plate systemlinked to a microfluidic system for process additions. Eachwell had a working volume of 0.5 mL with pH and OD mea-surement using PreSens patch technology (biolectorTM). Thecaustic for pH control addition was added from the micro-fluidic device allowing accurate 5 nL dispense volumes.

An alternative for mammalian cell culture screening andinitial process development is the Sim cell reactor systemfrom Seahorse Bioscience which has a cassette containing 6reactor chambers with 300 to 700 lL working volumes40

(Figure 3). Multiple cassettes are agitated by rotational agita-tion (20 rpm) and CFD confirmed this mimicked theexpected shear rate of a conventional stirred tank. This sys-tem is capable of housing up to 1500 cells within a singletemperature control incubator. The back of each cassette hasa gas permeable membrane for surface aeration achievingkLa values of 7 h�1. At scheduled times a robotic armremoves the cassette to a monitoring station where the DOand pH values are read from the immobilized patch sensorsand total cell count measured by light scattering. The cas-sette is routinely removed to the feeding station to allowautomated bolus additions of nutrient or acids/base for pHcontrol. The system has been demonstrated for process opti-mization of a CHO based antibody production where 180reactions were carried out simultaneously to evaluate theimpact of feed rate and pH on process performance.40 Pfizerpaired the reactors with an automated analytical system.Samples were routinely removed to a 96 well plate formatand analyzed for Mab titer using an Octet analyzer, while alab 90 microchip CE SDS system was used for integrity, pu-rity, and glycan heterogeneity measurements. The DOEshowed feed rate and pH were critical to cell viability, titer,and intact immunoglobulin G titer. The performance wasshown to be equivalent to that of a 3 L reactor with respectto mAb product and cell viability for a 13 day fed batch pro-cess.40 The system is well suited to mammalian cell cultureas the low oxygen transfer rates limit the application for mi-crobial systems.

Microreactors from the application of microfluidics

Microfluidic approaches using small channels and wellstypically fabricated in polymethylmethacylate (PMMA) andpolydimethlsiloxane (PDMS) have been implemented to gen-erate an inexpensive solution for microreactors. Applicationsbeing considered include strain screening and integration

with microarray chip analysis for tools to study gene expres-sion. A number of examples have reproduced lab scale mi-crobial batch processes achieving reasonable biomass levels(5–10 g L�1).66 The systems match stirred tank functionalitywith respect to pH control with monitoring via patch sensorswhereas aeration is by diffusion of oxygen through the per-meable materials of construction. An extensive review of themicrofluidic breakthroughs and shortcomings has recentlybeen provided by Schapper (2009). Some of the key advan-ces are summarized in this section (Table 3).

Zanzotto (2004)41 fabricated a bioreactor with a 5–50 lLvolume using PDMS on glass. The pH and DO measure-ments were made via patch sensors and optical density wasmeasured by transmittance through the culture. Temperaturewas maintained by placing the device in a temperature con-trolled chamber. Examples with E. coli used aeration by dif-fusion achieving kLa values around 60 h�1 and maximumoptical densities of 8, with equivalent performance to a labscale reactor (0.5 L). Improvements to this device includedincreasing the reactor volume to 150 lL and the incorpora-tion of magnetic driven stir bars to improve mixing.42 Thesystem was redesigned to enable rapid setup with a ‘‘plug-in-and-flow’’ connection to automatically align the optical andfluidic connections with the outside housing unit.67 This de-vice allowed the addition of acid, base, and glucose viamicrovalves and control circuits, however dissolved oxygenlimitation remained an issue. Lee et al. (2006)43 used a simi-lar design and provided a peristaltic oxygenating mixer toimprove the kLa to �360 h�1(Figure 4). For E. coli opticaldensities up to 40 were achieved and growth curve resultswere similar to a 4 L benchtop reactor.

Marharbiz et al. (2004)44 designed a device for 250 lLreactors platformed on plastic microtiter plates and printedcircuit boards. Closed loop control of each well for tempera-ture and pH was obtained with a small resistor heater andthermistor for temperature and, an ion selective field effecttransistor sensor chip and platinum reference electrode forpH. Electrolyte gas generation was utilized to create oxygenand carbon dioxide control in an electrolysis chamber belowthe culture chamber. The gas diffused through a slim siliconemembrane to provide DO control to the cultures. Agitationof the culture was provided by a small stainless steel beadand stirring of the reactor assemblies at 1750 rpm, whichachieved optical density levels\3 for E. coli.

Microreactors for animal cell culture have concentrated onimproving mixing efficiency while minimizing exposure todetrimental shear stress levels. Avoiding evaporation is alsoa concern that can lead to osmotic effects to the cells. Diaoet al. (2008)45 designed a reactor constructed from polya-crylic acid and a polymethylpentene membrane with twoelongated chambers connected by a smaller channel to forma U-shape. Pressure shuttling was used to transfer the culture(2.5 mL) between the two chambers, producing mixing andkLa values around 14.8 h�1. Sf-21 cell growth and produc-tion were enhanced compared to a 50 mL spinner flask. Asample port allowed samples to be taken as needed howeveronline pH measurement or control was not available.

A major challenge of bioprocess in microfluidic devices isthe resulting analytics. In most cases each microbioreactorhas to be sacrificed at every time point to provide sufficientmaterial for offline analysis.41,68 This could be overcome bylinking the microfluidic reactor with microarray assays thatrequire only a few microliters of sample. Currently only alimited set of analytics are available in microfluidic type

Figure 3. Microbioreactor array for cell culture (SimCellcourtesy of Seahorse BioSciences).40

Each reactor array is rotated up to 20 rpm on each station.


devices or microarray platforms which include ELISA, SDSPage, FACS, glycan Analysis, and DNA.69,70

Small-scale bioreactors

A number of groups have fabricated small-scale stirredtank reactors at the 10 to 100 mL scale which incorporategeometric similarity to large scale, and have the ability forprocess control of pH, dissolved oxygen, and substrate feed-ing (Table 4, Figure 5). A number of these examples haveshown predictive ability to pilot scale systems with respectto oxygen transfer, growth, and productivity for E. coliand B. subtilis cultures.32,47,69,71 The comparisons have beenbased upon either equal kLa,

49 or energy dissipation.48 Thelimitations of the evaluations were completed at low biomasslevels (\10 g L�1 dry cell weight) so the systems werenever truly challenged with respect to the ability to handlethe high cell densities expected in industry.

Higher biomass levels of 20 g L�1 dry cell weight for aE. coli process were achieved by a multireactor block systemcomprising of 48 reactors with 10 mL working volumes.49 Aunique magnetically driven gas induced impeller introducedgas bubbles through the impeller shaft achieved kLa valuesin the 700 to 1600 h�1 range. Liquid handling enabled thepH, DO, and turbidity to be measured at line by dispensing20 lL samples into microtiter plates containing the fluores-cence patches.49,50 Growth rates and biomass levels wereshown to be equivalent to the 3 L scale. Improvements tothe system led to fed batch capability of intermittent feedingand achieved B. subtilis biomass levels of 45 to 50 g L�1

dry cell weight.51 Oxygen limitation was avoided by using50% oxygen supplementation of the inlet air.

A number of commercial stirred tank systems are availablein the working volume range of 150 to 400 mL which cansupport fast growing microbial cultures. Multiple reactors,T

able

3.ComparisonofState

oftheArtSmallScale

ReactorTechnology:Microfluidic

TechnologyComparedto

DesiredCriteria


Development

Param

eter

Criteria

Microfluidic

Exam

ples

Celltype(biomasslevel

orcellcount)

Cellculture

(upto

5�

107cells/mL)

andmicrobial(O

Dupto

600)

E.coliOD

8E.coliOD

8E.coli\13g/L

E.coliOD\3

Sf-21cells5.3

�106cell/m

Lk La(/h)*

Cellculture

�15/hMicrobial

�400/h

60

60

360

150

5.7–14.8

Agitationtype

Stirred

withstirredtankgeometry

Diffusion

Magnetic

stir

(upto

1500rpm)

Orbital

shaking

300rpm

Orbital

shaking

800–1300rpm

(3mm

throw)

Orbital

shaking

500rpm

(3mm

throw)

Disposable

reactor

�orß

��

��

�Workingvolume

100mL

5–50lL

150lL

100lL

250lL

2500lL

pH

&DO

control

pH

range4to

8.5

DO

control�2

0%

ßß

pH

&DO

via

injectors

&oxygenatingmixers

pH

byISFET,DO

by

electrical

generation

ofO2andCO2

ß

Feedregim

ecapability

Multifeeding:linearramp,

constant,exponential

ßß

ßß

ß

Fastsetuptime

\2h

��

��

�Abilityto

runmultiple

reactionsin

parallel

�24reactions

��

��

�

Abilityto

integratedwith

automated

purification

Abilityto

takematerialforw

ard

topurify

3–5mgofproduct

ßß

ßß

ß

References

41

42

43

44

45

*Allk Lavalues

arebased

upondynam


Figure 4. Microfluidic application from Lee et al. 200643:

microbioreactor array module (a) photograph offour reactors integrated into a single module, (b)cross section of the reactor showing the peristalticoxygenating mixer tubes and fluid reservoir withpressure chamber, (c) top view of the reactor (500lm deep, 100 lL working volume) with optical sen-sors and fluid injectors (d) cross section showing thefluid injector metering valves.


Table

4.ComparisonofState

oftheArtSmallScale

ReactorTechnology:StirredReactorTechnologyComparedto

DesiredCriteria


Development

Param

eter

Criteria

Stirred

Tankmim

ics

Stirred

48Cell

ReactorBlock

Dasgip

AG

6ReactorSystem

Biostat

Q6þ

(Sartorius)

Medicel

Explorer15

reactors

(Medicel)

CloneScreener

32Reactor

(BiospectraAG)

AmbrT

M24

Reactor

System

(TAP)

Celltype(biomass

level

orcellcount)

Cellculture

(upto

5�

107cells/mL)

andmicrobial

(OD

upto

600,

[200g/L

dry

cellweight)

E.coli,B.subtilis,

\10g/L

dry

cellweight

E.coli20g/L

Bacillus50g/L

CellculturePichia,

E.coli350g/L

Wet

cellweight

Cellculture

E.coli

7g/L

dry

cell

weight

Cellculture

Pichia

200g/L

WCW

Cellculture

and

microbial

Cellculture

only

k La(/h)*

Cellculture

�15/h

Microbial�

400/h

Upto

480

Upto

1600

Upto

400

Upto

400

Upto

191

TBD

\30/h

Agitationtype

Stirred

withstirred

tankgeometry

50–3000rpm

200–7000rpm

100–2000rpm

100–2000rpm

100–1200rpm

100–1200rpm

100–1200rpm

�200rpm

Disposable

reactor

�orß

�ß

ßß

ßß

�Workingvolume

50–200mL

10–100mL

10mL

60–200mL

500–1000mL

150mL

400mL

10mL

pH

&DO

control

pH

range4to

8.5

DO

control�

20%

��

þO2gas

blending

�þ

O2gas

blending

�þ

O2gas

blending

�þ

O2gas

blending

�þ

O2gas

blending

�þ

O2gas

blending

Feedregim

ecapability

Multifeeding:linear

ramp,constant,

exponential

ßInterm

ittent

��

��

Interm

ittent

additions

Fastsetuptime

\2h

ßß

ßß

ßß

�Abilityto

runmultiple

reactionsin

parrallel

�24reactions

��

ß6units/system

ß6units/system

ß15units/system

�32reactors

�24reactors

Abilityto

integratwith

automated

purification

Abilityto

takematerial

forw

ardto

purify

3–5mgofproduct

ßß

��

��

�

References

32,46–48

49–51

Merck

internal

data

Merck

internal

data

Communication

withMedicel

Oy

andtheMedicell

Explorer

specfications

datasheet.

TheAutomation

Partnership

specifications

documentfor

theam

brT

M

system

.

*allk Lavalues

arebased

upondynam



usually groups of 6, are operated from a central control sys-tem (Multifors from Infors, Biostat Qþ from Sartorius stedimBiotech and Dasgip system16). These systems use standard labscale geometry and conventional glass pH probes and poly-graphic DO measurement for control. They all acquire steamsterilization within an autoclave and significant set up time andcleaning. The level of complexity of feed control strategyvaries between systems. For example the multifors has limitedfeeding capability, while the Dasgip and Sartorius enableadvanced feeding strategies of exponential or multivariate flowcontrol. This can be controlled by gravimetric feed control(Biostat Qþ, Sartorius) or by calibrated micropump (Dasgip)to deliver the desired low flow rates. Additional modules areavailable for vent gas analysis using sensors, electrochemical,and infrared for oxygen and carbon dioxide, respectively. Forcell culture these reactors have become the routine tool for pro-cess development. They replace the spinner flasks (magneti-cally stirred glass vessels) which had limited automation formultireactor parallel work when studying ranges for pH, tem-perature, and feed manipulation.

A number of automated systems have recently becomecommercially available that incorporate automated controlwith sampling and post fermentation conditioning. MedicelOy designed a 15 parallel fermentation system (MedicelExplorer) with a 150 mL working volume, independent con-trol of pH, DO, agitation (200 to 1200 rpm) and one continu-ous feed. Integrated vent gas analysis is available along withroutine automated reactor sample collection and online ODmeasurement. The system has been demonstrated for mam-malian cell culture with good batch to batch reproducibilityand similar performance to shakeflasks. Initial evaluationfor microbial work with Pichia generated 200 g L�1 wetcell weight broths using a 150 mL working volume at 30 �Cwith a single impeller. KLa values were achieved in the151 h�1 to 191 h�1 range (Communication with Medicel Oyand the Medicell Explorer specfications data sheet). Theneed for manual sterilization and setup of reactors will takearound 1 day to complete as disposable reactors are not cur-rently available.

A fully automated system is available from Biospectrawhich incorporates 32 bioreactor segments each consistingof a 400 mL working volume reactor, with four feeding ves-sels (acid/base and two feed reservoirs). Automated clean inplace and steam in place is performed along with automatedprobe calibration, inoculation, sampling, and harvest. Thecomplete system is housed in a 2 m � 2 m system with aweight of 3.5 tons and requires a multimillion dollar invest-ment. Initial applications have been applied to mammaliancell culture.

A high throughput construct screening tool for basicresearch, designed by The Automation Partnership (TAP) inconjunction with an industrial consortium, enabled the con-struct screening for protein production (The AutomationPartnership specifications document 15-A for Piccolo con-struct screening system). The reactor system was designedwith 384 � 10 mL working volume tubes with mixing pro-vided by an agitating finger rod. Culture conditions can bevaried with respect to temperature, and inducer concentra-tion. Constructs are grown in the tubes to a desired opticaldensity, measured online, and then automatically transferredthrough cell lysis and protein purification steps. This hasbeen applied to E. coli and insect cells where optical den-sities upto 20 have been achieved. The Automation Partner-ship has also recently developed an advanced automatedmicroscale bioreactor system for cell culture (The Automa-tion Partnership specifications document for the ambrTM sys-tem). (Figure 6). Twenty four disposable cell culture reactorsof 10 mL working volume are supported on a liquid han-dling deck. Gas additions of nitrogen and oxygen blendingare feasible along with closed loop pH control from sensorpatches mounted in the base of each reactor. Automated con-trol of sampling, additions and intermittent feeding regimesare possible using standard sterile liquid handling pipettetechnology inside a biosafety cabinet. The system is envi-sioned to support cell line evaluation and initial process de-velopment optimization. Potential limitations include thesmall working volume that may limit the availability of puri-fied product from each reactor, and the number of product

Figure 5. Example of a miniature stirred tank reactor system.

(Working volume of 10 mL).46,71


quality attributes that can be characterized. A microbial ver-sion of this system is currently not available.

Summary: Future Requirements for the NextGeneration of Advanced Reactor Systems

The need for the automated small-scale parallel reactorsystem to mimic large scale capability is well-under-stood.31,32 This would provide the confidence that the growthkinetics and protein expression can be expected to achievequantitative scale up. Significant impact would be provided,not only to process development, both upstream and down-stream, but also for strain selection and evaluation. Thisreview has shown that no single device has yet to meet allthe challenges of miniaturizing large scale process conditionswhile retaining the full functionality of conventional bioreac-tors for industrial processes (Tables 2–4). It is clear that ahigher degree of instrumentation for feedback control of pHand DO and feed regime strategies are essential. The simpli-fied systems of shakeflasks and microtiter plates provide sig-nificant high throughput capability. Yet they carry lessinstrumentation which limits the opportunity for data qualityand quantity. For this reason they remain as screening toolswithout implementation of robust pH and DO control. Someimprovement was made with the Applikon micro 24 wellplate system that enabled pH control. However the systemstill lacks continuous and independent nutrient feed capabil-ity and requires significant adjustments by the customer toimplement a particular project. Although some microfluidicdevices enable control of pH and DO, they lack the complexfeeding ability to achieve high cell densities. The small vol-umes also limit the ability for the full range of product qual-ity attributes necessary for mAb process development. Theminiaturized pH and DO monitoring from patch sensors hasimproved significantly over the last 10 years and is nowbecoming routine operation for the pH ranges of 5.5 to 8.5.Improvements to measure lower pH values of 3.5–5.0 wouldhave significant impact for microbial culture, particularlyPichia processing. In addition, a greater understanding ofpatch robustness and lot to lot variability would also expand

the application. Similar advances are needed to bring feed-back control for substrates and metabolites to the small-scalereactors such as online NIR and miniature LC/MS.

It is clear that a number of devices have recognized theimportance to replicate geometric similarity to the largescale bioreactors. Nevertheless, a greater emphasis is neededin the development of systems that can support high celldensities as the majority of the literature examples have onlyevaluated microbial processes at low biomass levels (opticaldensities \30). A few small-scale stirred tank systems haveshown promising progress (Dasgip, Biostat Qþ Sartorius,Medicell Explorer) with the use of microfeed pump technol-ogy along with gas blending to support the desired high celldensities. They also have sufficient volume to enable purifi-cation of product for the quality analysis. However thesesystems are still burdened with significant preparation andclean up time. This could be improved by a greater imple-mentation of presterilized disposable parts, of not only thereactor, but also presterile manifolds for additions and dis-posable reservoirs. This would move the technology to a‘‘plug & play’’ paradigm for minimizing downtime betweenruns. The application of disposables for microbial systems isclearly lagging behind the progress made for cell culturesuch as the SIM cell systems and ambr system from TAP.The improvements over the last decade for disposable mate-rials of construction and mould technologies should makethis task achievable.

Moving forward, this review has outlined the significantopportunity for the expanded use of automation to improvedevelopment throughput. Automation is needed to increasethe parallelism of the small-scale stirred tank systems, partic-ularly for microbial culture. Elegant solutions are needed forlinking the upstream parallel throughput with sample analy-sis and purification. This will provide the total solution foroptimization of overall process yield and product quality. Afew examples have shown cell culture integrated with auto-mated sample analysis38,40 but they have not included thenecessary extensive characterization of product quality (TheAutomation Partnership specifications document for theambrTM system). The authors envisage an automated solution

Figure 6. Cell culture automated microscale bioreactor system (ambrTM

from The Automation Partnership).

A total of 24 disposable reactors are controlled on a liquid handler. Each reactor has a 10 mL working volume with pH and DO measurement fromthe base of the reactor using patch sensors.


encompassing the integration of liquid handling decks forupstream and downstream purification. The upstream systemwould use disposable reactors for fast setup time with auto-mated media dispense, inoculation, and growth control ena-bling variable conditions and feed regimes. Automated samplecollection will need to be integrated to a purification deck,expanding upon the initial microscale methods for centrifuga-tion,27 disruption (if necessary),28 and chromatography.27,72

This will provide sufficient sample purity to complete productquality characterization. Significant effort will be required forthe development of micro scaledown purification methods,particularly for the primary recovery of high cell density mi-crobial broths where simple microwell manifold filtration73 isunlikely to be feasible. Fundamental engineering understand-ing for centrifugation and disruption microscale work will beessential. The longer term (10–15 years) will likely bring fur-ther throughput improvements from the introduction of micro-fluidic approaches into the automated development workflow.Early work has demonstrated feasibility of microfluidicapproaches to ion exchange chromatography at the 1.5 lLscale.74 It is also anticipated that standard LC column meth-ods will be replaced by microfluidic chip analysis, such asrecently demonstrated for DNA and RNA analysis.75,76

The authors believe the technology improvements outlinedin this review are essential for the future success of biologicsprocess development. The advances need to continue on allfronts: upstream, downstream, and analytical development.This review focused on the challenges confronting upstreamdevelopment with the vision of integration to purificationand analytical platforms. It is envisaged the integration ofparalleled, disposable, well controlled systems will make sig-nificant impact to improving process development efficiency(estimated at 4–5 fold). This will provide faster routes toscaleable robust processes, without sacrificing process pro-ductivity or product quality.

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Manuscript receivedMar. 13, 2010, and revision received Jun. 13, 2010.


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