MM embranechromatography — orthe synonymouslyused expressionmembrane adsorption

— is an essential process platformtechnology. After some 20 years ofdevelopment with drawbacks,disappointments, and crucialinnovations, the current generation ofmembrane adsorbers plays a centralrole in today’s biotech processes amidnumerous economic and regulatorychallenges. Efficient development anduse of this technology is an importantstrategic factor as the biotech industrymatures and faces consolidation.Modern membrane adsorbersaddress current trends in state-of-

the-art downstream processing: fulldevelopment pipelines withmanufacturing capacity limitations,process integration to overcomecomplex supply chain scenarios,growing cost awareness, high qualitydemands, and other driving forcestoward higher efficiency andproductivity. Multiple- and single-use membrane adsorbers build thebasis for the most efficient separationof biopharmaceuticals and theremoval of unwanted process-relatedor endogenous impurities. Variousapplication examples set the stage forfast purification-train assembly frommodular, predeveloped unitoperations allowing a standardized,seamless process transfer withreduced engineering costs andreliable economic calculations.

This is a short overview aboutthe present capabilities and

limitations of membrane adsorbertechnology in protein and viruspurification using Sartoriusmembrane adsorbers. For reviews ofmembrane chromatography, seereferences 1 and 2.

THE PROCESS VIEW

Of central concern in processperformance is the ability ofmembrane structures to cope withthe growing demands of thefeedstream in biotechmanufacturing. Such demands resultfrom a clear trend toward very large-scale applications that requireoptimized throughput and mustaccommodate significantly increasingexpression levels in the upstream. Incell culture, fermentation is settingthe pace with rapid progression ofcell line and technologydevelopment; routine production of

PRODUCT FOCUS: PROTEINS AND

VIRUS PRODUCTS

PROCESS FOCUS: DOWNSTREAM

PROCESSING: ION-EXCHANGE AND

AFFINITY CHROMATOGRAPHY

WHO SHOULD READ: PROCESS

DEVELOPMENT MANAGERS,DOWNSTREAM PROCESSING MANAGERS

KEYWORDS: MEMBRANE

CHROMATOGRAPHY, DIFFUSION

LIMITATION, PROTEIN PURIFICATION,ION-EXCHANGE CHROMATOGRAPHY,AFFINITY CHROMATOGRAPHY,CONTAMINANT REMOVAL, ADENOVIRUS

PURIFICATION, DNA-REMOVAL, VIRUS

REMOVAL

LEVEL: INTERMEDIATE

B I O P R O C E S S TECHNICAL

Membrane adsorber technology is entering biopharmaceutical production suitesPHOTO SUPPLIED BY THE AUTHOR

56 BioProcess International MAY 2004

Membrane AdsorbersA Cutting Edge Process Technology at the Threshold

Uwe Gottschalk, Stefan Fischer-Fruehholz, and Oscar Reif

glycosylated proteins in the 1–3 g/Lrange is becoming feasible. This isfurther reinforced by advances inmicrobial fermentation and access totransgenic options.

Innovative concepts are welcomebecause downstream processingcontributes up to 80% of the total production cost of abiopharmaceutical purification (3).Bringing products to market fasterand improving process economicshelps manufacturers competesuccessfully. For example, the batchyield of a product after tenproduction steps with a 95% yieldper step results in a purification yieldof 60% for the complete downstreamprocess. With 90% yield per step, theoverall yield drops to 35%. Further,with a supposed price of $250 perdose and a given batch yielding10,000 doses at 60% purificationyield, the decrease of value would befrom US$2.5 million to $1.45million — a loss of $1.05 million.

Modern large-scale downstreamprocesses for secreted biomoleculesderived from fermentation follow astreamlined concept of cell removal,capturing, intermediate purification,and polishing. A new trend is forthe capture step to play anincreasingly important role inremoving more than 90% of bulkimpurities (Figure 1).

It is now widely accepted that theideal capture step quickly isolates aproduct from the feedstream.Harvesting is usually the firstnonsterile process step. Because thefeed solution becomes contaminatedas it matures, speed in columnloading is extremely important.Primary selection criteria for mediaused in the capturing step thereforefocus on efficiency, yield,concentration factor, and clearanceof major contaminants. Because

dynamic capacity under physiologicalconditions is central to the screeningprocess, results are often optimizedfor conductivity and contact time(specific process parameters).

Additional polishing steps ensureclearance of viruses and otherinfectious agents and remove keyimpurities such as host cell proteins,media components, aggregates,product variants, nucleic acids,endotoxins, and proteases. In anycase, every batch of material mustmeet end-product specifications.Robust and reliable processes arethe basis for comparability ofdifferent production versions, scales,and manufacturing sites for complexbiotech products such asrecombinant proteins, monoclonalantibodies, and virus particles.

Trends in bioprocess engineeringand fermentation development areprompting continuous improvementin downstream processingoperations. Despite the complexityof biotech processes, there is roomfor innovation using integrated unitoperations, automation, and rationaldesign of facilities and equipment.During the recovery phase, thecombination of clarification (cellremoval) and capturing composes anideal scenario.

TECHNICAL BACKGROUND

OF MEMBRANE ADSORPTION

The limitations of preparative low-pressure chromatography result frompacking large-scale columns withcoarse resins. Large particles creatediffusion limitations (especially formacromolecules) and have a negativeeffect on resolution that increaseswith increasing flow rates.

Some 10 years ago, perfusionchromatography addressed thisproblem with newly designed resins.In perfusion chromatography, theconvective flow of the mobilechromatographic phase accesses thecore of particles (flow-throughpores) to provide high linear flowrates of more than 1000 cm/hduring routine processing. These areattempts to mimic the naturalhydrodynamic properties ofmembranes, although still at leastone order of magnitude below theiractual performance limits.

The highest convective flow ratesand mass transfer rates at low backpressures are reserved for membranesthat reflect breakthroughs in batchcycle times and therefore overallprocess performance. Membranepore sizes of 0.45 (or �3) µm aretwo orders of magnitude above thoseof gels. In addition, the ratio of bed

TTaabbllee 11:: Data of Sartobind ion-exchange MultiSep 50 cm (20 inch) standard modules

Membrane Membrane Nominal Nominal Nominal flux Module size bed height (mm) bed volume (L) membrane area (m²) number of layers (L/min �� 0.1 MPa)

MultiSep 15-50 4 0.56 2 15 10

MultiSep 30-50 8 1.1 4 30 5

MultiSep 60-50 16 2.1 8 60 2

FFiigguurree 11:: From seed to bulk: phases in biomanufacturing

MAY 2004 BioProcess International 57

surface to bed height is 10–100times larger than in columns. Thechromatographic adsorber bed ofSartobind MultiSep modules isformed by stacking 15, 30, and 60membranes to bed heights of 4, 8,and 16 mm. A 15-layer 560 mL (2 m2 membrane area) Q modulegenerates a flux of 10 L/min at 0.1 MPa (Table 1). Because of therelation between linear flow andpressure, flux may increase up to 60 L/min at 0.6 MPa.

Downscaled MA 75 Luer Lockunits (15 layers, 75 cm2, flow rate at0.1 MPa �27 mL/min) are usedfor the initial experiments. It ispossible to scale up directly toprocess 15-layer modules. Althoughit is easy to connect several units bythe Luer Lock connectors up to thedesired number of layers, it wouldbe advantageous to construct 30- and 60-layered small-scale unitsinstead. Membranes available at thistime offer high flow rates andsufficient binding capacities.Current options with �3 µm poresize include ion-exchange andiminodiacetic acid metal chelatemembranes. Affinity or couplingmembranes such as epoxy- andaldehyde-activated membranes orprotein A are available in the 0.45-µm pore size. The highestbinding capacities for proteins suchas lysozyme or bovine serumalbumin (BSA) are presentlyavailable on the strong ion-exchanger in the range of 30 mg/mL (0.8 mg/cm2).

A disadvantage at this time is thefact that the manufacturer produces

only one adsorber membrane withone pore size for each ligand. As thenumber of applications grows, wewill observe the same differentiationto a variety of membrane pore sizesthat we have seen in the history ofconventional chromatography media.

An intrinsic property ofmacroporous and microporousmembranes is that transportphenomena are convection driven(rather than dependent on the pre-and intraparticle diffusion of solutemolecules in beads). That factcompensates for the generallysmaller available surface and makesscale-up and processing of very largefeedstream volumes much easierthan with conventional packed bedchromatography. Binding sites inadsorbers are exposed to themolecules within short diffusiondistances (film diffusion), whereas90–95% of bead ligands will bereached only by large diffusiondistances and pore diffusion (Figure 2).

Studies using confocal microscopyon the intraparticle protein transportin porous bead-type adsorbentsdemonstrate that it takes hours evenunder static conditions before adiffusion-controlled process leads toprotein uptake in the core ofchromatography resins, whereas theinternal surfaces of adsorbermembranes in the flow-throughpores are accessible by convectivetransport at a much faster rate (4, 5).

Even second-generation adsorberson which polymer chains have beengrafted to multiply potential bindingsites (and therefore bindingcapacities) demonstrate only minor

contributions of additional rate-limiting diffusive effects. At extremelyhigh flow rates with three-dimensional adsorptive layerstructures, it is possible to keep themass transfer high without significantloss in resolution. The space–timeyield of such high-capacity adsorberscan be about six times higher, with aconstant dynamic capacity over awide range of flow rates comparedwith polymer-based chromatographyresins (6). The open membranestructure combines the advantages ofexcellent flow characteristics (andthus productivity) with the highselectivity of classical chromatography.

CAPTURE WITH

MEMBRANE ADSORBERS

Membrane chromatography achievesits full potential in the fast andefficient processing of large amountsof liquid. Membrane adsorbers meetall requirements needed in a capturestep, recovering large volumes withrather dilute proteins — in mostcases not the main constituents.

In most biotech processes, thehandling of a diluted feed stream isassociated with high manufacturingcosts. The most important task isbringing that volume down asquickly as possible. The feasibility ofmembrane adsorbers for fast andefficient capture has beendemonstrated by numerousexamples. Among these applicationsare the preparative purification ofhuman serum albumin from humanplasma (7), continuous large-scalepurification of bovine lactoferris (8),ion-exchange chromatography ofmonoclonal antibodies (9), large-scale purification of oligonucleotides(10), and virus purification (11). Inall those examples, the evaluatedmembrane adsorbers provedsuperior compared with traditionalchromatography in time-yieldperformance, flow-independentbinding capacity, and short cycletime.

Whether or not membranes arethe right choice for a bind-and-eluteseparation is assessed case by case,depending on a number of process-related parameters: product andmatrix, operation lifetime of

58 BioProcess International MAY 2004

FFiigguurree 22:: Comparison of existing transport phenomena in conventional beads and membrane adsorbers

sorbent, number of cycles, cost ofgoods, validation, cleaning effort,and so on. Because to datemembranes cost more than resinscapacity-wise (measured with smallproteins at low flow rates), theyneed to be operated as multiusedevices and in a format exploitingtheir hydrodynamic advantages.

Meanwhile, process type adsorbersystems based on cylindrical modulegeometry with up to 100 m2 arebecoming available (12). With acombination of different adsorbermodules in parallel or serialconnections, key parameters such asbreakthrough behavior and (low-)flow resistance can be optimized.Scale-up by combining individualmodules provides a large window ofoperation, reduces investment, andincreases flexibility.

Figure 3 shows a cross-section ofthe Sartobind MultiSep cylindricalmodule and a capsule. In thesemodules and capsules, themembrane adsorber is rolled up toform the chromatographic bed. Thefeed (red) enters the module fromthe top and is distributed by acentral dead-volume–reducing coreto the internal membrane channel.Then the solution passes themembrane layers axially. The eluatearrives at the external channel (blue)and is collected at the outlet. Flowis from inside to outside to preventany deterioration of the module athigh flow rates. By contrast, thecapsule is constructed similar to afiltration capsule and can bediscarded after a single use.

In a typical experiment withmultistage equipment, 60 g ofhemoglobin could be purified frombovine blood in a single step(adsorber size 16 m2, cycle timeseven minutes). The theoreticalproductivity of such a unit with a4.3-L bed approaches tons ofprotein per year. Up to 1000 cycleshave been performed on small-scaleequipment without loss ofperformance. Even elution withlinear gradients is easily possible byincreasing resolution as a functionof the number of membrane layers.

Adsorbers can reduce size

exclusion effects typically observedduring conventionalchromatography of molecules largerthan 100 kDa. Comparable butslightly lower capacities onmembranes measured with smallstandard proteins increase whenlooking at large molecules such asgenomic DNA or even viruses(Table 2).

DNA-binding capacity onmembranes is much higher (andfaster) than on beads and is acceptedfor economic DNA removal frompharmaceutic antibodies (13). Tomeasure that capacity, a monoclonalantibody dissolved in a 10-mMphosphate/250-mM NaCl buffer(pH 7.25, 25 mS/cm at a concen-tration of 0.63 mg/mL) was spikedwith 25 µg/mL of DNA isolatedfrom CHO cells. A total of 900 mLwas run through a Sartobind Q 100anion-exchange unit (area 100 cm2)at a flow rate of 30 mL/min. A smallcontinuous increase of the 254-nmline, starting at 620 mL of effluent,indicated breakthrough. The dynamicbinding capacity for the membrane(275 µm thick) was determined to be156 µg/cm2 or 5.6 mg/mL. Thedynamic binding capacity for ananion-exchange beat matrix — QSepharose FF (Amersham PharmaciaBiotech, Uppsala, Sweden) — in achromatography column of 7 cm by1 cm was determined at 0.6 mg/mLof DNA at a flow rate of 78 mL/h(13).

For viruses, membrane ion-exchange chromatography seems tobe the only practical option (11).The analytical ion-exchangechromatography of adenoviruses onmembranes is already a standardoffered by a number ofmanufacturers. The “Small-Scale

Adenovirus Purification Protocol”box presents a typical protocol forsmall-scale purification ofadenoviruses using Sartobind Q 5(5 cm2 area).

Those results are shedding adifferent light on cost

FFiigguurree 33:: Cross section of cylindric SartobindMultiSep Membrane Adsorber module system(A) and a SingleSep capsule for single-usecontaminant removal (B)

TTaabbllee 22:: Comparison of the dynamic binding capacity for DNA on a membrane adsorber and on

a bead-type strong anion exchange matrix.

Flow rate DNA Binding capacitymL/hr µg/mL mg/mL

Sartobind Q 100(100 cm2 � 2.8 mL) 1800 25 5.6

Q-Sepharose FF (8.6 mL) 78 25 0.6

(A)

(B)

60 BioProcess International MAY 2004

considerations. When a processrequires very high flow rates, andthe target molecule is large enough(or if it is a virus particle), aneconomic calculation clearly favorsmembrane adsorption technology.

Affinity membranes are asubgroup of membrane adsorbers.Here the thermodynamics ofadsorption are determined not onlyby the overall flow characteristics,but also by an underlying bioaffinityinteraction between ligand andtarget molecule. Because separationshould occur under physiologicalconditions, the recommendedassociation constants are abovecertain critical values. For affinity

chromatography of antibodies,protein A membrane adsorbers basedon polysulfone and regeneratedcellulose were evaluated in a recentstudy (14), which revealed goodselectivity for human IgG and areusability performance in repeatedadsorption-elution cycles.

Figure 4 presents the result of atypical repeated cycling experimentwith a laboratory-scale unit and amonoclonal antibody from 0.45 µmof prefiltered human plasma.Loading and elution is performedwithin seven minutes on a Sartobindrecombinant protein A analytical unit(three layers, 9.3 cm2). Elution wasachieved with 0.1 M glycine at pH2.3. The first large peak of each cyclerepresents the flow-through ofunbound proteins, mainly humanserum albumin; peak B representsthe eluted IgG fraction. Cycles 1–5and 96–100 are shown. The constantpeak sizes of the eluted IgG fractionalso represent a stable linkage of theprotein A ligand on the matrix.

POLISHING WITH

MEMBRANE ADSORBERS

If the target protein is the majorconstituent of the feed, then

impurities such as DNA, viruses, orhost cell proteins are very efficientlyremoved in a polishing step.

Established cell lines for theproduction of glycosylated proteinscontain infectious viruses or virus-like particles. In addition to thoseendogenous agents, mammaliancells are susceptible to process-related infections with adventitiousviruses. Several independent safetymeasures must be taken to ensurethat a downstream process can cleara potential virus load. Retrovirustiters must be determined in thebulk harvest, and regulations requirethat the purification processdemonstrate an overall virusreduction potential far above thatdetermined level in the ensuingvirus validation studies.Other criticalimpurities of biotech processesinclude DNA, infectious agents ofthe prion-disease type, and otherhost-cell-derived impurities such asendotoxins and proteins. When theprotein is the major constituent ofthe feedstream, unwantedcontaminants must be removedefficiently in a polishing step.

The traditional unit operation forremoval of trace contaminants suchas DNA, host cell proteins,endotoxins, and infectious agents hasbeen flow-through chromatography.Typically, traces of DNA requireonly a small amount of resin relativeto the volume of process solution.Because of flow-rate and pressurelimitations in conventionalchromatography, these steps requirelarge columns, with the associateddrawbacks of hardware investment,column packing, validation efforts,and additional handling. Bycomparison, a small, disposable in-line membrane adsorber unit withno regeneration and validationrequirements and a breakthroughcapacity independent of flow rate isclearly advantageous. Load-to-volume ratios of chromatographysteps versus membrane adsorption ina polishing situation differ by afactor of at least 100.

Several authors have reported aDNA breakthrough capacity formembrane adsorbers that wasconstant over a 4–9 pH range and

Figure 4: 100 Purification cycles of humanIgG on Sartobind protein A; each cyclecontains two major peaks: A = flow-throughis mainly serum albumin; B = IgG fractioneluted with 0.1 M glycine at pH 2.3

SMALL-SCALE ADENOVIRUS PURIFICATION PROTOCOL WITH SARTOBIND Q 5

1. Amplify adenovirus in HEK 293cells grown on three 100-mmdishes with 10–12-mL medium.

2. Once nearly all the cells aredetached, collect the mediumand cells in a 50-mL conicaltube. Keep at �20 °C untilpurification.

3. Thaw and freeze totally threetimes. Make sure thattemperature of solution will notbe higher than room temperature(RT) at any time in thawing.

4. Centrifuge at 3500g for 10–15minutes to pellet cell debris.

5. Prefilter through a 0.45-µmfilter

6. Add same volume (or weight)of adenovirus dilution buffer (20 mM Bicine 0.6 M NaCl, pH 8.4, adjusted with HCl, at RT)and filter medium and buffer.

7. Prewet one Sartobind Q 5(Luer Lock connectors) with 2–5 mL saline.

8. Filter through all the mediumand buffer (color is supposed tobe purple) with one Sartobind Q5 attached with 10-mL syringeand 18 gauge needle.

9. Wash with 30 mL adenoviruswashing buffer (10 mM Bicine0.4 M NaCl, pH 8.4 at RT) andremove all the liquid frommembrane.

10. Elute the adenovirus with 1 mL adenovirus elution buffer (10 mM Bicine 0.61 M NaCl, pH 8.2 at RT) by pushing slowlyusing a 1-mL syringe.

11. Count particles by measuringOD with 0.1% SDS PBS. Atypical yield will be 0.7 �1 �1012 particles/mL.

62 BioProcess International MAY 2004

64 BioProcess International MAY 2004

at conductivities �40 mS/cm, withcomplete removal of typical DNAlevels and a 23-fold process timereduction compared withchromatographic beads. Besides thesuperior pressure–flow relation,benefits included ease of handlingfor set-up and a considerable yieldimprovement (9, 13). In large-scaleexperiments of 2000-L and 12,500-L batches of a cell harvestedmonoclonal antibody purified by aProtein A column (Table 3), theDNA burden was cleared to thedetection limit with Sartobind Q(13). Excellent removal rates havealso been reported for proteases andendotoxins (15).

In addition, disposablemembrane systems can removetypical host-cell-protein loads evenunder worst-case conditions, asdemonstrated under normalizedeconomic conditions (cost per kg ofantibodies processed per hour).

Membrane adsorption representsa robust step for virus clearance.The virus-reduction potential ofstrong anion- and cation-exchangemembranes has been documented ina number of experiments, in one ofwhich a clearance validation for aregistered monoclonal antibody wasperformed. Typical model virusloads were reduced by about fourlogs under downscaled processconditions (16) as listed in Table 4.

LIMITATIONS AND FUTURE WORK

Despite some major limitations ofclassical chromatography — such asthe high pressure drop of a packedbed, long cycle times, and scale-upproblems with conventionalchromatographic supports — thistechnology represents the industrystandard for most downstreamseparations. Classical distributionchromatography with pulseinjection, high-end capacityseparations especially of lowmolecular weight components, andapplications that require high-endresolution with HPLC-typeequipment will probably remain inthe area of column chromatography.Complementing rather thanreplacing column chromatography isthe next logical step.

For membrane adsorbers, theefficiency of flow distribution, andhence use of the accessible surface,remains a challenge. Inefficient flowdistribution as well as lower surface-to-bed volume ratios lead to lowerbinding capacities of membraneadsorbers than with beads.Developments are progressing toovercome those limitations throughintelligent combinations of filterelements. Modern adsorbers showbreakthrough curves that areindistinguishable from packed-bedseparations. Despite broad successand applicability to variousseparation tasks, and although thefirst pharmaceutical made usingmembrane adsorbers has beenapproved by the FDA (16),membrane adsorption is still in itsinfancy — compared with morethan 60 years of practical experiencein chromatography — as aseparation technique.Chromatography and filtration —two mature and irreplaceable unitoperations — are coming together,

and membrane adsorption may bethe perfect synergistic match forboth capturing and polishing ofbiomolecules.

REFERENCES1 Ghosh, R. Review: Protein Separation

Using Membrane Chromatography,Opportunities and Challenges. J. Chromatogr. A 2002, 952: 13–27.

2 Klein, E. Affinity Membranes: A 10-Year Review. J. Membrane Science 2000 179:1–27,

3 Thömmes, J; et al. Isolation ofMonoclonal Antibodies from Cell ContainingHybridoma Broth Using a Protein-A CoatedAdsorbent in Expanded Beds. J. Chromatogr.A 1996, 752 (1–2): 111–122.

4 Ljunglöf, A; Thömmes, J. VisualisingIntraparticle Protein Transport in PorousAdsorbents By Confocal Microscopy. J. Chromatogr. A 1998, 813: 387–395.

5 Reichert, U; et al. Visualising ProteinAdsorption to Ion-Exchange Membranes ByConfocal Microscopy. J. Membrane Science2002, 199: 161–166.

6 Gebauer, KH; Thömmes, J; Kula,MR. Breakthrough Performance of High-Capacity Membrane Adsorbers in ProteinChromatography. Chem. Eng. Science 1997,52(3): 405–419.

TTaabbllee 33:: Sartobind Q used in process scale manufacturing of 2000 and 12,500 liter batches toclear DNA below detection limit

Average DNA DNA after Affinity Concentration

Number Purification Step after Sartobind QBatch Size of Batches (pg/mg protein) (pg/mg protein)

Sartobind Q MultiSep 15-06(15 membrane layers, 6 cm height, 70 mL bed volume) 2000 8 168 �8

Sartobind Q MultiSep 15-50(15 membrane layers, 50 cm height,560 mL bed volume) 12,500 3 187 �4

TTaabbllee 44:: Virus clearance under downscaled conditions with Sartobind Q

Size Clearance by Sartobind Q Clearance by Sartobind QViruses (nm) Run 1, Factor (log10) Run 2, Factor (log10)

SV-40: Simian Virus 40 45 1.25 � 0.46 1.34 � 0.43

Reo-3:Reovirus Type III 75–80 4.07 � 0.50 3.62 � 0.42

MuLV:Murine Leukemia Virus 80–110 3.80 � 0.39 4.40 � 0.56

PRV: Pseudorabies virus 150–250 3.97 � 0.44 3.88 � 0.38

BPIBPI RepRep����rts rts on you!Send news of product releases (or

setbacks), clinical trial results, researchbreakthroughs, advances in technology,association and worldwide government

initiatives, economic developmentinitiatives, and meeting reports to assistant

editor Leah Rosin at lrosin@bioprocessintl.com.

7 Gebauer, KH; Thömmes, J; Kula, MR. Plasma ProteinFractionation With Advanced Membrane Adsorbents. Biotech andBioeng. 1997, 53(3): 1–13.

8 Ulber, R; et al. Downstream Processing of Bovine Lactoferrinfrom Sweet Whey. Acta Biotechnol. 2001, 21(1): 27–34.

9 Knudsen, HL; et al. Membrane Ion-ExchangeChromatography for Process-Scale Antibody Purification. J.Chromatogr. A 2001. 907: 145–154.

10 Deshmukh, RR; et al. Large-Scale Purification of AntisenseOligonucleotides By High-Performance Membrane AdsorberChromatography. J. Chromatogr. A 2000 890: 179–192.

11 Karger, A; et al. Simple and Rapid Purification ofAlphaherpesviruses By Chromatography on a Cation ExchangeMembrane. J.Virological Methods 1998, 70: 219–224.

12 Demmer, W; Nussbaumer, D. Large Scale MembraneAdsorbers. J. Chromatogr. A 1999, 852: 73–81.

13 Walter, J K. Strategies and Considerations for AdvancedEconomy in Downstream Processing of Biopharmaceutical Proteins.In Bioseparation and Bioprocessing, Vol. 2, Subramanian, G, Ed.(Wiley VCH, 1998), 447–460.

14 Castilho LR; Anspach, FB; Deckwer, WD. Comparison ofAffinity Membranes for the Purification of Immunoglobulins. J.Membrane Science 2002, 207:, 253–264.

15 Belanich, M; et al. Reduction of Endotoxin in a ProteinMixture Using Strong Anion Exchange Membrane Adsorption.Pharmaceutical Technology 1996, 20(3): 142–150.

16 Galliher, P; Fowler, E. Validation of Impurity Removal by theCAMPATH-1H Biomanufacturing Process, presented at IBC’sBiopharmaceutical Production Week, Paradise Point Resort, SanDiego, CA, 12–15 November 2001. ��

Corresponding author Uwe Gottschalk is head of GMPprotein manufacturing for Bayer AG, Friedrich-Ebert-Str. 217-333, D-42096 Wuppertal, Germany, 49-202-36-7704,uwe.gottschalk@bayerhealthcare.com; Stefan Fischer-Fruehholz is product manager (stefan.fischerf@sartorius.com)and Oscar Reif is vice president of R&D and technology(oscar.reif@sartorius.com) at Sartorius AG, D 37070Goettingen, Germany.

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