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comprising porous beads made of a polysaccharide, mineral,or synthetic matrix derivatized with specific functional groups.

The functional groups exploit different separation principles,such as size, charge, hydrophobicity, or affinity for particularligands, to resolve target proteins. The resin may bind the targetprotein while eluting contaminants (bind-and-elute or capturemode) or may bind contaminants while eluting the target protein(flow-through or polishing mode) ( 11 ). The highest resolutionand therefore the highest purity is achieved when orthogonalprinciples are applied. For the purification of monoclonalantibodies, this usually involves a native or recombinant ProteinA capture step combined with a cation exchange column forintermediate purification and an anion exchange column operat-ing in flow-through mode to remove negatively chargedimpurities such as DNA, host cell proteins, endotoxins, andendogenous and adventitious viruses (Figure 1). However,

companies such as Medarex Inc. have developed a non-ProteinA platform for antibody purification with optimized yields andminimized cycle times (Figure 2). The evolution of this processinvolved replacement of the Protein A step with an equallyefficient cation exchange resin and reduction of the overallnumber of column chromatography steps from three to one byreplacing the anion exchange flow-through column with adisposable anion exchange membrane ( 12).

What makes the combination of resin and membrane chro-matography steps such a good idea? On closer inspection,capturing and polishing, although following different principles,are subject to the same limitations. However, the consequencesare different as discussed below.

Capturing. Despite the essential role of packed-bed chro-

matography in biomanufacturing, high-titer processes imposepractical limitations that suggest that the true bottleneck inrecovery processes is the first adsorptive column, reflectingpoorer performance with increasing scale ( 13 ). Very largecolumns can be as robust and reliable as smaller ones, but thereis no economy of scale with such devices because the additionalcost of resins, buffers, and other consumables outstrips anysavings made by increasing the productivity. Indeed, thebottleneck in process-scale chromatography negates any ad-vantages of scaling up earlier process units, since capture stepsare driven by mass rather than volume and savings madeupstream therefore do not translate into increased productivityduring purification. Larger columns also impact directly onfacility layouts, costs and infrastructure because the space and

buffer volumes for all steps, including preparation and cleaning,must be adapted to compensate. As a consequence, pool andbuffer volumes are serious limitations when it comes to theintroduction of high-titer processes into existing facilities, henceThe Bad.

The physical constraints of process-scale chromatographymust also be considered. The largest biochromatographycolumns in use today are 2 m in diameter and are operated ata 10- to 20-cm bed height, which is fast becoming a limitationin large-scale processes. The requirements to capture 100 kg of monoclonal antibody on a Protein A affinity column in a singlecycle may serve as an example. If one assumes an optimalloading capacity of 50 g/L, this would require 2,000 L of resin,which would need to be packed in a column with a diameter of

3.2 m at a bed height of 25 cm ( 4). The need for oversizedcolumns can be circumvented if several cycles are used toprocess a single batch (and this is common industrial practicefor the Protein A capture step for antibodies). This, in return,reduces the operational lifetime of the resin and requires morebuffer, therefore introducing additional costs. A column contain-ing 2,000 L of resin would require 50,000 L of buffer forequilibration, elution, and cleaning, and these buffers need tobe prepared, stored, and disposed of in an appropriate manner(4). As well as productivity and economic issues, large columnsalso suffer from scale-related packing problems such ashysteresis, edge-effects, and resin compression, which result inunpredictable fluid distribution and pressure drops. Hysteresisis a phenomenon in which uneven resin packing leads to

Figure 1. Typical process train for the manufacture of monocolonal antibodies.

Figure 2. Evolution of downstream processes for antibody purificationwith the protein A affinity step replaced with non-affinity methods.Reproduced with permission ( 49 ).

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differences in fluid flow rates throughout the column, especiallyaround the edges where looser packing density encourages themobile phase to flow faster and can, in the worst cases, resultin the so-called cheese-cake effect where the resin collapsesand leaves large gaps. Resin compression reduces its porosityand thus influences the pressure drop across the column. Otherrisks not so openly discussed include progressive fouling of theresin due to the aging of feed material during long load cyclesand/or insufficient cleaning accompanied by microbial con-tamination, hence The Ugly.

Although there is considerable debate about this issue ( 13 -15), one can conclude that bind-and-elute operations formonoclonal antibodies are approaching their physical limits. Forthe time being, there is no alternative to packed-bed chroma-tography for such operations due to its selectivity and ease of use. In the long run, limitations in productivity and thus processeconomy may better be addressed by low-technology alterna-tives as discussed later.

Polishing. The limitations of column chromatography out-lined above also apply to flow-through applications, where theobjective is to capture impurities rather than the product. Flow-through anion exchange chromatography as used in the purifica-tion of monoclonal antibodies illustrates the problem well. Inresin-based anion exchange media, the transport of solutes totheir binding sites relies on pore diffusion, but the contaminantsare often large molecules, e.g., DNA and viruses, which do notreadily diffuse into the pores (Figure 3a). This causes masstransfer resistance and lowers the column efficiency becauselarge molecules can only bind to the outer surface of the beadand longer residence times are required to find binding ligandsinside the resin particle ( 16 , 17 ). This challenge may beaddressed by using a greater column bed height and/or reducingthe linear flow rate, both of which impact on overall productiv-

ity. Therefore, to keep up with process demand, most traditionalpolishing steps operate at a flow rate of between 100 and 150cm/h and use significantly oversized columns to accommodatethis. For flow-through applications, these limitations can beovercome by changing the chromatographic support structurefrom a resin to a membrane, a topic explored in detail later.

What Are the Alternatives?

Various alternatives have been put forward either to replacecolumn chromatography or to reduce the load of impurities inthe feedstream so that one or more chromatography steps canbe eliminated. Some of these alternatives apply to capture stepswhereas others represent innovate filtration and chromatographyformats that apply to polishing operations.

Examples of simple technologies that have been revisitedrecently include flocculation, precipitation, two-phase extraction,and crystallization ( 4, 13 , 18 , 19). Flocculation and precipitationcan be used in combination with conventional cell separationtechniques such as centrifugation and microfiltration to removeresidual particulates and soluble impurities that might otherwiseincrease the burden on downstream polishing steps, thereforeallowing the number of chromatography processes to be reduced.Flocculation is particularly useful for removing sub-micrometer-

sized particulates that increase the turbidity of the feedstreamand usually need to be removed with an interstitial dead-endfiltration step to prevent column fouling. Polymers can alsopersuade small particles to clump together, facilitating theirremoval by centrifugation, but innocuous inorganic alternativessuch as calcium chloride and potassium phosphate have alsobeen used successfully ( 20 , 21 ). Crystallization is anotherinexpensive technology that in some cases can simultaneouslypurify, concentrate, and stabilize a recombinant protein andprovide a useful delivery mechanism ( 22 ). Several commercialprocesses for therapeutic protein manufacture involve a crystal-lization step that replaces chromatography ( 23 , 24 ), althoughapplications in antibody purification are currently limitedbecause of low yields, the inherent complexity of the process,

and difficulties with process control. Some engineering-basedsolutions have also been implemented, including the deploymentof radial flow chromatography and simulated moving bedchromatography to increase throughput while reducing bufferusage ( 25 ).

Examples of higher-end technologies for the replacement of column chromatography include the use of charged ultrafiltrationmembranes and membrane-adsorbers. The separation of proteinsby charged ultrafiltration membranes was first reported byNakao and colleagues ( 26 ) who used polyethersulfone ultrafil-tration membranes bearing either positive or negative chargesto separate myoglobin and cytochrome c by setting the bufferpH near the pI of one or other of the proteins, allowing efficientseparation even though the proteins were of similar sizes. Thebasis of protein separation using charged filtration devices hasbeen studied intensively by Zydney and van Reis ( 27 , 28 ), whohave developed positively charged ultrafiltration membranes thatcan separate antibodies from CHO cell impurities including hostcell protein ( 29 ). The most obvious progress has been made inthe development of alternative chromatography formats suchas monoliths and membrane absorbers ( 30 , 31 ). Here weconcentrate on the use of membrane chromatography ( 32 ) as apotential solution to circumvent the limitations of resins forflow-through applications.

Membranes are already integral to many bioprocesses becausethey can be used as disposable modules, but thus far theirprincipal application has been filtration rather than chromatog-

raphy. Disposables are becoming more important in biopro-cessing as confirmed in the industry survey discussed above(6 ). For many unit operations, particularly filtration and media/ buffer storage, disposable devices have been in common usefor quite some time because they save on cleaning and validationcosts. However, disposables have other benefits: they save time,provide flexibility, and streamline process development ( 33 ).The ability to replace each module completely makes it easierto assemble process trains for new products in existing premiseswithout worrying about cross-contamination, although there canbe additional validation burden because of leached materials.

Interest in membrane chromatography is growing because of the success of disposable membrane filters, but there is still alack of appreciation of the many advantages membrane devices

Figure 3. Mechanistic comparison of solute transport in (a) packed-bed and (b) membrane chromatography. Thick arrows represent bulk convection, thin arrows represent film diffusion and curly arrowsrepresent pore diffusion.

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unit operation for capturing molecules of < 200 kDa, especiallywhen peak cutting, gradients, etc. are required for the separationof closely related species. Selectivity should be similar for bothconcepts, as long as the same ligand is used. However, for largermolecules (including most of the anticipated contaminants inantibody manufacture) there is higher capacity and fasterprocessing, which is why membranes are better for flow-throughapplications in biomanufacturing (Figure 6).

Head to Head: Counting the Cost

The important remaining question is how membrane devices

compare with columns in terms of cost, both fixed (capital) costsand variable (running) costs. Capacity and disposability arecritical factors to consider when calculating unit operation costsfor new processes. Although membrane devices clearly have ahigher throughput, a direct comparison of resins and membranesbased on volume shows that disposable membranes are currentlymore expensive. This must be balanced, however, against thereduced size of membrane devices, which also reduces bufferrequirements, makes the process time shorter, and brings alongall the other benefits of a disposable technology ( 33 ).

A 10-year cost model ( 44 ) showed that Q-membrane chro-matography was economically unfeasible compared to Q-resincolumns at a process capacity of 500 g/m 2 (equivalent to about1.8 kg/L) mainly due to the cost of membrane devices. The

model was based on an upstream CHO platform featuring a15,000-L bioreactor with a yield of 1 g/L antibody. Thisgenerates a load of 13 - 15 kg of antibody per batch, whichwould require a 220-L column or a 1.6-L membrane devicebased on typical performance standards. The model assumedthat up to 40 batches could be run in a year, with the columnresin replaced after each 100 cycles. Therefore, the columnwould need to be repacked with resin four times during theprocess lifetime, whereas 400 membrane devices would be

required over the same period. The model suggested thatcapacity would need to increase above 2 kg/m 2 (7.2 kg/L) tobecome competitive. Capital costs for chromatography hardwarewere not considered.

Another cost model ( 45 , 46 ) suggests that membrane chro-matography could break even with resins at a load of just 2kg/L. With a load capacity of 10 kg/L, the membrane-basedprocess costs only one-fifth as much as an equivalent operationusing resins (Figure 7). This cost of goods model was based onthe use of 10 in. Q ion-exchange membrane devices (180 mLvolume). The values of 10 kg/L and 2 kg/L were consideredtypical for a relatively pure (late stage) feed stream afterintermediate polishing and a less pure (earlier stage) feed stream,after clarification and capture by Protein A chromatography.

The unique aspect of the model was its consideration andseparation of all direct and indirect costs into four majorcategories: capital equipment, consumable equipment andmedia, consumable chemicals and materials, and labor. Thefixed capital cost was the most significant in the case of columnchromatography (nearly 19,000 EUR per batch) while that of membrane chromatography is less than 2,500 EUR. Consum-ables were important in both forms of chromatography, withresins accounting for the bulk of costs in column chromatog-raphy and disposable devices accounting for most of the costsin membrane chromatography. As might be anticipated, the costof consumable equipment and media is higher for membranechromatography because the membrane device needs to bereplaced after each batch while the column resin can be cleaned

Table 1. Scale-Up with SingleSep Q Membrane Chromatography a

frontal surfacearea (cm 2)

scale-up factorfor flow rate

rec flow rate(L/min)

bed vol(mL)

min static bindingcapacity (g) (release test)

dynamic capacityat 10% (mg/mL)

dynamic capacityat 100% (mg/mL)

nano 2.4 1 0.03 1 0.03 22.5 395 in. 160 66 1.9 70 2.0 19.5 3010 in. 450 187 5.0 180 5.3 20.5 29.520 in. 900 375 10 360 10.5 20.5 3530 in. 1350 562 15 540 15.8 20.5 37.5mega 4050 1687 45 1620 47

a Parameters such as frontal surface area, bed volume, flow rate and static binding capacity scale up in a linear fashion (assuming constant bed height of 4 mm). Normalized dynamic BSA binding capacity remains constant at a given breakthrough (values shown at 10% and 100%; see also Figure 5). Data fromSartorius-Stedim Biotech.

Figure 5. Dynamic binding capacities of Q membrane chromatography devices represented by breakthrough values as percentage of total load(C / C 0) against membrane volume (mL). Individual curves represent selected lots of different sized devices ranging from nano (1 mL) to 30 in. (540mL) (see keys).

Table 2. Membrane Chromatography Spiking Study with FourModel Viruses a

virus b size (nm) LRV run 1 LRV run 2virus

recovery (%)

MVM 16 - 25 6.03 ( 0.21 6.03 ( 0.20 100Reo-3 75 - 80 7.00 ( 0.31 6.94 ( 0.24 100MuLV 80 - 110 5.35 ( 0.23 5.52 ( 0.27 > 70PRV 150 - 250 5.58 ( 0.28 5.58 ( 0.22 100

a Test substance was a human monoclonal antibody (5 - 9 g/L), pH 7.2;4 mS/cm; 1% spike; 450 - 600 cm/h. Data from ref 50. b MVM: minutevirus of mice. Reo-3: reovirus Type III. MuLV: murine leukemia virus.PRV: pseudorabies virus.

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and regenerated and the costs therefore spread over 100 cycles.However, the consumption of membrane is lower at the higherloading capacity since fewer capsules need to be used. So,whereas at 2 kg/L load the consumable equipment representsnearly 60% of the entire process cost, when capacity is increasedto 10 kg/L this falls to less than 20%. Overall, disposables areonly 11% more expensive than column chromatography at thehighest loading capacity considered in the model. The use of other consumables is much higher in the case of columnchromatography because the resin needs to be washed andregenerated and the large size of the column demands highervolumes, and the cost of labor is approximately four-fold higherbecause of buffer preparation, cleaning, validation, maintenanceof equipment, and quality work.

Conclusions

Higher upstream productivity presents a challenge to chro-matographic unit operations, and there is no unified solutionfor the capturing and polishing steps. While capturing will

remain the domain of packed-bed columns with little (butgrowing) substitution pressure from low-technology alternatives,polishing is currently in a technology transition phase. Mem-brane chromatography offers a cost-effective alternative totraditional packed-bed chromatography in flow-through opera-tions, such as polishing for the removal of viruses andcontaminants in antibody manufacture. They are particularlyattractive at larger scales, where columns suffer from the risingcosts of resins and buffers and fall foul of scale-related packingissues that reduce column efficiency. With devices up to 5 L,membrane adsorbers can polish 100-kg batches of antibodiesand can thus be operated in high-titer processes while providingother advantages through their disposability. The impact of disposable membrane chromatography on virus clearance pro-

cedures represents an important technological advantage, sincestudies have shown LRVs of > 5 with model viruses even athigh loads without the need for carry-over studies. Virus elutionstudies demonstrated an adsorptive mechanism that is orthogonalto any filtration process based on size exclusion. Membranechromatography thus provides an advantage that is now increas-ingly important given the new EMEA guidelines on virusclearance studies, which indicate that at least two orthogonalsteps for the clearance of enveloped and non-enveloped virusesare mandatory for phase 1 studies ( 47 ).

Since the advantages of membrane chromatography have onlybeen realized in polishing applications so far, the open questionis how to address other bottlenecks, particularly the capture stepin antibody manufacture. More specifically, what is the rolefor packed-bed chromatography in future processes? Fourreviews ( 4, 8, 13, 18 ) published over the past 2 years come tovirtually the same conclusion on this topic: yes, packed bedchromatography is still the workhorse in bioseparation and willremain as the standard for at least the next 5 years in this

conservative industry, but it cannot really cope with thechallenges provided by increasing fermentation titers. Becauseof its selectivity, packed-bed chromatography has never been anecessary evil. As the central enabling technology, it has laidthe foundations for downstream processing as an independentdiscipline. However, although it is here to stay for the foresee-able future, it is now slowly becoming part of the problem andnot the solution. Even if we are able to bind 100 kg of amonoclonal antibody to a single column, it is questionablewhether this is the best solution from an economic and qualitystandpoint, since older enabling technologies such as precipita-tion could now provide an equally acceptable and much moreeconomic alternative. Following good initial recovery of theproduct from the biomass, downstream unit operations could

Figure 6. Comparative advantages of resin and membrane chromatography for the absorbance of (a) small and (b) large molecules. Orange shapesrepresent resin chromatography, and green shapes represent membrane chromatography.

Figure 7. Comparative results from a cost model comparing traditional and membrane chromatography ( 22 , 23 ), showing each component (labor,materials, consumables, and capital charges) as a percentage of the total cost of column chromatography (which is fixed arbitrarily at 100% so thatthe savings brought about by membrane chromatography can be shown as a percentage cost reduction per batch). Costs break even at a loadcapacity of 2 kg/L (a) and at 10 kg/L (b) membranes cost less than 30% per batch as compared to running a column.

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(46) Lim, J. A. C.; Sinclair, A.; Kim, D. S.; Gottschalk, U. Economicbenefits of single-use membrane chromatography in polishing. Acost of goods model. BioProcess. Int. 2007 , 5, 60- 64.

(47) EMEA. Guideline on virus safety evaluation of biotechnologicalinvestigational medicinal products. EMEA/CHMP/BWP/398498/ 2005-corr. EMEA: London, 2006.

(48) Gottschalk, U. The Renaissance of protein purification. Biopharm. Int. 2006 , 19 (suppl 2), 8 - 9.

(49) Arunakumari, A. Downstream design considerations for efficientbatch processing of high titer cell culture processes for the production

of monoclonal antibodies. BioProcess International European Con-ference and Exhibition, Paris, France April 24 - 25, 2007.

(50) Zhou, J.; Tressel, T.; Solamo, F.; Dermawan, S.; Hong, T.;Gottschalk, U.; Reif, O.; Pastor, A.; Mora, J.; Hutchison, F.; Murphy,M. A new Sartobind Q scale-down model for process-scale antibodypurification. J. Chromatogr. A 2006 , 1134 , 66 - 73.

Received November 20, 2007. Accepted March 24, 2008.

BP070452G

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