58 BioProcess International OCTOBER 2004

TT he sequencing of the humangenome and the rise ofproteomics have increasedthe numbers of potential

therapeutic targets. Biotechnologycompanies need to increaseproductivity, decrease discovery andproduction costs, and use technologiesthat easily transfer across departments ifthey wish to remain competitive. Themost important tools are those forseparation (purification) of targetsubstance(s). They should be easy to useand offer an identical performance andpurification profile no matter wherethey are implemented — in discovery,production, or quality assurance (QA).

CIM Convective Interaction Mediashort monolithic columns are just such aunifying technology. Produced in shapes

and sizes from microliter to liter scale,they represent an evolutionary approachto meeting biochromatographicseparation requirements in research andproduct development. Able to withstand1M NaOH with no loss of capacity orresolution, these easily scalable columnshave been optimized for analysis andcGMP production of complexbiomolecules ranging fromoligonucleotides and plasmid DNA(pDNA) to proteins and viruses.

For increasing productivity ofchromatographic processes, CIMcolumns offer high flow rates; ease ofuse; very short column lengths (a fewmillimeters for laboratory units to a fewcentimeters for large industrial units);extremely high support capacity for bigbiomolecules (e.g., 10 mg plasmidDNA/mL, 20 mg genomic DNA/mL, 25mg tomato mosaic virus/mL); and fastand simple scale-up. Chromatographicsupports used for screening on themicroliter scale have the samecharacteristics as those used in cGMPproduction. Research and development(R&D) and production scientists canprepare and optimize processes quicklyand easily to save time and moneywhile accelerating drug discovery andproduction. Short column lengths (andhence product contact times) minimizeconformational changes andinactivation of biomolecules. In allinstances, performance is consistent andreliable.

MONOLITHS IN

BIOCHROMATOGRAPHY

Methacrylate monoliths were firstdescribed in 1990 (1), and the first CIMproducts became available in 1998 (2).So a wealth of experience hasaccumulated in applying thistechnology to biomolecular discoveryand production and in qualityassurance/validation activities. Themedium is a short, continuous, andhomogenous methacrylate or styrene-divinylbenzene rigid polymeric blockcross-linked with uniform, three-dimensional, interconnected channelswith 62% porosity. Macrochannels areabout 1500 nm in diameter, andmesochannel diameters are >100 nm. The larger channelscontribute to a column’s low backpressure, even when operating atelevated flow rates; the smaller onesprovide a large surface area for a highdynamic binding capacity. CIMmonolithic columns are resistant tochemical and thermal stress. Each

PRODUCT FOCUS: ALL COMPLEX

BIOMOLECULES

PROCESS FOCUS: DOWNSTREAM

PROCESSING

WHO SHOULD READ: R&D,MANUFACTURING, AND QA/QCPERSONNEL

KEYWORDS: CHROMATOGRAPHY,MONOLITHIC COLUMNS, SCALE-UP, VALIDATION,ANALYTICAL METHODS

LEVEL: INTERMEDIATE

V E N D O R Voice

Photo 1: 0.34-mL and 2-mL CIM disks

Revolutionizing BiotherapeuticDiscovery and Production A New Generation of ChrA New Generation of Chromatographic Supporomatographic SupportstsDarryl G. Glover, Miloš Barut, Aleš Podgornik, Matja Peterka, Aleš Štrancar

monolith can withstand temperatures of4 °C (39 °F) to >120 °C (248 °F), theworking pH range is 1–14, and columnscan easily withstand 1M NaOH (rangesfor some chemistries may be slightlydifferent).

As single blocks of porous materialwith highly interconnected channels,CIM monolithic chromatographicsupports have a very fast mass transfer(due to convective flow). The shortlength of the column enhances thespeed of separation and reducesbackpressure, unspecific binding, andproduct degradation. Further, thesupports exhibit low nonspecificbinding of biomolecules, therebypreventing minor changes inbiomolecular structure. The channelsize and surface have been optimizedso that the biomolecule can easilyaccess ligands for binding. The supportshave an extremely high capacity forvery large molecules, ensuring that theycan easily permeate the matrix. Thesefeatures decrease the purification timeby an order of magnitude, which in turnreduces purification costs. Therefore

CIM supports combine the separationpower, capacity, and sampledistribution of conventional porousparticle chromatographic columns withthe convective mass transport ofmembrane technology.

CIM monolithic chromatographicsupports can be prepared for analyticaland QA use (disks, minidisks, 96-wellplates, and syringes) and for production(tubes) (Table 1). The analytical diskscan be used for proteomics, screening,and as precolumns for MS–MS. Thetubes are used for preclinical, pilot, andfull cGMP production. The process ofscaling up from screening to small-scalepurification, pilot-scale purification, andthen production is facilitated by theidentical performance and purificationprofiles of all short monoliths (no matterwhat size or shape). Once methodoptimization is completed, scale-upfrom the laboratory to full-scaleproduction takes only a few days,resulting in significant time and costsavings.

Both small and large columns areeasy to operate and adjustable to meet

the needs of biochromatographers. Ittakes seconds to set up or change theconfiguration of a column, and the

Figure 1: Multidisk CIM disk monolithiccolumns

CIM Monolithic columns serve as a unifyingtechnology.

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Phone 518-891-2356 Fax 518-891-5753www.bionique.com

Safe Cells is a Registered Trademark of Bionique Testing Laboratories, Inc.

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60 BioProcess International OCTOBER 2004

columns have a fast equilibration andregeneration. There is no packing ofparticles, as is necessary withconventional columns, and no need toworry about air bubbles because theyare not trapped in the monolithicstructure but are washed out with themobile phase. As Figure 2 illustrates(with 3.4–800 mL/min flow rates), theseparation quality does not change withthe speed of the separation. The supporthas no diffusion limitations because theflow-through channels make all poresaccessible to the biomolecules. Theyoperate at high flow rates in allchromatographic modes on analyticaland preparative scales. They arecompatible with any peristaltic pump orconventional LC/HPLC or FIA system.

The working lifetime of a CIMsupport varies depending on the samplepurity. One thousand injections of astandard protein mixture wereperformed with no loss of resolution ordynamic binding capacity, and thesupport was regenerated after every 200injections. The data from this internalstudy were published in a companybrochure available from BIASeparations.

CIM supports come in a completerange of chemistries, including ionexchange (anion, cation), reversedphase, hydrophobic interaction,affinity/immunoaffinity, and epoxy. ACIM CDI (1,1-carbonyldiimidazolemodification) and a CIM IMAC(immobilized metal affinity column)support will soon be available. Theability to bind a wide range of enzymes,immunoglobulins, and peptides makesthem ideally suited to serve asbioreactors, as affinity columns, and foron-line and off-line in-process control.The existing chemistries haveapplications for virus purification, virusconcentration, and vaccine production;pDNA production, monoclonalantibody isolation; biomoleculediscovery; purification of basic andacidic proteins; and oligonucleotideand peptide separations andbioconversions.

APPLICATIONS: DISCOVERY,PRODUCTION, QA VALIDATION

The wide variety of potentialapplications for CIM monolithiccolumns range from laboratory analysis

and discovery to cGMP production. Thecolumns can be used to improve thedetection of environmental hazardssuch as infectious plant viruses (3) orzinc contamination of ground water (4),and they can screen for geneticallymodified foods (5). The supports haveapplications for concentrating humanviruses to improve their detection byRT–PCR (6).

In drug discovery, the supports havepotential uses in proteomics. BeyondGenomics (Waltham, MA,www.beyondgenomics.com) recentlyreported an application that depletedabundant proteins in plasma and serumsamples (7); others have used them forHIS–TAG protein isolation (8, 9).Applications in affinity chromatographyrange from synthesizing peptides on aCIM support and then using it forscreening (10), to trypsinimmobilization on either an epoxy (11)or CDI-activated chemistry (12), to L-histidine immobilization for thebinding of IgGs (13).

Bartolini et al. covalentlyimmobilized human recombinantacetylcholinesterase (hrAChE) onto aCIM EDA (ethylenediamine) disk toobtain a microimmobilized-enzymereactor to perform on-linechromatographic studies (14). Theyachieved a decrease in analysis timeand demonstrated the support’spotential for rapid drug screening. Asthey pointed out, the only four drugscurrently approved for Alzheimer’streatment are acetylcholinesteraseinhibitors. This approach for Alzheimer’scould also be used for other diseasestates in which activation or inactivationof an enzyme is required for treatment.This could significantly improve theability to screen and identify the mostsuitable candidates in a shorter timethan current methods allow.

Lim et al. recently reportedimmobilizing bovine pancreatic elastaseto a CIM disk to cleave human plasmaderived inter-alpha inhibitor protein intofragments (15). Those fragments weresmaller than could be achieved usinghyaluronidase and led to theidentification of the part of the inter-alpha inhibitor that was reactive to anovel monoclonal antibody (MAb69.31) that may have benefits in cancertreatment. Platonova et al. used

Figure 2: Scale-up of a standard proteinmixture from a CIM DEAE researchcolumn (1 disk) to an industrialpreparative column (800 mL). Sample =myoglobin (first peak), conalbumin(second peak), trypsin inhibitor (thirdpeak); Mobile phase = Buffer A: 20mmol/L Tris-HCl buffer, pH 7.4; Buffer B: A+ 0.7 mol/L NaCl (27). Y axes = absorbanceat 280 nm (mAU).

.

polynucleotide phosphorylase fromThermus thermophilus immobilized onCIM for the preparation of highlyselective affinity immunosorbents (16).That enzymic preparation was placed ina flow-type bioreactor and examinedfor the ability to synthesizepolyriboadenylate from ADP and tocarry out its reverse phosphorolysis.

Analytical Applications: CIMcolumns have been used for differentanalytical applications and in-processcontrols such as the fast analysis ofhuman IgG on a protein A CIM diskmonolithic column (17). To ensureproduct quality and early detection ofcontaminants in commercialimmunoglobulin concentrates, a fastconjoint liquid chromatography (CLC)method was developed and successfullyvalidated (18). In this process, two CIMProtein G disks and one CIM quaternaryamine (CIM QA) disk were inserted intoone CIM housing, enabling theseparation of transferrin, albumin, andIgG in a single step and in only fourminutes. As a final example,immobilized glucose oxidase (GOX)was used for the on-line monitoring of

glucose produced during a fermentationprocess (19).

Preparative and IndustrialApplications: The most beneficial usesfor CIM supports are for preparative andindustrial-scale applications. Hall et al.used an anion exchange CIM supportfor the separation of a PEGylated formof myelopoietin (MPO), an engineeredhematopoietic growth factor (20). Theycompared CIM with Q and SP-Sepharose columns for preparativepurifications. The use of either

monolithic or Sepharose-based supportsfor preparative chromatographyproduced highly purified PEGylatedMPO, with the monolithic mediademonstrating peak resolution andrepeatability at flow rates of 1 and 5 mL/min. This resulted in runtimes as much as fivefold shorter thanfor the Sepharose separations.

At Octapharma Austria(www.octapharma.com), a pilot FactorIX production line is being established(21). The method was developed very

Table 1: Different formats of CIM supports

Area of Use Monolith Format Volume Flow Rates

Diagnostics Syringe Varies —96-well plate 0.005–0.06 mL —

Screening Minidisks 0.06 mL 0.5–2 mL/min

R&D, QA Disks* 0.34 mL 10 mL/min

Pilot scale Tube 8 mL 40 mL /min80 mL 250 mL/min

Production Tube 800 mL 2 L/min8 L 10 L/min

* CIMGEL is also available for the packing of traditional 16 and 26 mm columns. However, the maximumflow rate is restricted to the column manufacturer’s specifications.

62 BioProcess International OCTOBER 2004

quickly and was easily transferred toindustrial-scale production. The resultswere highly reproducible in terms of thespecific activity and yield of Factor IXafter purification at a separation speed20 times faster than with traditionalcolumns. With the CIM monolithiccolumns, the specific activity of theeluate was five times higher than wasseen during the equivalent purificationstep using a conventional column.

BIA Separations codeveloped aplasmid DNA production process inconjunction with Boehringer Ingelheim,Austria (www.boehringer-ingelheim.at),to produce plasmids for gene therapy(22). CIM diethyl aminoethyl (DEAE)columns (drug master file 15838) werefound to have a dynamic bindingcapacity of 7.9 mg pDNA/mL. That wasat least 30 times higher than thetraditional methacrylate and Sepharoseparticle resins and 230 times higher

than the silica-particle-based columnstested (23, 24). The key is that thisprocess is done without RNAase andhas resulted in the production of greaterthan 98% ccc (covalently closedcircular) pDNA. The very high capacityof CIM supports and the short cycletimes contributed to a 20-fold increasein productivity. Combining thehydrophobic-interaction (HIC) andanion-exchange steps into one cGMPtube monolithic column could possiblyfurther reduce time and equipmentcosts by establishing an industrialconjoint liquid chromatographymethod.

Virus Concentration andPurification: Preliminary work usingplant virus models has shown that CIMhas a tremendous potential for virusconcentration and purification. Thecapacity for viruses is extremely high,ranging from tenths of a mg/mL to 25

mg of tomato mosaic virus/mL of CIMsupport (25).

According to Alois Jungbauer at thedepartment of biotechnology, Universityof Natural Resources, Vienna, Austria:“There are two advantages to purifyingor concentrating live or attenuatedviruses with CIM: Viruses maintain theirinfectivity and immunogenicity, and theproductivity of such processes can bean order of magnitude or more higherwhen compared to traditionaltechniques. One project currently underdevelopment is the production of acombined SARS and influenza vaccinedone in conjunction with Green HillsBiotechnology, Austria(www.greenhillsbiotech.com). Themanufacturing of this vaccine will bebased on an entirely new productionprocess and use CIM supports for thepurification step.

The above was only a brief review ofa few applications in which CIMsupports have been used, but it is fair to

Equation 1: A simple equation for scaling up/down an optimized gradient (27)

Column Gradient Time Column Void Volume Flow Rate Column Length

Column1 (Starting) tg1 V1 F1 L1

Column2 (Target) tg2 V2 F2 L2

Table 2: Monolithic support parameters used for scale up/down calculation (27)

Monolith Volume Void Volume Flow Rate Column Length Disk (mL) V (mL) F (mL/min) L (cm)

1 0.34 .21 10 0.32 0.68 .42 10 0.63 1.02 .63 10 0.94 1.36 .84 10 1.2

Monolith Volume Void Volume Flow Rate Column Length Tube (mL) V (mL) F (mL/min) L (cm)

8 8 6 40 0.6580 80 78 250 1.6

800 800 670 2,000 2.18000 8000 Data Pending 10,000 2.2

Equation 2: An example showing the use of Equation 1 and parameters in Table 2 forscaling up from 80mL to 800mL (Figures 2 and 3)

tg2 = ? V2 = 670mL F2 = 2000mL L2 = 2.1cm

tg1 = 2 mins V1 = 78 mL F1 = 250 mL/min L1 = 1.6cm

V2 F1 L1

V1 F2 L2� � ��� �tg2 = tg1

Photo 3: 80-, 800-, and 8000-mL CIM tubemonolithic supports

Photo 2: 8-, 80- and 800-mL CIM tubemonolithic columns

say that the only limit to other potential applications is theimagination of the scientists using the material and itsperformance during method development. In addition, CIMcould possibly become a part of diagnostic tests that wouldmore quickly and easily detect diseases that could then betreated with biotechnology-based drugs purified by thesupports.

METHOD DEVELOPMENT

Conventional columns and monolithic columns follow thesame chromatographic principles. Therefore, methodsdeveloped for conventional columns should be easilytransferable to monolithic columns. However, maximalperformance of CIM columns requires optimization of liquidchromatography system. They include the need for LC systemsto work at high flow rates and have low system back pressures,fast detector response time, fast gradient formation time, andfast data acquisition.

When CIM monoliths are used for laboratory gradientseparations, it is preferable to use an LC system that includestwo LC pumps connected by capillaries and a mixing tee tothe injection valve or autosampler, a filling loop for thesample injection, a detector, and a recorder or dataacquisition system. Each component has a direct influence onthe separation quality when fast analyses are required forR&D and QA:

Capillaries: The inner diameter of the capillaries stronglyaffects the peak shape. Using smaller diameters results insharper peaks, increases the quality of the separation, andraises the back pressure of the LC system, especially at highflow rates. As a compromise we recommend using capillarieswith an inner diameter of 0.5 mm.

Mixing tee: A low dead volume mixing tee (e.g., 10 µL)should be used for fast gradient separations. This will improvethe peak quality and resolution.

Detector: To obtain optimal performance from a CIM diskmonolithic support(s), the detector response time should beset to the lowest possible value. For most UV detectors this is0.1 second.

Acquisition rate: The acquisition rate depends on theanalysis time. Typical analysis times in the case of CIM diskmonolithic columns are just a few minutes. To obtain optimalresolution for separations of less than one minute, theacquisition rate should be 5 to 10 Hz.

It is important to note that when using CIM tubemonolithic columns, you should ensure only that the LCsystem can work at high flow rates because the above systemfeatures do not influence these larger systems.

Because CIM columns are easy to use and allow fastseparation, fast equilibration, and fast regeneration, methoddevelopment can be done quickly and easily. Instead of oneto four runs per day using traditional columns, it is possible toperform 100 or more tests per day with different chemistries,conditions (buffers), and gradients, thus significantly speedingup method development. A process that requires multiplechemistries (Figure 1) can be done in the same housing andcan be set up in seconds using CLC mode (2, 18). Differentchemistries (e.g. affinity and ion-exchange chromatography)can be combined in a single column housing yielding a one-

step purification process. An example of this is the workperformed by Tennikova et al. in the area of affinitychromatography (26). Her group immobilized four differentantigens to four CIM disks, each with a capacity of 3–5mg/mL of support and an adsorption capacity of 1.5–2.0 mgIgG/mL. All four disks were placed in the same housing, anda one step adsorption was performed. Then each disk wasindividually placed into the housing, and desorption of thespecific IgG’s of interest was performed. The entireproceduretook around two minutes to complete, and the immobilizeddisks were stable for 12 months.

Because the analytical and scale-up columns behave inexactly the same way, the CLC method can be scaled up forproduction by taking advantage of the column’s “tube-in-a-tube” design. Equivalent tube layers can be produced thatmatch the order, chemistry, and performance of the disksused, yielding a one-step production process in a singleindustrial column.

METHOD TRANSFER: SCALE-UP/DOWN

The ability to scale a chromatographic method up or downquickly and easily is an extremely beneficial feature for achromatographic medium to offer. One commonly facedproblem with transferring chromatographic methods fromsmaller to larger particle-based columns is the variation instationary phases, especially in the particle size. This changecommonly influences the resolution power of the stationaryphase. The chromatographic method must be adjusted topreserve separation quality. To avoid this problem, the CIMmonolithic structure, especially its pore size distribution, iskept the same regardless of the column size. Therefore, thetransfer of the chromatographic method from the laboratoryto production is as simple as assembling the column for use.

Because CIM monolithic supports are optimized for theseparation of large molecules, gradient chromatographicmethods are normally applied. With this in mind, Milavec etal. derived a very simple equation for scaling up/down anoptimized gradient (Equation 1) (27). The elegance of thisequation is that the only truly variable parameter is thegradient time (tg). The flow rate (F) becomes variable onlywhen a rate less then the maximum (shown in Table 2) is used.

An example demonstrating the use of that equation forscaling up from 80 mL to 800 mL (Photos 2, 3) is shown inEquation 2. Applying the formula you get tg2 = 2 min(670/78) (250/2000) (1.6/2.1), which results in a new gradienttime (tg2) of 1.6 minutes. Thus, it takes only a few seconds tocalculate the new gradient time and proceed with youroptimized method on your scaled up or down CIMmonolithic column. It is important to note that some finetuning of the gradient may be required after method transferbecause extra column effects can lead to band broadening(Figure 2).

A UNIFYING TECHNOLOGY

CIM convective interaction media — short, layered monoliths— are a new generation of chromatographic supports. They arebiocompatible and mechanically, chemically, and thermallystable. The main advantages of these novel media are

• Properties are unaffected by flow;they can therefore be prepared indifferent formats with no loss ofresolution or capacity even withapplication of extremely high flow rates.

• Media offer extremely highcapacity for very big biomolecules (e.g.10 mg of plasmid DNA/mL, 20 mg ofgenomic DNA/mL, 25 mg of tomatomosaic virus/mL media).

• Because the chromatographicsupport used for screening on the µLscale has the same characteristics as theone used for cGMP production, scale-up is fast and simple

• A very short column length (someare only a few mm) minimizesconformational changes andinactivation of biomolecules andreduces unspecific binding.

This unifying technology can beapplied across all departments. It savestime and money and increasesproductivity by an order of magnitude.Therefore, CIM technology canpotentially revolutionize drug discoveryand production by speeding up thedevelopment, QA, and productionprocesses of biotechnology companies.

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Darryl G. Glover is manager ofinternational marketing, Miloš Barut ishead of production, Aleš Podgornik ishead of research and development,Matja Peterka is head of molecularbiology, and corresponding author AlešŠtrancar is managing director at BIASeparations, Teslova 30, SI-1000 Ljubljana, Slovenia;ales.strancar@biaseparations.com;www.biaseparations.com,www.monoliths.com.

66 BioProcess International OCTOBER 2004