WO2002060553A1 - Isolement de nanoparticules - Google Patents

Isolement de nanoparticules Download PDF

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Publication number
WO2002060553A1
WO2002060553A1 PCT/EP2002/000764 EP0200764W WO02060553A1 WO 2002060553 A1 WO2002060553 A1 WO 2002060553A1 EP 0200764 W EP0200764 W EP 0200764W WO 02060553 A1 WO02060553 A1 WO 02060553A1
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Prior art keywords
nanoparticles
matrix
particles
solution
supeφorous
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PCT/EP2002/000764
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English (en)
Inventor
Per-Erik Gustavsson
Lars Hagel
Per-Olof Larsson
Raf Lemmens
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Amersham Biosciences Ab
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Publication of WO2002060553A1 publication Critical patent/WO2002060553A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10351—Methods of production or purification of viral material

Definitions

  • the present invention relates to a process for isolation of nanoparticles from undesired impurities in a solution. More specifically, the present process is based on adsorption of such nanoparticles to an especially advantageous matrix material.
  • the target substance is then a protein of an essentially spherical geometry and with a surface similar among the target proteins throughout the population.
  • proteins are of a reasonably good diffusivity and many effective carrier materials have been suggested, which rely on the diffusion of protein into a gel structure to binding groups, such as ion exchanging groups or other adsorptive or affinity groups.
  • binding groups such as ion exchanging groups or other adsorptive or affinity groups.
  • Such effects may be shielding or enhancing with respect to the separation performance, but will in any case entail problems due to the difficulties in predicting possible outcomes.
  • One particularly problematic situation arises when the macro-structure is not uniform among the particles in a population, such as in plasmid purification.
  • the same plasmid, i.e. plasmids having the same chemical identity will normally exist in different conformations, and transformation between said conformations will not occur freely.
  • plasmids having the same chemical identity will normally exist in different conformations, and transformation between said conformations will not occur freely.
  • PerSeptive BioSystems suggests a method of purification of plasmid DNA by anion perfusion chromatography on a matrix comprising through- pores. More specifically, part of the chromatographic flow passes through each individual particle improving its mass transport properties.
  • all separation methods using such flow through particles will include an inherent limitation as regards the flow rate, since for an efficient separation to take place, the molecules must be allowed a sufficient time to diffuse into the smaller pores, the size of which are in a range of about 0.15 ⁇ m. Accordingly, there is still a need within this field of an even more efficient and flexible method for isolation of nanoparticles, such as plasmids.
  • WO 97/19347 relates to a chromatographic separation method and a device comprising a medium with fast kinetics, with high efficiency, with good mechanical properties and with low back pressures.
  • the method and device are suitable for separation of large biomolecules, exemplified as proteins, peptides, nucleic acids, oligonucleotides, cells or viruses.
  • the improvement provided by WO 97/19347 is related to the macroporous cross-liked organic polymer, which is prepared by the so- called HIPE (High Internal Phase Emulsion) technique.
  • USP 4,699,717 discloses a process for chromatographic separation of nucleic acid using surface modified carrier materials that similar to the above mentioned WO specification contain cavities, which in the experimental part of this patent specification are of sizes up to 0.4 ⁇ m.
  • the physical appearance of such cavities is illustrated in Fig 1 thereof, from which it appears clearly that the cavity is of a depth which is essentially corresponding to, or slightly superior to, the diameter of said cavity.
  • cavities are of a different geometry than pores, the depth of which usually exceeds their diameter several times. In any case, these cavities would be too small for nanoparticles, such as large nucleic acids e.g. plasmids.
  • the example given with plasmids should involve plasmid bound to the outer surface, since the cavities are too small to allow any appreciable entrance of plasmids.
  • USP 6,143,548 discloses a method of purification of active adenovirus and A AN, which method has been improved compared to the prior art in order not to damage the virus.
  • the design considerations taught relate to the objective of minimising or eliminating damage to the virus by contact with various chromatographic materials.
  • the approaches are said to be intended to obviate the effect of openings or pores in such materials.
  • the chromatographic materials suggested comprise pores of a size of up to about 1.2 ⁇ m, which is sufficient for the largest known spheroidal viruses.
  • the process is also a batch-type technique, since by this method the virus particles are less likely to enter the pores in the beads where they can become damaged.
  • WO 01/07597 also discloses a chromatographic process, which is run with batch conditions.
  • US 5 057426 teaches a method of separation of long-chain nucleic acids from other substances in solutions containing nucleic acids and other materials wherein a matrix denoted a "highly porous" silica gel is used.
  • this method has not been shown to provide a sufficient capacity, and therefore there is still a need of improvements within this field.
  • the object of the present invention is to provide a process for isolation of nanoparticles, which avoids one or more of the above-discussed drawbacks.
  • one object of the present invention is to provide a process for isolation of nanoparticles on a porous matrix, which is more efficient due to the large dynamically available binding area thereof.
  • Another object of the invention is to provide such a method, wherein the high shear damage normally caused by high flow rates through conventional chromatography matrices is avoided.
  • one object of the present invention is to provide a method for plasmid- purification, which is particularly useful in large-scale operation.
  • the objects of the invention are more specifically obtained by the process as defined in the appended claims.
  • Figure 1 A illustrates, in subsequent optical slices, how plasmid DNA under batch conditions has been bound to the surface of supe ⁇ orous agarose beads with a pore size of 4 ⁇ m under batch conditions.
  • Figure IB is an enlargement of an optical section in the middle of the bead, which shows the binding of plasmid DNA to the outer surface of the bead.
  • Figure 2 A and 2B show the same type of prototype bead as under Figure 1, with binding of plasmid DNA on the surface and some plasmid DNA in the supe ⁇ ores under dynamic conditions in accordance with the present invention.
  • Figure 3 A shows a prototype agarose particle with a particle size 180-106 ⁇ m, wherein the size of the supe ⁇ ores is 15 ⁇ m.
  • Fig 3B shows the results of use of such a prototype as an ion exchanger (hydrophilic Q exchanger), with incubation with plasmid DNA under dynamic conditions
  • nanoparticle is understood herein to include large molecules and molecule aggregates, such as virus, plasmids, cell organelles, membrane fragments and inclusion bodies. Such nanoparticles are typically of a size within the range of about 30-1000 nm.
  • dynamic conditions as compared to static conditions means herein that the process is a chromatographic procedure, wherein the solution comprising the molecules to be isolated is brought to pass over a column packed with a suitable adsorbent or brought in contact with the adsorbent in "expanded bed” mode, as compared to incubation with the adsorbent in batch conditions.
  • eluant is used herein with its conventional meaning in chromatography, i.e. a solution capable of perturbing the interaction between the solid phase (adsorbent matrix) and product (nanoparticle) and promoting selective dissociation of the product from the solid phase.
  • a first aspect of the present invention is a process for isolation of nanoparticles from a solution, comprising the steps of
  • the supe ⁇ orous matrix is comprised of particles of an average supe ⁇ ore diameter of up to about 25 ⁇ m and the adso ⁇ tion step is run under dynamic conditions.
  • the present invention describes for the first time the unexpected advantages of using a supe ⁇ orous matrix under conditions of dynamic flow.
  • the process according to the invention is an isolation of nanoparticles expressed in cells, and, consequently, it also comprises a first step of disintegrating the cells to provide the solution comprising nanoparticles.
  • Such disintegration is performed e.g. by lysis, such as alkaline lysis, according to standard protocols (see e.g. Maniatis, T, Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY).
  • the present process comprises the further step of eluting the nanoparticles by contacting a suitable eluant with said matrix.
  • the elution step can be performed as a dynamic or batch procedure.
  • the nature of the eluant will depend upon the matrix material used in the column, as discussed in more detail below. (For a general review of chromatographic separation, see e.g. Protein Purification Handbook, 1999, Amersham Pharmacia Biotech AB, Uppsala, Sweden)
  • the process according to the invention is utilised e.g. for purification of nucleic acids for use in gene therapy and laboratory studies related to gene therapy.
  • the present process will provide isolated nanoparticles in the form of viruses or plasmids of acceptable gene therapy grade. More specifically, it is predicted that in a near future, there will be an increasing demand of virus and plasmids in large quantities for use in gene therapy as carriers or vectors of genetic material. As mentioned above, the previously described methods for isolation of such carriers have not been satisfactory to this end, and the process according to the present invention is thus the first to enable large scale processing of nanoparticles for medical and diagnostic use.
  • a further application of the invention is in the preparation of vaccine, in which case molecular aggregates, which are of interest as carriers of immunogenic structures are isolated by the present process. In this case, it is especially important to be able to efficiently isolate well-characterised aggregates having defined properties.
  • Yet another field of application of the invention is in the context of inclusion bodies, i.e. condensed protein aggregates, which is often the form proteins are obtained in during an efficient expression thereof in a cell system.
  • the average supe ⁇ ore diameter of the supe ⁇ orous matrix particles used in the present process may be of a value of up to about 25 ⁇ m, for example a diameter within a range of up to 10, such as about 8, or within a range of 10-20, such as about 15 ⁇ m.
  • the supe ⁇ ore diameter is up to about 20-30 or even about 30-40 ⁇ m.
  • the term "supe ⁇ orous" relates to particles wherein the pores are large enough so as to constitute an essential part of the structure of the particles, i.e. to penetrate the particles to a much deeper extent than in normal particles for chromatography, which are more or less equally porous straight through the particle.
  • the pores of a normal particle are much smaller than the size of a nanoparticle. The consequence of this is that a nano- particle only has access to the outer surface of a particle, or to a very thin outer layer due to statistical fluctuation of the pore diameter.
  • the lower limit of average supe ⁇ ore diameter of the supe ⁇ orous matrix particles used in the present process may be about 4 ⁇ m, but under certain conditions, it may be even lower, such as about 2 ⁇ m or even about 1 ⁇ m. Conditions that may need to be considered when working at these extremes of the diameter range is e.g. the size of the nanoparticle used, the flow rate etc. As regards the nanoparticle size, as the skilled person in this field will realise, a too small pore will not allow a sufficient penetration. On the other hand, a too large pore may also be undesired, since then a reduction in available pore surface area may be expected.
  • the method of preparing the matrix can have an influence on the separation properties, since some methods may give rise to broader pore size distributions or more inhomogeneous pore size distribution over the particle radius pores of a slightly more "pointed” nature than others.
  • this last- mentioned factor is not easily predicted and therefore the question whether or not a supe ⁇ orous matrix in this lower range region is working for a defined pu ⁇ ose is most advantageously tested by routine experiments, which are easily performed by a skilled in this field.
  • Binding properties i.e. to which extent the desired nanoparticle has entered the supe ⁇ ores and been bound to the matrix surface can for example be performed as described in the section "Experimental part" below.
  • the present invention relates to a separation matrix in which the supe ⁇ ores are penetrated and bind nanoparticles to at least about 25%, preferably at least about 50% and most preferably at least about 75%, such as about 90% or even 95% of the maximum binding capacity.
  • the skilled person can similarly to what is discussed above easily decide on a suitable flow rate for each selected combination of supe ⁇ orous matrix desired nanoparticle.
  • the flow rate cannot be too high, since then the particles may collapse and/or the nanoparticles may be broken due to high shear forces, and it must also be sufficiently low to allow the desired mass transfer to take place.
  • the nanoparticles will not be able to penetrate the supe ⁇ ores to a sufficient degree.
  • US 5 057 426 relates to a method of separation of long-chain nucleic acids from other substances in solutions containing nucleic acids and other materials wherein a matrix denoted a "highly porous" silica gel is used. It appears that "highly porous” is defined as pores of a diameter of about 0.05-2.5 ⁇ m in particles having a size of about 15-250 ⁇ m. Accordingly, the proportion between pore diameter and particle size disclosed therein is in general much smaller than that of the present invention.
  • the pore diameter of the US 5 057 426 matrix will enable binding of nanoparticles only to the surface layers thereof, and not in the pores, which renders their properties very different from those of the present supe ⁇ orous particles.
  • the available binding area of said matrix will be considerably smaller than that according to the present invention, wherein the porosity allows binding throughout essentially all of the particles, as illustrated in the drawings.
  • the nanoparticles are adsorbed to a supe ⁇ orous matrix comprised of particles of a mean size in the range of about 50-300 ⁇ m, e.g. within a range of 50-100, 100-200 or 200-300 ⁇ m, and especially in a range of about 106-180 ⁇ m.
  • the particles can advantageously be prepared in any size for which commercially available sieve equipment is exist, such as 250, 212, 180, 150, 125, 106, 90, 75, 63, or 45 ⁇ m.
  • the super pore diameters of the particles used according to the invention are in the area of about 1/9 of the particle diameter.
  • the particles used in accordance with the present invention can easily be designed by conventional methods based on values of supe ⁇ ore and particle size as mentioned above. Materials and binding function will be discussed in more detail below. For a definition of the method of preparation used in the examples below, see P.-E. Gus- tavsson, P.-O. Larsson, J. Chromatogr. A 734 (1996) 231-240, wherein verification was by size exclusion experiments with 0.5 ⁇ m latex particles and microscopy.
  • the present invention shows to be advantageous for the isolation of plasmids.
  • the invention combines a large surface area with the possibility of adsorbing e.g. plasmids in the matrix' pores with a very low shear stress, using chromatography separation performed under dynamic conditions on a matrix of supe ⁇ orous particles. This opens the possibility of retaining a plasmid in its supercoiled form, which is the preferred conformation for gene therapy pu ⁇ oses.
  • Plasmids isolated in accordance with the invention can be of any origin. Most commonly, microorganisms like bacteria, such as E.coli, are used for culturing the plasmids, but the use of host cells is not limited and can be prokaryotic or eukaryotic cells.
  • the host cells harbouring the plasmid can be cultivated in a number of ways well known in the art, e.g. in incubator, bioreactor, fermentor etc.
  • the plasmid isolated according to the invention can be of virtually any size, e.g. in the range of about 2kb up to about 10 kb. At the time of the filing of the present application, commercial plasmids are often of a size of about 300-1000 nm. As an upper limit, the isolation of cos- mi ds and artificial chromosomes is also encompassed, the size of which may be up to about 50 kb and 500 kb, respectively.
  • Plasmids can be of a high copy number or low copy number and can carry any gene, either genomic or synthetic, encoding protein or peptide of interest, from any source.
  • the culturing of the host cells, as well as the exploitation of the plasmid for gene therapy, is well known in the state of the art.
  • the cells After culturing the host cells containing the plasmid, the cells are recovered by e.g. centrifugation or filtration.
  • the cell can be stored, for example in a freezer, or processed immediately.
  • lysis thereof is advantageously performed by alkaline lysis.
  • the lysate may then be treated with metal ions, such as of divalent alkaline earth metal ions, to precipitate impurities and specifically RNA and chromosomal DNA.
  • the solution can be applied to the column.
  • the nanoparticle isolated according to the present invention is a virus, such as an adenovirus, an adeno-associated virus (AAV), a He ⁇ es Simplex virus (HSV), a retrovirus, etc.
  • the size of the nanoparticles is in a range of about 20-500 nm.
  • the virus may initially be present in any solution, which depending on the production technology used may comprise a large number of proteins and possibly cell debris.
  • One method of producing viruses is by injecting a small number of viruses in a fertilised chicken egg and allowing the virus to multiply for a number of days, after which the contents of the egg is collected. Thus, in this case the virus produced will need to be purified from the content of the egg.
  • virus can also be produced in cell culture. If the virus is non-lytic, it has to be purified from the culture medium contents (e.g. fetal calf serum, proteins, growth hormones, vitamins, salts, cellular excretion products). If the virus on the other hand is lytic, cells are lysed by the virus, which will mean that all cell content (proteins, genomic DNA, cell organelles) and cellular debris are present in the solution. The further processing thereof can then be performed as described above in relation to plasmids grown in cells.
  • the culture medium contents e.g. fetal calf serum, proteins, growth hormones, vitamins, salts, cellular excretion products.
  • the virus on the other hand is lytic, cells are lysed by the virus, which will mean that all cell content (proteins, genomic DNA, cell organelles) and cellular debris are present in the solution. The further processing thereof can then be performed as described above in relation to plasmids grown in cells.
  • the adsorbing or binding groups present on the matrix material can be ion exchanging groups, affinity groups, hydrophobic interaction groups, etc. Since the nanoparticles contemplated by the present invention are predominantly negatively charged, positively charged binding groups constitute suitable ligands for the present isolation. However, other types of ligands can alternatively be used.
  • a matrix useful in the present method can be made of a native polymer such as agarose or a synthetic polymer such as polystyrene/divinylbenzene. In a specific embodiment, the matrix used according to the invention is made from an inorganic material, such as silica. Many such matrix materials are known to the skilled person in this field and various methods are available for producing porous matrices of a desired porosity thereof.
  • the matrix material used is provided with anion exchanging groups.
  • the anion group attached to the matrix can vary from quaternary amino groups (Q), quaternary aminomethyl- (QMA), quaternary aminoethyl- (QAE), triethyl aminomethyl-(TEAE), triethyl aminopropyl- (TEAP), polyethyleneimine- (PEI), diethyl aminoethyl- (DEAE), polyaminoethyl groups (PAE) and others.
  • the anion exchange groups are bound to the base matrix via extenders such as described in SE 9700383-4.
  • extenders such as described in SE 9700383-4.
  • the positive effect caused by such an extender is believed to depend on the fact that the extender will provide the inner surfaces (pore surfaces) and/or outer surfaces of the matrix beads with a flexible polymer layer which is permeable to macromolecules and other molecules are allowed to pass the bed. This will cause an increase in the effective interaction volume as well as in the steric availability of the anion exchange groups. This in turn will increase the mass transfer rate as well as the total capacity.
  • Suitable extenders should be hydrophilic and contain a plurality of groups selected from e.g. hydroxy, carboxy, amino, repetitive ethylene oxide (-CH 2 CH 2 0-), amido etc.
  • the extender may be in the form of a polymer.
  • Hydrophilic polymeric extenders may be of synthetic origin or of biological origin. Typical synthetic polymers are polyvinyl alcohols, polyacryl- and polymethacrylamides, polyvinyl ethers etc. Typical bio- polymers are polysaccharides, such as starch, cellulose, dextran, agarose etc.
  • the preferred polymeric extenders are often water-soluble in their free state, i.e. when they are not attached to the base matrix. The length (size) of the optimal extender will depend on several factors, such as number of attachment points to the base matrix, type of extender, type and size of anion groups etc.
  • polymeric extenders for which attachment and/or cross-linking is possible at several monomeric units, it is believed that larger extenders are preferred. It is believed that the most suitable polymers should contain at least 30 monomeric units, which for polysaccharides like dextran indicates a M w > 5000 g/mole.
  • the base matrix of the beads may be of organic or inorganic nature. Usually it is a polymer, such as glass, a synthetic polymer or a biopolymer.
  • the base matrix may be a hydrophilic polymer such as styrene-divinyl benzene copolymer, which has been hy- drophilised on inner and/or outer surface by being coated with the appropriate hydrophilic polymer or by other means.
  • the base matrix may be a water- insoluble hydrophilic polymer, e.g. agarose, cellulose, dextran, starch, etc. which has been cross-linked to give the desired porosity and stability, if necessary.
  • a preferred base matrix in the present invention is based on cross-linked agarose with dextran as extenders.
  • the eluant used in the present process depends on the nature of the adsorber used in the matrix as well as on the nanoparticles bound thereon.
  • the principle for choosing a suitable eluant is easily made by the skilled person in this field, see e.g. Protein Purification Handbook, 1999, Amersham Pharmacia Biotech AB, Uppsala, Sweden).
  • One advantage with the present invention is the relatively high flow rates that are contemplated by the supe ⁇ orous matrix, which exceed the flow rates obtainable simply by its gravitational force. It is especially advantageous to be able to use such flow rates without impairing nanoparticles of specific conformations, such as supercoiled plasmids. This feature renders the process especially advantageous in the context of plasmid isolation on an industrial scale.
  • the present process may be performed with the matrix as an expanded bed or as a packed bed.
  • packed bed adso ⁇ tion the adsorbent is packed in a chromatographic column and all solutions used during a purification process flow through the column in the same direction.
  • expanded bed adso ⁇ tion however, the adsorbent is expanded and equilibrated by applying a liquid flow through the column.
  • a stable fluidised expanded bed is formed when there is a balance between particle sedimentation or rising velocity and the flow velocity during application of the sample and washing steps.
  • the adsorbent is sedimented and behaves like a packed bed adsorbent.
  • the present invention relates to a kit for isolation of nanoparticles, which comprises a supe ⁇ orous matrix in a chromatographic column.
  • the matrix is comprised of particles of a supe ⁇ ore diameter of at least about 4 ⁇ m, such as about 5-10 or 10-20 ⁇ m, and may be of a value of up to about 25 ⁇ m, such as about 20-30 or even about 30-40 ⁇ m.
  • the matrix particles are of a mean size in the range of about 50- 300 ⁇ m, e.g. within a range of 50-100, 100-200 or 200-300 ⁇ m, and especially in a range of about 106-180 ⁇ m.
  • the particles can advantageously be prepared in any size for which commercially available sieve equipment is exist, such as 250, 212, 180, 150, 125, 106, 90, 75, 63, or 45 ⁇ m. Further details regarding the nature of adsorbent coupled to the supe ⁇ orous matrix kit, uses etc are as discussed above in relation to the process.
  • Figure 1 A displays following two-dimensional confocal microscopy images (Leica TCS SP confocal scanning laser microscope, argon-krypton laser) obtained after the plasmid DNA with fluorescent dye (TOTO-3, dimeric cyanine nucleic acid stain from Molecular Probes; T-3604)) is incubated with a supe ⁇ orous agarose particle of 50 ⁇ m with a pore size of 4 ⁇ m under batch conditions for 90 minutes (see example).
  • fluorescent dye TOTO-3, dimeric cyanine nucleic acid stain from Molecular Probes; T-3604
  • Figure IB more clearly shows the lack of plasmid DNA in the pores of the bead.
  • Figure 2 A shows the presence of the same stained plasmid DNA as in the above Figure bound in the supe ⁇ ores under dynamic conditions according to the invention in packed bed with supe ⁇ orous agarose particles with a size ranging from 45 to 75 ⁇ m and supe ⁇ ores of 4 ⁇ m, as described in example 1.
  • Figure 2B an enlargement of the confocal microscopy image shown in Figure 2A.
  • Figure 3 A shows a confocal image of prototype agarose particle with a particle size 180-106 ⁇ m, wherein the size of the supe ⁇ ores is 15 ⁇ m.
  • Fig 3B shows the results of use of such a prototype as an ion exchanger, more specifically a hydrophilic Q exchanger, with incubation with plasmid DNA under dynamic conditions.
  • Figure 3B shows a fluorescence intensity profile obtained with plasmid DNA visualised with TOTO-3. With dynamic conditions, plasmid DNA utilised the whole particle volume for adso ⁇ tion, and completely entered the supe ⁇ ores.
  • Supe ⁇ orous agarose beads were prepared by a double emulsification procedure.
  • One hundred ml of an agarose solution (Sepharose quality) (6%, w/v) was prepared by heating a suspension of agarose in water to 95-100°C in a microwave oven, and keep- ing it at that temperature for 1 min. During the warm-up period, care was taken to keep the agarose powder well suspended with occasional shaking.
  • the solution was transferred to a thermostatic (60°C) stirred glass reactor and a mixture containing 3.0 ml of Tween 80 (Merck-Schuchardt, Kunststoff, Germany) and 50 ml of cyclohexane (Merck, Darmstadt, Germany) (60°C) was added.
  • the mixture was emulsified by stirring at 1000 rpm for 4 min (emulsion 1).
  • a thermostatic solution 60°C containing 300 ml of cyclohexane and 12 ml of Span 85 (sorbitane trioleate; Fluka, Buchs, Switzerland) was added.
  • the reactor was stirred at 600 ⁇ m. After 1 min the reactor thermostat setting was changed to 25°C.
  • the temperature decreased below approximately 40°C, the agarose solidified into supe ⁇ orous spherical particles.
  • the particles were isolated on a sieve and washed with water, ethanol-water (50:50, v/v) and finally water.
  • the particles were sized wet with graded metal screens [Gustavsson et. al. J. Chromatogr. A 734/2 (1996) 231-240].
  • the supe ⁇ orosity will be dependent on the ratio of organic phase and agarose phase in the preparation procedure of supe ⁇ orous particles.
  • the supe ⁇ ore diameter can be varied independently on the supe ⁇ ore volume and vice versa.
  • RNA and plasmid DNA are prepared using the conventional alkaline lysis protocol followed by anion exchange chromatography and an isopropanol precipitation. Pelleted bacteria are first resuspended, and subsequently lysed under alkaline conditions. After neutralisation, proteins, cell debris and genomic DNA is removed by centrifugation. The supernatant is then loaded on an ion-exchanger column and eluted with a continuous salt gradient, after which the plasmid DNA is precipitated by the addition of isopropanol followed by centrifugation.
  • the starting material is a 500 ml overnight bacterial cell culture.
  • the suspension is centrifuged at 10.000 ⁇ m in a Sorvall GSA-rotor for 15 minutes at 4°C (lO.OOOg).
  • the supernatant is removed by carefully decanting, and the centrifuge tubes are stored in inverted position on a piece of paper tissue for 5 minutes to remove all remains of the supernatant.
  • the bacterial pellet is then resuspended by pipetting repeatedly up and down in 50 ml of a buffer pH 8,0 containing 50 mM Tris-HCl, 10 mM EDTA, and 100 ⁇ g/ml RNase A.
  • a lysis buffer consisting of 200 mM NaOH and 1% SDS is added, and the solutions are thoroughly mixed by gentle inversion of the tube. Lysis is performed by incubation for 5 minutes at room temperature. After this incubation period, 50 ml of a cold neutralisation buffer (3 M KAc pH 5,5) is added to the viscous solution and again the solutions are mixed gently but thoroughly by inverting the tube about five times. White fluffy material containing genomic DNA, proteins and cell debris with SDS is formed, and the lysate becomes less viscous. The fluffy material is removed by 30 minutes centrifugation at 4°C at 11.000 ⁇ m is a Sorvall GSA rotor (20.000g). The supernatant is promptly transferred to a fresh tube and recentrifuged under the same conditions for 15 minutes, after which the supernatant is collected.
  • a column packed with silica gel with DEAE anion exchanger groups is washed with a sufficient volume of equilibration buffer (750 mM NaCl, 50 mM MOPS pH 7,0, 15% isopropanol). The supernatant from the last centrifugation step is then applied to the column. After washing the column with large amounts of 50 mM MOPS pH 7,0 containing 1 M NaCl and 15% isopropanol, the plasmid DNA is eluted from the column by a buffer containing 1.25 M NaCl, 50 mM TRIS pH 8,5 and 15% isopropanol.
  • the DNA is precipitated by the addition of 0.7 volumes of isopropanol at room temperature.
  • the solution is mixed and subsequently centrifuged at 4°C at 11.000 ⁇ m is a Sorvall SS-34 rotor (15.000g).
  • the supernatant is carefully removed, and the pellet is washed by adding 7 ml of room temperature 70% ethanol. Again the mixture is centrifuged at 4°C at 11.000 ⁇ m is a Sorvall SS-34 rotor (15.000g).
  • Cautiously the supernatant is removed and the tube is left to air-dry for 20 minutes.
  • DNA pellet is resuspended in 50 ⁇ l of TE-buffer (10 mM Tris-HCl pH 8,0 with lkh mM EDTA).
  • TE-buffer 10 mM Tris-HCl pH 8,0 with lkh mM EDTA.
  • the obtained solution containing plasmid DNA is used for incubation with the superporous beads.
  • HR5/2 columns (5 mm diameter, 20 mm length; Amersham Pharmacia Biotech) are packed with several types of derivatized agarose beads with a particle size of ranging between 45 and 180 ⁇ m and supe ⁇ ores of 4 or 15 ⁇ m diameter.
  • the columns are installed on an Akta Explorer 10 (Amersham Pharmacia Biotech).
  • the columns In a first stage, the columns are washed with 1 M NaOH, at a speed of 90 cm/h, followed by an equilibration with 5 column volumes of equilibration buffer (20 mM Tris-HCl, pH 8,0, 10 mM EDTA). Blank samples are taken by opening the columns and transferring 20 ⁇ l of agarose beads to different tubes.
  • a 50 ⁇ l sample of the plasmid DNA preparation (1:4 diluted with H 2 0) is loaded on the columns at the same flow rate as described above. Again, the columns are opened and a 20 ⁇ l sample is taken for confocal scanning laser microscopy.
  • Supe ⁇ orous agarose particles Provided agarose particles with particle size 45-75 ⁇ m, size of the supe ⁇ ores 4 ⁇ m, hydrophilic Q exchanger
  • Supe ⁇ orous agarose particles Provided agarose particles with particle size 106-180 ⁇ m, size of the supe ⁇ ores 15 ⁇ m [Gustavsson et. al. J. Chromatogr. A 734/2 (1996) 231-240]; hydrophilic Q exchanger)
  • TOTO-3 reagent 50 ⁇ l TOTO-3 reagent was diluted in TE-buffer to 20 ml
  • TE-buffer 10 mM TRIS, 1 mM EDTA, pH 7.5
  • Plasmid DNA was adsorbed under dynamic conditions in a column experiment to supe ⁇ orous agarose particles (in 20 mM TRIS-HC1, 10 mM EDTA pH 8.0). 16 ⁇ l gel slurry (1 :1 in TOTO-3 solution) was incubated end-over-end for 2.5 hours. The particles were then analysed by confocal microscopy.

Abstract

La présente invention se rapporte à un procédé d'isolement de nanoparticules à partir d'une solution. Ledit procédé consiste à utiliser une solution contenant les nanoparticules; à faire adsorber les nanoparticules par des groupes adsorbants disposés sur une matrice superporeuse; et à éventuellement laver la colonne avec une solution appropriée; l'étape d'adsorption au moins se déroulant dans des conditions dynamiques. Les nanoparticules isolées selon le procédé décrit ci-dessus peuvent être des plasmides ou des virus. Le procédé de la présente invention est particulièrement adapté à un isolement à grande échelle.
PCT/EP2002/000764 2001-01-29 2002-01-25 Isolement de nanoparticules WO2002060553A1 (fr)

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EP1679117A2 (fr) * 2005-01-10 2006-07-12 HaemoSys GmbH Sytème d'adsorption pour éliminer les virus et les composés viraux de fluides, en particulier du sang et du plasma sanguin

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003041830A3 (fr) * 2001-10-12 2003-11-20 Protista Internat Ab Gel macroporeux, sa preparation et son utilisation
US7547395B2 (en) 2001-10-12 2009-06-16 Protista Biotechnology Ab Macroporous gel, its preparation and its use
EP1679117A2 (fr) * 2005-01-10 2006-07-12 HaemoSys GmbH Sytème d'adsorption pour éliminer les virus et les composés viraux de fluides, en particulier du sang et du plasma sanguin
EP1679117A3 (fr) * 2005-01-10 2007-12-26 HaemoSys GmbH Sytème d'adsorption pour éliminer les virus et les composés viraux de fluides, en particulier du sang et du plasma sanguin

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