(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`1111111111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(43) International Publication Date
`26 June 2003 (26.06.2003)
`
`PCT
`
`(10) International Publication Number
`WO 03/051483 Al
`
`(51) International Patent Classification7:
`C12N 15/10, C07H 1108
`
`BOlD 15/08,
`
`1000 Ljubljana (SI). PODGORNIK, Ales [SI/SI]; Rimska
`25, 1000 Ljubljana (SI).
`
`(21) International Application Number:
`
`PCT/EP02114314
`
`(74) Agents: HAMMANN, Heinz eta!.; Boehringer Ingelheim
`GmbH, 55216 Ingelheim am Rhein (DE).
`
`(22) International Filing Date:
`16 December 2002 (16.12.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`01130067.0
`
`18 December 2001 (18.12.2001)
`
`EP
`
`(71) Applicants (jor all designated States except US):
`BOEHRINGER
`INGELHEIM
`INTERNATIONAL
`GMBH [DE/DE]; Postfach 200, 55216 Ingelheim am
`Rhein (DE). BIA SEPARATIONS D.O.O. [SI!SI];
`Teslova 30, 1111 Ljubljana (SI).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SC, SD, SE,
`SG, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VC, VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, Fl, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, SI, SK,
`TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`(72) Inventors; and
`(75) Inventors/Applicants (jor US only): NECINA, Ro(cid:173)
`man [AT/AT]; Schillgasse 32118, A-1210 Vienna (AT).
`URTHALER, Jochen [AT/AT]; Johannesstrasse 25112,
`A-2344 Maria Enzersdorf (AT). STRANCAR, Ales
`[SI!SI]; Planina 45, 5270 Ajdovscina (SI). JANCAR,
`Janez [SI!SI]; Simon Jenko 12, 1230 Domzale (SI).
`MERHAR, Mojca [SI!SI]; Za Humcem 8, 1331 Dolenja
`vas (SI). BARUT, Milos [SI/SI]; Dolennjska cesta 54,
`
`Published:
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`iiiiiiii
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`-iiiiiiii --
`== -iiiiiiii
`iiiiiiii ----
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`~
`QO
`~ ----------------------------------------------------------------------------
`,..-.1 (54) Title: METHOD AND DEVICE FOR ISOLATING AND PURIFYING A POLYNUCLEOTIDE OF INTEREST ON A MAN(cid:173)
`lf) UFACTURING SCALE
`0 ..........
`~ (57) Abstract: A process for isolating and purifying a polynucleotide on a munufacturing scale uses a chromatographic separation
`process comprises a combination of two different chromatographic steps selected from hydrophobic interaction chromatography,
`0 polar interaction chromatography and anion exchange chromatography. In at least one of the two steps the chromatographic support
`> is a porous monolithic bed. Chromatographic device ad its use for isolating and purifying a polynucleotide of interest, in particular
`~ plasmid DNA, on manufacturing scale.
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`1
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`MTX1019
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`METHOD AND DEVICE FOR ISOLATING AND PURIFYING A
`POLYNUCLEOTIDE OF INTEREST ON A MANOF ACTURING SCALE
`
`5
`
`The present invention pertains to a method and device for isolating and purifying a
`
`polynucleotide of interest on a manufacturing scale.
`
`The developments in molecular and cell biology in the last quarter of the 20th
`
`10
`
`century have led to new technologies for the production of complex biomolecules,
`
`which are increasingly used in human health care in the areas of diagnostics,
`
`prevention and treatment of diseases. At first, research was mainly directed to
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`protein molecules, while lately some of the most revolutionary advances have been
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`made with polynucleotides in the field of gene therapy and nucleic acid vaccines.
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`15
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`Representative members ofpolynucleotides are messenger RNA (mRNA),
`
`genomic DNA (gDNA), and plasmid DNA (pDNA). Polynucleotides are a
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`heterogeneous group of molecules in terms of size, shape, physico-chemical
`
`properties and biological function. Common to all of them are their building blocks
`
`(A,G,C,T,U) and their high negative charge under physiological conditions.
`
`20
`
`Polynucleotides can be single- or double-stranded. Similarly to proteins, they are
`
`able to build structures and multimers. Some species, e.g. mRNA, are very labile in
`
`their natural environment, in celllysates or even in purified form, partly due to the
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`presence ofDNases and RNases. Due to their size gDNA and pDNA are very
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`sensitive to mechanical and shear forces. Therefore, polynucleotide-containing
`
`25
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`solutions need to be handled very carefully and gently.
`
`One of the most important and at the same time most expensive steps in the
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`production ofpolynucleotides is their isolation and purification (down-stream
`
`processing). More than 50% of the total costs of the production process are
`
`incurred by these operations. Precipitation, extraction, ultrafiltration, and liquid
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`chromatographic techniques are most widely used for these purposes. Typically,
`
`these methods are combined with each other. Liquid chromatography is the most
`
`powerful method, which allows manufacturing of a product suited for therapeutic
`
`use.
`
`5 During the last few years, gene therapy has become a promising therapeutic
`
`approach in human medicine. DNA plasmid-based treatment is considered an
`
`alternative to classical chemical drugs or proteins recovered from recombinant
`
`cells. Potential applications of plasmid DNA (pDNA) are in the treatment of
`
`acquired and genetic disease and in vaccines. The plasmid carries information that
`
`10
`
`allows expression of a protein of interest in the targeted human cells; in addition it
`
`contains regulatory elements that control expression in the host cell. pDNA is
`
`formulated for ex vivo or in vivo administration, jointly with viral or non-viral
`
`gene delivery vehicles.
`
`Due to the increasing amounts of pDNA required for preclinical and clinical trials,
`
`15
`
`there is a demand for processes to be performed on a manufacturing scale. These
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`production processes must fulfil regulatory requirements (FDA, EMEA) and
`
`should be economically feasible.
`
`At present, E. coli is the most commonly used production host, but other bacterial
`
`hosts, yeasts, mammalian and insect cells may also be used as host cells. A high
`
`20
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`copy number per cell and stable maintenance during the fermentation are crucial
`
`for a robust process with high yield. Fermentation is performed in batch or fed(cid:173)
`batch mode. Since fed batch processes reach higher cell densities (OD > 1 00) they
`
`are considered to be superior for large-scale production. After fermentation the cell
`
`broth is harvested by centrifugation and optionally frozen. For downstream
`
`25
`
`processing of pDNA cells are (thawn and) disintegrated. A combination of column
`
`chromatography and/or precipitation steps is utilized for purification ofpDNA.
`
`After a fmal sterile filtration, the pDNA bulk is aliquoted and stored under proper
`
`conditions.
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`In manufacturing processes, the requirements for pDNA are different from those
`
`for recombinant proteins, because the two types of macromolecules differ
`
`significantly in their physico-chemical properties. Plasmids are negatively charged
`
`5
`
`over a wide pH range, are large and have a long, thin shape. A typical plasmid
`contains between 5 and 20 kilo base pairs which corresponds to 3 x 106-13 x 1 06
`Da and several thousand A. Its shape is responsible for its sensitivity against
`mechanical stress.
`
`There are several different forms ofpDNA. The supercoiled or covalently closed
`
`circular ( ccc) form is the most stable form. The degree of supercoiling is dependent
`
`1 0
`
`on the environmental conditions, such as temperature and pH. The open circular
`
`( oc) (or nicked) form is produced by breaking a single strand. Breakage of both
`
`strands can be caused by chemical and physical stress and produces the linear form.
`
`Since ccc pDNA is considered the most potent and stable form ofpDNA, the final
`
`product obtained from the production process should contain more than 90% of tins
`
`15
`
`form. The amount of ccc pDNA is considered as one of the most important
`
`characteristics for the quality of the pDNA preparation.
`
`Critical points in manufacturing scale production of plasmid DNA are cell lysis,
`
`subsequent clarification of the cell lysate, purification by a combination of different
`
`techniques, such as chromatography, extraction and precipitation.
`
`20
`
`Plasmid DNA, due to its size and shape, is very sensitive to shear forces.
`
`Therefore, lysis of cells must not be performed by using a high-pressure technique
`
`such as homogenization. Chemical and enzymatic e.g. using (lysozyme) methods
`
`cause minimal mechanical stress and minimal irreversible changes of the plasmid.
`
`During cell lysis under alkaline conditions, cells are subjected to NaOH and SDS.
`
`25
`
`Subsequent neutralization to pH 5.5 causes flocculation of cell debris, proteins, and
`
`gDNA. Very often RNAse is added to digest RNA into small pieces in order to not
`
`interfere during the downstream process. After addition ofNaOH and SDS, the
`
`solution becomes hlghly viscous. Mixing without destroying the plasmid is
`
`difficult. Usually glass bottles containing the viscous solution are mixed very
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`gently by hand. Some processes use optimized tanks and stirrers or a combination
`
`of different mixers in order to overcome these problems.
`
`The isolation and purification of large polynucleotides like plasmid DNA is
`
`hampered by the low performance of commercially available chromatographic
`
`5
`
`sorbents, which are mainly based on highly porous particles. Most of the
`
`chromatographic supports were tailor-made for the high adsorption capacity of
`
`proteins with particle pore diameter of typically 30-400 nm (protein diameter
`
`typically <5 nm). In these processes, large polynucleotide molecules like pDNA
`
`(with a size of30 to >300 nm in diameter) adsorb only at the particle surface,
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`10
`
`leading to capacities approximately 50 times lower than those achieved with
`
`proteins. The design and application of new chromatographic supports and devices
`
`that would allow high capacity polynucleotide purification is therefore crucial to
`
`move gene therapy forward.
`
`As a rule, liquid chromatography is a rather slow process. It often causes
`
`15
`
`significant product degradation and requires expensive separation media, large
`
`volumes of solvents, a long process time and high investments for buffer tanks and
`
`other chromatography-related equipment. Diffusional constraints within the large
`
`pores of the porous particle in particular limit the speed of separation, especially of
`
`larger molecules, as they cause a rapid reduction in resolution with increasing
`
`20
`
`elution velocity in the case of conventionally packed columns. On the other hand,
`
`the efficient isolation of labile, valuable biomolecules requires a fast, reliable and
`
`affordable separation process under mild conditions.
`
`Most processes available for pDNA purification are conducted on a small
`
`laboratory scale. They mainly involve cell lysis using enzymes like RNAse or
`
`25
`
`lysozyme, extraction with organic solvents and ultracentrifugation in density
`
`gradients. Most of these processes are time consuming and not scalable.
`
`Furthermore, due to the usage of material that is not certified for application in
`
`humans, the usage of enzymes and toxic reagents such as phenol, CsCl, CsBr, etc.,
`
`these processes are not suitable to meet approval by regulatory authorities.
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`fu the last few years there have been several attempts to develop manufacturing
`
`scale processes for pDNA purification. US 5,981,735 describes a process for large(cid:173)
`
`scale pDNA purification based on anion exchange and gel permeation
`
`chromatography. For this process, the use ofRNAse during the cell lysis is
`
`5
`
`suggested. The first chromatography step represents an anion exchange expanded
`
`or fluidized bed packed with porous particles (capacities 0,05 - 0,1 mg pDNA/ml).
`
`To produce a pharmaceutical grade plasmid DNA a second purification step is
`
`needed using high-resolution anion exchange column packed with porous particles
`
`with optional use of Triton X-100 or Tween 20 to remove impurities. Final
`
`10
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`polishing is achieved by gel permeation chromatography.
`
`US 6,197,553 describes a process for lar~e-scale plasmid purification using two
`
`chromatographic steps, i.e. an anion exchange chromatography as the first step and
`
`reversed phase chromatography as the second step. Major drawbacks are, besides
`
`using porous particles in both chromatography steps, usage ofRNAse and, during
`
`15
`
`the cell lysis, usage of lysozyme. fu addition, toxic organic solvents are required
`
`for the second chromatographic step.
`
`US 6,242,220 discloses a process for purification of ccc pDNA using only one
`
`chromatography step, i.e. an anion exchange column. Besides using a porous
`
`particle chromatographic column the drawback is the use of nuclease to cleave the
`
`20
`
`gDNA, ocDNA and linear DNA.
`
`The established processes are oflimited usefulness due to various drawbacks:
`
`All these processes are based on chromatography using various types of porous
`
`particles optimized for protein purification and exhibit low or very low capacities
`
`for pDNA, furthermore, they are slow in the separation process. Therefore, large
`
`25
`
`chromatographic columns are necessary for purification ofpolynucleotides in gram
`
`scale. For equilibration, washing and elution large buffer volumes are necessary.
`
`Some of the resins are for single-use only and need to be replaced after each run.
`
`Column emptying, cleaning and packing need to be taken into account for every
`
`run. This has a significant input on the process time, costs of goods and the size of
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`equipment such as tanks for buffer and wastewater treatment. Limited separation
`
`capability of the applied chromatographic resins requires a combination with
`
`extraction or precipitation steps. As these processes require usage of organic, toxic
`
`solvents several safety issues need to be addressed; these also need to be taken into
`
`5
`
`account for most of the detergents. Several established processes require the usage
`
`of animal-derived enzymes. Usage offlammable, organic solvents requires
`
`explosion-proofed production areas, which contribute to a major part to the
`
`production costs. In addition, precautions for the safety of patients, operators and
`
`the protection of the environment need to be implemented.
`
`10 An alternative to using porous particles is the use of membrane technology, which
`
`reflects technological advances in both membrane filtration (ultrafiltration) and
`
`fixed-bed liquid chromatography (Heath CA, Belfort G (1992) Adv Biochem
`
`Eng/Biotechnol47:45; Zeng XF, Ruckenstein E (1999) Biotechnol Progr 15:1003).
`
`Ultrafiltration membranes (filters) are employed mainly as »cut off« devices; they
`
`15
`
`can separate biomolecules whose sizes differ by one order of magnitude or more.
`
`When affinity, ion exchange, hydrophobic interaction or reversed phase ligands are
`
`coupled to such membranes (filters), an increase in selectivity can be achieved. The
`
`chromatographic interactions in the membrane are usually similar to those in the
`
`porous particulate material. They mainly differ in their hydrodynamic properties.
`
`20 Membrane-based chromatography can generally be distinguished from porous
`
`particle-based chromatography in that the interaction between a solute (for example
`
`proteins or pDNA) and a matrix (immobilized ligand) does not take place in the
`
`dead-end pores of a porous particle, but mainly in the flow-through pores ofthe
`
`membrane. While the mass transport in dead-ended pores necessarily takes place
`
`25
`
`by diffusion, the liquid moves through the pores of the membrane by convective
`
`flow, dramatically reducing the long diffusion time required by conventional
`
`particle-based chromatography. As a consequence, membrane separation processes
`
`are generally very fast, in fact at least one order of magnitude faster than columns
`
`packed with the corresponding porous particles. In addition, most of the active sites
`
`30
`
`are exposed at the surface of the flow-through channels, which can result in higher
`
`capacities for big molecules.
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`Since membranes are very thin beds (usually a few mm stack of several thin sheets
`
`with large diameter) as compared to chromatographic columns packed with porous
`
`particles, reduced pressure drop is found along the chromatographic unit, allowing
`
`increased flow rates and consequently higher productivity (Heath CA, Belfort G
`
`5
`
`(1992) Adv Biochem Eng/Biotechnol47:45; Zeng XF, Ruckenstein E (1999)
`
`Biotechnol Progr 15:1003).
`
`Many membrane separations are performed by using a conventional filtration
`
`apparatus; others are configured for compatibility with existing chromatography
`
`pumps and detectors. Regardless of the configuration of the apparatus and the type
`
`10
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`of matrix, the problem of uniform flow distribution from a relatively thin pipe to a
`
`large area has to be solved, as well as the problem of recollecting the eluate at the
`
`other end of tht:) device with minimal back -mixing and distortion of zones, to
`
`improve the resolution power of the membrane device. Another problem is in their
`
`relatively large dead volume within the unit resulting in large band spreading
`
`15 which in consequence lowers the resolution power of the membrane
`
`chromatography units.
`
`Apart from a predominantly diffusive transport, the problem of particulate
`
`separation media is their inability to completely fill the space within the
`
`chromatographic column, the latter problem being valid for membranes as well.
`
`20
`
`This results in peak broadening and decreased column efficiency.
`
`An even higher degree of continuity, than with membranes was achieved with the
`
`introduction of monoliths.
`
`A monolith is a continuous bed consisting of a single piece of a highly porous
`
`solid material where the void volume is decreased to a minimum (Tennikova TB,
`
`25
`
`Svec F (1993) J Chromatogr 646:279). The most important feature ofthis medium
`
`is that all the mobile phase is forced to flow through the large pores of the medium.
`
`As a consequence, mass transport is enhanced by convection and has a positive
`
`effect on the separation. Three types ofm:onolithic supports are currently
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`commercially available
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`Silica gel based monolithic beds: These columns are solid rods of silica monolith
`
`that have been prepared according to a sol-gel process. This process is based on the
`
`hydrolysis and polycondensation of alkoxysilanes in the presence of water-soluble
`
`polymers. The method leads to "rods" made of a single piece of porous silica with
`
`5
`
`a defined bimodal pore structure having macro (of about 2 !liD) and mesopores (of
`
`about 0,013 J.Lm) when smaller rods intended for analytical purposes are prepared.
`
`The main feature of these columns is about 80% porosity, which is 15% more than
`
`columns packed with standard particulate packing. As a result, the pressure drop
`
`along the column is one-third to one-fifth of that on columns packed with 3 !liD or
`
`10
`
`5 !liD beads. Scale-up columns, which are suitable for laboratory and semi(cid:173)
`
`industrial purification have macro pores of about 4 !liD and mesopores of about
`
`0,014 !liD and this allows even higher flow rates to be used then in case ofthe
`
`analytical ones (Nakanishi K, SagaN (1991) JAm Ceram Soc 74:2518; Cabrera K,
`
`Wieland G, Lubda D, Nakanishi K, SagaN, Minakuchi H, Unger KK (1998)
`
`15
`
`Trends Anal Chern 17:50).
`
`Polyacrylamide based monolithic beds are made of swollen polyacrylamide gel
`
`compressed in the shape of columns. Their technology relies on the polymerization
`
`of advanced monomers and ionomers directly in the chromatographic column. In
`
`the presence of salt, the polymer chains form aggregates into large bundles by
`
`20
`
`hydrophobic interaction, creating voids between the bundles (irregularly shaped
`
`channels) large enough to permit a high hydrodynamic flow. Following
`
`polymerization, the bed is compressed by connecting it to an HPLC pump adjusted
`
`to a flow rate equal or higher than that used in subsequent runs. The obtained bed
`
`can be regarded as a rod or plug permeated by channels in which the eluent can
`
`25
`
`pass upon application of pressure. The polymer chains form a dense, homogeneous
`
`network of nodules consisting of microparticles with an average diameter of 2 Jlill.
`
`The channels between the nodules are large enough to permit a high hydrodynamic
`
`flow beds (Hjerten S, Liao J-L, Zhang R (1989) J Chromatogr 473:273; Liao J-L,
`
`Zhang R, Hjerten S (1991) J Chromatogr 586:21).
`
`30 Rigid organic gel based monolithic beds: These supports are prepared by free
`
`radical polymerization of a mixture of a polymerizable monomer, optionally with
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`functional groups, such as glycidyl methacrylate, ethylene dimethacrylate, a
`
`crosslinking agent, a radical chain initiator, such as 2,2' -azobisisobutyronitrile, and
`
`porogenic solvents ( cyclohexanol and dodecanol) in barrels of an appropriate mold
`
`(Svec F, Tennikova TB (1991) J Bioact Compat Polym 6:393; Svec F, Jelinkova
`
`5 M, Votavova E (1991) Angew Macromol Chern 188:167; Svec F, Frechet JMJ
`
`(1992) Anal Chern 64:820) in the case of glycidyl methacrylate-co-ethylene
`
`dimethacrylate (GMA-EDMA) monoliths. Another method uses free radical
`
`polymerization of a mixture of styrene and divinylbenzene (as a cross-linking
`
`reagent) using 2,2'-azobisisobutyronitrile as an initiator and porogenic solvents
`
`10
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`(dodecanol and toluene) (Merhar M, Podgornik A, Barut M, Jaksa S, Zigon M,
`
`Strancar A (2001) J Liq Chromatogr 24:2429). After polymerization, the formed
`
`block of polymer is washed, e.g. with methanol, followed by a methanol-water
`
`mixture (50:50) and distilled water to remove porogenes and residual monomers
`
`from the polymer. After this, the monolithic bed is ready for derivatization to
`
`15
`
`achieve a desired chemistry or immobilization ofligands. GMA-EDMA monoliths
`
`have active epoxide groups which can easily be further modified using various
`
`chemicals, e.g. diethyl amine, propane sulfone for ion exchange chromatography,
`
`e.g. butyl groups for hydrophobic interaction chromatography and any desired
`
`protein ligand for affinity chromatography. Alternatively, the epoxide groups
`
`20
`
`containing monolith material can be modified to obtain polar groups on the surface,
`
`e.g. by using acids, e.g. sulfuric acid; to obtain the monolith material in hydrolized
`
`form that carries OH groups. Depending on the adsorption and elution conditions, a
`
`monolith carrying polar groups, e.g. hydroxyl (OH) groups or amino (NH2) groups,
`
`is suitable for being used in a variety of adsorption principles, e.g. the so-called
`
`25
`
`"normal phase" chromatography (Dorsey JG, Foley JP, Cooper WT, Barford RA,
`
`Barth HG (1990) Anal. Chern. 62:324 R) or the so-called "hydrophilic interaction"
`
`chromatography (Alpert AJ (1990) J. Chromatogr. 499:17 ), the so-called
`
`"cohydration/cosovent exclusion promoted chromatography" (Validated
`
`Biosystems: Purification Tools for Monoclonal Antibodies, Cagnon P (1996), 1 03)
`
`30
`
`or the so-called "hydrogen bond chromatography" (Fujita T, Suzuki Y, Yamauti J,
`
`Takagal1ara I, Fujii K., Yamashita J, Horio T (1980) J. Biochem. (Tokyo), 87
`
`(1):89).
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`ill the following, a chromatography method that uses a support, e.g. a porous
`
`monolithic support, that carries polar groups on its surface, is termed "polar
`
`interaction chromatography" ("PIC").
`
`5
`
`h1 adsorptive chromatographic modes the slope of the capacity factor k', defined as
`
`the molar ratio of the separated compound in the stationary phase and the mobile
`
`phase, plot versus the composition ofthe mobile phase is very steep. Up to a
`
`certain composition ofthe mobile phase, the k' value may become so high that the
`
`protein is bound to the stationary phase only and does not move along the column.
`
`10 Reaching a defined point, a small change of the mobile phase composition causes a
`
`rapid decrease ink' to a value near zero. At this point, the protein dissolves in the
`
`mobile phase and passes through the column practically without any retention. ill
`
`other words, the macromolecule remains adsorbed at the top ofthe column until the
`
`eluting power of the mobile phase reaches the point at which a small change in the
`
`15
`
`composition of the mobile phase causes the movement of the protein without any
`
`retention. One can also speak about selective elution of the compound. As a result
`
`of this process, even very short colunms can provide very good separations and
`
`very good recovery, while longer columns might cause problems due to unspecific
`
`binding, product degradation and minor changes in the structure of the biomolecule
`
`20 which increase with the length of the column (Tennikova TB, Belenkii BG, Svec F
`
`(1990) J Liq Chromat~gr 13:63; Strancar A, Barut M, Podgomik A, Koselj P, Josie
`
`D, Buchacher A (1998) LC-GC illt 10:660).
`
`On the basis of this postulation, new types of monolithic columns were developed
`
`in the shape of disks and, for the scale-up applications, in the shape of tubes
`
`25
`
`(WO 96/06158; US 5,972,218; EP 777725) and so-called "tube-in-a-tube"devices
`
`as disclosed in WO 99/44053. The disclosure of these references is incorporated by
`
`reference. "Monolithic columns" are available under the trade name CIM® disk
`
`monolithic columns and CIM@ tube monolithic columns.
`
`Scale up of particle columns containing particles that range in size from a few
`
`30 micrometers up to 100 micrometers can be achieved by packing ofthese small
`
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`wo 03/051483
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`PCT/EP02/14314
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`11
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`particles in larger columns. In contrast thereto, large-scale monolithic columns are
`
`obtained by producing a large block of a polymer casted in a proper cartridge
`
`(monolith holder). An issue to be addressed is preparing such a monolith with a
`
`uniform structure. In the following, this process will be elucidated, using as an
`
`5
`
`example methacrylate monoliths prepared by polymerization.
`
`When the polymerization temperature is reached, the initiator decomposes and
`
`oligomer nuclei start to form. The solubility of the polymers in the reaction mixture
`
`decreases during the growth phase and they start to precipitate. In terms of their
`
`thermodynamic properties, the monomers are better solvating agents for the
`
`10
`
`polymer than the porogenes. Consequently, the precipitated nuclei are swollen in
`
`the presence of the monomers. Since the monomer concentration is higher than in
`
`the surrounding solution, polymerization in the nuclei is kinetically preferred. In
`
`the absence of mixing, due to higher density, insoluble nuclei sediment and
`
`accumulate at the bottom of the mould. Initially, they form a very loose structure,
`
`15 which is highly porous. In the course of polymerization, nuclei continue to grow
`
`and crosslink until the final structure is achieved. As can be deduced from the
`
`above description, the pore size distribution ofthe polymer depends on its chemical
`
`composition and the polymerization temperature. Namely, the temperature defines
`
`the degradation rate of the initiator and, therefore, also the number of nuclei formed
`
`20
`
`in a given time. Since the amount of the monomers is constant, the lower number
`
`of nuclei formed at lower temperatures within a defined volume corresponds to
`
`their larger size, thus, to larger pores between the clusters of growing nuclei. In
`
`contrast, at higher polymerization temperatures, at which the decomposition of
`
`initiator occurs much faster, the number of growing nuclei is much larger and, as a
`
`25
`
`consequence, the formed pores are much smaller. Therefore, the polymerization
`
`temperature is a powerful tool for the control of pore formation.
`
`Polymerization of a methacrylate-based monolith is an exothermic process.
`
`Therefore, heat is released during the reaction. With no mixing being employed
`
`and with a size ofthe mould in a range of centimeters, the released heat cannot
`
`30
`
`dissipate fast enough. As a consequence, an increase ofthe temperature inside the
`
`reaction mixture occurs. In a mould of e.g. 50 mm, a temperature increase in the
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`12
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`range of 100°C was observed and a 25°C temperature differential was recorded
`
`across the radius of the column. Pore size distribution measurements revealed that
`
`the pores in the middle of the polymer are larger than on the outer part, resulting in
`
`an inhomogeneous pore size distribution. Obviously, the preparation of large
`
`5
`
`volume monoliths is limited due to the exothermic nature of the polymerization and
`
`due to a pronounced temperature influence on the pore size distribution. To
`
`overcome these problems, the so-called "tube-in-a-tube" design, as disclosed in
`
`WO-A-99/44053, which is herewith incorporated by reference, was developed.
`
`Instead of gradually adding the polymerized mixture to form a single large volume
`
`10 monolith, this approach is based on the construction of a monolithic mmulus of a
`
`required radius but limited thickness. However, since it is possible to construct the
`
`annuluses where the outer diameter of a smaller monolith is equal to the inner
`
`diameter of a larger one, a large volume monolithic unit can be constructed by
`
`inserting two or several aimuluses one into another as shown in Fig. 15, forming a
`
`15
`
`so-called "tube-in-a-tube" system. In this way, a monolithic unit of required
`
`volume and uniform pore size distribution can be obtained. Furthermore, the voids
`
`between the annuluses can be filled with the reaction mixture and polymerization is
`
`allowed to proceed for a second time. Sin,ce the voids are very thin, no increase in
`
`temperature during the course of the reaction is expected.
`
`20
`
`This approach was at first verified by the construction of an 80 ml "tube-in-a-tube"
`
`monolithic column. The monolithic column was characterized by low
`
`backpressures even at high flow rates (below 2.5 MPa at the flow rate of250
`
`mllmin). One interesting feature is that in contrast to conventional radial columns
`
`of large diameter and a small bed thickness, the bed in this case had an outer
`
`25
`
`diameter that was 35 mm while the inner diameter was only 1.5 mm. Due to this,
`
`the linear velocity of the mobile phase increases more then 23 times from the outer
`
`to the inner surface of the column. In the case of conventional porous particle
`
`supports, such changes in the linear velocity would generally result in a
`
`pronounced deterioration ofthe column efficiency. However, the characteristics of
`
`30
`
`the monoliths were found to be flow independent, therefore the change in linear
`
`velocity should not have any influence either on the resolution or on the binding
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`capacity. This was proved by the separation of a protein mixture as well as by
`
`measuring the dynamic binding capacity determined at different flow-rates. This
`
`concept was further verified by construction and characterization of the 800 ml
`
`monolithic column.
`
`5
`
`Plasmid DNA separation using disc monolithic columns was first described by
`
`Giovannini et al (Giovannini R, Freitag R, Tennikova TB (1998) Anal Chern
`
`70:3348). The authors reported that by using optimized conditions a pure pDNA
`
`sample can be separated into 3 peaks which presumably correspond to supercoiled
`
`( ccc ), open circular and linearized pDNA. Separations under gradient and isocratic
`
`10
`
`conditions were stud