the clarification of bio reactor cell cultures for bio pharmaceuticals

8/2/2019 The Clarification of Bio Reactor Cell Cultures for Bio Pharmaceuticals

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he clarification of biological products such as recombi-nant therapeutic proteins, monoclonal antibodies, and

vaccines requires choosing a sequence of operations thatensures high yield, product consistency, and repro-ducibility.Depth,or pad, filtration is gaining acceptance as onetechnique for the removal of cells, cell debris, colloids, insolu-ble precipitants,aggregates, and other materials found in mam-malian-cell and bacterial fermentation broths. One or moredepth filters can be used in a clarification sequence to optimizethe filtration with each successive filter, thereby removing pro-gressively smaller particles by means of mechanical sieving andadsorption. Because optimized depth filtration can play suchan important role in enhancing the efficiency of the entire clar-ification process, this article primarily addresses depth filtra-

tion technologies and applications. The authors wish to em-phasize secondary clarification of effluent from variousbioreactor types and methods of primary clarification and theeffects of the various bioreactor types on the sizing of down-stream sterile filters. The authors also describe considerationsthat should govern the design of the filtration stream as a whole.

The choice of technology for any given process must dependon the fluid properties of the stream as well as requirements forintegration with downstream processes such as chromatogra-phy and ultrafiltration. The goals of clarification include highyield, product consistency, and reproducibility.

The clarification process can be separated into primary re-

covery, secondary clarification, prefiltration, and sterile filtra-tion. The process as a whole is designed to handle high partic-ulate loading and the wide particle-size distribution found inbiological streams (see Figure 1). The efficiency and capacityof the total process is improved by distributing the particle load-ing across several steps.

Primary recovery, the first phase of the process, removes thebulk of large particles, whole cells, and cell debris. This recov-ery step is usually performed by tangential-flow filtrationmicrofiltration (TFFMF), centrifugation, or, in some cases,depth filtration. Secondary clarification treats the product ofprimary recovery and is used for the removal of colloids, lipids,

DNARNA, residual cells, and other particles not removed inthe primary recovery process. Secondary filtration typically in-cludes one to several sequences of depth filtration designed to

62 Pharmaceutical Technology MARCH 2003 www.pharmtech.com

The Clarification ofBioreactor Cell Cultures forBiopharmaceuticalsDavid Yavorsky, Robert Blanck,* Charles Lambalot, and Roger Brunkow

David Yavorsky, PhD, is an R&D

manager for clarification technologies,

Robert Blanckis a clarification market

manager, and Charles Lambalot is a

product manager of clarification filtration, all

at Millipore Corporation, 290 Concord Rd.,

Billerica, MA 01821, tel. 781.533.8989,

[email protected]. Roger

Brunkow is a bioprocessing consultant at

Brunkow Consulting, LLC.

*To whom all correspondence should be addressed.

TClarification processes are critical stepsin the production of biological productsbecause they directly affect yield,

product consistency,and

reproducibility.Depth, or pad,filtration

is a technology commonly used to

remove whole cells, cell debris, and

colloids found in mammalian-cell and

bacterial fermentation broths. Because

optimized depth filtration can play such

an important role in enhancing the

efficiency of the entire clarification

process, this article primarily addresses

depth filtration technologies and

applications. By implementing the

strategies discussed in this article,

process compression can be achieved,

thus improving yield, lowering

operating costs, and reducing the

process footprint.


2/864 Pharmaceutical Technology MARCH 2003 www.pharmtech.com

filtration step before sterile fil-tration is designed to removetrace bioburden and colloids,thereby protecting the sterile fil-ter. After sterilization, the ster-ile product is then captured and

purified using chromatography,ultrafiltration, and other pu-rification processes.

The keys to an optimal sec-ondary filtration are the qual-ity of effluent from the primaryseparation and the effect thatthe product of secondary fil-tration has on downstream ster-ile filters. Although multiplelinked stages are common

within secondary filtration to achieve product clarity, each unit

process has the potential for yield loss. One of the process de-velopers goals is to achieve the highest level of product recov-ery and contaminant removal with the fewest number of unitprocesses.

Various technologies such as TFFMF and normal-flow depthfiltration (NFF) may be used for primary recovery before sec-ondary filtration. The choice of technology will have an effecton the selection and sizing of depth filtration. Figure 2 showsthe possible combinations of unit processes for the effective re-covery of a biological drug product.

For large-volume processes (typically2000-L fermenters),either TFFMF or centrifugation may be used as the primary

recovery step.For small-volume streams (2000-L fermenters),depth filtration may be used as a primary recovery device, butits sole use may be limited by the large amount of solids accu-mulating in the filter media. For streams with high solids, theamount of material retained by the depth filter may be too largeto handle practically. In some cases, a pretreatment device suchas a settling tank or coarse screen may be used immediately afterthe bioreactor, and in other cases a complete bypassing of thesecondary filtration may be possible with the use of certain tightTFFMF membranes in the 0.1-m range.

Cell culture variability

In todays bioreactor processes, higher concentrations of cellsand longer fermentation times result in higher drug titers andgreater product yields. These bioreactor conditions reduce cellviability, increase cell debris, and raise concentrations of or-ganic constituents in the cell broths.The amorphous, colloidalnature of these components tends to complicate the separationprocess. The choice of a clarification technology also shouldtake into account any requirements for integration with down-stream processes such as chromatography and ultrafiltration.

The characteristics of cell fermentation broths vary accord-ing to the type of cell expression system and the type of biore-actor used. Figure 3 shows the relative difficulty that various

bioreactor types contribute to clarification. Fed-batch and per-fusion-style reactors show lower cell viability, higher cell den-sities, higher turbidity, and greater concentrations of colloids

Table I: Types of normal-flow filters used in clarification processes.

Type of Filter Filter Media Process Characteristic

Lenticular depth filter Single-layer cellulose General clarification;

(positive charge) with filter aid usually requires multiple filters.

Double-layer cellulose Improved capacity

with filter aid and retention.

Double-layer cellulose Highest retention of particles

with filter aid and and colloids. Reduces the total

membrane filter number of required filters.

Pleated prefilter Membrane, High retention efficiency for

glass microfiber, membrane protection; low capacity

polypropylene in high colloid streams.

Wrapped depth filter Polypropylene Moderate contaminant capacity.

Poor retention of colloids.

Use for small batches.

Primaryrecovery

Secondaryclarification

Pre-filtration

Sterilefiltration

Bioreactor

Solution

ProductBioburdenParticlesColloidsWater

Solubleentities

Bioburdenparticles

Bioburdenand

colloids

Tracebioburden

andcolloids

Additionaltraces of

bioburdenand colloids

Product

Figure 1: Typical filtration of a biological product.

Primary recovery

Large-volumebioreactors

Tangential-flow

filtration

CentrifugationBioreactor

Secondary filtration

Tight MF_No depth filtration

Loose MF_Reduced depth filtration

Depthfiltration

Depthfiltration

Clarifieddrug

Depthfiltration

Small-volumebioreactors

Figure 2: Primary recovery and secondary clarification scenarios for

removal of cells, cell debris, and contaminants from a bioreactor

process stream.

remove progressively smaller particles. The range of depth fil-tration may remove particles by size exclusion from 0.6 to 0.2m, and the use of a depth filter with adsorber properties fur-

ther removes small particles not removed by size exclusion.Ad-ditional filtration steps may be performed to sterilize the drugproduct before downstream purification. The use of a pre-



and polynucleic acids than traditional batch reactors. Thesebioreactor types present new challenges for clarification andsecondary filtration that can be addressed with filtration andmechanical separation technologies.

Primary recoveryTFFMF. TFFMF is a membrane filtration process that sepa-rates particles using size exclusion. Membrane TFFMF gener-ally produces the highest product clarity because the membraneprovides a positive size exclusion barrier to particles larger thanthe pore-size rating of the membrane. The filtration processuses a microfiltration membrane that has a membrane pore sizethat can range from 0.1 to 0.65 m. The process is highly effi-cientit removes as much as 99.5% of all whole cells, cell de-bris, and other particulates. The selection of tight membranesin the 0.10.2-m range can facilitate the removal of lipids and

other colloids, thereby minimizing or eliminating the need forfurther filtration devices downstream.The process of bulk cell removal in primary clarification de-

termines to a large extent the particle load on the subsequentsecondary filtration step. TFFMF provides the most consis-tent separation in terms of particle size by limiting the upperend of the range to the nominal pore size of the membraneused. The TFFMF system must be designed to prevent rup-ture and fragmentation of cells in the recirculation loop. Forthis reason, secondary clarification or prefiltration may be re-quired after TFF-MF to reduce the small-particle load beforesterile filtration.

The TFFMF membrane barrier ensures high removal effi-ciency throughout the process run and is resistant to variabil-ity in the process feed. This efficiency and uniformity allows for

a more consistent feed to the secondary depth filters and en-sures lot-to-lot consistency in the clarified product. This con-sistency, in turn, helps avoid costly product loss and reducedefficiency in downstream purification or sterilization equip-ment. TFFMF devices offer advantages in scale-up because ofthe modularity of the device. Small-scale devices are available

for some types of TFFMF devices that can be used to initiateprocess development work with laboratory-scale volumes.Centrifugation.Centrifuges separate particles by variances in

density by subjecting the stream to a centrifugal force. Cen-trifugation typically is capable of handling high concentrationsof insoluble material in the feed but may be limited in its abil-ity to produce a clear product,and as a result will require moresecondary filters than TFFMF. Some centrifuges can concen-trate the cell mass to40% by volume and have advantages intheir ability to process large volumes with high cell-mass con-centrations. For some newer centrifuge types, the levels and du-rations of mechanical stress on cells may be low, thus mini-

mizing cell lysis and the release of cell fragments and otherintracellular debris.Depending on the centrifuge design, some disadvantages can

be found in the centrate quality, the ability to scale up pre-dictably, high maintenance costs, and the high initial expenseof the equipment. Centrifugation produces a measurably dif-ferent effluent than TFFMF does. Conventional large-scaledisk-stack centrifuges with a 10,00012,000-g operating ca-pacity are not effective in the removal of particles1 m, andtherefore yield a product stream more heavily solid-laden thanthat produced by microfiltration. As particles decrease in size,removing them efficiently decreases exponentially. A study by

Kempken and Preissmann showed that high numbers of smallparticles (ranging from 1 to 6 m) were not effectively removedby the centrifugation process (1). Because this small-particleloading may have serious effects on downstream chromatog-raphy or other purification, the study suggests that at least onesecondary clarification step be used after the centrifuge. Cen-trifuges can be applied to the removal of very high cell densi-ties, but their use as the sole clarification step is often limited.

Secondary clarificationTypes of NFF depth filters. Secondary clarifying depth filters aremost commonly cellulosic filters in a lenticular stacked-disk

cartridge design. Depth filters function by retaining particleswithin the porous matrix of the media. The available filtersrange from 0.1 to10m, the choice of which should be basedon the process steps that immediately precede and follow thedepth filtration.These high-area depth filters provide the neededcontaminant capacity to cost-effectively handle the high levelsof colloidal particles present in the fluid stream.

As cell culture fluids have high colloidal content, depth fil-ters are commonly used in stages by using a more open filterahead of the grade that will provide the needed protection tothe downstream step. Some depth filters combine sequentialgrades of media into one filter along with a membrane prefilter

layer. These depth filters provide a high level of protection totrailing downstream membranes or chromatography columns.The multimedia, multilayer design minimizes the number of

Low solids,low colloids

Easy to clarify

High solids,high colloids

Difficult to clarify

Cell-culturemedia

Batchbioreactor

Fed-batchbioreactor

Perfusionbioreactor

Bacterial oryeast lysate

Figure 3: Cell-culture characteristics from various bioreactor types.

Perfusionbioreactor

Spin filter

Depth filter 0.45-m

sterile filter

Cell harvest 70-80 NTU

Biomass

300-400 NTU

Figure 4: Major process steps for product harvest using a perfusion

bioreactor.



unit operations while making more efficient use of the filtermedia through improved flow distribution.

Other types of filters used for primary recovery are wrappedpolypropylene depth filters. These have been successfully ap-plied in some low-volume primary clarification applicationsand should be limited to processes in which cell density is low

and viability is high. Low effective filtration areas and inade-quate retention of colloids preclude the successful applicationof these filters in most large-volume fermentation broth clari-fications. Table I describes several depth filtration products andtheir process characteristics.

Performance of normal-flow filtration. Normal-flow filter per-formance is typically judged according to two performance cri-

teria: capacity and retention. Capacity is simply the volume offluid that can be processed at a maximum differential pressure.Capacity measurements can be acquired by using either con-stant flow (a maximum pressure limit) or constant pressure (aminimum flow limit) test conditions. Retention, on the otherhand, has traditionally been determined according to the pre-

filters dominant role in bioprocessing, which is the protectionof downstream sterilizing-grade filters (typically 0.22m). Theretention of the depth filter is generally expressed as the processcapacity of the trailing sterile filter or filters (i.e., the processvolume for a maximum P at a prescribed flux rate). There-fore, scalability in secondary clarification implies that accuratesizing of both the depth filters and the companion sterile filterat full process scale is required.

After secondary clarification, cartridge prefilters may be re-quired to remove trace components that foul the relatively ex-pensive sterile filters.The ideal situation is the selection of pri-mary or secondary clarification devices that significantly reduce

or completely eliminate the need for such prefilters.Scaling secondary filtration. The scale-up of a secondary filterto perform a given service is expected to follow in proportionto the available frontal area of the filter mediadoubling thefilter area should double the capacity or volume of fluid thatcan be processed within an acceptable range of variation. Un-fortunately, this ideal case is rarely, if ever, encountered in ac-tual production, and one must be mindful of a number of vari-ables that influence the design of full-scale prefiltration systems.

The accuracy to which full-scale depth filter performancecan be predicted from small-scale testsno matter how wellcontrolled the processcan never be greater than the variabil-

ity of the filter itself.The underlying presumption of the processof filter scale-up is that if all operating parameters are held con-stant, the filter media at any scale (or area) will behave identi-cally, if not predictably. If media use is somehow affected by thedevices geometry or size, these variations can be comfortablyaddressed with a correction factor that is largely insensitive toprocess conditions.

The consistency of depth filter media also influences scale-up.The cellulosic depth filter media is composed of three mainmaterials: refined cellulose fibers, an inorganic filter aid (suchas diatomaceous earth), and a charged (cationic) polymericbinding agent. The manufacture of cellulosic filter media (a

wet-laid process) does not lend itself to the creation of a per-fectly uniform porous matrixvariations in sheet thickness,density (both lateral and longitudinal), and composition areinherent in the process when measured using the square-centimeter (rather than square-meter) scale.

Laboratory-scale tests have shown thateven when draw-ing from the same feed lot and operating under identical con-ditionscapacities for small-scale depth filter devices can varyas much as 30% within a single test set. The scale-up com-parison between laboratory- and process-scale devices (lentic-ular stacked disks) indicates that deviations in capacity as greatas50% are possible even under tightly controlled test condi-

tions. When scaling up to different device geometries (from flatdisk to lenticular disk stacks), the variations in capacities areno greater than those witnessed for repeat runs on the same

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laboratory-scale device. Thus, the change in flow patterns thatoccurs when scaling up to different device designs does not ap-pear to influence performance as much as the variability of thefilter media itself.

Given this variability, multiple repeat tests must be run toobtain a statistically meaningful result,which especially applies

to laboratory-scale operations in which the prefilter media sam-ple is small and the probability is relatively high that any singlesample will misrepresent one or more average properties of themedia as a whole.Multiple laboratory-scale tests (3) run witha controlled feed, and tightly prescribed operating conditionswill provide the most accurate measure of performance as wellas an estimate of the precision to which scale-up performancecan be predicted.

Sizing methods for secondary filtration. Depth filtration gener-ally invokes two separation mechanisms: mechanical sievingand adsorption. Adsorptive capture often is effective in the re-moval of small particles from cell culture (1 m) by making

use of the native net-negative electrical charge that most bio-logical solids carry at neutral pH conditions. Depth filter mediasuch as the cellulosic composite can be modified to carry a pos-itively charged (cationic) surface that will bind suspended par-ticles by simple electrostatic attraction.

Adsorptive capture, unlike mechanical sieving, typically pro-duces no measurable change in the operating pressure of thefilter. The capacity or service life of the filter is instead deter-mined according to the number of binding sites available. Anysizing or test method that ignores the quality of the filter efflu-ent by focusing exclusively on pressure build-up can yield mis-leading results.

For many filter types, a bench-scale, short-duration, con-stant-pressure test (commonly referred to as the Vmax method)will yield a reasonable estimate of the filter area required forscale-up (2). The conventional pore-plugging model that un-derlies the Vmax method allows one to linearly extrapolate themaximum volume of fluid that the filter can process from theinitial flow decay rate.Although sufficiently accurate and con-venient for the evaluation of most surface or membrane filters,the Vmax method has been shown to be inappropriate for depthfilter media, which do not generally comply with this operat-ing model.

In most bioprocess applications in which depth filters are

used, the flow rate per unit cross-sectional area (or flux rate)will have a significant effect on the capacity of the filterthehigher the flow rate, the lower the capacity. Capacityis definedas the volume of fluid that can be processed at a maximum al-lowable pressure differential and is generally expressed in litersper square meter (L/m2) of filter frontal area. For this reason,atest method that allows for a variable flow rate over time, asVmax does,will generally result in an underestimation of filtercapacitythe initial surge in feed flow that occurs in a constantpressure test can load the filter inefficiently and thereby createpremature clogging, as documented by Yavorsky and McGee(3). Moreover, the Vmax method does not conveniently permit

filtrate quality monitoring, which is critical to properly assessdepth filter performance in which adsorption is the dominantmechanism of particle capture.

To get an accurate profile of performance for a given depthfilter medium, a constant-flow test method (referred to as thePmax method) should be applied. In this method, the feed flowrate to the filter is held steady, and the differential pressure ismeasured against throughput until the maximum allowable op-erating pressure is reached. In addition, the Pmax method al-

lows for the effluent quality to be monitored either accordingto filtrate turbidity or,more precisely, according to the pressureresponse of a trailing sterile filter. By using flow control and fil-trate monitoring, the Pmax method provides a truer measureof depth filter capacity and retention than is achievable by theVmax method. Pmax testing should be conducted at several se-lected flow rates throughout a range that includes the lowestpractical flow rate for full-scale processing. In this manner, aproduction system sizing that is based on a specific batch vol-ume and processing time can be accurately calculated at the ac-tual flux rate required.

Considerations for hardware design. Full-scale process systems

are constructed with complex piping networks between largefilter housings. As the size and number of filters in a housingincrease, the flow through the filter elements changes and is af-fected by hydraulic head and dynamically changing pressureprofiles as the filtration continues. Flushing, steaming, clean-ing, and filtration are all affected by the size and complexity ofthe system. Cleaning and preparation steps can require a sig-nificant amount of time and labor, especially if steaming,cool-ing, assembly, and disassembly are required. The clarificationstep can easily become an obstacle or limiting step in the har-vesting of a fermenter.

Large clarification systems may require significant areas in a

fermentation suite as well as enough headroom to disassemblehousings. In addition, if disposable elements are used for clar-ification in a large-scale operation, room must be available forhandling the new and used elements. Significant crowding ofequipment may occur, which can impede operations and safety.The handling of solid waste must also be considered in the en-vironmental effect and the operational cost computation.

Safety. The final, but very important, factors in the operationof the clarification system are safety and containment.The safetyaspects range from the proper design and fabrication of theequipment for steaming, to the physical aspects of removing thespent filters from the housing, to the containment of the live ge-

netically engineered organisms.The design of the housings,pip-ing, and connections for operation at elevated temperatures andpressures becomes more critical with increasing housing size.As housing diameter and height increase, a special design effortmust be made to ensure system safety. The physical operationsrequired to remove the spent filters, which can weigh20 kg,can cause severe back injury in operational personnel if they arerequired to manually lift the cartridges from the housing. Notonly are the cartridges heavy, but they also may be hot aftersteaming to decontaminate the solids collected.

When designing a system, one must also take containmentinto account. For example, the system design should not limit

the capability to decontaminate new filters with heat or spentfilters with steam. Any failure to address this problem becomesevident after construction is complete and start-up is in progress.



The temptation to shorten the steaming cycle or reduce the tem-perature may compromise decontamination.

Secondary clarification: case studiesCommonly available stacked-disk cellulosic-pad prefilters have

proven to be a versatile and effective tool for a variety of clari-fication duties in the downstream processing of mammalian-cell cultures. The following case studies describe three applica-tions in which cellulosic-pad prefilters have been applied tocell-culture secondary clarification.

Perfusion bioreactor harvest. In this example (see Figure 4), amammalian-cell culture is sustained during an extended 3060-day period, and the soluble protein product is harvested con-tinuously from two effluent streams. The primary product har-vest takes place through an integrated spinning basket filter thatuses a fine screen to separate the broth from the cells.The cellsare returned directly to the bioreactor. The coarse structure of

the spin filters yields a harvest fluid that contains substantialamounts of cell debris and other suspended solids as reflectedby its relatively high turbidity. To maintain a stable and pro-ductive cell population, the bioreactor is also periodically purged(the purge volume represents roughly one-third of the fluidharvested through the spin filter). These two effluents are sub-sequently batch-clarified and sterile-filtered each in turn througha depth filtersterile filter train. Figure 4 shows the major processsteps for product harvest.

A series of laboratory-scale trials were conducted to identifythe best prefilter in combination with a select sterile filter thatwould yield the highest overall system capacity (liters processed

per unit prefilter area). The prefilters chosen represented an as-sortment of bilayer cellulosic-pad filters and one high-charge(cationic) single-layer pad. The total system pressure was trackedin relation to the volume of fluid processed and limited to amaximum of 20 psid. Figure 5 shows the results.

The single-layer high-charge capacity pad showed little ca-pacity for the heavier biomass material because of its tight porestructure, which easily blocked the front surface. Several of thebilayer pad combinations also reached the 20 psid system limitafter processing only moderate volumes of either the harvestfluid and/or biomass material. However, one particular dual-layer pad filter optimized for this application outperformed all

contenders.Microfiltration permeate polishing. The use of TFFMF in cell-culture clarification produces a high-quality product stream.In cases where a 0.450.65-m membrane is used, further pol-ishing may be required because some suspended solids are stillpresent in the permeate that will significantly diminish theprocess capacity of a typical 0.2-m sterile filter (see Figure 6).

In this example, a manufacturing process was being devel-oped to produce a monoclonal antibody starting with a fed-batch Chinese hamster ovary cell culture. When the culture isterminated, the fluid is first clarified using a cassette-type TFFsystem with a nominal 0.65-m membrane. Samples of this

permeate were then processed using several laboratory-scalecellulosic-pad prefilters, each paired with a trailing 0.22-msterilizing filter.

500

400

300

200

100

0Prefiltercapacity(L/m2)at20psid

ManufacturerAdual-layer

high-retentionmedia

ManufacturerAsingle-layer

high-chargemedia

ManufacturerAdual-layer

medium-retentionmedia

Millistak+B1HC

Millistak+C0HC

flux rate: 155 L/m2/h

>500 L/m2

Figure 5: Clarification of perfusion bioreactor harvests using various

cellulosic-pad depth filters.

BioreactorTangential

flowfiltration-

micro-filtration

Depthfiltration

Sterilefiltration0.22 m

Clarifiedproduct

Figure 6: Primary clarification of bioreactor feed with TFFMF

followed by depth filtration.

700

600

500

400

300

200

100

0

Sterilefilter(0.22m)capacity(L/m2)

ManufacturerAsingle-la

yer

high-chargeme

dia

ManufacturerAdual-la

yer

high-retentionme

dia

ManufacturerBsingle-la

yer

high-retentionme

dia

Millistak+A1

HC

Prefilter flux: 37 LMHSterile filter flux: 200 LMH

Figure 7: Sterile filter protection with various cellulosic-pad depth

filters.

Bioreactor CentrifugeDepth

filtration

Sterilefiltration0.22 m

Clarifiedproduct

Cells anddebris

Centrate

Figure 8: Primary clarification of bioreactor feed with centrifugation

followed by depth filtration.



Because of the small sizes and low concentrations ofparticles contained in the MF permeate, none of the cel-lulosic-pad prefilters exhibited any significant pressurebuild-up through the performance teststhe capacity ofthe secondary clarification step was determined insteadby the capacity of the sterile filter. The pad prefilters be-

have much like an adsorbent bed, wherein the effluent (orfiltrate) quality initially is high until the media is satu-rated with adsorbed material.At this point, there is a break-through or abrupt rise in filtrate particles.Figure 7 showsthe measured capacities of the trailing sterile filter for eachof the pad prefilters tested at a pressure differential of 20psid. These tests were conducted at a constant flow rateand a prefiltersterile filter area ratio of approximately5:1. Again, the dual-layer graded-density pad filter withmembrane demonstrated greater performance.

Centrate clarification. A scale-up study conducted on atypical recombinant protein production process demon-

strates the accuracy with which the results of laboratory-or pilot-scale trials can be used to predict performanceat full-scale production. In this study, the cell culture waspreclarified by centrifugation, as shown in Figure 8. Thecentrate was further clarified using a prefilter, and thefiltrate then delivered to a 0.22-m cartridge filter forsterilization.

35

30

25

20

15

10

5

0

cell culture: 1.54 PCV, 60% viabilityfeed turbidity: 80_100 NTUprefilter flux: 100_170 LMHprefilter: Millistak+ B1HC

sterile filter: 0.22-m Durapore

Prefilterandsterilefilterpressure

Process scaleLab scalePilot scale

0 50 100 150 200 250 300 350 400

Prefilter capacity (L/m2)

test scale prefilter area sterile filter arealab 23 cm2 4 cm2

pilot 650 cm2 200 cm2

process 1.8 m2 0.69 m2

Figure 9: Prefilter and sterile filter scale-up.

Circle/eINFO 53


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Constant-flow tests were conducted with the prefilter andsterile filter operating in tandem, and the combined systempressure was monitored in relation to throughput or capacity.Figure 9 records the results of multiple tests run at three pre-filter device sizes.

As shown, filter capacities at the three scales of testing are in

reasonably close agreement.Scale-up from laboratory to processsize (a factor of 782:1 in prefilter frontal area) yielded filter ca-pacities that deviate 35%. The moderately higher capacitiesachieved with the pilot- and process-scale prefilters can be at-tributed to better flow distribution (and, consequently, betterfilter media use) in the multiplestacked cell devices.

This example illustrates the utility and accuracy of theconstant-flow (Pmax) method for evaluating clarification depthfilter media. The constant-flow method not only mimics con-ventional process conditions but also allows for simultaneoustesting of prefilter and sterile filter pairings. The disparity be-tween laboratory- and full-scale device performance appears

to mirror the variability of the prefilter media itself.

Conclusion

The development of a clarification process requires integratingseveral unit processes such as centrifugation, TFFMF, depthfiltration,and sterile filtration.Optimization of the total processrequires an understanding of the effects of the unit processeson each other. The challenges are to select equipment that meetsthe increasing complexity of process fluids produced by todaysmore efficient bioreactors. Increases in drug titer, cell density,cell debris, and cell lysis products add difficulty to the clarifi-cation process and confound the selection of separation and

filtration devices.As process-scale selections are made, consideration shouldbe given to equipment design, ease-of-use,and cleanability.Thiswill ensure efficient changeover and operator safety in handlingspent filters.

To develop a clarification process, a robust integration of clar-ification steps is important to ensure the cost-efficient pro-cessing of cell-culture fluids. Successful product scale-up re-quires achieving uniform filtration consistency and compressingthe number of unit processes. A range of filter devices is read-ily available to facilitate laboratory trials, pilot production, andfull-scale processing. Through the implementation of a well-

devised scale-up work plan that assesses several clarificationoptions, one can confidently select and size clarification filtersto protect downstream unit operations while reducing operat-ing costs.

References1. R. Kempken and B.W. Preissmann, Assessment of a Disk Stack Cen-

trifuge for use in Mammalian-Cell Separations, Biotechnol. Bioeng.46 (1995).

2. F. Badmington et al.,Vmax Testing for Practical Microfiltration TrainScale-Up in Biopharmaceutical Processing, BioPharm 8 (7), 4852(1995).

3. D.P. Yavorsky and S. McGee, Selection and Sizing of ClarificationDepth Filters, Genetic Eng. News 22 (9), 4445 (2002).PT

the clarification of bio reactor cell cultures for bio pharmaceuticals

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