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Improving Biopharmaceutical Process Scaleup & Tech Transfer

Special RepoRt

SPonSored By

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Introduction *

Eli Lilly on Bioreactor Operational Excellence: Best Practices from Scaleup to Control

CliCk here p. 3

Engineers from CH2M Hill and Lockwood Green on How to Succeed at Bioprocess Scaleup

CliCk here p. 10

Duncan Lowe discusses the application of Process Analytical Technologies (PAT) at Amgen CliCk here p. 16

Hamilton Company Resources

CliCk here p. 18

Additional Process Control & Tech Transfer Resources

CliCk here p. 18-19

CONTENTS

in biopharmaceutical development, scaleup and scaledown are critical to developing a robust process and maintaining product quality. technology must be correctly transferred, a multi-disciplinary process that requires knowledge of real world materials and equipment, potential sources of variability, and critical quality attributes for the product.

the pharmaceutical Quality by Design framework, iCh Q10, FDA’s 2004 process Analytical technology (pAt) Guidance and 2011 process validation Guidance can help manufacturers develop a strategy for process and analytical method transfer, validation and continuous quality improvement. however, for any biopharmaceutical process, the foundation for all of this work is frequent and accurate process measurement, typically using dissolved oxygen, ph and conductivity sensors.

this e-resource summarizes best practices for biopharmaceutical process scaleup, as well as technology and analytical method transfer from experts at pharmaceutical manufacturing and engineering companies, while it also highlights the role that Do, ph and conductivity sensors play in ensuring process robustness.

introDuCtion

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the modern cell culture bioprocess has been successfully scaled up to volumes greater than 25,000l through sound engineering fundamentals and thorough process understanding. this hasn’t happened by accident, but rather by bioprocessing professionals taking a systematic approach to characterizing the bioreactor’s capabilities and tendencies, developing robust and reliable scale-up procedures, and establishing and maintaining proper control criteria. those manufacturers that identify and document operational best practices for a cGMp cell culture plant also tend to be those that sustain operational success and deliver high-quality biopharmaceuticals to patients in a timely and reliable manner.

identifying and documenting bioreactor operation best practices allows for more robust processing by helping to properly educate the operations, engineering and technical staff who oversee the bioreactor processes. shared learning helps to reduce the amount of “tribal knowledge” that exists within a group and to maintain high levels of operational excellence even in times of employee turnover, with the end result being a sustainable and reliable supply of biopharmaceuticals.

With these ideas in mind, we have set out to document best practices that we have learned for bioprocessing, most notably in the areas of equipment design and overall process control.

BioReactoR opeRational excellence:

Best Practices from Scale-up to Controlin biomanufacturing, knowledge of scale-up, processing and control should be shared and docu-mented in order to achieve and sustain operational excellence.

By BRian J. StampeR and cillian mccaBe, BiopRoceSS ReSeaRch and development, eli lilly and company

BIoreaCTor deSIgnstarting off with the proper bioreactor design can resolve many process issues before they arise. one key bioreactor design issue that should not be underestimated is the importance of geometric similarity between bioreactors: maintaining aspect ratios, impeller sizing ratios, impeller spacing ratios and baffle size and location will greatly increase the probability of success at scale. A properly designed bioreactor can lead to reduced qualification and process validation timeframes, as well as increased apparent process robustness and operational success.

Another key aspect to bioprocess scale-up success is the design of the sparger. often, two spargers are installed in the production bioreactors while only one sparger is used for the seed bioreactors. While various types of spargers have been utilized within the industry, we have successfully implemented the use of drilled pipes and sparge stones. the drilled pipe yields large bubbles and a lower kla (which will be discussed below), while the sparge stone yields small bubbles and a very high kla such that a greater amount of oxygen

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can be delivered into the cell broth for the same gas flow rate. Figures 1 and 2 illustrate the sparger location and two sparger types.

the ability of the bioreactor to deliver oxygen to the cells is defined by the mass transfer relationship shown in equation 1. the change in oxygen concentration is controlled by kLa, the average saturation oxygen concentration of the bubbles,, dissolved oxygen concentration ([O2]dissolved) and the oxygen uptake by any cells present (OUR).

the kLa can be mapped as a power law function of the power/volume and superficial gas velocity. understanding of the kLa allows for estimation of the our capacity of the bioreactor, prediction of required oxygen flow rates and prediction of the time-course profile of the dissolved carbon dioxide levels.

Based on process needs and sparger capabilities, the process engineer must determine the preferred configuration for the bioreactors. if process OUR needs are sufficiently low, the default configuration can be the use of one drilled pipe in the seed bioreactors, and two drilled pipes in the production bioreactors. the combination of a drilled pipe and sparge stone may also be used, but the oxygen transfer ability afforded by the sparge stone is typically not necessary. use of the sparge stone should be avoided if possible due to the increased operational complexity associated with bioreactor set-up, manual changes in gas flows during a process to maintain dissolved carbon dioxide levels, increased foaming and potential cleaning concerns.

FiguRe 1. illustration of the sparger location within a bioreactor (not drawn to scale).

FiguRe 2. Comparison of bubble sizes erupting from sparge stone and drilled pipe (reproduced from www.mottcorp.com).

SpaRge Stone dRilled pipe

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MIxIng CharaCTerIzaTIonWhen scaling up a free suspension cell culture bioreactor, a thorough understanding of the mixing characteristics is essential. if the mixing inside the bioreactor is appropriately controlled, then the cells will experience an environment very similar to that of the bench-scale bioreactor and will therefore be much more likely to behave as they did in the scale-down bioreactors.

the literature shows that many methods of scale-up have been considered, including matching power/volume, impeller blade tip speeds, bulk mixing reynolds numbers and bulk mixing times. Due to the nature of these various parameters, it is not possible to maintain them all during scale-up under one set of conditions.

experience has shown that maintaining a similar power/volume (P/V) at the various bioreactor sizes greatly increases the probability that the mixing within the bioreactor will be appropriate. P/V is a function of the impeller geometry, the agitation rate and working volume, as shown in equation 2, where is the density, n is the number of impellers, Np is the impeller type power number, n is the agitation rate, Di is the impeller diameter and V is the liquid volume.

to further characterize the mixing, the bulk mixing time at various agitation rates can be measured via ph or conductivity, or calculated using commercially available models. this is performed to determine the length of time required for the bulk liquid to become 99% homogeneous with respect to ph or conductivity.

Combining the results of the kla mapping, mixing time determination and P/V calculations can lead the process engineer to choose the appropriate agitation setpoint to

enable successful scale-up. once the agitation setpoint is determined, the sparging scheme can be designed to ensure the dissolved oxygen in the bioreactor is maintained while carbon dioxide is effectively stripped from the bioreactor and foam accumulation is kept to a minimum.

BIoreaCTor ConTrol and alarMIngMammalian cell culture based bioprocesses require monitoring and control of the bioreactor environment to ensure consistent bioprocess performance. the parameters requiring control include temperature, agitation, dissolved oxygen (Do) and ph. other parameters such as cell density, nutrient concentration, desired and undesirable culture by-products are controlled indirectly via medium and feed formulation and can be greatly affected by the physical and chemical parameters. neglecting control of these parameters could potentially impact final product quality, so online measurements can be employed to maintain the culture in an optimal state. Bioreactor control schemes entail a series of steps as outlined below.

Measure of response variable

Comparison of measured value to process setpoint

Activation of control scheme

parameter monitoring and control requires the use of an appropriate analytical device, an appropriate sampling method and a control system which can act appropriately to the information it receives. online monitoring control systems are rapid, non-invasive and minimize potential for contaminant introduction and may be performed

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inside or outside of the bioreactor but must be connected directly to the bioreactor interior. Conventional parameters subject to online analyses include ph, Do concentrations, agitation, backpressure and temperature. Measurements of additional chemical parameters including cell density and viability, waste metabolites, nutrients and product concentration have historically been measured offline, although many technologies are becoming available that enable online measurement.

online control employs probes, each of which have a sensor whose function is to gather information relevant to the biological state of the culture. this information is then converted into an electrical signal that can be amplified, recorded and analyzed so as to drive the applicable control scheme. in light of this, it is important that this information is pertinent to the current or future state of the bioprocess and be quickly generated and processed with minimal manual intervention. the sensors should be selected based on the following criteria: potential to cause contamination, robustness and reliability of sensor elements, specificity for parameter being measured and insensitivity to the harsh environment of the bioreactor. Maintaining reproducible and acceptable product quality and productivity, while minimizing downtime, are the primary business drivers behind an effective bioprocess monitoring and control strategy.

effective bioreactor control may entail monitoring more than just the primary control parameters. For example, we once encountered a situation in which the ph stayed within the control range, but caustic was being fed to the tank even though the base controller had an output of zero such that the controller was not trying to feed caustic. our investigation led to the discovery that the tubing from the caustic vessel to the bioreactor was not installed properly in the peristaltic pump, and caustic was leaking past the pump and into the bioreactor.

Another problem we experienced entailed hyperoxygenation of one of the seed bioreactors. this led to decreased growth, viability and increased specific lactate production. investigation of the incident led to the discovery that oxygen was leaking into the process air line. the Do probes had been calibrated per ticket instructions but the oxygen leak led to false readings and improper Do control of the bioreactor. the only indicator, other than cell culture performance, of these false readings was the nano-Amp readings of the Do probes, which were found to be much higher than normal.

TeMPeraTure ConTroltemperature is a key parameter requiring monitoring and control throughout bioprocesses to ensure an actively growing and productive mammalian cell population. in general, an accuracy of ± 0.5°C is considered adequate for cell culture, although transient excursions may exceed that range with no impact to product quality or cell culture performance. typical bioreactor temperature measurement devices are resistance temperature devices (rtDs), which are highly accurate, reproducible and only moderately expensive. the response time of these devices is in the order of several seconds. these rtDs rely on the fact that the platinum core wire conductance varies with temperature to quantify temperature. in the rtD temperature sensor control scheme, the signal is amplified, linearized and transmitted to a controller whereupon it is compared to a setpoint. Based on this continuous comparison, the bioreactor’s temperature is regulated by adjusting the temperature of the jacket surrounding the bioreactor. if and when temperature deltas are recorded, the temperature of the jacket is adjusted appropriately through use of heat exchangers.

dISSolved oxygen ConTrolMammalian cell cultures require oxygen for the production of energy from organic carbon sources — e.g., glucose.

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Given oxygen’s poor solubility in water-based solutions, the control of oxygen flow is carefully regulated to ensure it does not become a rate-limiting factor in the process. in contrast, a hyperoxygenated bioreactor air supply can irreversibly and adversely impact culture performance.

Due to fluctuating cell concentrations and the associated fluctuating oxygen consumption rate, the quantity of dissolved oxygen (Do) in culture medium is in a state of dynamic equilibrium. At a constant temperature, the Do concentration in the culture media (Cl) is proportional to the amount of oxygen in the vapor phase within the media (CG) in a manner that is dependent on temperature and media composition (represented by henry’s law constant, h in the equation below).

Cl = hCG

Amperometric Do probes are typically used, which measure the reduction of oxygen at a cathode and the formation of silver chloride at the anode with an electrolyte solution bridging the gap between the nodes. Given the nature of amperometric Do probes, it is necessary for these probes to be allowed to polarize prior to their use. A calibration is then performed, and in the event that the probe falls outside of the acceptable calibration range, the probe membrane body and electrolyte are replaced.

Ph ConTrolAlong with temperature and dissolved oxygen control, effective ph control is vital to ensure process success given the sensitivity and potential cellular damage that may occur if ph control remains unchecked. Although cell culture media typically provides substantial buffering of ph, mammalian cell metabolism routinely decreases the culture ph due to the production of lactate and carbon dioxide, both of which are acidic in nature. excessive hydrogen ion concentration may alter normal cell metabolism and proliferation by impairing substrate uptake and product

release. in addition, it is possible that the bioactivities of some secreted monoclonal antibodies or therapeutic peptides could be ph sensitive.

typically, the ph probes on the bioreactors are calibrated while connected to the transmitters on the bioreactor that is destined for use and prior to installation into the tanks. typical calibrations are conducted using two buffers, with a calibration check performed in an intermediate buffer. Failed calibrations are typically due to damaged ph probes, but may also be attributed to faulty cables or transmitters.

once calibrated, ph probes have occasionally been observed to generate incorrect readings, due to probe drifting, slowed response time or impaired sensitivity. these erroneous readings are typically attributed to sensor membrane alterations due to extreme temperature swings and fouling from media and cellular components. As a result, a policy for re-standardization of the probes may need to be developed using an orthogonal ph measurement method as the gold standard.

effective ph control can be achieved through use of two separate piD loops, where one is the acid controller and one is the caustic controller. in a bicarbonate-buffered system, the acid controller controls the carbon dioxide flow and is configured such that the carbon dioxide flow ramps up very quickly when the process value is above set point and instantly turns off when the acid controller set point is reached. the liquid caustic controller utilizes a pulse width modulator (pWM) to control the amount of time the caustic peristaltic pump is on or off. the set point on the peristaltic pump is set manually per manufacturing ticket instructions and the controller only turns the pump on and off. the frequency of measurement and pulse addition and duration can be altered to effect varying levels of control by tuning the control loop. Due to the high ph of the caustic feed, it should be fed into the bioreactor through a sub-surface port to facilitate quick dispersion into the culture.

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dISSolved CarBon dIoxIde ConTrolDissolved and evolved (i.e., headspace) carbon dioxide levels can be indicative of cellular metabolism and are thus routinely monitored as indicators of culture performance. in general, the mammalian cell cultures display sensitivities to extremes of dissolved carbon dioxide (pCo2) be they low or high. high pCo2 levels have been reported in the literature as an inhibitor of growth and metabolism and can impact product quality characteristics such as glycosylation of the protein product.

several parameters can affect the pCo2 levels, including ph set point, temperature, sodium bicarbonate concentration, cellular metabolism, caustic addition to the medium and gas flows. each of these parameters must be considered carefully to enable successful pCo2 control. ph, temperature and bicarbonate concentrations are typically not adjusted during a process to control pCo2, but rather gas flows and caustic addition are controlled to maintain the pCo2 within the desired target range. the gas flows can be chosen to strip out the desired amount of dissolved carbon dioxide as experience has shown the carbon dioxide levels are influenced more by total gas flow through the bioreactor rather than kla.

if the culture ph has drifted to the acidic side of the dead band, increasing the airflow strips out carbon dioxide potentially leading to an overall reduction of caustic addition. the reduced amount of caustic can lead to a lower pCo2 at the end of the culture when lactate levels typically decrease. however, if the ph is on the basic side of the dead band such that the Co2 is being fed, increasing the airflow will only lead to increased Co2 flow and will not affect the pCo2.

BaCkPreSSure ConTrolthe stainless steel bioreactors are maintained under positive pressure to create an environment that is more conducive to axenic operation. A backpressure setpoint is

generated by maintaining a constant overlay process air flow into the headspace of the bioreactor. the backpressure can then be controlled via a piD control loop that operates a flow control valve on the vent line. to avoid safety concerns associated with over-pressurization, rupture discs may be incorporated into all pressurized stainless steel vessels to act as pressure relief devices. in addition to the bioreactor headspace, positive pressure should be maintained on all transfer lines within the sterile boundary and any associated auxiliary stainless steel vessels used for additions to the bioreactors.

Backpressure can also be used as the driving force to govern bioreactor-to-bioreactor transfers and bioreactor-to-primary recovery transfers. Care should be given to ensure the transfer is fast enough to not allow cells to settle during the transfer, but not so fast as to subject the cells to excessive shear. the transfer time can be dictated by the pressure drop and pipe dimensions.

alarM STraTegythe alarm strategy should be configured to alert the operators that the process is deviating from its acceptable range, but should also provide early warnings such that the operator can respond in time to prevent loss of the batch. to this end, multiple levels of alarming may be implemented. the first level of alarms can be set to include the normal variability present within a control loop such that if the alarm is activated, operations can assume that an unexpected excursion has occurred but will have time to take pre-emptive action before the process is negatively impacted. the final level of alarms should be set to match the acceptable ranges listed in the process flow chart specific to a process. While determining the alarm strategy, care should be taken to apply alarms only to the appropriate parameters. if excessive alarming occurs, operators may begin to not respond effectively to alarms to the extent that important alarms may be missed.

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Use of offline DataAdditional information regarding culture health and performance may be obtained offline using analysis of aseptic samples of the culture. typical offline testing will yield information regarding cell numbers and cell viability using an automated cell counter system. in addition, a blood gas analyzer can be used to determine the levels of relevant parameters including lactate, glucose, pCo2 and pH. the offline samples may be used primarily for informational purposes and not linked into automated response systems to drive bioreactor control changes. However, offline measurements may be used by technical services to monitor the process and instruct operators to, for example, restandardize the pH probes should a drift from setpoint be observed.

Bioreactor feeDing strategythe basal medium may be added to the bioreactor from a disposable bag via peristaltic pump, or for larger volumes from stainless steel media make-up tanks via pressure transfer. For the tank transfers, the media is typically sterilized in-line via two hydrophilic filters, a pre-filter followed by a sterilizing grade filter. the filters are steam sterilized in line simultaneously with the transfer path and are cooled prior to media transfer.

nutrient feeds are typically prepared in disposable bags. the nutrient feed typically consists of multiple stock components that need to be well mixed and may require pH adjustment. upon one nutrient feed into a bioreactor, a high pH excursion was observed in the bioreactor which was later attributed to poor mixing of the nutrient feed components. the nutrient feed bag was subsequently re-

designed to allow for better mixing within the bag to avoid the pH change in the bioreactor.

nutrient feeds are delivered to the bioreactors at pre-determined times during the cell culture process. these feeds are manually added to the bioreactors, with very little automation associated with them. in fact, the automation is configured such that the near-to block valve on the nutrient feed line is always open during culture phase to maintain positive pressure on the line. therefore, the peristaltic pump head is the only block on the line, such that the operator can initiate the feed simply by turning on the pump. the nutrient feeds may be slightly acidic, so a typical concern associated with addition of the feed is a change in pH in the bioreactor. to account for this, the addition flow rate of the feed is dictated, as well as instructions to the operators to stop the feed if the pH exceeds the acceptable range.

aBoUt the aUthorsCillian McCabe has a first-class honours degree in Biotechnology from national university of ireland (nui), Galway, and a phD in “Gene therapy Approaches to the treatment of type 1 Diabetes Melitus” from the school of Medicine at nui. He joined eli Lilly & Co. in 2007 and has supported Bioprocess Development and Manufacturing operations both in the u.s. and ireland as part of Lilly’s Manufacturing science & technology functional group.

Brian stamper has a B.s. in Biochemistry from indiana university (Bloomington, ind.) and an M.s. in Biological engineering from purdue university (West Lafayette, ind.). He joined eli Lilly in 2001 as a development scientist in Bioprocess Development and transitioned to Bioprocess operations in the Clinical trial Material supply pilot plant in 2005 as a manufacturing associate.

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the ability to produce valuable end products via batch biological processes, using such diverse microorganisms as e. coli and other bacteria, algae, mammalian cells, transgenic plants and other host cells, is proving to be a versatile and promising synthesis route for a growing slate of end products (Figure 1). Because such processes rely on the fermentation of renewable carbohydrate feedstocks (from cane, beets, corn, grain and other sources), they have the potential to offer an environmentally friendly and less costly alternative to conventional synthesis routes that are based on petroleum-based feedstocks, which face supply and price pressures (Figure 2).

While fermentation-based syntheses were once reserved for producing high-value specialty chemicals and biopharmaceuticals (whose end products may command prices on the order of $400,000 for a 5-ml vial), bioprocess routes now are gaining increasing attention for commodity products. For instance, commercial-scale bioprocess facilities are already producing: vaccines and therapeutic pharmaceuticals (such as Amgen’s epogen and Wyeth’s Mylotarg), food products (l-phenylalanine, a building block for nutrasweet), and food-grade additives (such as the algae-derived fatty acid DhA & ArA from Martek Biosciences, which is used as a nutritional additive). other specialty and commodity biochemical facilities are in the works. For example, Bp and Dupont are teaming up to commercialize bio-based butanol as a gasoline blendstock in 2007 (Cp, August, p. 15).

SCale-uP ChallengeSWhile the scale-up of any chemical process can involve a host of issues, the challenges are compounded when the process involves batch fermentation. Due to the typical fragility of the engineered microorganisms, large-scale fermentation vessels must be designed with the ability to:

Succeed at bioprocess scale-upWhile fermentation-based syntheses were once reserved for producing high-value specialty chemicals and biopharmaceuticals, bioprocess routes now are gaining increasing attention for commodity products.

By John l. ShaW, p.e., and Scott a. RogeRS, p.e., ch2mhill lockWood gReene

FiguRe 1. Biprocessing provides an increasing variety of products, including some commodities, with many more likely.

Present

Future

Biological Chemical Factory

Bioprocessing for the Future

Petrolleum RefiningChemicals &

Intermediates

Polymers

Fibers

Bioprocessing

Renewable Crops

Coatings

Pharmaceuticals, solvents, crop protection chemicals

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• remove the heat buildup that results from metabolic processes;

• manage agitation and mixing with minimal shear damage;• effectively control the highly variable liquid flowrates and

turndowns that are associated with batch fermentation; and• execute safeguards and sterilization techniques to guard

against potential contamination.

the engineering challenges are more acute when the fermentation process will be used to make commodity-type chemicals. Because such products don’t command premium prices like higher-end specialty chemicals, food additives and biopharmaceuticals, their production facilities often are forced to make engineering tradeoffs in the face of capital, maintenance and operating cost constraints and leaner profit margins.

Additional challenges arise because emerging (non-mature or unproven) synthesis routes often exhibit a high degree of change throughout the scale-up and design stages. With relatively limited project experience with a given route to draw upon, the design team must anticipate and manage changes to the design and construction specifications to minimize cost creep and keep the project on schedule.

one of the most commonly made mistakes during the design of bio-based manufacturing processes is the failure to adequately integrate the experience, expertise and proven techniques developed by the pilot-plant engineers, facility microbiologists and chemists into the criteria for the overall flowsheet, equipment specifications, process and instrumentation diagrams, and waste-handling systems. the members of the design, operations and maintenance teams that will set these criteria are generally added closer to startup and face tremendous task and time constraints to be ready for commercial operation, diminishing their availability for technology-transfer efforts. however, during the specification of commercial-scale equipment and

controls, it is crucial to study and adapt the administrative and manual tasks carried out during pilot-scale operations, such as those related to closed-vessel policies, material handling, cleaning, waste handling and other operational aspects. A well-integrated team approach, with a common project view of the need to balance cost constraints against sterility needs, is essential.

“BIo” ISSueSWhen producing pharmaceuticals and food additives, product contact streams are regulated by the u.s. Food and Drug Administration and, possibly, by the u.s. Department of Agriculture in the case of biosynthesis. By comparison, chemical facilities are only regulated to keep the genetically engineered organism out of the surrounding environment.

experience shows that the performance characteristics of various organisms — even those already in use to produce

FiguRe 2. Crops and agricultural wastes offer a source of renewable feedstocks and more control of raw material costs.

Carbohydrates Product

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a given product — can be improved through ongoing bioengineering efforts. As a result, “next generation” bugs (such as those with enhanced metabolic activity or less sensitivity to process conditions) are constantly being pursued, to increase the yield of the target product, decrease the batch cycle time, reduce the amount of effluents or undesired byproducts, and cut energy consumption.

While such improvements are worthwhile, they may necessitate changes in equipment or utilities. For example, increased metabolic rates can enhance throughput, provided the higher heat generation can be controlled within the required temperature band, and agitation and delivery systems suffice to deliver needed nutrients and oxygen to the more quickly multiplying organisms.

a CoMMon fraMeworkirrespective of the microorganisms used or the end products produced, most fermentation-based facilities employ the same basic production blocks. And all commercial-scale bioprocess facilities can be roughly divided into two sections:

1. the upstream biosynthesis operation, where the desired end product is made, typically involves highly proprietary methods and calls for rigorous sterility requirements.

2. the downstream portion employs a site-specific mix of widely used chemical-engineering unit operations to extract and purify the target product, and appropriately dispose of all waste streams.

the particular engineering requirements (and challenges) associated with each of these two distinct portions differ, but must be tightly integrated during process design to ensure the most-cost-effective plant operation.

Fermentation. each of multiple fermentation vessels required by a commercial-scale facility will have its own particular design and operating requirements. these

include: the need to introduce the fermentation broth, sterile air (both to maintain the required dissolved oxygen levels and provide air lift for low-shearing mixing inside the vessel) and sterilized nutrients (such as vitamins, amino and fatty acids, minerals and even antibiotics that ensure the health and maximize the productivity of the microorganisms). When air lift in the vessel can’t provide sufficient mixing, the fermenter may be equipped with low-shear agitation devices.

Fermentation vessels must also be designed to ensure adequate heat-removal capabilities (to handle heat produced by the metabolic processes) and promote cooling as needed (to maintain the narrow temperature range that can be tolerated by the bioengineered organisms). sufficient safeguards must also be in place (both through design elements and operating procedures) to guard against contamination and cell mutation. these include double-block and bleed valves, and steam-in-place (sip)/clean-in-place (Cip) systems. Meanwhile, the variable flow rates associated with different stages of the organism’s metabolism and growth cycle, and the required cleaning cycles have tremendous design implications for the turndown necessary for process parameters (including flow and pressure). All of these factors complicate the internal geometry (in terms of baffles and agitators, for instance) of the vessel, as well as the number, location and type of tank nozzles and ports needed.

in addition, commercial-scale fermentation vessels must be equipped with a variety of advanced instruments, sensors and transmitters to monitor everything from pressure, level and temperature inside the fermenter to ph, dissolved oxygen and nutrient levels within the fermentation broth. in some cases, the number of in-line monitoring devices needed can be reduced by using external lab sampling or indirect relationships between key operating parameters. the appropriate number and location of the analytical instruments and in-process checks must also be reconciled against capital and operating cost constraints, and

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sterilization concerns (an increasing number of instrument ports raises the contamination risk to the bioreactor).

Because fermentation vessels and other specialized equipment components often require long lead times, the ability to make a firm commitment to the desired design specifications in advance — and withstand the urge to make subsequent changes later — will help to control costs and minimize delays.

production and recovery of derived product. During fermentation, the desired product ends up in the fermentation broth (excreted from the microorganisms or within the cell body). At the end of each batch fermentation, the microorganisms are destroyed; the product is separated and purified; and the dead cell bodies, unreacted carbohydrate feedstock/nutrients and byproducts are removed, concentrated and neutralized prior to disposal.

purification and concentration. A combination of classical separation operations (such as filtration, evaporation, ion exchange, distillation, crystallization, liquid/liquid extraction, spray drying and direct reaction) are typically used to purify and concentrate the product from the fermentation broth. increasingly, newer separation techniques (such as liquid chromatography, liquid and catalytic membranes, and supercritical fluid extraction) are finding a role in commercial-scale bioprocess operations.

Waste handling. the dead cell bodies and other solid waste, and the high biochemical-oxygen-demand (BoD) aqueous streams produced throughout the process must be disposed of properly. they must be concentrated and neutralized prior to disposal; an aqueous waste-pretreatment facility is almost always required to reduce the bioload prior to discharge to a landfill or publicly owned treatment works (potW). the specific handling and disposal requirements are ultimately dictated by the biosafety classification of the microorganisms in the waste stream.

residual high-BoD aqueous waste streams result and are typically treated in onsite aerobic or anaerobic digesters prior to being sent offsite to a potW. Aerobic digestion is economically applied for BoD up to 10,000 ppm. Anaerobic digestion is generally used from 8,000 ppm and up, and almost always when BoD exceeds 15,000 ppm. (Anaerobic systems will require further processing when discharging directly to a river but are commonly used for city sewer discharge.)

PITfallS and oPPorTunITIeSseveral issues deserve particularly close attention during the design and construction of bioprocess facilities, to streamline the overall process, minimize rework and contain costs.

sterility considerations and tradeoffs. the microorganisms that are used during biological-treatment processes at water- and sewage-treatment plants are notoriously hardy and can handle widely varying operating conditions. By comparison, microorganisms that have been genetically modified to yield a desired pharmaceutical, food or chemical product are typically viable over only a very narrow range of operating conditions and cannot withstand large or sudden variations in temperature, ph, dissolved oxygen, nutrient level, agitation rate and other critical operating parameters. the extreme sensitivity of these highly evolved yet fragile organisms — which has earned some the nickname “metabolic cripples” — creates unique design and operating challenges, particularly when it comes to maintaining close control over all of the critical operating parameters within vessels whose capacities generally exceed 50,000 gallons.

As noted earlier, the microorganisms in a given fermentation broth are highly susceptible to the presence of impurities. therefore, it is critical to guard against potential contamination. the most common sources of contamination are invasion by phage infections (rogue cells unique to each facility) and mutations within the bioengineered organism population. such occurrences not only lead to the disposal of the valuable fermentation batch but also call for an immediate shutdown for sterilization and cleanout.

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Master cell cultures are typically created and maintained offsite to minimize the possibility of mutations and phage infections. nonetheless, the facility’s approach for sterilization, cross-contamination prevention and routine cleaning must be identified early to ensure the piping and equipment configuration meets the intended cycle time and sterilization objectives.

When it comes to addressing sterility concerns, bioprocess facilities that produce high-value specialty chemicals and pharmaceuticals often can easily justify the very best sterile equipment and components. however, commodity biochemical facilities will not be able to afford such luxuries as large-pipe-diameter diaphragm and sanitary valves, sanitary tubing, specialized aseptic fittings, removable components and instruments, automated sip/Cip systems, and ultra pure water for process use. indeed, bioprocess facilities producing lower-margin commodity chemicals are often forced to balance budget-related engineering tradeoffs without compromising process sterility. this can be accomplished by using appropriate lower-cost equipment components (e.g., standard industrial valves with welded pipe connections) with more rigorous sip/Cip procedures and valve preventative maintenance.

the compressed air system is one important example of an element demanding close attention. it provides the huge volumes of air used to deliver oxygen and air-lift capabilities to the fermentation vessel. Designing systems that adequately filter airborne contaminants and bacteria, and remain dried (to avoid entrained condensate carrying bacteria through filters) requires design rigor. this is particularly crucial for facilities operating in hot, humid climates. retrofitting after the fact is very costly.

extreme operating variances. Due to the cyclic nature of batch fermentation, bioprocess facilities typically have enormous tankage requirements and highly transient

flow rates, which call for very complex pump and valve assemblies (both in terms of the number of components needed and their necessary turndown capabilities). to cost-effectively achieve turndown rates as high as 15:1 (flow requirements that can vary from 1,500 gpm to 100 gpm from a single device), bioprocess facilities often contain a significant number of pumps equipped with variable-frequency drives (vFDs) and extensive control-valve systems, both of which increase capital costs and software-programming effort.

operating costs. recycle and energy reuse are the standard for successful biologic commodity products. Commercial-scale fermentation facilities handle enormous volumes of water and steam (with varying composition and temperature) from fermentation, purification, evaporation and cleaning systems. Dynamic/transient mathematical modeling programs offer an invaluable tool to help designers identify the water and energy pinches and, thus, to strategically combine water streams of varying composition and temperature to achieve maximum water and energy recovery. such modeling can ensure that the overall flowsheet has the appropriate number and size of tanks and piping arrays to maximize water and energy reuse. With non-mature processes, significant changes in these areas can be expected as new information surfaces throughout design.

the location of the facility provides another opportunity for cost minimization. For instance, the ability to co-locate a fermentation-based manufacturing plant near a low-cost source of carbohydrates, such as a facility that is processing grain, corn, beets, sugar cane and other farm-derived products, can help to reduce both raw-material and transportation costs.

Bioprocesses require removal of large volumes of water. so, considerable operating savings can be realized by

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opting for today’s highly efficient separations technologies, such as evaporation with mechanical vapor recompression and multiple-effect evaporators. in general, low-energy evaporation is essential in commodity bioprocesses to keep operating costs aligned with product costs. however, getting from vendors design information essential in sizing utilities and building takes time; expect three to four months to pass before usable information is received. this, along with appropriate expediting resources, should be factored into the original design schedule and staffing.

proper design of the waste-disposal facilities can also help to contain operating costs. typically, the initial solids separation (cell/product) is done in complex unit operations, and the solids are further dried using standard press, plate, belt or drum dryers before being sent to a landfill for disposal.

Biosafety. the u.s. environmental protection Agency and the national institutes of health have issued guidelines for handling of many of the commercial microorganism strains. in addition, in the u.s., toxic substances Control Act regulations establish procedures for commercializing new or modified strains.

the biosafety classification of the microorganism used in the fermentation process will determine what level of containment is required for operations such as sampling, offgas venting and waste disposal to minimize the potential for biohazard risk to personnel and the environment. Certain criteria must be met for the containment and deactivation of the microorganism — using “any combination of engineering, mechanical, procedural, or biological controls designed and operated to restrict environmental release of viable microorganisms from the structure.”

long-lead equipment. While the design and operation of liter-scale fermentation vessels used during bench- and pilot-scale testing is relatively easy, considerable complexity

enters the picture when the process makes the jump to commercial-scale capacities. such facilities typically require a mix of AsMe- and Api-specified vessels. Fermentation vessel design must account for the high pressures and temperatures needed for sterilization as well as the large capacities required, up to several hundred thousand gallons. such large-scale vessels require field fabrication, with internal finishes often calling for extensive hand polishing and passivation. Material lead times and site construction presence must be factored in during the planning stages. other long-lead equipment typically include large vendor packages needed for purification — such as filtration skids, distillation and extraction columns, mechanical recompression equipment and spray dryers. insufficient planning related to transportation, vessel fabrication and erection, welding and finishing — particularly when the work onsite could interfere with the timely execution of foundation preparation and other construction activities at the site — is often to blame for delayed startup.

SuCCeSSful SCale-uPthe identification and genetic engineering of a suitable organism, followed by prudent piloting certainly is crucial to success with bio-based manufacturing. so, too, is effective technology transfer from the development effort and adequate attention to practical design issues. As is so often the case, a scale-up strategy that combines integrated teamwork with solid engineering efforts can go a long way to minimize costly rework and delays, and help today’s promising manufacturing routes based on renewable feedstocks to achieve their full commercial-scale potential on time and on budget.

John l. shaw, p.e., is a senior technology manager for Ch2Mhill lockwood Greene, spartanburg, s.C. e-mail him at john.shaw@ch2m.com.

scott A. rogers, p.e., is a process engineer for Ch2Mhill lockwood Greene, spartanburg, s.C. e-mail him at scott.rogers@ch2m.com.

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Biomolecules are complex, which translates into complex biomanufacturing processes. A bill of materials for an antibody process can have up to 90 items, notes Duncan low, scientific executive director at Amgen, “and that’s not counting the cell culture materials.” Biologics manufacturing is “a little bit like Macbeth,” he notes, “where the witches are adding eye of newt and tongue of frog.”

low spoke november 9 at ispe 2009 in san Diego on how pAt can be used comprehensively in bioprocessing, upstream and down, to help manufacturers exercise greater control over operations and simplify some of the complexity.

Critical quality attributes (CQA’s) for biologics range from product-related variants and process-related impurities to drug composition/strengths and adventitious agents. Most of the pAt work in biologics has centered upon the first two items, low noted, but is branching out into the latter two.

uPSTreaM aT aMgenupstream, “the key is to have good control over growth conditions and nutrients,” he said. Cell culture media are inherently complex and a major source of variation, and their control is getting increasingly difficult, low said.

Beyond the CQA’s mentioned above, there are also many variables which must be monitored from batch to batch in a bioreactor, including pressure, temperature, dissolved oxygen, ph, lactose, glucose, etc. nir, raman, mass spectroscopy, and other tools all work towards assessing how processing conditions contribute to the growth of cell culture.

these pAt tools also contribute to the establishment of a knowledge space, design space, and ultimately a control system and control space. even upon commercialization of a product, biologics manufacturers must continue to leverage pAt to optimize production, said low.

PaT By paul thomaS, SenioR editoR

Editor’s Note: The following is a report based on a prior ISPE conference presentation.

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Multivariate data analysis (MvDA)—made possible by data collected from pAt applications—has proved to be a powerful asset for manufacturers as well, he noted. one of the benefits that Amgen has realized through MvDA, low said, is understanding what raw materials are truly impacting the process and contributing to cell culture growth. this kind of knowledge was not possible before implementing comprehensive upstream pAt tools and methodologies. “We can establish a correlation between certain media components and growth,” he continued. the MvDA is “more diagnostic now than for control, but we’ve had some good results.”

For example, Amgen has found that control of seeding density reduces titer variability. “this allows us to move within our operating specifications and work more closely to the upper levels, which can result in higher titers,” low noted.

MvDA provides “correlative understanding,” but low cautioned that this should not be equated with “cause and effect.”

to conclude his upstream thoughts, low stated: “i wouldn’t advocate generating loads of data just for the sake of doing it, but there is usefulness in these tools.”

lookIng downSTreaMBiologics manufacturers’ downstream purification goals include concentrating product, removing gross impurities, recovering active protein, and inactivating and removing adventitious agents. pAt, too, can greatly assist these efforts, low said.

Biomanufacturers may not realize the importance of pAt tools for equipment monitoring, he added, as well as the benefits of Quality by Design-influenced predictive modeling and process design.

Monitoring and controlling process chromatography is a significant challenge, low noted, in that the speed of process chromatography usually impedes real-time

monitoring. Manufacturers have traditionally sought to collect fractions and conduct offline analysis, or include a delay loop for monitoring—“not optimal,” low conceded—and so a more advanced solution would be to develop predictive models to monitoring chromatography operations. this is something that Amgen is experimenting with currently. one predictive model might result in a plot of % purity vs. elution volume, for example.

A dissolved oxygen transition analysis (which Amgen has found correlates well with offline hetp) has proven effective for looking at the fitness for purpose of chromatography columns, low said.

BIo PaT of The fuTureAmgen and biologics manufacturers in general are just scratching the surface of what pAt and QbD can do for their upstream and downstream processes, low concluded. in the future, these tools and strategies may assist manufacturers in gaining even more understanding of their complex processes, by measuring or monitoring, for example: sub-visible aggregates, protein variants, process-derived impurities, and even immunogenicity. Meanwhile, the fundamental pAt tools such as hplC, Ms, nir, and GC, will be found in more and more control points throughout bioprocesses.

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addITIonal ProCeSS ConTrol & TeCh TranSfer reSourCeS

A Framework for technology transfer to satisfy the requirements of the new process validation Guidancehttp://www.pharmamanufacturing.com/articles/2012/064.html

scaleup and technology transfer as part of pharmaceutical Quality systemshttp://www.fda.gov/downloads/Drugs/DevelopmentApprovalprocess/Manufacturing/uCM291604.pdf

tools for improving process Quality and robustnesshttp://www.pharmatek.com/pdf/pteku/ptek-u_nov10-2010.pdf

tech transfer: Do it right the First timehttp://www.pharmamanufacturing.com/articles/2010/007

is Wireless ready for Bioprocess Monitoring ….and Control?http://www.pharmamanufacturing.com/articles/2011/079.html

haMIlTon CoMPany reSourCeS

Biopharmaceutical process sensor selection tool http://www.hamiltoncompany.com/products/sensors/c/1008/

application note:

sensor Application in a scale Down simulator for studying the impact of industrial scale inhomogeneities on Microbial Cultureshttp://www.hamiltoncompany.com/downloads/695097r00%20polilyte%20plus%20ArC%20And%20visiferm%20Do%20ArC%20in%20pFr.pdf

Roche application note:

Dissolved oxygen sensor in Microbial Fermentationhttp://www.hamiltoncompany.com/downloads/695098r00%20App%20note%20visiferm%20roche%20en.pdf

application note:

how GeA Diessel Gmbh pses ph-, Do and Conductivity Measurement in a process Fermentation planthttp://www.hamiltoncompany.com/downloads/695099r00%20An%20ArC%20in%20GeA%20fermentation%20plants%20en.pdf

video demonStRation:

ArC sensors for Wireless Bioprocess Analysishttp://www.pharmamanufacturing.com/multimedia/2010/016.html

video:

next-Generation Wireless Bioprocess sensors http://www.youtube.com/watch?v=vc5i_sxpWkw

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aBouT haMIlTon CoMPanyFor more than 30 years, the name hamilton has been associated worldwide with uncompromised quality in precision fluid measuring and analytical products as well as in fully automated analytical processes. the same competence has led the sensor technology Group to design a line of high quality ph, redox Conductivity and oxygen electrodes for laboratory and process measurement.

hamilton Company4970 energy Wayreno, nv 89502

phone: 775-858-3000www.hamiltonCompany.com

addITIonal readIng

scaleup and technology transfer as part of pharmaceutical Quality systems millili, g. (meRck)

http://www.fda.gov/downloads/Drugs/DevelopmentApprovalprocess/Manufacturing/uCM291604.pdf

tools for improving process quality and robustness meiSSneR, d. (BiopRoceSS SolutionS, llc)

http://www.pharmatek.com/pdf/pteku/ptek-u_nov10-2010.pdf