A Review of Advanced Small-Scale Parallel Bioreactor Technology for Accelerated Process Development: Current State and Future Need

Rachel Bareither and David Pollard

Biologics New & Enabling Technologies, Biologics Development, Merck Research Laboratories, Merck & Co. Inc., Rahway, NJ 07065 DOI 10.1002/btpr.522 Published online November 9, 2010 in Wiley Online Library (wileyonlinelibrary.com).

The pharmaceutical and biotech industries face continued pressure to reduce development costs and accelerate process development. This challenge occurs alongside the need for increased upstream experimentation to support quality by design initiatives and the pursuit of predictive models from systems biology. A small scale system enabling multiple reactions in parallel (n ! 20), with automated sampling and integrated to purication, would provide signicant improvement (four to vefold) to development timelines. State of the art attempts to pursue high throughput process development include shake asks, microuidic reactors, microtiter plates and small-scale stirred reactors. The limitations of these systems are compared to desired criteria to mimic large scale commercial processes. The comparison shows that signicant technological improvement is still required to provide automated solutions C that can speed upstream process development. V 2010 American Institute of Chemical Engineers Biotechnol. Prog., 27: 214, 2011 Keywords: bioreactor, process development, integrated, automation

Introduction
Importance of bioprocesses and the need to control development costs The application of biotechnology for the commercialization of pharmaceuticals continues to increase in importance as biologics research becomes [50% of pharmas pipeline portfolio. Over 600 biologics are under development each year with the majority comprising of monoclonal antibodies (mAbs) providing the fastest growth of new therapeutics with a global expectation of around !$49 B by 2013.1 This increasing inuence of biologics development comes at a time of continued pressure on the pharma industry from the challenges of cost pressures from healthcare providers, revenue reduction from patent expiration, pipeline concerns and increasing generics competition.1,2 The focus of cost control applies not only to manufacturing but also to development where costs have risen exponentially during the last decade.2 The increasing size and complexity of clinical trials contributes to these costs, but equally there is a need to lower drug supply development costs, and reduce the time from discovery to market.3 Companies recognize the need to pursue new technological methods to gain development efciency and potential competitive advantage.

Accelerating process development by high throughput bioreactor technology The high throughput methods implemented for strain development, using 96 well plate screens with integrated analytical automation, has shifted the rate limiting step toward
Correspondence concerning this article should be addressed to D. Pollard at david.pollard@merck.com.
2

strain evaluation and process development. Figure 1 demonstrates the intensive upstream experimentation required for process development of a biologic. This is a signicant resource burden, requiring integration with purication, to overcome the issues of process inuence to product quality and obtaining sufcient process understanding for robustness and scalability. The remainder of this introduction provides further clarity to the justication for technology implementation to accelerate development. Improving Process Efciency: Cost of Goods and Development Timelines. The currently approved mAbs are primarily produced in mammalian cell lines, typically either Chinese Hamster Overy (CHO) or Murine myeloma cells (NS0, SP2/0).4 In 2006, the average production cost for a mAb was determined in the range of $300 to $3000 per gram.5 In the late 1990s the upstream bioreactor production titer was the main driver for process economics with 600 mg L1 as an average titer.5 Titers greater than 2 g L1 are now common place while titers in the 5 to 10 g L1 range are achievable.6,7 At these titers the process economics shift toward the purication costs and deter the need for future upstream titer improvement.6 This puts increasing emphasis on improving product development timelines and achieving desired product quality attributes.8 The use of faster growing microbial systems offers the ability to reduce development timelines and production costs compared to slower growing mammalian cell lines. The replacement of mammalian cell culture with glycoengineered Pichia pastoris libraries, that replicate the most essential glycosylation pathways found in mammals, is now becoming a commercial reality.810 mAb titers of !1 g L1 have routinely been expressed in Pichia with highly uniform human N-linked glycans and productivities of [50 mg h L1 are likely to be achievable.8 This capability adds to the existing microbial expression host
C V 2010 American Institute of Chemical Engineers

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

This example is for protein/mAb expression from Pichia process taking up to 1.53 years of development time.

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 ne chemicals.15 This assists to invigorate the demand for improved technology to speed process development.16 Maintaining Product Quality and Process Reproducibility throughout the Development Phases. The therapeutic efciency of many antibodies is inuenced from the N-linked glycosylation which modulate the interaction with specic Fc receptors, and determine antibody dependent cytotoxicity (ADCC) function.17 These complex glycoproteins are susceptible to heterogeneity and modications, such as variants of N terminal pyroglutamate and C-terminal lysine, methionine oxidation, disulphide bond scrambling, aggregation, and fragmentation. It is well-established that the combination of the upstream process (expression host and culture conditions) and the interaction with the primary recovery and purication processes will inuence the product quality. It is understood that multifactorial statistical experimentation is essential to investigate the complex relationships, especially as optimum conditions for titer often compete with the conditions for desired quality attributes. Using this approach has led to many examples of improved product quality, such as the impact of media composition process conditions (dO2, dCO2, pH, substrate feed rates) and use of additives to inuence the glycosylation during expression.1820 This experimental bur-

den requires high intensity resources, especially for long durations with cell culture (1021 days processes) and would clearly benet from high throughput automated technology. Increased Experimentation to Support Cellular Function Models. In the coming years the acceleration of process development will be assisted from the use of predictive models of complex cellular behavior.21 Over expression is often rate limited by an enzyme of a complex multicompartment process, where the kinetics are poorly understood. Establishing the interconnection between genomic sequences to phenotype function leads to identifying rate the limiting steps. Signicant effort has been made to advance the technical methods required for systems biology, such as the cost reduction of microarrays22 combined with improved methods for proteomics,23 metabolic ux analysis24 metabolite analysis25 and stoichiometric network analysis. These advances have recently enabled the genome wide analysis of cell physiology to become routinely applied to process development. To ultimately build effective cellular function models demands the systems biology tools to be applied to industrially relevant manufacturing conditions to understand the impact of feed rates, physical and chemical stresses. The requirement of large data sets dictates the majority of the work to be carried out at lab scale with scale down technology that mimics the industrial fed batch high cell density processes. This signicant upstream experimental burden is only achievable with effective automated reactor technology that allows parallel cultures (n ! 20). The development of such automated small-scale fermentation

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The microcolumns were operated in a simple step gradient mode with load, wash, and elution steps that enabled a comparable performance to lab scale.27 These methods were used to rapidly understand the upstream impact on the quality attributes, aggregation, conformation, and purity, for the production of virus-like particles.27 Similar micromethods were implemented for the centrifugation and periplasmic release of Fab fragments from E. coli, where post induction fermentation feed rate inuenced fragment antibody leakage from the periplasmic space.30 In summary this review goes beyond previous work (Ref. 29, 31, 32) by providing not only a detailed update on the current state of the art but also, the denition of an ideal automated system using process criteria from large scale examples. Industrial quantitative and qualitative criteria for an automated small-scale parallel bioreactor for both mammalian and microbial cell culture are presented (Table 1). This is compared to the current state of the art technology (Table 24) which establishes the technological deciencies needed to be overcome to reach the desired future state.

Criteria for Automated Small-Scale Parallel Bioreactor System

Figure 2. Fermentation scalability for monoclonal antibody expression by Pichia pastoris process.
3L ~, 30L l, 1200L n. Comparison of biomass level, oxygen uptake rate and product titer. Product quality with respect to Nglycans, O-glycans, integrity and purity were also found to be similar (data not shown).

technology tools to assist this work lags behind the advancement of the systems biology technology. Scale Up Evaluation and Late Stage Process Characterization. Optimization begins with the baseline lab scale process using operating constraints that mimic the limitations expected at large scale. This will nd key stress factors and parameters that inuence cell growth, yield, and product quality and identify areas for further optimization. The development process must include pilot scale runs to verify the scalability and validate the lab scale down process (Figure 1). During late stage development, process validation includes critical process characterization and robustness assessment. Extensive statistical DOEs determine the acceptable ranges for critical operational parameters and dene the edge of failure (Figure 1). This design space denition forms a key part of the new quality by design paradigm.26 It typically requires at least 5060 experiments for fermentation characterization of a process taking up to 68 months to complete. An effective scaledown model is required for this work and is currently performed at the 1 to 30 L scale where fermentation kinetics, product titer, and quality are representative of a large scale batch (Figure 2). Integrating Upstream with Downstream Purication. Finally, to streamline development for the increasing experimentation demand, the linking of high throughput fermentation to automated purication would clearly provide benets. A few examples have shown this advantage with microscale purication steps operated on standard liquid handling decks. Methods have included low volume, high pressure cell disruption with a microuidizer or adaptive focused acoustics along with microcentrifuges and micropipette chromatography columns (\100 lL) (PhyTips, PhyNexus).2730

Upstream process development work is usually carried out at the lab scales of 1 to 30 L with 68 reactors available per project. A typical approach is iterative rounds of statistical design of experiments with each round evaluating 5 to 10 parameters requiring 2030 experiments. This would usually require up to 4 weeks to complete for a microbial process and up to 812 weeks for a 10 day mammalian cell process. The application of an automated small-scale parallel reactor system enabling up to 2024 reactors, to be operated at any one time by a single researcher, would clearly have a signicant impact to the process development timeline. The system would need to be capable of mimicking large scale processes of high cell densities and ideally be integrated with purication to enable an overall process development capability of the total process. The desired criteria for such an automated reactor system, with dual capability for microbial and mammalian cell culture, are outlined in Table 1 and reect the signicant technology challenge. The diversity of the operating differences between mammalian and microbial culture, such as shear sensitivity and oxygen demand are clearly well-established. A common approach across the expression systems is the use of fed batch feeding regimes to maximize productivity by building high cell densities, typically upto three times higher than batch phase. In general the feeding regime strategy can vary from simple bolus additions, to xed continuous feeds, or more advanced exponential feed control. A continuous feed of concentrated growth limiting substrate can minimize catabolic regulation, oxygen limitation, and heat generation that can often occur during unlimited batch processes with excess carbon. The feed rate of a slowly metabolized carbon source, such as glucose, glycerol or methanol, often below the maximum uptake rate, is used to optimize the specic growth rate. This in turn builds biomass to high cell densities and maximizes product formation. Examples include the controlled glucose feeding to prevent prevention of acetate accumulation for E. coli,52 ethanol inhibition of S. cerivisae and methanol inhibition of the AOX 1 promoter for Pichia.8 Often product secretion is growth associated so that specic productivity is a function of

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Table 1. Criteria for an Ideal Small Scale Automated Parallel Reactor System Based Upon Parameter Ranges Achieved for Current Industrial Processes Process Characteristic Growth rate (/h) Doubling time (h) Cell density Oxygen update rate (OUR) (mmol/Lh) Power per unit vol (Pg/V) kLa (/h) Dissolved oxygen (%air saturation) Agitation (rpm) Dissolved CO2 Temp ( C) pH pH control mechanism Fed batch capability Advanced automation required for feed control Cell Culture 0.0410.075 1524 h 106107 cells/mL \5 mmol/Lh 0.8 to 2 W/L 115/h [20% 50150 rpm Yeast 0.54 0.55 h 200500 g/L wet cell weight Upto 300 mmol/Lh 2 to 10 W/L 200400/h [20% 1003000 rpm E. coli 0.14 0.14 h 200500 g/L wet cell weight Upto 300 mmol/Lh 2 to 10 W/L 200400/h [20% 1003000 rpm Desired agitation expected at 100 mL working volume Comments

Require vent gas analysis for online process measurement

3580 mmHg (possible \5% \5% toxicity [110 mmHg) 32 to 38  C 18 to 30 C 18 to 37 C 6.8 to 7.15 4 to 8 67.5 Acid or caustic addition Caustic or CO2 addition 2090 pg/cell/day 0.5 to 9 g/Lh Feeding strategies (linear ramp, exponential, constant, bolus additions) and nutrient additions by event triggers or feeding via pH stat or DO stat

Geometric similarity to stirred tank Cycle time Sufcient working volume to enable parallel processing and automated sampling At least 24 reactors run in parallel with fast set up time Set up time Integrated with automated small scale purication

Example of MeOH feed initiation triggered upon OUR drop after glycerol depletion. Equal HL/Di, DT/Di, impeller spacing, bafes : number & spacing Duration upto 7 days 12 mL for primary screening 50150 mL for development Duration \ 4 days Acceptable to allow daily manual removal of accumulated samples Enable sufcient volume for product quality analysis after 1st chrom step

Duration of 20 days

[20 reactors allows single partial factorial DOE with 46 parameters to be run by 1 scientist \2h to setup using disposable reactors and feed manifolds Sample processing to enable puriciation sufcient to analyze product quality.

Apply microscale purication methods of centrifugation, ltration and chromatography

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

specic growth rate. This requires careful optimization of limited substrate feed to achieve the correct balance of growth and production rates to maximize productivity.53 An alternative strategy to carbon limited feeding is to control the growth rate by limited dissolved oxygen, usually set to zero, to control the balance of growth rate to specic productivity.14 In this case the carbon usually remains in residual concentration while the operating conditions (agitation, airow rate) control the oxygen uptake rate (OUR) which ultimately controls the balance of growth rate to specic productivity. Feed on demand systems have also been implemented such as dissolved oxygen stat54 or pH stat55 approaches which avoid accumulation of toxic residual carbon levels. Using a reactor conguration with geometric similarity to large scale would enable a number of key assumptions to be maintained during scale up. This includes equal aspect ratio allowing the prediction of hydrostatic pressure and oxygen solubility. Similar principles for achieving oxygen transfer and mixing can be maintained including the calculation of power input. It also enables similar uid dynamics and ow

properties that inuence mass transfer and mixing. This is a key factor as a number of small-scale reactor research groups have shown that different methods of agitation can produce different ow regimes, which in turn can impact the ability to reproduce large scale cultivation conditions. For example, using reactor systems with orbital shaking are more prone to surface aeration inuences. For this reason it is recommended to evaluate clone selection using the same reactor technology as used at large scale. For accelerated process development short set up times (\2 h) between experiments is desired and this could be aided by using presterilized disposable reactors. This compares to the current practice of requiring 12 days to complete clean up, setup, sterilization, and maintain a set of autoclavable benchtop reactors (n of 6). Automated sampling procedures speed process development by providing high throughput sample collection and release time for researchers to carry out other development activities. The improved sampling frequency, such as overnight data collection, increases the understanding of rate kinetics and accelerates the knowledge

6
Table 2. Comparison of State of the Art Small Scale Reactor Technology: Shake Flask and Microtiter Plate Technology (Mtp) Compared to Desired Criteria for High Throughput Process Development Cell culture only

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Pichia & E. Coli [250 g/L WCW [150 using O2 blending

Orbital shaking 8001300 (3 mm throw)

MTP 24 Well Plate (Micro 24 Applikon) (Microbial)

 56 mL pH [5.5or DO

 

(Volume too small) 39

build of the process, which ultimately improves the success of meeting the desired process targets. Sufcient broth volume is needed to enable sampling for process characterization including product titer and quality analysis. Therapeutic proteins or monoclonal antibodies around 35 mg of puried product is needed for full characterization which includes assays for O glycan, N glycan, LC MS, sialic acids, potency, aggregates, residual DNA, and purity from SDS PAGE gels. The purication usually includes the rst stage of chromatography required, such as protein A for a mAb, to provide sufcient purity for the quality assays. The automation of these methods onto standard liquid handling formats would provide signicant speed improvement to process development.

20 rpm circular rotation

SIM Cell (Seahorse BioScience)

 0.3 to 0.7 mL pH 6.5 to 7.3 Intermittent additions  

(Volume too small) 40

CHO cells 32 106 cells / mL \30

Orbital shaking 500 rpm (3 mm throw)  56 mL pH [5.5or DO

MTP 24 Well Plate (Micro 24 Applikon) CHO Cells

Current State of the Art for Small-Scale Parallel Reactors

(Volume too small) 38

Orbital shaking 8001300 rpm (3 mm throw)  0.10.5 mL

E. coli 7 g/L dry cell weight 500828

 

(Volume too small) 37,36,37

This section summarizes the current state of the art for reactor applications as compared to the desired system criteria outlined in the previous section (Table 1). It will become apparent that no single device has solved all the challenges of miniaturization to mimic large scale process conditions while maintaining the full functionality of conventional bioreactors. The applications cover a range of systems from shakeasks, microuidic reactors, microtiter plates and small-scale stirred tank reactors. Their key specications and performance are outlined in Table 24. Shakeasks

MTP 96 deep Well

E. coli \10 g/L

Orbital shaking 300 rpm

MTP 96 Shallow Well

 0.050.1 mL

 

(Volume too small) 34

Shake asks continue to be the common tool of choice of most microbial manufacturing processes for growing sufcient culture inoculum between the frozen vial and the rst seed stage reactor or directly into the production vessel. They are convenient and simple to use with the availability of disposable asks with vented caps from the 10 mL to 3 L format. The reliance on surface aeration and orbital shaking for agitation makes reduced oxygen transfer a major limitation when compared to stirred tank reactors. Oxygen transfer can be maximized by the use of bafed asks with high agitation and minimizing the working volume.33 For example, a 20 mL working volume in a 250 mL ask with the standard 5 cm orbital throw enabled kLa values upto 150 (h1)34 (Table 2). At Merck optical densities values up to 45 have been achieved for Pichia inoculum seed train expansion. This typically uses 400 mL volume for a 3L ask (250 rpm, 5 cm throw) with growth eventually becoming pH limited. The application of shake asks to process development has been limited by not only the oxygen transfer issue but also difculty to maintain feed capability, control pH, DO, and the lack of similarity to stirred tanks. Shake ask development work has been shown for slow growing cultures, such as strain selection and media design of fungal natural products,56 yeasts, or animal cell culture.57 The use of stackable incubators enables statistical Plackett Burman type screening experiments with 50 to 100 asks with sufcient volume for time course proling.58 The application of uorescence dye patches has made some improvement to pH and DO monitoring. Dynamic luminescence quenching of the specic uorophore, such as from ruthenium oxide, by oxygen or protons concentrations is described by the Stern-Volmer equation. The dye is incorporated into a patch which is adhered to the base of the ask

140160

Orbital shaking 150300 rpm

 10 to 3000 mL

PichiaOD 40

Shake Flask

 

 

Cell culture (upto 5 107 cells/mL) and microbial (OD upto 600) Cell culture 15/h Microbial 400/h Stirred with stirred tank geometry

33, 34 * All kLa values are based upon dynamic gassing out methodology.

150/h

Fast set up time Ability to run multiple reactions in parrallel Ability to integrated with automated purication References

Cell type (biomass level or cell count) kLa (/h)*

Feed regime capability

Agitation type

Disposable reactor Working volume pH & DO control

Parameter

 100 mL pH range 4 to 8.5DO control !20% Multifeeding: linear ramp, constant, exponential \2 h !24 reactions

Ability to take material forward to purify 35 mg of product

Criteria

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or coated onto the tip of a ber optic linked probe.34 Presterilized shake asks containing patches for DO and pH measurement are now commercially available (Sensolux from Sartorius stedim biotech and SFR from PreSens). The asks are integrated with a shaker tray which houses the optical ber sources for reading the patch sensors. The pH monitoring is currently limited to a range of 5.5 to 8.5. This patch technology is now becoming common place for disposable mammalian cell culture reactor systems.4,57 Offgas analysis for oxygen uptake and carbon dioxide evolution rates is also available.59 Some rudimentary shakeask feeding systems have been applied to E. coli high producer screening strategies.60 Alternative approaches of controlling nutrient levels include the application of feed beads for the slow release of glucose61 and enzyme based gel storage systems for the slow diffusion of starch which is enzymatically catalyzed to glucose.62 Despite these attempts the shakeask remains primarily an inoculum expansion tool and screening tool at the 10 to 20 mL scale. Where possible the majority of screening work has transferred to the micro titer plate format.

Microtiter plates Over the last decade the microtiter plate has become an established screening tool alternative to shakeasks. The technology has been easily integrated into standard lab automation liquid handling decks and the measurements for pH, OD, and DO are routinely possible via the incorporation of uorescence patch sensors into the base of each well. For bioprocess strain selection the 96 well plates are routinely used for primary screening (Figure 1). The agitation by shaking applied to microtiter plates generates a centrifugal induced rotational movement similar to that of the shakeasks whereby the gas transfer is solely by surface aeration. The gas liquid exchange area is the most limiting factor and development efforts have focused on improvements through agitation.35 Surface tension is another important factor in this regime, which counteracts the ow and movement of the liquid g-forces arising from orbital shaking and is impacted by the culture media components. The combination of issues complicates the modeling of the process specic oxygen transfer and uid mixing regime. These factors have shown that a satisfactory oxygen transfer rate can be achieved despite a poor degree of mixing within the bulk of the liquid.35 For conventional 96 well plates (100 lL volume) the kLa values are limited to those for shakeasks (140160 h1: 300 rpm, 5 cm orbital throw) (Table 2).35 Higher agitation results in spilling and splashing of the contents due to the shallow well design. The use of deep well (2 mL) 96 well plates with 300 to 500lL working volume has become the standard for microbial screening which is sufcient volume for analytical analysis of the desired titer attribute (Figure 1). High agitation rates (800 to 1400 rpm) are used in combination with a short orbital throw of 3mm, generating kLa values equivalent to stirred tanks (kLa 500800 h1, OTRs 100200 mmol O2 L1h1) (Table 2). These conditions were developed from the knowledge gained from a number of groups.35,36,63 including work at Merck, that conrmed the OTR to be proportional to shaking amplitude and frequency. It is also known that the OTR is inversely proportional to the height/ ll volume at the high shaking speeds 8001000 rpm and short orbital throws (3 mm).35,63 Minimizing the

working volume of the well expands the available head space to maximize the agitated liquid height. Such efforts have shown to gain a 10-fold improvement in available surface area for gas liquid transfer.34 In addition, the squarewell plates provide 30 to 50% higher oxygen transfer (kLa 828 h1) compared to round wells at 800 rpm.63 The corners of the well were found to act as bafes limiting the vortex and increasing turbulence. The high surface to volume ratio can lead to evaporation so gas permeable sealing membranes are routinely applied. A number of the studies demonstrated predictive scale up from 96 deep well plate (2 mL) scale to lab scale stirred tank (310 L scale).35,36,63,64 Micheletti et al. 2006 showed microscale reproducibility of transketolase over expression from E. coli from 2 mL to 1.4 L scale based upon constant kLa. The microwell generated maximum kLa values of 288 h1 at 1000 rpm with biomass levels limited to less than 7 g L1 dry cell weight63 (Table 2). Similarly, mammalian cultures of CHO cells were successfully scaled from 800 lL to 100 mL shakeask using mean energy dissipation rates.63 Improved biomass levels were achieved using the 24 deep well plate system (micro 24) supplied by Applikon. The round well bafess plate has a total well-volume of 10 mL with a 3 to 5 mL working volume using orbital short throw agitation. The single reactor plate is placed inside a reactor unit that controls temperature, DO, and pH across the plate. Each well has a sintered sparger point at the base of the well including uorescence patch sensors for pH, DO, and OD measurements. The technology has become established in a number of industrial screening processes as the next step after the primary 96 well deep plate screening38,65 (Figure 1). Chen et al. 2009 implemented the technology for screening high producing CHO cells at Genentech.38 They demonstrated similar performance as a 2-L stirred tank reactor with respect to kLa ([30 h1), doubling time (1.5 to 1.7 days), metabolite production rate (lactate : 2.522.86 pmol per cell day), antibody production (1.5 to 1.6 g L1), and quality attributes. Surface aeration was relied upon as direct sparging produced foaming issues. The manual addition of carbonate was required as the mechanism for stripping CO2 as N2 gassing was not sufcient to reach the desired pH setpoint. The dual control of both pH and DO control was not possible. They also combined multiple reactor units with the proprietary robotics technology into a single biosafety cabinet.38 The automated system enabled the simultaneous loading, sampling and feeding of cells for 24 independent reactions. A number of industrial examples used the micro 24 system for microbial cultures of, E. coli, Pichia, and Saccharomyces with automated pH and DO control.37,64,65 Performance was found to match lab scale (up to 20 L scale) for nutrient consumption rates and biomass formation. Isett et al. 2007 showed well to well reproducibility across the plate for DO and pH control, using ammonia.39 Air sparge kLa values of 30 h1 supported biomass levels of OD 10 (dry cell weight \10 g L1). KLa values were improved to 150160 h1 using 100% oxygen. The feeding regime was limited to manual bolus addition of MeOH feed to all wells regardless of demand. The combined use of oxygen blending and manual MeOH additions produced Pichia wet cell weight up to !250 g L1. Other changes included the use of gas permeable membranes to replace the well lter caps, which became blocked from foaming. The resulting Pichia processes showed reproducibility with titer and glycan quality for Mab production from 3 mL to 2000 L.65

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Figure 3. Microbioreactor array for cell culture (SimCell courtesy of Seahorse BioSciences).40
Each reactor array is rotated up to 20 rpm on each station.

An important challenge is how to control the pH of a small volume reactor and particularly how to deliver a small volume addition of acid or base. Buchenauer et al. 200937 addressed this issue with a 48 well microtiter plate system linked to a microuidic system for process additions. Each well had a working volume of 0.5 mL with pH and OD measurement using PreSens patch technology (biolectorTM). The caustic for pH control addition was added from the microuidic device allowing accurate 5 nL dispense volumes. An alternative for mammalian cell culture screening and initial process development is the Sim cell reactor system from Seahorse Bioscience which has a cassette containing 6 reactor chambers with 300 to 700 lL working volumes40 (Figure 3). Multiple cassettes are agitated by rotational agitation (20 rpm) and CFD conrmed this mimicked the expected shear rate of a conventional stirred tank. This system is capable of housing up to 1500 cells within a single temperature control incubator. The back of each cassette has a gas permeable membrane for surface aeration achieving kLa values of 7 h1. At scheduled times a robotic arm removes the cassette to a monitoring station where the DO and pH values are read from the immobilized patch sensors and total cell count measured by light scattering. The cassette is routinely removed to the feeding station to allow automated bolus additions of nutrient or acids/base for pH control. The system has been demonstrated for process optimization of a CHO based antibody production where 180 reactions were carried out simultaneously to evaluate the impact of feed rate and pH on process performance.40 Pzer paired the reactors with an automated analytical system. Samples were routinely removed to a 96 well plate format and analyzed for Mab titer using an Octet analyzer, while a lab 90 microchip CE SDS system was used for integrity, purity, and glycan heterogeneity measurements. The DOE showed feed rate and pH were critical to cell viability, titer, and intact immunoglobulin G titer. The performance was shown to be equivalent to that of a 3 L reactor with respect to mAb product and cell viability for a 13 day fed batch process.40 The system is well suited to mammalian cell culture as the low oxygen transfer rates limit the application for microbial systems. Microreactors from the application of microuidics Microuidic approaches using small channels and wells typically fabricated in polymethylmethacylate (PMMA) and polydimethlsiloxane (PDMS) have been implemented to generate an inexpensive solution for microreactors. Applications being considered include strain screening and integration

with microarray chip analysis for tools to study gene expression. A number of examples have reproduced lab scale microbial batch processes achieving reasonable biomass levels (510 g L1).66 The systems match stirred tank functionality with respect to pH control with monitoring via patch sensors whereas aeration is by diffusion of oxygen through the permeable materials of construction. An extensive review of the microuidic breakthroughs and shortcomings has recently been provided by Schapper (2009). Some of the key advances are summarized in this section (Table 3). Zanzotto (2004)41 fabricated a bioreactor with a 550 lL volume using PDMS on glass. The pH and DO measurements were made via patch sensors and optical density was measured by transmittance through the culture. Temperature was maintained by placing the device in a temperature controlled chamber. Examples with E. coli used aeration by diffusion achieving kLa values around 60 h1 and maximum optical densities of 8, with equivalent performance to a lab scale reactor (0.5 L). Improvements to this device included increasing the reactor volume to 150 lL and the incorporation of magnetic driven stir bars to improve mixing.42 The system was redesigned to enable rapid setup with a plug-inand-ow connection to automatically align the optical and uidic connections with the outside housing unit.67 This device allowed the addition of acid, base, and glucose via microvalves and control circuits, however dissolved oxygen limitation remained an issue. Lee et al. (2006)43 used a similar design and provided a peristaltic oxygenating mixer to improve the kLa to $360 h1(Figure 4). For E. coli optical densities up to 40 were achieved and growth curve results were similar to a 4 L benchtop reactor. Marharbiz et al. (2004)44 designed a device for 250 lL reactors platformed on plastic microtiter plates and printed circuit boards. Closed loop control of each well for temperature and pH was obtained with a small resistor heater and thermistor for temperature and, an ion selective eld effect transistor sensor chip and platinum reference electrode for pH. Electrolyte gas generation was utilized to create oxygen and carbon dioxide control in an electrolysis chamber below the culture chamber. The gas diffused through a slim silicone membrane to provide DO control to the cultures. Agitation of the culture was provided by a small stainless steel bead and stirring of the reactor assemblies at 1750 rpm, which achieved optical density levels \3 for E. coli. Microreactors for animal cell culture have concentrated on improving mixing efciency while minimizing exposure to detrimental shear stress levels. Avoiding evaporation is also a concern that can lead to osmotic effects to the cells. Diao et al. (2008)45 designed a reactor constructed from polyacrylic acid and a polymethylpentene membrane with two elongated chambers connected by a smaller channel to form a U-shape. Pressure shuttling was used to transfer the culture (2.5 mL) between the two chambers, producing mixing and kLa values around 14.8 h1. Sf-21 cell growth and production were enhanced compared to a 50 mL spinner ask. A sample port allowed samples to be taken as needed however online pH measurement or control was not available. A major challenge of bioprocess in microuidic devices is the resulting analytics. In most cases each microbioreactor has to be sacriced at every time point to provide sufcient material for ofine analysis.41,68 This could be overcome by linking the microuidic reactor with microarray assays that require only a few microliters of sample. Currently only a limited set of analytics are available in microuidic type

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Sf-21 cells 5.3 106 cell/mL 5.714.8

Orbital shaking 500 rpm (3 mm throw)  2500 lL

 

Table 3. Comparison of State of the Art Small Scale Reactor Technology: Microuidic Technology Compared to Desired Criteria for High Throughput Process Development

Orbital shaking 8001300 rpm (3 mm throw)  250 lL pH by ISFET, DO by electrical generation of O2 and CO2

E. coli OD \3

150

Microuidic Examples

 100 lL pH & DO via injectors & oxygenating mixers

 

44

45

Magnetic stir (upto 1500 rpm)

Figure 4. Microuidic application from Lee et al. 200643: microbioreactor array module (a) photograph of four reactors integrated into a single module, (b) cross section of the reactor showing the peristaltic oxygenating mixer tubes and uid reservoir with pressure chamber, (c) top view of the reactor (500 lm deep, 100 lL working volume) with optical sensors and uid injectors (d) cross section showing the uid injector metering valves.

E. coli \13 g/L

Orbital shaking 300 rpm

360

 

43

E. coli OD 8

devices or microarray platforms which include ELISA, SDS Page, FACS, glycan Analysis, and DNA.69,70
 150 lL   42

60

Small-scale bioreactors A number of groups have fabricated small-scale stirred tank reactors at the 10 to 100 mL scale which incorporate geometric similarity to large scale, and have the ability for process control of pH, dissolved oxygen, and substrate feeding (Table 4, Figure 5). A number of these examples have shown predictive ability to pilot scale systems with respect to oxygen transfer, growth, and productivity for E. coli and B. subtilis cultures.32,47,69,71 The comparisons have been based upon either equal kLa,49 or energy dissipation.48 The limitations of the evaluations were completed at low biomass levels (\10 g L1 dry cell weight) so the systems were never truly challenged with respect to the ability to handle the high cell densities expected in industry. Higher biomass levels of 20 g L1 dry cell weight for a E. coli process were achieved by a multireactor block system comprising of 48 reactors with 10 mL working volumes.49 A unique magnetically driven gas induced impeller introduced gas bubbles through the impeller shaft achieved kLa values in the 700 to 1600 h1 range. Liquid handling enabled the pH, DO, and turbidity to be measured at line by dispensing 20 lL samples into microtiter plates containing the uorescence patches.49,50 Growth rates and biomass levels were shown to be equivalent to the 3 L scale. Improvements to the system led to fed batch capability of intermittent feeding and achieved B. subtilis biomass levels of 45 to 50 g L1 dry cell weight.51 Oxygen limitation was avoided by using 50% oxygen supplementation of the inlet air. A number of commercial stirred tank systems are available in the working volume range of 150 to 400 mL which can support fast growing microbial cultures. Multiple reactors,

E. coli OD 8

Diffusion

 550 lL

60

Cell culture (upto 5 10 cells/mL) and microbial (OD upto 600) Cell culture 15/ h Microbial 400/h Stirred with stirred tank geometry

 

41 * All kLa values are based upon dynamic gassing out methodology.

Cell type (biomass level or cell count) kLa (/h)*

Disposable reactor Working volume pH & DO control

Parameter

Agitation type

Feed regime capability

Fast set up time Ability to run multiple reactions in parallel Ability to integrated with automated purication References

Multifeeding: linear ramp, constant, exponential \2h !24 reactions

 or 100 mL pH range 4 to 8.5 DO control !20%

Ability to take material forward to purify 35 mg of product

Criteria

10

Table 4. Comparison of State of the Art Small Scale Reactor Technology: Stirred Reactor Technology Compared to Desired Criteria for High Throughput Process Development

Parameter E. coli, B. subtilis, \10g/L dry cell weight Cell culturePichia, E. coli 350 g/L Wet cell weight Cell culture Pichia 200 g/L WCW E. coli 20 g/L Bacillus 50 g/L Cell culture E. coli 7g/L dry cell weight

Criteria

Stirred Tankmimics

Stirred 48 Cell Reactor Block

Dasgip AG 6 Reactor System

Biostat Q6 (Sartorius)

Medicel Explorer15 reactors (Medicel)

Clone Screener 32 Reactor (Biospectra AG) Cell culture and microbial

AmbrTM 24 Reactor System (TAP) Cell culture only

Cell type (biomass level or cell count)

kLa (/h)* Upto 480 2007000 rpm  10100 mL   32,4648 4951 Merck internal data   Merck internal data  6 units/system 1002000 rpm 1002000 rpm 1001200 rpm Upto 1600 Upto 400 Upto 400 Upto 191

TBD 1001200 rpm

\30/h 200rpm

Agitation type

1001200 rpm

Disposable reactor Working volume pH & DO control

Feed regime capability

Cell culture (upto 5 107 cells/mL) and microbial (OD upto 600, [200 g/L dry cell weight) Cell culture 15/h Microbial 400/h Stirred with stirred tank geometry 503000 rpm  or 50200 mL pH range 4 to 8.5 DO control ! 20% Multi feeding: linear ramp, constant, exponential \2 h ! 24 reactions 10 mL  O2 gas blending Intermittent 6 units/system 60200 mL  O2 gas blending  5001000 mL  O2 gas blending  150 mL  O2 gas blending  15 units/system  Communication with Medicel Oy and the Medicell Explorer speccations data sheet. 

400 mL  O2 gas blending   32 reactors

 10 mL  O2 gas blending Intermittent additions   24 reactors  The Automation Partnership specications document for the ambrTM system.

Fast set up time Ability to run multiple reactions in parrallel Ability to integrat with automated purication

Ability to take material forward to purify 35 mg of product

References

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

* all kLa values are based upon dynamic gassing out methodology.

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11

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

(Working volume of 10 mL).46,71

usually groups of 6, are operated from a central control system (Multifors from Infors, Biostat Q from Sartorius stedim Biotech and Dasgip system16). These systems use standard lab scale geometry and conventional glass pH probes and polygraphic DO measurement for control. They all acquire steam sterilization within an autoclave and signicant set up time and cleaning. The level of complexity of feed control strategy varies between systems. For example the multifors has limited feeding capability, while the Dasgip and Sartorius enable advanced feeding strategies of exponential or multivariate ow control. This can be controlled by gravimetric feed control (Biostat Q, Sartorius) or by calibrated micropump (Dasgip) to deliver the desired low ow rates. Additional modules are available for vent gas analysis using sensors, electrochemical, and infrared for oxygen and carbon dioxide, respectively. For cell culture these reactors have become the routine tool for process development. They replace the spinner asks (magnetically stirred glass vessels) which had limited automation for multireactor parallel work when studying ranges for pH, temperature, and feed manipulation. A number of automated systems have recently become commercially available that incorporate automated control with sampling and post fermentation conditioning. Medicel Oy designed a 15 parallel fermentation system (Medicel Explorer) with a 150 mL working volume, independent control of pH, DO, agitation (200 to 1200 rpm) and one continuous feed. Integrated vent gas analysis is available along with routine automated reactor sample collection and online OD measurement. The system has been demonstrated for mammalian cell culture with good batch to batch reproducibility and similar performance to shakeasks. Initial evaluation for microbial work with Pichia generated 200 g L1 wet cell weight broths using a 150 mL working volume at 30  C with a single impeller. KLa values were achieved in the 151 h1 to 191 h1 range (Communication with Medicel Oy and the Medicell Explorer speccations data sheet). The need for manual sterilization and setup of reactors will take around 1 day to complete as disposable reactors are not currently available.

A fully automated system is available from Biospectra which incorporates 32 bioreactor segments each consisting of a 400 mL working volume reactor, with four feeding vessels (acid/base and two feed reservoirs). Automated clean in place and steam in place is performed along with automated probe calibration, inoculation, sampling, and harvest. The complete system is housed in a 2 m 2 m system with a weight of 3.5 tons and requires a multimillion dollar investment. Initial applications have been applied to mammalian cell culture. A high throughput construct screening tool for basic research, designed by The Automation Partnership (TAP) in conjunction with an industrial consortium, enabled the construct screening for protein production (The Automation Partnership specications document 15-A for Piccolo construct screening system). The reactor system was designed with 384 10 mL working volume tubes with mixing provided by an agitating nger rod. Culture conditions can be varied with respect to temperature, and inducer concentration. Constructs are grown in the tubes to a desired optical density, measured online, and then automatically transferred through cell lysis and protein purication steps. This has been applied to E. coli and insect cells where optical densities upto 20 have been achieved. The Automation Partnership has also recently developed an advanced automated microscale bioreactor system for cell culture (The Automation Partnership specications document for the ambrTM system). (Figure 6). Twenty four disposable cell culture reactors of 10 mL working volume are supported on a liquid handling deck. Gas additions of nitrogen and oxygen blending are feasible along with closed loop pH control from sensor patches mounted in the base of each reactor. Automated control of sampling, additions and intermittent feeding regimes are possible using standard sterile liquid handling pipette technology inside a biosafety cabinet. The system is envisioned to support cell line evaluation and initial process development optimization. Potential limitations include the small working volume that may limit the availability of puried product from each reactor, and the number of product

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

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 from the base of the reactor using patch sensors.

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

Summary: Future Requirements for the Next Generation of Advanced Reactor Systems
The need for the automated small-scale parallel reactor system to mimic large scale capability is well-understood.31,32 This would provide the condence that the growth kinetics and protein expression can be expected to achieve quantitative scale up. Signicant impact would be provided, not only to process development, both upstream and downstream, but also for strain selection and evaluation. This review has shown that no single device has yet to meet all the challenges of miniaturizing large scale process conditions while retaining the full functionality of conventional bioreactors for industrial processes (Tables 24). It is clear that a higher degree of instrumentation for feedback control of pH and DO and feed regime strategies are essential. The simplied systems of shakeasks and microtiter plates provide signicant high throughput capability. Yet they carry less instrumentation which limits the opportunity for data quality and quantity. For this reason they remain as screening tools without implementation of robust pH and DO control. Some improvement was made with the Applikon micro 24 well plate system that enabled pH control. However the system still lacks continuous and independent nutrient feed capability and requires signicant adjustments by the customer to implement a particular project. Although some microuidic devices enable control of pH and DO, they lack the complex feeding ability to achieve high cell densities. The small volumes also limit the ability for the full range of product quality attributes necessary for mAb process development. The miniaturized pH and DO monitoring from patch sensors has improved signicantly over the last 10 years and is now becoming routine operation for the pH ranges of 5.5 to 8.5. Improvements to measure lower pH values of 3.55.0 would have signicant impact for microbial culture, particularly Pichia processing. In addition, a greater understanding of patch robustness and lot to lot variability would also expand

the application. Similar advances are needed to bring feedback control for substrates and metabolites to the small-scale reactors such as online NIR and miniature LC/MS. It is clear that a number of devices have recognized the importance to replicate geometric similarity to the large scale bioreactors. Nevertheless, a greater emphasis is needed in the development of systems that can support high cell densities as the majority of the literature examples have only evaluated microbial processes at low biomass levels (optical densities \30). A few small-scale stirred tank systems have shown promising progress (Dasgip, Biostat Q Sartorius, Medicell Explorer) with the use of microfeed pump technology along with gas blending to support the desired high cell densities. They also have sufcient volume to enable purication of product for the quality analysis. However these systems are still burdened with signicant preparation and clean up time. This could be improved by a greater implementation of presterilized disposable parts, of not only the reactor, but also presterile manifolds for additions and disposable reservoirs. This would move the technology to a plug & play paradigm for minimizing downtime between runs. The application of disposables for microbial systems is clearly lagging behind the progress made for cell culture such as the SIM cell systems and ambr system from TAP. The improvements over the last decade for disposable materials of construction and mould technologies should make this task achievable. Moving forward, this review has outlined the signicant opportunity for the expanded use of automation to improve development throughput. Automation is needed to increase the parallelism of the small-scale stirred tank systems, particularly for microbial culture. Elegant solutions are needed for linking the upstream parallel throughput with sample analysis and purication. This will provide the total solution for optimization of overall process yield and product quality. A few examples have shown cell culture integrated with automated sample analysis38,40 but they have not included the necessary extensive characterization of product quality (The Automation Partnership specications document for the ambrTM system). The authors envisage an automated solution

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14. Chew L. Pseudomonas uorescens. In Production of Recombinant Proteins. G. Gellissen, Editor. Weinheim, Germany: Wiley VCH; 2005:4566. 15. Yajun Y, Liao JC. Engineering metabolic systems for production of advanced fuels. J Ind Microbiol Biotechnol. 2009;36: 471479. 16. Knocke C, Vogt J. Biofuelschallenges & chances. How biofuel development can benet from advanced process technology. Eng Life Sci. 2009;9:9699. 17. Li H, dAnjou, M. Pharmacological signicance of glycosylation in therapeutic proteins. Curr Opin Biotechnol. 2009;20: 678684. 18. Zhu M, Goyal A, Rank D, Gupta S, Boom T, Lee S. Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody fusion protein. Biotechnol Prog. 2005;21:7077. 19. Mostafa S, Gu X. Strategies for improved dCO2 removal in large scale fed batch cultures. Biotechnol Prog. 2003;19:4551. 20. Bahrami A, Shojaosadati S, Khalizadeh R, Mohammadian J, Masoumain M. Prevention of human granulocyte colony stimulating factor protein aggregation in recombinant Pichia pastoris fed batch fermentation using additives. Biotechnol Appl Biochem. 2009;52:141148. 21. Park JH, Lee S, Kim Y, Kim H. Application of systems biology for bioprocess development. Trends Biotechnol. 2008;26:404412. 22. Jaluria P, Chu C, Betenbaugh M, Shiloach J. Cells by design: a mini review of targeting cell engineering using DNA microarrays. Mol Biotechnol. 2008;39:105111. 23. Nice E, Rothacker J, Weinstock J, Lim L, Catimel B. Use of multidimensional separation protocols for the purication of trace components in complex biological samples for proteomics analysis. J Chrom A. 2007;1168:190210. 24. Tyo K, Alper H, Stephanopoulos G. Expanding the metabolic engineering toolbox: more options to engineer cells. Trends Biotechnol. 2007;25:132137. 25. Milburn M. Using metabolic proling technology to advance cell culture development. BioPharm Int. 2009;June:2840. 26. Rathore AS, Winkle H. Quality by design for biopharmaceuticals. Nat Biotechnol. 2009;27:2634. 27. Wenger MD, DePhilips PD, Price CE, Bracewell DG. An automated microscale chromatographyc purication of virus like particles as a strategy for process development. Biotechnol Appl Biochem. 2007;47:131139. 28. Wenger MD, DePhilips P, Bracewell DG. A microscale yeast cell disruption technique for integrated process development strategies. Biotechnol Prog. 2008;24:606614. 29. Micheletti M, Lye GJ. Microscale bioprocess optimization. Curr Opin Biotechnol. 2006;17:611618. 30. Tustian A. Speeding the design of bioprocesses. Chem Eng Prog. 2007;104:S8S13. 31. Marques M, Cabral J, Fernandes P. High throughput in biotechnology: from shake asks to fully instrumented microfermentors. Recent Patents Biotechnol. 2009;3:124140. 32. Betts J, Baganz F. Miniature bioreactors: current practices and future opportunities. Microbial Cell Factories. 2006;5:21. 33. Lotter S, Buchs J. Utilization of specic power input measurements for optimization of culture conditions in shaking asks. Biochem Eng J. 2004;17:195203. 34. Wittman C, Kim HM, John G, Heinzle E. Characterization and application of an optical sensor for quantication of dissolved oxygen in shake-asks. Biotechnol Lett. 2003;25:377380. 35. Deutz WA. Microtiter plates as mini bioreactors. Trends Microbiol. 2007;15:469475. 36. Zhang H, Lamping SR, Pickering SCR, Lye GJ, Shamlou PA. Engineering characterization of a single well from 24 well and 96 well microtitre plate. Biochem Eng J. 2008;40:138149. 37. Buchenauer A, Hofmann M, Funke M, Buchs J, Mokwa W, Schnakenber A. Micro-bioreactors for fed batch fermentations with integrated online monitoring and microuidic devices. Biosensors Bioelectron. 2009;14111416. 38. Chen A, Chitta R, Chang D, Amanullah A. Twenty four well plate miniature bioreactor system as a scale-down model for cell culture process development. Biotechnol Bioeng. 2009;102:148160. 39. Isett K, George H, Herber W, Amanullah A. Twenty four well plate miniature bioreactor high throughput system: assessment

encompassing the integration of liquid handling decks for upstream and downstream purication. The upstream system would use disposable reactors for fast setup time with automated media dispense, inoculation, and growth control enabling variable conditions and feed regimes. Automated sample collection will need to be integrated to a purication deck, expanding upon the initial microscale methods for centrifugation,27 disruption (if necessary),28 and chromatography.27,72 This will provide sufcient sample purity to complete product quality characterization. Signicant effort will be required for the development of micro scaledown purication methods, particularly for the primary recovery of high cell density microbial broths where simple microwell manifold ltration73 is unlikely to be feasible. Fundamental engineering understanding for centrifugation and disruption microscale work will be essential. The longer term (1015 years) will likely bring further throughput improvements from the introduction of microuidic approaches into the automated development workow. Early work has demonstrated feasibility of microuidic approaches to ion exchange chromatography at the 1.5 lL scale.74 It is also anticipated that standard LC column methods will be replaced by microuidic chip analysis, such as recently demonstrated for DNA and RNA analysis.75,76 The authors believe the technology improvements outlined in this review are essential for the future success of biologics process development. The advances need to continue on all fronts: upstream, downstream, and analytical development. This review focused on the challenges confronting upstream development with the vision of integration to purication and analytical platforms. It is envisaged the integration of paralleled, disposable, well controlled systems will make signicant impact to improving process development efciency (estimated at 45 fold). This will provide faster routes to scaleable robust processes, without sacricing process productivity or product quality.

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