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EP-4737898-A2 - SEPARATION AND ISOLATION OF NUCLEIC ACIDS USING AFFINITY LIGANDS BOUND TO A SOLID SURFACE

EP4737898A2EP 4737898 A2EP4737898 A2EP 4737898A2EP-4737898-A2

Abstract

A method of isolating and separating a target macromolecule, such DNA (double stranded or single stranded), RNA (double stranded or single stranded), messenger RNA, or other oligonucleotide or oligonucleoside, from a sample by binding the target macromolecule to an affinity ligand that is bound to a surface is disclosed. The method may be employed in chromatography or any other of the separation sciences.

Inventors

  • Hurst, Alistair J.
  • LEVISON, DEREK W.K.
  • MOELLER, UWE

Assignees

  • EMP Biotech GmbH

Dates

Publication Date
20260506
Application Date
20201124

Claims (15)

  1. A method of separating a target macromolecule from a sample, comprising the steps of: a. selecting an affinity ligand that will bind to the target macromolecule, b. binding the affinity ligand to a surface to create a coupled surface-affinity ligand; c. placing the coupled surface-affinity ligand into a container; d. introducing the sample containing the target macromolecule to the coupled surface-affinity ligand and causing the coupled surface-affinity ligand to incubate with the sample for a residence time, wherein the target macromolecule binds to the affinity ligand; and e. separating the coupled surface-affinity ligand bound to the target macromolecule from the sample that has the target macromolecule removed therefrom; wherein the target macromolecule is a nucleic acid or a fragment thereof; wherein the surface is a solid surface, and optionally wherein the container is a chromatography column, bowl, cylinder, conical-shaped vessel, or vat; wherein the affinity ligand is an intercalator, a minor groove binder, a major groove binder, or any combination thereof, optionally wherein the affinity ligand does not bind to any proteins in the sample; and wherein the affinity ligand is a compound which has been modified to include a linker group capable of binding the affinity ligand to the surface, wherein the compound is selected from the group consisting of (a) an acridine selected from GelGreen (10,10'-(6,22-dioxo-11,14,17-trioxa-7,21-diazaheptacosane-1,27-diyl)bis(3,6-bis(dimethylamino)acridin-10-ium) iodide), acridine orange (N,N,N',N'-Tetramethylacridine-3,6-diamine) and derivatives thereof, amsacrine, and acriflavins (3,6-Diamino-10-methylacridin-10-ium chloride) and derivatives thereof, and optionally proflavine; (b) DAPI (4',6-diamidino-2-phenylindole); (c) a phenanthridine selected from Ethidium bromide, Propidium iodide, Propidium monoazide, and GelRed (5,5'-(6,22-dioxo-11,14,17-trioxa-7,21-diazaheptacosane-1,27-diyl)bis(3,8-diamino-6-phenylphenanthridin-5-ium) iodide); (d) a cyanine of benzothiazole-quinolines or a cyanine of benzoxazole-quinolines; (e) a phenothiazine selected from Methylene and derivatives thereof, or Dicarboxymethylene Blue NHS ester (DCMB-SE); and (f) an anthroquinone selected from anthracyclines and derivatives thereof, Daunorubicin, Doxorubicin, Mitoxantrone, Losoxantrone, Pixantrone, Pirarubicin, anthroquinone analogs, optionally anthraquinone-2-amidopentyl carboxylic acid NHS ester; and wherein step a comprises: (i) selecting an intercalator, a minor groove binder, a major groove binder, or any combination thereof that will bind to the target macromolecule; and (ii) modifying the intercalator, minor groove binder, major groove binder, or combination thereof to include a linker group to produce the affinity ligand capable of binding to the surface.
  2. The method of claim 1, wherein the target macromolecule is double stranded DNA, single stranded DNA, double stranded RNA, single stranded RNA, double stranded messenger RNA, single stranded messenger RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA) protein containing an oligonucleotide or oligonucleoside, lipid containing an oligonucleotide or oligonucleoside, other oligonucleotide or oligonucleoside, any fragment thereof, or any combination thereof.
  3. The method of claim 1, wherein the target macromolecule is DNA, RNA, any fragment thereof, or any combination thereof, optionally wherein the target macromolecule is double stranded DNA or double stranded RNA.
  4. The method of claim 1, further comprising the step of: f. collecting an eluent that is substantially free of the target macromolecule; or of f. eluting and recovering the target macromolecule from the coupled surface-affinity ligand.
  5. The method of claim 1, wherein the affinity ligand is a cyanine of benzothiazole-quinolines or benzoxazole-quinolines selected from the Sybr Green family of dyes, optionally Sybr Green I ( N',N '-dimethyl- N -[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]- N -propylpropane-1,3-diamine), Sybr Green II, Sybr Gold, and Sybr Safe (( Z )-4-((3-Methylbenzo[ d ]thiazol-2(3 H )-ylidene)methyl)-1-propylquinolin-1-ium 4-methylbenzenesulfonate)), the TOTO ™ family of dyes and derivatives thereof, the YOYO ™ family of dyes and derivatives thereof, the YO-PRO ™ family of dyes and derivatives thereof, the TO-PRO ™ family of dyes and derivatives thereof, the POPO ™ family of dyes and derivatives thereof, the BOBO ™ family of dyes and derivatives thereof, the LOLO ™ family of dyes and derivatives thereof, the JOJO ™ family of dyes and derivatives thereof, Thiazole Orange and derivatives thereof, Oxazole Yellow and derivatives thereof, Pico Green and derivatives thereof, and LightCycler ® Green and Red family of dyes.
  6. The method of claim 1, wherein the affinity ligand is a modified cyanine of a benzothiazole-quinoline, or a modified cyanine of a benzoxazole-quinoline.
  7. The method of claim 6, wherein the affinity ligand is the cyanine of benzothiazole-quinoline, or cyanine of benzoxazole-quinoline that has been modified to include a spacer ending in an epoxy, carboxy, halide or amino group.
  8. The method of claim 6, wherein the affinity ligand is a modified cyanine of benzothiazole-quinoline or a modified cyanine of a benzoxazole-quinoline that has been modified to include an epoxy group, carboxy group, or spacer ending in an amino or halide, and wherein the spacer contains 1-30 atoms.
  9. The method of claim 6, wherein the affinity ligand is a modified cyanine of a benzothiazole-quinoline, or a modified cyanine of a benzoxazole-quinoline selected from Sybr Green family of dyes, optionally Sybr Green I ( N',N' -dimethyl- N -[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]- N- propylpropane-1,3-diamine), Sybr Green II, Sybr Gold, and Sybr Safe (( Z )-4-((3-Methylbenzo[ d ]thiazol-2(3 H )-ylidene)methyl)-1-propylquinolin-1-ium 4-methylbenzenesulfonate)), the TOTO ™ family of dyes and derivatives thereof, the YOYO ™ family of dyes and derivatives thereof, the YO-PRO ™ family of dyes and derivatives thereof, the TO-PRO ™ family of dyes and derivatives thereof, the POPO ™ family of dyes and derivatives thereof, the BOBO ™ family of dyes and derivatives thereof, the LOLO ™ family of dyes and derivatives thereof, the JOJO ™ family of dyes and derivatives thereof, Thiazole Orange and derivatives thereof, Oxazole Yellow and derivatives thereof, Pico Green and derivatives thereof, and LightCycler ® Green and Red family of dyes, and which has been modified to include a spacer ending in an epoxy, carboxy, halide or amino group, wherein the spacer contains 1-30 atoms.
  10. The method of claim 1 or 6, wherein the affinity ligand is Thiazole Orange, Sybr Green, or Oxazole Yellow.
  11. The method of claim 1, wherein the solid surface is a bead, membrane, particle, mesh, polymer, glass, metal, ceramic, silica, polysaccharide, monolith, or any other material used as a resin in chromatography, optionally wherein the solid surface includes a functionalized group.
  12. The method of claim 11, wherein the solid surface includes a functionalized group and wherein the functionalized group comprises an epoxy, a carboxy, an aldehyde, or an amino group.
  13. The method of claim 11, wherein the solid surface is an amino-agarose bead, or wherein the solid surface is an aldehyde membrane.
  14. The method of any of claims 1-13, wherein the method is used in chromatography.
  15. The method of claim 1, wherein the target macromolecule is DNA and the sample contains DNA and other nucleic acids, or wherein the target macromolecule is RNA and the sample contains RNA and other nucleic acids.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/939,934, filed on November 25, 2019. The entire contents of the foregoing application are incorporated by reference herein in its entirety. FIELD OF THE INVENTION This disclosure relates to a method of separating, isolating and removing target macromolecules, such as DNA and RNA, from a feed stream, or generically, a sample, using specifically selected affinity ligands bound to a surface. BACKGROUND OF THE INVENTION Chromatography, as it is generally used, is a technique for the separation of various components of a sample mixture. In a liquid chromatography system, a sample followed by an elution fluid is injected into a chromatographic separation column. The separation column contains a packing or matrix medium or material which interacts with the various components of the sample to be separated. The composition of the separating medium depends on the fluid being directed therethrough to effect the desired separation. As the sample and elution fluids pass through the separating medium, the various components of the sample travel at different rates through the separating medium as a result of differential interactions. These components emerge separated in the outlet or effluent from the separation medium. Various types of the vertical and horizontal flow separation columns are known in the art. With the need for high performance chromatography, horizontal flow type chromatographic columns were developed. Such horizontal or radial flow columns are described in, e.g., U.S. Patent Nos. 4,627,918 and 4,676,898. In the horizontal or radial flow type columns, the sample and elution fluids are introduced via a distributor to the outer periphery or circumferential wall or surface of the separating medium or matrix, and the fluids pass horizontally or radially inwardly through the separation medium to a central or collection port and then elute from the column at different times and at different rates. Later, chromatographic columns and methods were developed for direct processing of crude feeds for isolation of biologically active materials, including cell/fermentation harvest, tissue extracts, and plasma/blood. The large bead chromatography media are packed into a standard, low pressure chromatography column in which end-plate screens are replaced with large pore screens (60-180 µm pores). The large pores prevent column blockage. Because particle sizes are large, the cellular material flows between the beads in the interparticle lumen, while the soluble product is captured by functional groups on the beads. Traditionally, downstream processing of biologics from cell culture/fermentation harvests has required two major operations: recovery and purification. Recovery involves the removal of cellular and other particulate materials by centrifugation and/or microfiltration, as well as an initial volume reduction step, typically ultrafiltration. Since conventional chromatography media are rapidly fouled by cell debris, particle-free feed must be prepared for the purification operation. In certain purification processes of (therapeutic) biological preparations (such as monoclonal antibodies), the sample/product is produced via a living cell system (mammalian, bacterial, moss, algae, plant, etc.) and the product is either secreted into the feed stream by the cells or the cells are broken up to release the product into the surrounding liquid. However, in all of these production systems, the product is not available as a single component pure product, but is a very complex mix of the desired product and "contaminants," which includes host cell protein (HCP) and genomic DNA and RNA. During purification, HCP and DNA/RNA must be removed from the purified product to levels that are below limits set by regulatory authorities (such as the FDA). This purification is usually achieved by anion ion exchange methods. During filtration and purification of samples containing proteins, it may be difficult to separate the proteins from other cell components and host proteins without degenerating the protein of interest, and also to isolate the target proteins. A common method is through precipitation; however, this may lead to denaturation or degeneration of the protein which may result in a loss of protein function. Refolding of the protein often leads to a loss in activity. Fast Protein Liquid Chromatography (FPLC) is a common method employed in protein purification. Without having to denature the protein, using high pressure or aggressive pH-buffers, proteins may be purified using solely specific interactions between the buffer and the protein of interest for isolation. There remains a need for a system for removal and isolation of DNA and RNA, both double and single stranded, from a feed stream. SUMMARY OF THE INVENTION A method of separating a target macromolecule from a sample is disclosed, comprisin