Multivalent halide salts for use in hydrophobic interaction chromatography

Abstract

The present invention relates to the use of multivalent halide salts in hydrophobic interaction chromatography. The present invention further relates to a method for purifying biomolecules using hydrophobic interaction chromatography with multivalent halide salts. The present invention further relates to a use of a chromatography support in a method according to the invention. The present invention further relates to a Kit for the purification of biomolecules comprising multivalent halide salts. The present invention also relates to a method for purifying biomolecules, use of a multivalent halide salt for purifying biomolecules, use of a chromatography support for purifying biomolecules, and use of a kit for purifying biomolecules.

Description

[0001] MULTIVALENT HALIDE SALTS FOR USE IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY FIELD OF THE INVENTION

[0002] The present invention relates to the use of multivalent halide salts in hydrophobic interaction chromatography. The present invention further relates to a method for purifying biomolecules using hydrophobic interaction chromatography with multivalent halide salts. The present invention further relates to a use of a chromatography support in a method according to the invention. The present invention further relates to a kit for the purification of biomolecules comprising multivalent halide salts. The present invention further relates to a method for purifying biomolecules, use of a multivalent halide salt for purifying biomolecules, use of a chromatography support for purifying biomolecules, use of a kit for purifying biomolecules.

[0003] BACKGROUND OF THE INVENTION

[0004] The production of plasmid DNA (pDNA) is a critical process step in the development and manufacturing of DNA and mRNA vaccines as well as gene therapy viral vectors. The production process usually involves fermentation, followed by lysis, several filtration and chromatographic steps to obtain the pDNA purity required by regulatory agencies.

[0005] The usual process includes that, after fermentation, bacterial cells are harvested and lysed with caustic reagent and detergent (typically NaOH / SDS) to release the target pDNA, but also other cellular components obtained as impurities, such as endotoxins, host-cell RNA (hcRNA), host-cell proteins and host-cell DNA (hcDNA). After neutralization of alkaline lysate, CaC12 can be used to precipitate a large amount of host-cell RNA, but this step never achieves 100% RNA removal (Eon Duval 2003). Residual RNA is chromatographically removed from DNA with anion exchange chromatography, resulting in highly enriched DNA.

[0006] Following the capture step, the pDNA commonly undergoes a “polishing” step to isolate the desired supercoiled (SC) isoform, as well as to remove residual endotoxins and residual proteins. Polishing is typically achieved with hydrophobic interaction chromatography (HIC). The technique utilizes differences in hydrophobicity between different DNA isoforms under certain binding or elution conditions. The efficiency of this separation is highly dependent on the type of salt used in the process. Kosmotropic salts, such as ammonium sulfate (NH4)2SO4, sodium sulfate, potassium phosphate and sodium or potassium citrate are used due to their ability to effectively remove residual RNA, unwanted plasmid isoforms, residual endotoxin, and proteins (Gagnon (2020), Freitas (2009)).

[0007] It is known to use kosmotropic salts as mobile phase modifier for hydrophobic interaction chromatography. Kosmotropic salts are thought of as a type of salt that stabilizes water structure and increases the ordering of water molecules around hydrophobic surfaces. These salts have a strong ability to promote hydrophobic interactions and stabilize macromolecules like proteins, often by increasing the strength of hydrogen bonds within the water network. Kosmotropic salts include, but are not limited to, ammonium sulfate, sodium sulfate, potassium phosphate, sodium citrate, and potassium citrate. In hydrophobic interaction chromatography, kosmotropic salts are often used in the mobile phase to enhance the hydrophobic interactions between the biomolecules and the stationary phase, promoting their binding to the column. As the salt concentration is reduced, the hydrophobic interactions weaken, leading to the elution of the bound molecules. A commonly used mobile phase modifier in hydrophobic interaction chromatography is ammonium sulfate.

[0008] However, the use of ammonium sulfate poses serious challenges due to the difficulties associated with the disposal of ammonium sulfate. When ammonium sulfate enters natural bodies of water, it leads to eutrophication of the water ecosystem, promoting unwanted growth of algae and other organisms which may lead to a disruption of the ecosystem. Use of ammonium sulfate may therefore be associated with significant environmental issues, and there is a need in the art for environmentally friendly alternatives.

[0009] SUMMARY OF THE INVENTION

[0010] Against this background, it is a first object of the present invention to provide a mobile phase modifier for hydrophobic interaction chromatography applications that overcomes the disadvantages lined out above while maintaining satisfactory functionality and quality of the chromatography results achieved.

[0011] It is a second object of the present invention to provide improved means and methods for purifying biomolecules such as pDNA, specifically SC pDNA.

[0012] The first object named above is solved in accordance with the first aspect of the present invention by a use of at least one multivalent halide salt as mobile phase modifier in hydrophobic interaction chromatography. In the first aspect of the invention, the environmentally problematic ammonium sulfate is exchanged for less problematic salts such as CaC12 as loading salt for HIC, with significant environmental benefits over ammonium sulfate.

[0013] The second object is solved in accordance with the second aspect of the present invention by a method for purifying biomolecules, the method comprising:

[0014] (a) mixing a sample comprising the biomolecules and one or more impurities with the multivalent halide salt to precipitate one or more of the impurities; and

[0015] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support.

[0016] The object named above is further solved in accordance with the second aspect of present invention by use of a multivalent halide salt for purifying biomolecules in a hydrophobic interaction chromatography; use of a chromatography support for purifying biomolecule; and use of a kit for purifying biomolecules.

[0017] In the second aspect, the present invention describes a biomolecule purification process which harnesses the two-fold function of multivalent halide salts such as CaC12 as precipitating agent, and its unexpected property as a loading salt for hydrophobic interaction binding of the biomolecule or the impurities to a support. For example, after a multivalent halide salt (such as CaC12) RNA reduction following the lysis step, the CaC12-comprising sample can de directly incubated with a hydrophobic interaction support to either capture the biomolecule or to bind impurities, by using the multivalent halide salt as the loading salt to promote selective binding of pDNA or impurities onto the support.

[0018] The benefits of the invention according to the second aspect are several-fold: The number of unit operations can be reduced and at least one chromatographic step can be removed in the process (figure 10 presents the classical pDNA process (fig. 10 A) compared to an exemplary process according to the second aspect of the invention, e.g., a pDNA purification process using CaC12in the lysate as binding buffer for a hydrophobic interaction step such as the hydrophobic interaction chromatography (HIC) capture step (fig. 10 B). Additionally, the process volume can be reduced (e.g., 6-10-fold), the selective binding results in highly pure (e.g., >95%) biomolecule in a single chromatographic step, and ammonium sulfate does not need to be used in the process. Also the endotoxin removal to below the limit of quantification (<LOQ) can be achieved in a single chromatographic step.

[0019] DETAILED DESCRIPTION

[0020] Although certain embodiments of the present invention are described in detail below, it is to be understood that this invention is not limited to the particular embodiments, methodologies, protocols and reagents described herein as these may vary within the scope set by the claims. It is also to be understood that terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which is defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0021] In the following description, certain elements of the present invention will be described. These elements may be discussed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples, features and particular embodiments should not be construed to limit the present invention to only the explicitly described embodiments or to the explicitly described combination of features. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by this description unless the context indicates otherwise.

[0022] The above objects are achieved by the following embodiments in accordance with the first aspect of the invention:

[0023] 1. Use of at least one multivalent halide salt as mobile phase modifier in hydrophobic interaction chromatography.

[0024] 2. Use according to embodiment 1 of the first aspect, wherein the at least one multivalent halide salt comprises divalent and / or trivalent metal cations, preferably Mg, Ca, Sr, Al, Fe, and / or Ba, more preferably Mg and / or Ca cations.

[0025] 3. Use according to embodiment 1 or 2 of the first aspect, wherein the at least one multivalent halide salt comprises F anions, Cl anions, Br anions, I anions, or At anions, or any combination thereof, preferably F and / or Cl anions.

[0026] 4. Use according to any one of the preceding embodiments of the first aspect, wherein the at least one multivalent halide salt is MgC12 and / or CaC12. 5. Use according to any one of the preceding embodiments of the first aspect, wherein the at least one multivalent halide salt is present at a total maximum concentration of 0.25 M - 5.0 M.

[0027] 6. Use according to any one of the preceding embodiments of the first aspect, wherein the at least one multivalent halide salt is present in at least one buffer having a pH value in the range from 2.5 - 11.

[0028] 7. Use according to any one of the preceding embodiments of the first aspect, wherein the mobile phase comprises less than 0.5 mol / L kosmotropic salt, preferably less than 0.05 mol / L kosmotropic salt, more preferably wherein the mobile phase is essentially free from kosmotropic salt.

[0029] 8. Use according to any one of the preceding embodiments of the first aspect for the purification of biomolecules, wherein preferably the biomolecules are selected from the group consisting of nucleic acids, for example plasmid DNA, polypeptides, viruses, and virus-like particles.

[0030] 9. Method for purifying biomolecules using hydrophobic interaction chromatography, the method comprising:

[0031] (a) passing a sample comprising the biomolecules through a chromatography support, wherein the sample further comprises at least one multivalent halide salt at a first concentration, and

[0032] (b) passing a first solution through the chromatography support, wherein the first solution comprises the at least one multivalent halide salt at a second concentration,

[0033] (c) wherein the second concentration is lower than the first concentration.

[0034] 10. Method according to embodiment 9 of the first aspect, wherein the biomolecule is a nucleic acid, preferably plasmid DNA, and / or a polypeptide. 11. Method according to embodiment 9 or 10 of the first aspect, wherein the sample additionally comprises a buffer to maintain the pH in the range of 2.5 - 11.

[0035] 12. Method according to any one of embodiments 9 to 11, wherein the at least one multivalent halide salt is present in the sample at a total concentration of 0.25 - 5.0 M.

[0036] 13. Method according to any one of embodiments 9 to 12 of the first aspect, wherein the at least one multivalent halide salt is present in the sample at a total concentration of 0.25 - 5.0 M,

[0037] (a) wherein preferably, passing the first solution through the chromatography support comprises applying a decreasing salt gradient of the at least one halide salt, wherein preferably the concentration of the multivalent halide salt is decreased from an initial concentration of 0.25 - 5 M to a final concentration of 0 - 1 M, wherein preferably, the decrease is carried out in a continuous or step-wise fashion.

[0038] 14. Method according to any one of embodiments 9 to 13 of the first aspect, wherein the chromatography support is suitable for hydrophobic interaction chromatography or reverse-phase chromatography, preferably wherein the chromatography support comprises neutral ligands selected from the group consisting of alkyl, aryl, cycloalkyl, hydroxy, epoxy, ether, amide and ester , and / or multimodal hydrophobic-hydrogen bonding ligands such as pyridine, further preferably wherein the chromatography support is a porous particle support, a membrane support, a monolith support or a nanofiber support.

[0039] 15. Use of a chromatography support in a method according to any one of embodiments 9 to 14 of the first aspect.

[0040] 16. Use according to embodiment 15 of the first aspect, wherein the chromatography support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl. 17. Kit for the purification of biomolecules comprising:

[0041] (a) a chromatography support comprising hydrophobic ligands, and (b) at least one multivalent halide salt.

[0042] 18. Kit according to embodiment 17 of the first aspect, wherein the at least one multivalent halide salt is present in a buffer solution, preferably in the form of a stock solution.

[0043] 19. Kit according to embodiment 17 or 18 of the first aspect, wherein the hydrophobic ligands are selected from the group consisting of alkyl, aryl and cycloalkyl or any combinations thereof.

[0044] The above objects are achieved by the following embodiments in accordance with the second aspect of the invention:

[0045] 1. Method for purifying biomolecules, the method comprising:

[0046] (a) mixing a sample comprising the biomolecules and one or more impurities with at least one multivalent halide salt to precipitate one or more of the impurities; and

[0047] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support.

[0048] 2. Method according to embodiment 1 of the second aspect, wherein in step (b) impurities bind to the hydrophobic interaction support, optionally by using a solid phase adsorption or powder assisted adsorption.

[0049] 3. Method according to embodiment 1 or 2 of the second aspect, wherein the hydrophobic interaction support comprises a particle support, a nanoparticle support, a fiber support, a nanofiber support, a gel support, a sheet support, a film support, a membrane support, a monolith support, or any combination thereof. Method according to any of the embodiments 2-3 of the second aspect, wherein the hydrophobic interaction support comprises a nanoparticle or particle support or any combination thereof, optionally selected from powders, granules, or particles or any combination thereof.

[0050] Method according to any of the embodiments 2-4 of the second aspect, wherein the hydrophobic interaction support comprises porous, non-porous, or semi-porous material or any combination thereof.

[0051] Method according to any of the embodiments 2-5 of the second aspect, wherein the hydrophobic interaction support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl or any combinations thereof; optionally comprising butyl ligands.

[0052] Method according to any of the embodiments 2-6 of the second aspect, wherein the hydrophobic interaction support comprises ligands selected from the group consisting of polymers, polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins, polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene-ABS, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, or polyisobutylene-PIB or any combination thereof.

[0053] Method according to any of the embodiments 2-7 of the second aspect, wherein in step (b) the sample and the support are agitated to promote binding of the impurities to the hydrophobic interaction support, optionally by aeration, stirring, shaking, swirling, churning, rocking, or any combination thereon. 9. Method according to any of the embodiments 2-8 of the second aspect, further comprising a removal step of the hydrophobic interaction support, optionally by filtration, centrifugation, sedimentation, or the like.

[0054] 10. Method according to any of the embodiments 2-9 of the second aspect, wherein the hydrophobic interaction support is washed with a washing solution containing the multivalent halide salt, optionally wherein the multivalent halide salt has the same concentration as in the sample from step (b).

[0055] 11. Method according to any of the embodiments 2-10 of the second aspect, further comprising

[0056] (c) passing the sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support.

[0057] 12. Method according to embodiment 11 of the second aspect, wherein the chromatography is a mixed-mode chromatography, hydrophobic interaction chromatography, an ion exchange chromatography, a size exclusion chromatography, an affinity chromatography, an immobilized metal affinity chromatography or the like.

[0058] 13. Method according to embodiment 11 or 12 of the second aspect, further comprising eluting the biomolecules to obtain purified biomolecules.

[0059] 14. Method according to any of the embodiments 2-13 of the second aspect, wherein step (b) is a flow-through step.

[0060] 15. Method according to embodiment 1 of the second aspect, wherein in step (b) the biomolecules bind to the hydrophobic interaction support.

[0061] 16. Method according to embodiment 15 of the second aspect, wherein the concentration of the multivalent halide salt is adjusted prior the incubation, optionally wherein the concentration of the multivalent halide salt is increased; optionally wherein the concentration of the multivalent halide salt is increased following the flow-through step.

[0062] 17. Method according to embodiment 15 or 16 of the second aspect, further comprising in step (b):

[0063] (bl) passing the sample through the hydrophobic interaction support, wherein the sample further comprises the multivalent halide salt at a first concentration, and

[0064] (b2) passing a first solution through the support, wherein the first solution comprises the multivalent halide salt at a second concentration,

[0065] wherein the second concentration is lower than the first concentration; optionally wherein step (b) comprises steps and embodiments described for the method for purifying biomolecules according to the first aspect, for example in embodiment 9 of the first aspect.

[0066] 18. Method according to any of embodiments 15-17 of the second aspect, wherein the hydrophobic interaction support is suitable for hydrophobic interaction chromatography, preferably wherein the chromatography support comprises neutral ligands selected from the group consisting of alkyl, aryl, cycloalkyl, hydroxy, epoxy, ether, amide and ester, and / or multimodal hydrophobic-hydrogen bonding ligands such as pyridine.

[0067] 19. Method according to any of the preceding embodiments of the second aspect, wherein the multivalent halide salt comprises divalent and / or trivalent metal cations, preferably Mg, Ca, Sr, Al, Fe, and / or Ba, more preferably Mg and / or Ca cations.

[0068] 20. Method according to any of the preceding embodiments of the second aspect, wherein the multivalent halide salt comprises F anions, Cl anions, Br anions, I anions, or At anions, or any combination thereof, preferably F and / or Cl anions.

[0069] 21. Method according to any of the preceding embodiments of the second aspect, wherein the multivalent halide salt is MgC12 and / or CaC12. 22. Method according to any of the preceding embodiments of the second aspect, wherein in step (a) the multivalent halide salt is present at a concentration of at least about 0.25 M, optionally from about 0.5 M to about 3 M, preferably from about 0.75 M to about 3 M.

[0070] 23. Method according to any of the preceding embodiments of the second aspect, wherein in step (b) the multivalent halide salt is present at a concentration of at least about 0.25 M, optionally from about 0.5 M to about 3 M, preferably from about 0.75 M to about 3 M.

[0071] 24. Method according to any of the preceding embodiments of the second aspect, wherein in step (a) the sample has a pH value in the range from about 4 to about 9, preferably pH is about 4.5-5.5, preferably about 5.

[0072] 25. Method according to any of the preceding embodiments of the second aspect, wherein in step (b) the sample has a pH value in the range from about 4 to about 9, preferably pH is about 4.5-5.5, preferably about 5.

[0073] 26. Method according to any of the preceding embodiments of the second aspect, wherein step (b) is not preceded by an anionic exchange chromatography, optionally wherein the method does not comprise an anionic exchange chromatography.

[0074] 27. Method according to any of the preceding embodiments of the second aspect, wherein the impurities comprise cellular components, optionally wherein the cellular components comprise RNA, endotoxins, proteins, open-circular plasmid DNA, linear pDNA, or genomic DNA or any combination thereof.

[0075] 28. Method according to any of the preceding embodiments of the second aspect, wherein the impurities are derived from lysed cells producing the biomolecule.

[0076] 29. Method according to any of the preceding embodiments of the second aspect, wherein the biomolecules are selected from the group consisting of nucleic acids, optionally DNA, polypeptides, viruses, and virus-like particles or combinations thereof, optionally wherein the biomolecules comprise plasmid DNA, optionally supercoiled plasmid DNA.

[0077] 30. Method according to any of the preceding embodiments of the second aspect, wherein the sample comprises less than 0.5 mol / L ammonium sulfate, optionally less than 0.05 mol / L ammonium sulfate, more optionally wherein the mobile phase is essentially free from ammonium sulfate.

[0078] 31. Use of a multivalent halide salt for purifying biomolecules in a hydrophobic interaction chromatography, optionally according to a method of any one of embodiments 1 to 30 of the second aspect.

[0079] 32. Use of a chromatography support for purifying biomolecules in a method according to any one of embodiments 1 to 30 of the second aspect.

[0080] 33. Use according to embodiment 32 of the second aspect, wherein the chromatography support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl, preferably butyl ligand.

[0081] 34. Use of a Kit for purifying biomolecules optionally in a method according to any one of embodiments 1 to 30 of the second aspect, the kit comprising:

[0082] a hydrophobic interaction support;

[0083] at least one multivalent halide salt; and

[0084] a chromatography support comprising ligands for hydrophobic interaction chromatography, anion exchange chromatography or mixed mode chromatography.

[0085] 35. Use of a kit according to embodiment 34 of the second aspect, wherein the multivalent halide salt is present in a buffer solution, preferably in the form of a stock solution.

[0086] 36. Use of a kit according to embodiment 34 or 35 of the second aspect, wherein the hydrophobic interaction support comprises ligands selected from the group consisting of polymers, polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins, polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene-ABS, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, or polyisobutylene-PIB or any combination thereof.

[0087] Definitions

[0088] The terms indicated for explanation of the invention have the following meaning, unless otherwise indicated in the description or the claims. Additional definitions are set forth throughout the detailed description.

[0089] Terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0090] The terms “about” or “approximately” as used herein denotes a range of ±10% of a reference value. For examples, “about 10” defines a range of 9 to 11. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context.

[0091] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0092] In the context of the present invention, the term “biomolecule” is intended to be understood as an organic molecule that is produced by a living organism or a synthetic procedure. For example, a biomolecule may be a nucleic acid such as DNA or RNA, or a polypeptide such as a peptide or a protein.

[0093] Unless expressly specified otherwise, the term “comprising” is used in the context of the present disclosure to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as specific embodiments of the present invention that each time the term “comprising” is used, this shall also encompass the possibility of no further members being present, i.e., for the purpose of such specific embodiments “comprising” can be understood as having the meaning of “consisting of’.

[0094] In the context of the present invention, the term “sample” is intended to be understood as a mixture of biomolecules that is introduced into a support (e.g., chromatography support) for separation and purification based on their hydrophobic properties. Generally, the sample further comprises a buffer containing salt, which in accordance with the present invention is a multivalent halide salt. For example, a sample may further comprise impurities and / or contaminants such as endotoxins or other biomolecules which need to be removed during the separation and purification process.

[0095] In the context of the present invention, the terms “support” and “chromatography support” are intended to be understood as the solid matrix or material that serves as the foundation for the stationary phase e.g., within a chromatography column. The support is typically functionalized with hydrophobic ligands that interact with the sample’s hydrophobic molecules, facilitating their separation based on differences in hydrophobicity. For example, the support may be present in the form of a monolith, a membrane, packed or loose granules, fibers, or a porous gel. The chromatography support is also referred to herein as “support”.

[0096] The term "plasmid" refers to an extrachromosomal, mostly circular DNA capable of being expressed in a given cell. Plasmids can also be engineered by standard molecular biology techniques (Sambrook et al., Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), N.Y.). In the context of the present invention, the term “halide” is intended to be understood as a compound comprising a halogen in form of a negatively charged ion (anion). Halogens include fluorine (F), chlorine (Cl), bromine (Br), iodine (I), and astatine (At). Halides therefore comprise one or more of the anions F’, CF, Br, I’, and At".

[0097] In the context of the present invention, the term “salt” is intended to be understood as an ionic compound consisting of positively charged cations and negatively charged anions that are held together by ionic bonds. A salt can be present in complexed form, e.g. in form of a hydrate, or can be water-free. In the context of the present invention, a “halide salt” is a salt comprising an anion derived from a halogen.

[0098] In the context of the present invention, the term “multivalent” is intended to be understood as the property of a metal to form multivalent cations, i.e. cations having a valency of more than 1. For example, a multivalent cation may be divalent (i.e.: have a charge of +2), trivalent (i.e.: have a charge of +3), tetraval ent (i.e.: have a charge of +4), or higher.

[0099] Examples of multivalent cations include, but are not limited to, Mg2+, Ca2+, Al3+, and Fe3+.

[0100] In the context of the present disclosure, the term “multivalent halide salt” is intended to be understood as an ionic compound consisting of a multivalent cation and at least one halide anion. Examples include MgC12, CaC12, CaBr2, and FeC13.

[0101] In the context of the present disclosure, the term “at least one multivalent halide salt” is intended to be understood as exactly one multivalent halide salt or a mixture of 2 or more multivalent halide salts, such as a mixture of MgC12 and CaC12.

[0102] In the context of the present disclosure, the term “hydrophobic interaction chromatography” (HIC) is intended to be understood as a type of liquid chromatography used to separate and purify nucleic acids, polypeptides, and other biomolecules based on their hydrophobicity. A hydrophobic interaction chromatography generally comprises a mobile phase and a stationary phase. For example, the mobile phase may be a flowing liquid and the stationary phase may consist of a solid chromatography support onto which hydrophobic ligands are covalently attached. For example, the mobile phase may contain an aqueous solution with varying concentrations of salt. In the context of the present invention, the term “mobile phase modifier” is intended to be understood as a chemical agent added to the mobile phase of a chromatography to influence the interaction between the stationary phase, the mobile phase, and the compound of interest. For example, the mobile phase modifier may reduce the hydrophobic interactions between the compound of interest and the stationary phase, facilitating the elution or flow-through of the compound from / through the chromatography support. Alternatively, the mobile phase modifier may enhance the hydrophobic interactions, thereby promoting the retention of the target molecule in the stationary phase while allowing other compounds to flow through the column.

[0103] The first aspect of the invention

[0104] The first object is solved in accordance with the first aspect of the present invention by a use of at least one multivalent halide salt as mobile phase modifier in hydrophobic interaction chromatography.

[0105] The research leading up to the invention has shown that multivalent halide salts, which are traditionally classified as chaotropic salts, can be successfully used as a mobile phase modifier in hydrophobic interaction steps such as hydrophobic interaction chromatography. This result is especially surprising since chaotropic salts are known to disrupt the structure of water and weaken the hydrophobic interactions within and between molecules, which is why multivalent halide salts have not been considered for use in hydrophobic interaction chromatography. In accordance with the present disclosure, it is generally preferred that the multivalent halide salt is used as the primary and / or only mobile phase modifier.

[0106] Using multivalent halide salts as mobile phase modifiers in hydrophobic interaction steps (such as chromatography) has the advantage that waste streams can be discarded without the risk of environmental damage due to eutrophication. At the same time, a dynamic binding capacity in HIC applications that is comparable to the one obtained with ammonium sulfate can be achieved by using multivalent halide salts, and they may be used to selectively retain and elute certain types of biomolecules, i.e. to separate a compound of interest from unwanted impurities such as endotoxins, for example. The object named above is further solved in accordance with the first aspect of present invention by a method for purifying biomolecules using hydrophobic interaction chromatography, the method comprising:

[0107] (a) passing a sample comprising the biomolecules through a chromatography support, wherein the sample further comprises at least one multivalent halide salt at a first concentration, and

[0108] (b) passing a first solution through the chromatography support, wherein the first solution comprises the at least one multivalent halide salt at a second concentration, wherein the second concentration is lower than the first concentration.

[0109] In the context of the present invention, the first solution may be understood as a wash or elution solution. In general, the first solution is an aqueous solution comprising at least one multivalent halide salt at a second concentration or multiple second concentrations (e.g., in case of a salt gradient) which is / are lower than the first concentration.

[0110] Therein, it is appreciated that a skilled person is capable of routinely optimizing the first and second concentrations depending on the binding and eluting behavior of the biomolecule of interest and the binding and eluting behavior of other components of the sample such as impurities and contaminants.

[0111] For example, the first concentration may be selected to allow the biomolecule of interest to flow through the chromatography support while more hydrophobic impurities / contaminants are bound by the chromatography support (flow-through mode). In this case, the chromatography support may subsequently be flushed with a washing solution comprising the at least one multivalent halide salt at approx, the first concentration, preferably at the same concentration as present in the sample, to remove any remaining unbound biomolecule of interest from the support. Subsequently, any specifically and non-specifically bound molecules such as impurities or contaminants may be removed from the chromatography support by flushing the chromatography support with a washing solution free from multivalent halide salt and / or a sanitizing solution which may comprise sodium hydroxide, for example. For example, the first concentration may be selected to allow the biomolecule of interest to bind to the chromatography support while less hydrophobic impurities / contaminants pass through the chromatography support without binding to it (bind-elute mode). In this case, the chromatography support may subsequently be flushed with a washing solution comprising the at least one multivalent halide salt at approx, the first concentration (same concentration as the sample) or a higher concentration to remove any unbound impurities from the support. Subsequently, the biomolecule of interest is eluted from the support by passing through the first solution which contains the multivalent halide salt at a second concentration which is lower than the first concentration, e.g. a constantly lower concentration or a decreasing salt gradient. Without wishing to be bound by theory, it is currently assumed that the biomolecule is precipitated onto the support when present in a solution comprising the high first concentration of multivalent halide salt and is solubilized - and thus eluted from the support - in a solution comprising the lower second concentration of multivalent halide salt. After eluting the biomolecule of interest, any specifically and non-specifically bound molecules such as impurities or contaminants may be removed from the chromatography support by flushing the support with a washing solution free from multivalent halide salt and / or a sanitizing solution which may comprise sodium hydroxide, for example.

[0112] For example, before passing the sample through the chromatography support, the support may be flushed with a preconditioning buffer comprising the at least one multivalent halide salt, preferably at approx, the first concentration.

[0113] The object named above is further solved in accordance with the present invention by a use of a chromatography support in a method according to the invention.

[0114] In this respect, any suitable chromatography support described herein may be used in the hydrophobic interaction chromatography method according to the invention.

[0115] The object named above is further solved in accordance with the present invention by a kit for the purification of biomolecules comprising a chromatography support comprising hydrophobic ligands, and at least one multivalent halide salt. In this respect, any chromatography support described herein that is suitable for hydrophobic interaction chromatography may be part of the kit according to the invention. Moreover, it is generally appreciated that the multivalent halide salt is present in the kit in a sufficient amount or concentration that allows for carrying out a method according to the invention using the kit, in particular a method where the at least one multivalent halide salt is used as mobile phase modulator in a hydrophobic interaction chromatography workflow.

[0116] Various embodiments of the use of multivalent halide salts in HIC, the HIC method, the use of a chromatography support in the HIC method and the Kit are described in the following. The individual embodiments are in each case individually applicable to the above-named aspects of the present invention. The individual embodiments may furthermore be combined with each other at will.

[0117] In an exemplary embodiment, the at least one multivalent halide salt comprises divalent and / or trivalent metal cations, preferably Mg, Ca, Sr, Al, Fe, and / or Ba, more preferably Mg and / or Ca cations.

[0118] For example, the multivalent halide salt may comprise metal cations from the elements in the second or 13thgroup of the periodic table, as well as from transition metals (i.e.: group 3 to 12) from the 4thperiod. Exemplary cations include Mn2+, Fe2+, Fe3+, Co2+, Co3+, Ni2+, Cu2+, Zn2+, Al3+, Ba2+, Sr2+, Be2+, Ca2+, and Mg2+.

[0119] It is generally appreciated that the multivalent halide salt needs to have a sufficient solubility in water to be used according to the invention.

[0120] In an exemplary embodiment, the at least one multivalent halide salt comprises F anions, Cl anions, Br anions, I anions, or At anions, or any combination thereof, preferably Cl anions.

[0121] For example, a suitable multivalent halide salt may be selected from the group consisting of CaC12, MgC12, MgBr2, CaBr2, CaI2, MgI2, FeI2, FeBr3, BaBr2, and BaI2.

[0122] In one embodiment, the at least one multivalent halide salt is MgC12 and / or CaC12. In an embodiment, the at least one multivalent halide salt is present at a total maximum concentration of 0.25 M - 5.0 M.

[0123] In the context of the present disclosure, the term “total (...) concentration” is intended to be understood as the sum of concentrations of all multivalent halide salts. For example, if 2 M MgC12 and 1 M CaC12 are present, the total concentration is 3 M multivalent halide salt.

[0124] In the context of the present disclosure, the term “maximum concentration” is intended to be understood as the highest concentration value in which the multivalent halide salt is used. For example, if the multivalent halide salt is used in a first solution at a concentration of 4 M and in a second solution at a concentration of 2 M, the maximum concentration is 4 M. For example, in a decreasing salt gradient starting at 4 M and ending at 0.8 M, the maximum concentration is 4 M.

[0125] For example, the total maximum concentration may correspond to the concentration of a saturated solution of the at least one multivalent halide salt in water.

[0126] In the research leading up to the invention, it was found that in the context of hydrophobic interaction chromatography, the dynamic binding capacity of biomolecules such as plasmid DNA (pDNA) is dependent on multivalent halide salt concentration in the mobile phase. For example, at neutral pH, a dynamic binding capacity allowing for selecting supercoiled pDNA over open circular pDNA may be achieved in a concentration range of 1.5 to 2.5 M MgCh or 1.25 to 2 M CaC12.

[0127] In an exemplary embodiment, the at least one multivalent halide salt is present in at least one buffer having a pH value in the range from 2.5 - 11.

[0128] In this context, the term “buffer” is meant to include any aqueous solution, which may further comprise a buffering compound and / or a biomolecule of interest (also referred to as a “sample”).

[0129] In the research leading up to the invention, it was found that in the context of hydrophobic interaction chromatography, the dynamic binding capacity for biomolecules such as pDNA achieved by use of multivalent halide salts depends in part on the pH value. For example, at an acidic or alkaline pH value, the at least one multivalent halide salt may provide a high biomolecule binding capacity at a lower concentration than when used at neutral pH.

[0130] For example, the pH value may be selected depending on the biomolecule of interest, e.g. according to an isoelectric point of said biomolecule.

[0131] For example, if the at least one multivalent halide salt is present in more than one buffer, these pH values of these buffers may differ from one another or may be the same.

[0132] In an exemplary embodiment, the mobile phase comprises less than 0.5 mol / L kosmotropic salt, preferably less than 0.05 mol / L kosmotropic salt, more preferably wherein the mobile phase is essentially free from kosmotropic salt.

[0133] In the context of the present disclosure, the term “kosmotropic salt” is intended to be understood as a salt whose ions have a tendency to stabilize and order water molecules, promoting stronger water-water interactions, wherein the anion is generally understood to have the primary influence. The degree of kosmotropic influence is determined by the commonly known Hofmeister series. Kosmotropic salts are traditionally used in hydrophobic interaction chromatography. Exemplary kosmotropic salts include, but are not limited to, ammonium sulfate, sodium sulfate, potassium phosphate, sodium citrate, and potassium citrate. Therein, it is generally appreciated that the concentration of kosmotropic salt corresponds to the concentration of kosmotropic anions (sulfate, phosphate, citrate, pyrophosphate) in the mobile phase.

[0134] In this context, the term “essentially free” is intended to be understood in that the mobile phase containing multivalent halide salt contains only trace amounts of kosmotropic salt or none at all, to the extent that the remaining kosmotropic salt has no significant impact on the chemical, physical, or functional properties of the mobile phase. For example, “essentially free” may mean that the mobile phase contains kosmotropic salt in a concentration of 10 mM or less, 5 mM or less, or 1 mM or less. Surprisingly, the research leading up to the invention has shown that mixtures of kosmotropic salts and the multivalent halide salts of the present invention are not preferred in concentrations sufficient for hydrophobic interaction chromatography, because they often result in precipitation of insoluble species. For example, a mixture of calcium chloride (CaC12) and the kosmotropic salt ammonium sulfate leads to the precipitation of calcium sulfate even at starting concentrations as low as 0.1 M which are insufficient for HIC applications.

[0135] In an exemplary embodiment, the invention may be used for the purification of biomolecules, wherein preferably the biomolecules are selected from the group consisting of nucleic acids, for example plasmid DNA, polypeptides, viruses, and virus-like particles.

[0136] In the context of the present disclosure, the term “nucleic acid” is intended to be understood as polynucleotides consisting of ribonucleic acids (RNA) or deoxyribonucleic acids (DNA). For example, nucleic acids may be of natural or synthetic origin. For example, nucleic acids may encompass different topological forms such as supercoiled, open circular or linear constructs. A particular example of a nucleic acid is a plasmid, which is a circular polynucleotide that is usually present in supercoiled form.

[0137] In the context of the present disclosure, the term “polypeptide” is intended to mean linear or branched chains of amino acids linked together by peptide bonds. For example, a polypeptide may have a specific three-dimensional structure or may be unfolded, i.e. have a non-specific structure. A particular example of a polypeptide is a protein, i.e., a polypeptide which performs a biological function.

[0138] In the context of the present disclosure, the term “virus” is intended to mean a submicroscopic infectious agent composed primarily of genetic material (either DNA or RNA) encased within a protein coat (capsid). Some viruses further comprise an additional lipid envelope.

[0139] In the context of the present disclosure, the term “virus-like particle” (VLP) is intended to refer to a molecular structure that closely resembles a virus but lacks the viral genome, making it non-infectious. For example, a virus-like particle may be composed of viral proteins that can self-assemble into structures that mimic a virus's capsid or envelope but do not contain the nucleic acid required for replication.

[0140] In an exemplary embodiment of the chromatography method according to the invention, the biomolecule is a nucleic acid, preferably plasmid DNA, and / or a polypeptide.

[0141] In an exemplary embodiment of the chromatography method according to the invention, the sample additionally comprises a buffer to maintain the pH in the range of 2.5 - 11. It is understood that the pH is measured at room temperature (20 °C).

[0142] Therein, the compound and concentration of the buffer compound is not crucial. It is, however, preferred that the buffering compound does not include a kosmotropic anion such as sulfate or phosphate as these tend to precipitate with the multivalent halide salts according to the invention.

[0143] In an exemplary embodiment of the method according to the invention, the at least one multivalent halide salt is present in the sample at a total concentration of 0.25 - 5.0 M.

[0144] In an exemplary embodiment of the method according to the invention, also referred to herein as “bind-elute mode”, the at least one multivalent halide salt is present in the sample at a total concentration of 0.25 - 5 M. For example, when passing the sample through a chromatography support, the biomolecule of interest is retained on the support while impurities and contaminants end up in the flowthrough or bind to the chromatographic support.

[0145] In this embodiment, it is preferred that passing the first solution through the chromatography support comprises applying a decreasing salt gradient of the at least one halide salt, whereby impurities and contaminants are gradually removed from the chromatography support while the biomolecule of interest stays bound to the support. Once the salt concentration reaches a certain value which depends on the biomolecule of interest, it separates from the support and can be collected in the flowthrough. By further decreasing the salt concentration, the remaining impurities and contaminants are at least partially removed from the support. In this embodiment, it is preferred that the concentration of the multivalent halide salt is decreased from an initial concentration of 0.25 - 5 M to a final concentration of 0 - 1 M. Therein preferably, the decrease is carried out in a continuous (e.g.: linear, exponential or sigmoidal decrease of the salt concentration) or step-wise (e.g.: steps of 0.2 M salt concentration) fashion.

[0146] In an exemplary embodiment of the method according to the invention, the chromatography support is suitable for hydrophobic interaction chromatography or reverse-phase chromatography. Therein, it is preferred that the chromatography support comprises neutral ligands selected from the group consisting of alkyl, aryl, cycloalkyl, hydroxy, epoxy, ether, amide and ester, and / or multimodal hydrophobic-hydrogen bonding ligands such as pyridine. It is further preferred that the chromatography support is a porous particle support, a membrane support, a monolith support or a nanofiber support.

[0147] For example, the chromatography support may be a hydrophobic resin, e.g. comprising porous particles. As described herein, a hydrophobic resin is any material to which the target biomolecule (e.g., an oligonucleotide) will bind such that it can be separated from impurities and contaminants in a method according to the invention. For example, the hydrophobic adsorbent may include hydrophilic carbohydrates, such as cross-linked agarose and synthetic copolymer materials. According to the present invention, the hydrophobic adsorbent comprises either phenyl or hexyl. For example, Hexyl650C is a suitable hydrophobic adsorbent. For example, a hydrophobic adsorbent may comprise butyl. Furthermore, the hydrophobic adsorbent may be packed into a column or similar structure at any bed height.

[0148] In an exemplary embodiment, the chromatography support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl.

[0149] In the context of the present invention, the term "alkyl" is intended to refer to a fully saturated branched or unbranched hydrocarbon moiety. For example, the alkyl may comprise 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some embodiments, an alkyl comprises from 6 to 20 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, or n-decyl.

[0150] In the context of the present invention, the term "alkenyl" is intended to refer to an unsaturated hydrocarbon group which may be linear or branched and has at least one carbon-carbon double bond. Alkenyl groups with 2-6 carbon atoms may be preferred. The alkenyl group may contain 1, 2 or 3 carbon-carbon double bonds, or more. Examples of alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and the like.

[0151] In the context of the present invention, the term "alkynyl" is intended to refer to an unsaturated hydrocarbon group which may be linear or branched and has at least one carbon-carbon triple bond. Alkynyl groups with 2-6 carbon atoms may be preferred. The alkynyl group may contain 1, 2 or 3 carbon-carbon triple bonds, or more. Examples of alkynyl groups include ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like.

[0152] In the context of the present invention, the term "aryl" is intended to refer to monocyclic, bicyclic or tricyclic aromatic hydrocarbon groups having from 6 to 14 carbon atoms in the ring portion. In one embodiment, the term aryl refers to monocyclic and bicyclic aromatic hydrocarbon groups having from 6 to 10 carbon atoms. Representative examples of aryl groups include phenyl, naphthyl, fluorenyl, and anthracenyl. The term "aryl" is also intended to refer to a bicyclic or tricyclic group in which at least one ring is aromatic and is fused to one or two non-aromatic hydrocarbon ring(s). Nonlimiting examples include tetrahydronaphthalene, dihydronaphthalenyl and indanyl. An "arylalkyl" is intended to refer to an aryl group linked via an alkylene linker to the reminder of the molecule. An "alkaryl" is intended to refer to an alkyl group linked via an arylene linker to the reminder of the molecule.

[0153] In the context of the present invention, the term "cycloalkyl" is intended to refer to a fully saturated cyclic hydrocarbon moiety. For example, the cycloalkyl may comprise 3 to 12 carbon atoms, more preferably 3 to 8 carbon atoms, or 3 to 6 carbon atoms. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be substituted or unsubstituted, and the ring may be either monocyclic or polycyclic.

[0154] In an exemplary embodiment of the kit according to the present disclosure, the at least one multivalent halide salt is present in a buffer solution, preferably in the form of a stock solution.

[0155] For example, the buffer solution, or preferably the stock solution, may comprise the at least one multivalent halide salt at a concentration sufficient for carrying out a hydrophobic interaction chromatography when mixed with the sample to be used.

[0156] In an exemplary embodiment of the kit according to the present disclosure, the hydrophobic ligands are selected from the group consisting of alkyl, aryl and cycloalkyl or any combinations thereof.

[0157] The second aspect of the invention

[0158] In a second aspect of the invention, a method for purifying biomolecules is provided, the method comprising:

[0159] (a) mixing a sample comprising the biomolecules and one or more impurities with at least one multivalent halide salt to precipitate one or more of the impurities; and

[0160] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the at least one multivalent halide salt with a hydrophobic interaction support.

[0161] The research leading up to the invention has shown that multivalent halide salts, traditionally used for precipitating impurities, can be successfully used in a subsequent hydrophobic interaction step such as a hydrophobic interaction chromatography to remove remaining impurities. This result is surprising since the further removal of impurities were thought to require a more complex process such as an adjustment of the binding conditions and an anion-exchange chromatography. Chaotropic salts were known to disrupt the structure of water and weaken the hydrophobic interactions within and between molecules, and therefore multivalent halide salts have not been considered for use in hydrophobic interactions, especially in a sample containing impurities remaining after the RNA precipitation. The present invention showed that the hydrophobic interaction can be successfully used even in samples containing impurities following the precipitation.

[0162] The research has unexpectedly shown that the hydrophobic interaction step can be used to bind and elute the target biomolecules without requiring any further removal of remaining impurities. This is unexpected as the remaining impurities were though to prevent sufficient binding of the target molecule to the hydrophobic interaction support.

[0163] As used herein, the biomolecules which should be purified using the disclosed method is also referred to as “target biomolecules”, “target biomolecule”, “biomolecule of interest” or “biomolecules of interest”. In some embodiments, these target biomolecules nucleic acids, such as RNA or DNA. In some embodiments, these target biomolecules are pDNA, in particular SC pDNA.

[0164] It is preferred that the at least one multivalent halide salt is used as the primary and / or only mobile phase modifier during the hydrophobic interaction step.

[0165] In some embodiments, incubating the sample with the hydrophobic interaction support comprises passing the sample through the support to allow for binding of the impurities or the biomolecules to the support. In some embodiments, incubating the sample with the hydrophobic interaction support consists of passing the sample through the support to allow for binding of the impurities or the biomolecules to the support.

[0166] In some embodiments, impurities bind to the hydrophobic interaction support in step (b). In this context, it is understood that the step further reduces the concentration of impurities in the sample. However, some impurities may remain in the sample.

[0167] In some embodiments, the hydrophobic interaction support can be used in various forms (e.g., as resin, ground monolith or other form of hydrophobic media or other particulate material with hydrophobic properties, such as, but not limited to, CIM C4 HLD, CIM C4A, CIM OH, CIM SDVB, CaptoPhenyl, Phenyl Sepharose, Octyl Sepharose™ High Performance, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl, Macro-Prep™ Methyl, Macro-Prep™ t-Butyl, WP Hl-Propyl (C3)™, Toyopearl™ ether, Toyopearl™ phenyl, Toyopearl™ butyl, ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen Octyl FF, and Tosoh Hexyl). In some embodiments, the hydrophobic interaction support can comprise various polymers (e.g. polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins , polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene-ABS, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, polyisobutylene-PIB). The hydrophobic interaction support can be added and optionally mixed in multivalent halide salt-treated lysate by stirring, shaking, aeration or other means of movement, to enhance precipitation of impurities prior to a capture step via the same mechanism (multivalent halide salt at a specific concentration and pH promotes binding of impurities to the hydrophobic interaction support, but not target biomolecules, thereby protecting the support from binding the impurities and thereby increasing the binding capacity for target biomolecules). This multivalent halide salthydrophobic interaction pretreatment can be used prior to the target biomolecules capture by any chromatographic approach, including, but not limited to anion exchange chromatography (AEX), HIC, multimodal chromatography (MMC), size exclusion chromatography (SEC), immobilized metal affinity chromatography (IMAC) or other target biomolecules capture techniques known in the art.

[0168] In some embodiments, the hydrophobic interaction support can be used in multiple forms (resin, ground monolith or other form of hydrophobic media or other particulate material with hydrophobic properties, such as, but not limited to, CIM C4 HLD, CIM C4A, CIM OH, CIM SDVB, Phenyl Sepharose, CaptoPhenyl, Phenyl Sepharose, Octyl Sepharose™ High Performance, Fractogel™ EMD Propyl, Fractogel™ EMD Phenyl, Macro-Prep™ Methyl, Macro-Prep™ t-Butyl, WP Hl-Propyl (C3)™, Toyopearl™ ether, Toyopearl™ phenyl, Toyopearl™ butyl, ToyoScreen PPG, ToyoScreen Phenyl, ToyoScreen Butyl, ToyoScreen Hexyl, HiScreen Butyl FF, HiScreen Octyl FF, and Tosoh Hexyl). In some embodiments, the hydrophobic interaction support can comprise various polymers (polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins , polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene-ABS, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, polyisobutylene-PIB). The hydrophobic interaction support can be added and optionally mixed in CaC12-treated lysate by stirring, shaking, aeration or other means of movement, to enhance clearance of residual endotoxin, residual host-cell RNA and residual host-cell proteins prior to a capture step via the same mechanism (CaC12 at a specific concentration and pH promotes binding of ETX, RNA, protein and the like to the hydrophobic interaction support, but not pDNA, thereby protecting the capture column from binding the impurities and thereby increasing the binding capacity for SC pDNA). This CaC12- hydrophobic interaction pretreatment can be used prior to the pDNA capture by any chromatographic approach, including, but not limited to anion exchange chromatography (AEX), HIC, multimodal chromatography (MMC), size exclusion chromatography (SEC), immobilized metal affinity chromatography (IMAC) or other pDNA capture techniques known in the art.

[0169] Without wishing to be limited by theory, such sample pretreatment avoids the need for packaging columns with hydrophobic interaction media while providing full benefit of hydrophobic interaction selectivity for impurities, by performing a flow-through step in solution. After binding, hydrophobic interaction support can be removed from the sample with centrifugation, sedimentation or filtration e.g., by using appropriate filter pore size to prevent passing of particles. This approach can particularly be favorable for continuous lysis coupled with continuous filtration. In some embodiments, the hydrophobic interaction support is provided in a flow-through cartridge to be used in conjunction with continuous lysis and filtration. In some embodiments, step (b) comprises or is a flow-through step. In some embodiments, a solid phase adsorption or powder assisted adsorption is used in step (b).

[0170] In some embodiments, the sample is derived from a previously performed cell-lysis. For example, the method can comprise:

[0171] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0172] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0173] (a3) mixing the sample comprising the biomolecules and the impurities with at least one multivalent halide salt to precipitate one or more of the impurities; and

[0174] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support.

[0175] The lysis can be performed using lysis agents known to the person skilled in the art. For example, the lysis can be an alkaline lysis, optionally using NaOh and / or sodium dodecyl sulfate (SDS). The lysis step can comprise a neutralization step, using a neutralization agent known to the person skilled in the art. An exemplary neutralization agent is potassium acetate (CH3COOK).

[0176] In an exemplary embodiment, the multivalent halide salt comprises divalent and / or trivalent metal cations, preferably Mg, Ca, Sr, Al, Fe, and / or Ba, more preferably Mg and / or Ca cations. For example, the multivalent halide salt may comprise metal cations from the elements in the second or 13thgroup of the periodic table, as well as from transition metals (i.e.: group 3 to 12) from the 4thperiod. Exemplary cations include Mn2+, Fe2+, Fe3+, Co2+, Co3+, Ni2+, Cu2+, Zn2+, Al3+, Ba2+, Sr2+, Be2+, Ca2+, and Mg2+. In an exemplary embodiment, the at least one multivalent halide salt comprises F anions, Cl anions, Br anions, I anions, or At anions, or any combination thereof, preferably Cl anions.

[0177] For example, a suitable multivalent halide salt may be selected from the group consisting of CaC12, MgC12, MgBr2, CaBr2, CaI2, MgI2, FeI2, FeBr3, BaBr2, and BaI2. It is generally appreciated that the multivalent halide salt needs to have a sufficient solubility in water to be used according to the invention. The precipitation can be achieved using any suitable multivalent halide salt that can be selected from the group consisting of CaC12, MgC12, MgBr2, CaBr2, CaI2, MgI2, FeI2, FeBr3, BaBr2, and BaI2. In some embodiments, the multivalent halide salt comprises MgC12 and / or CaC12. In some embodiments, the multivalent halide salt comprises CaC12.In some embodiments, the multivalent halide salt is MgC12 and / or CaC12 (e.g., multivalent halide salt can be a mixture of MgC12 and CaC12). In some embodiments, the multivalent halide salt is CaC12.

[0178] In some embodiments, the sample is derived from a previously performed cell-lysis. For example, the method can comprise:

[0179] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0180] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0181] (a3) mixing the sample comprising the biomolecules and the impurities with the multivalent halide salt to precipitate one or more of the impurities; and

[0182] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support.

[0183] In some embodiments, the sample is derived from a previously performed cell-lysis. For example, the method can comprise:

[0184] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0185] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0186] (a3) mixing the sample comprising the biomolecules and the impurities with MgCh to precipitate one or more of the impurities; and

[0187] (b) incubating the sample comprising the biomolecules, any remaining impurities, and MgCh with a hydrophobic interaction support.

[0188] In some embodiments, the method can comprise: (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0189] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0190] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities; and

[0191] (b) incubating the sample comprising the biomolecules, any remaining impurities, and CaC12 with a hydrophobic interaction support.

[0192] In some embodiments, the sample is filtrated following the lysing step.

[0193] In some embodiments, the hydrophobic interaction support comprises a particle support, a nanoparticle support, a fiber support, a nanofiber support, a gel support, a sheet support, a film support, a membrane support, a monolith support, or any combination thereof.

[0194] In some embodiments, the hydrophobic interaction support comprises a nanoparticle or particle support or a combination thereof. The hydrophobic interaction support can be selected from powders, granules, or particles or any combination thereof. For example, the hydrophobic interaction support can comprise or consist of a polymer powder, polymer granules, or polymer particles, or any combination thereof.

[0195] In some embodiments, the hydrophobic interaction support comprises a porous, non-porous, or semi-porous material or any combination thereof.

[0196] In some embodiments, the hydrophobic interaction support has a high surface area to optimize the interaction surface between the target molecules (e.g. impurities or the target biomolecules) and the hydrophobic ligands.

[0197] In some embodiments, the hydrophobic interaction support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl or any combinations thereof; optionally comprising butyl ligands. In some embodiments, the hydrophobic interaction support comprises ligands selected from the group consisting of polymers, polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins, polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene- AB S, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, or polyisobutylene-PIB or any combination thereof.

[0198] In some embodiments, the ligands comprise or consist of polymers. In some embodiments, the ligands comprise or consist of polysulfone (PES). In some embodiments, the ligands comprise or consist of polystyrene (PS). In some embodiments, the ligands comprise or consist of polyethylene (PE). In some embodiments, the ligands comprise or consist of polypropylene (PP). In some embodiments, the ligands comprise or consist of polyvinyl chloride (PVC). In some embodiments, the ligands comprise or consist of polyvinyl fluoride (PVF). In some embodiments, the ligands comprise or consist of poly(methyl methacrylate) (PMMA). In some embodiments, the ligands comprise or consist of polyoxymethylene (POM). In some embodiments, the ligands comprise or consist of polycarbonate (PC). In some embodiments, the ligands comprise or consist of nylon. In some embodiments, the ligands comprise or consist of polyethylene terephthalate (PET). In some embodiments, the ligands comprise or consist of epoxy resins. In some embodiments, the ligands comprise or consist of polyvinylidene chloride (PVDC). In some embodiments, the ligands comprise or consist of polyvinylidene fluoride (PVDF). In some embodiments, the ligands comprise or consist of polyphenylene sulfide (PPS). In some embodiments, the ligands comprise or consist of acrylonitrile butadiene styrene (ABS). In some embodiments, the ligands comprise or consist of poly(n-butyl methacrylate) (PnBMA). In some embodiments, the ligands comprise or consist of polytrifluoroethylene. In some embodiments, the ligands comprise or consist of polybutadiene. In some embodiments, the ligands comprise or consist of polychlorotrifluoroethylene (PCTFE). In some embodiments, the ligands comprise or consist of polydimethylsiloxane (PDMS). In some embodiments, the ligands comprise or consist of fluorinated ethylene propylene (FEP). In some embodiments, the ligands comprise or consist of hexatriacontane. In some embodiments, the ligands comprise or consist of paraffin. In some embodiments, the ligands comprise or consist of polytetrafluoroethylene (PTFE). In some embodiments, the ligands comprise or consist of hexafluoropropylene. In some embodiments, the ligands comprise or consist of polyisobutylene (PIB). In some embodiments, the ligands comprise or consist of any combination of the above.

[0199] In some embodiments, in step (b) the sample and the support are agitated to promote binding of the impurities to the hydrophobic interaction support, optionally by aeration, stirring, shaking, swirling, churning, rocking, any combination thereof, or any suitable agitation known to the person skilled in the art.

[0200] In some embodiments, step (b) can comprise iterative steps. In some embodiments, the method comprises two or more steps of incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with the hydrophobic interaction support. For example, the sample can be incubated with the support e.g., in a flow-through step, wherein the obtained flow-through can again be incubated with the hydrophobic interaction support. In some embodiments, the incubation step can be repeated 2, 3, 4, or 5 times to further reduce the concentration of impurities in the sample. In some embodiments, the incubation step can be repeated at least 2, at least 3, at least 4, or at least 5 times. In some embodiments, the incubation step can be repeated at least 6, at least 7, at least 8, or at least 9 times.

[0201] In some embodiments, the multivalent halide salt concentration is adjusted in the obtained flow-through. In some embodiments, the multivalent halide salt concentration is increased in the obtained flow-through, e.g., in order to adjust the conditions needed for binding the target biomolecule to a chromatography support (bind and elute).

[0202] The incubation in step (b) can be performed using any suitable incubation time. In some embodiments, the sample can be incubated with the hydrophobic interaction support for about 1-240 minutes. In some embodiments, the sample can be incubated with the hydrophobic interaction support for about 1-120 minutes. In some embodiments, the sample can be incubated with the hydrophobic interaction support for about 1-60 minutes. In some embodiments, the sample can be incubated with the hydrophobic interaction support for 1-5 minutes. In some embodiments, the sample can be incubated for about 10-30 minutes. In some embodiments, the incubation period may range from about 45-60 minutes. In some embodiments, the sample can be incubated for about 90-120 minutes. In some embodiments, the sample can be incubated for about 180-240 minutes. In some embodiments, the sample can be incubated for more than 240 minutes. In some embodiments, the sample can be incubated for 10 minutes, 15 minutes, or 30 minutes. In some embodiments, the sample can be incubated for 45 minutes, 60 minutes, or 75 minutes.

[0203] The mixing in step (a) can be performed using any suitable incubation time. In some embodiments, the sample can be mixed with the multivalent halide salt for about 1-240 minutes. In some embodiments, the sample can be mixed for about 1-120 minutes. In some embodiments, the sample can be mixed with for about 1-60 minutes. In some embodiments, the sample can be mixed for 1-5 minutes. In some embodiments, the sample can be mixed for about 10-30 minutes. In some embodiments, the mixed period may range from about 45-60 minutes. In some embodiments, the sample can be mixed for about 90-120 minutes. In some embodiments, the sample can be mixed for about 180-240 minutes. In some embodiments, the sample can be mixed for more than 240 minutes. In some embodiments, the sample can be mixed for 10 minutes, 15 minutes, or 30 minutes. In some embodiments, the sample can be mixed for 45 minutes, 60 minutes, or 75 minutes.

[0204] In some embodiments, the method comprises removing the hydrophobic interaction support, optionally by filtration, centrifugation, sedimentation, or any suitable step known to the person skilled in the art. For example, the hydrophobic interaction support can be removed by filtering the sample through a 0.45 pm filter 8such as a PES filter) to remove the support (e.g., CIMasphere particles) and obtain fine filtrate. The method can comprise: (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0205] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0206] (a3) mixing the sample comprising the biomolecules and the impurities with at least one multivalent halide salt to precipitate one or more of the impurities; (bl) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support, optionally to bind one or more impurities to the support; and

[0207] (b2) removing the hydrophobic interaction support to obtain a recovered sample.

[0208] In some embodiments, the recovered sample comprises the biomolecules and the multivalent halide salt. The recovered sample may further comprise impurities wherein the concentration of impurities is lower compared to the sample prior the incubation step.

[0209] In some embodiments, the hydrophobic interaction support is washed with a washing solution containing the multivalent halide salt, optionally wherein the multivalent halide salt has the same concentration as in the sample from step (b). The support may be washed with a washing solution comprising the multivalent halide salt at about the same concentration as present in the sample in step (b) to remove unbound target biomolecules.

[0210] In some embodiments, before passing the sample through the support, the support can be flushed with a preconditioning buffer solution that contains at least one multivalent halide salt. Preferably, the multivalent halide salt in the solution has the same concentration as in the sample.

[0211] In some embodiments, the method comprises:

[0212] (c) passing the sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support.

[0213] In some embodiments, the method comprises:

[0214] (a) mixing the sample comprising the biomolecules and the impurities with at least one multivalent halide salt to precipitate one or more of the impurities;

[0215] (b) incubating the sample comprising the biomolecules, remaining impurities, and the multivalent halide salt with a hydrophobic interaction support to bind the impurities to the support; and

[0216] (c) passing the sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support. In this context it is understood that the support in step (b) and (c) can be different supports.

[0217] In some embodiments, the multivalent halide salt concentration does not need to be adjusted before incubating the sample with the hydrophobic interaction support. In some embodiments, the salt concentration of the sample does not need to be adjusted between step (a) and (b). In some embodiments, the salt concentration of the sample is not adjusted between step (a) and (b). In some embodiments, the salt concentration of the sample is adjusted between step (b) and (c).

[0218] In some embodiments, the method comprises:

[0219] (a) mixing the sample comprising the biomolecules and the impurities with at least one multivalent halide salt to precipitate one or more of the impurities;

[0220] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support;

[0221] (b2) removing the hydrophobic interaction support to obtain a recovered sample; and (с) passing the recovered sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support.

[0222] In some embodiments, the method comprises:

[0223] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0224] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0225] (аз) mixing the sample comprising the biomolecules and the impurities with at least one multivalent halide salt to precipitate one or more of the impurities;

[0226] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support;

[0227] (b2) removing the hydrophobic interaction support to obtain a recovered sample; and (c) passing the recovered sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support.

[0228] In some embodiments, the method comprises: (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0229] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0230] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0231] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and CaC12 with a hydrophobic interaction support;

[0232] (b2) removing the hydrophobic interaction support to obtain a recovered sample; and (c) passing the recovered sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support.

[0233] In some embodiments, the precipitation is an RNA precipitation. Meaning, in some embodiments, the step of mixing the sample comprising the biomolecules (e.g., nucleic acids such as SC pDNA) and the impurities with the multivalent halide salt (e.g., CaC12) results in an RNA precipitation.

[0234] In some embodiments, the sample is not further purified (e.g. by further removal of impurities) following the precipitation of the impurities and before incubating the sample with the hydrophobic interaction support.

[0235] In some embodiments, the sample is filtered following the precipitation of the impurities and before incubating the sample with the hydrophobic interaction support.

[0236] In some embodiments, the chromatography in step (c) is a multimodal chromatography, hydrophobic interaction chromatography, an ion exchange chromatography, a size exclusion chromatography, an affinity chromatography, an immobilized metal affinity chromatography or the like. In some embodiments, the chromatography is a multimodal chromatography. In some embodiments, the chromatography is a hydrophobic interaction chromatography. In some embodiments, the chromatography is an ion exchange chromatography. In some embodiments, the chromatography is an anion exchange chromatography. In some embodiments, the chromatography is a cation exchange chromatography. In some embodiments, the chromatography is not a multimodal chromatography. In some embodiments, the chromatography is not an anion exchange chromatography. In some embodiments, the chromatography is selected form an anion exchange chromatography (AEX), a HIC, a multimodal chromatography (MMC), a size exclusion chromatography (SEC), ab immobilized metal affinity chromatography (IMAC) or other target biomolecules capture techniques known in the art.

[0237] In some embodiments, step (c) comprises eluting the biomolecules to obtain purified biomolecules.

[0238] In some embodiments, the method comprises:

[0239] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0240] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0241] (a3) mixing the sample comprising the biomolecules and the impurities with the multivalent halide salt to precipitate one or more of the impurities;

[0242] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support;

[0243] (b2) removing the hydrophobic interaction support to obtain a recovered sample;

[0244] (cl) passing the recovered sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support; and

[0245] (c2) eluting the biomolecules to obtain purified biomolecules.

[0246] In some embodiments, the method comprises:

[0247] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0248] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0249] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0250] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and the CaC12 with a hydrophobic interaction support;

[0251] (b2) removing the hydrophobic interaction support to obtain a recovered sample; (cl) passing the recovered sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support; and

[0252] (c2) eluting the biomolecules to obtain purified biomolecules.

[0253] In some embodiments, the method comprises:

[0254] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0255] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0256] (a3) mixing the sample comprising the biomolecules and the impurities with multivalent halide salt to precipitate one or more of the impurities;

[0257] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and multivalent halide salt with a hydrophobic interaction support;

[0258] (b2) removing the hydrophobic interaction support to obtain a recovered sample (such as a recovered flow-through); optionally adjusting the multivalent halide salt concentration; (cl) passing the sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support; and

[0259] (c2) eluting the biomolecules to obtain purified biomolecules, optionally by using a elution solution comprising a lower multivalent halide salt concentration compared to the multivalent halide salt concentration in the recovered flow-through.

[0260] In some embodiments, the method comprises:

[0261] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0262] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0263] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0264] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and CaC12 with a hydrophobic interaction support;

[0265] (b2) removing the hydrophobic interaction support to obtain a recovered sample (such as a recovered flow-through); optionally adjusting the CaC12 concentration; (cl) passing the recovered sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support; and

[0266] (c2) eluting the biomolecules to obtain purified biomolecules, optionally by using a elution solution comprising a lower multivalent halide salt concentration compared to the CaC12 concentration in the recovered flow-through.

[0267] In some embodiments, the method comprises:

[0268] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0269] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0270] (a3) mixing the sample comprising the biomolecules and the impurities with multivalent halide salt to precipitate one or more of the impurities;

[0271] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and multivalent halide salt with a hydrophobic interaction support;

[0272] (b2) removing the hydrophobic interaction support to obtain a recovered sample (such as a recovered flow-through); optionally adjusting the multivalent halide salt concentration; (cl) passing the sample through the hydrophobic interaction support, wherein the sample further comprises the multivalent halide salt at a first concentration; and

[0273] (c2) passing a first solution through the support, wherein the first solution comprises the multivalent halide salt at a second concentration, wherein the second concentration is lower than the first concentration; and

[0274] In some embodiments, the method comprises:

[0275] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0276] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0277] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0278] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and CaC12 with a hydrophobic interaction support; (b2) removing the hydrophobic interaction support to obtain a recovered sample (such as a recovered flow-through); optionally increasing the CaC12 concentration;

[0279] (cl) passing the recovered sample through the hydrophobic interaction support, wherein the sample further comprises the CaC12 at a first concentration; and

[0280] (c2) passing a first solution through the support, wherein the first solution comprises the CaC12 at a second concentration, wherein the second concentration is lower than the first concentration.

[0281] In some embodiments, the method comprises:

[0282] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0283] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0284] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0285] (bl) incubating the sample comprising the biomolecules, any remaining impurities, and CaC12 with a hydrophobic interaction support;

[0286] (b2) removing the hydrophobic interaction support to obtain a recovered sample (such as a recovered flow-through), increasing the CaC12 concentration;

[0287] (cl) passing the recovered sample through the hydrophobic interaction support, wherein the sample further comprises the CaC12 at a first concentration; and

[0288] (c2) passing a first solution through the support, wherein the first solution comprises the CaC12 at a second concentration, wherein the second concentration is lower than the first concentration; and

[0289] In step (c2) the purified biomolecules can be obtained (eluted).

[0290] In some embodiments, the concentration of the multivalent halide salt is adjusted prior to passing the sample through a chromatography support to bind the biomolecules to the support. In some such embodiments, the concentration of the multivalent halide salt is increased. In some embodiments, in step (b) the biomolecules bind to the hydrophobic interaction support. For example, in step (b) the sample is incubated with the support, allowing the biomolecules to pass and bind the support.

[0291] In some embodiments, the method comprises:

[0292] (a) mixing the sample comprising the biomolecules and the impurities with the multivalent halide salt to precipitate one or more of the impurities, followed by adjusting the multivalent halide salt concentration;

[0293] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support; and

[0294] (c) eluting the biomolecules to obtain purified biomolecules.

[0295] In some embodiments, the method comprises:

[0296] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0297] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0298] (a3) mixing the sample comprising the biomolecules and the impurities with the multivalent halide salt to precipitate one or more of the impurities; optionally adjusting the multivalent halide salt concentration;

[0299] (b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support; and

[0300] (c) eluting the biomolecules to obtain purified biomolecules.

[0301] In some embodiments, the method comprises:

[0302] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0303] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0304] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0305] (b) incubating the sample comprising the biomolecules, any remaining impurities, and CaC12 with a hydrophobic interaction support; and (c) eluting the biomolecules to obtain purified biomolecules.

[0306] In some embodiments, the concentration of the multivalent halide salt is adjusted prior the incubation, optionally wherein the concentration of the multivalent halide salt is increased.

[0307] In some embodiments, the method further comprising in step (b):

[0308] (bl) passing the sample through the hydrophobic interaction support, wherein the sample further comprises the multivalent halide salt at a first concentration; and (b2) passing a first solution through the support, wherein the first solution comprises the multivalent halide salt at a second concentration,

[0309] wherein the second concentration is lower than the first concentration.

[0310] Without wishing to be bound by theory, it is currently assumed that the biomolecule is precipitated onto the support (or bind the support) when present in a solution comprising the higher first concentration of multivalent halide salt and is solubilized - and thus eluted from the support - in a solution comprising the lower second concentration of multivalent halide salt. After eluting the target biomolecule, any specifically and non-specifically bound molecules such as impurities or contaminants may be removed from the chromatography support by flushing the support with a washing solution free from multivalent halide salt and / or a sanitizing solution which may comprise sodium hydroxide, for example.

[0311] It is appreciated that a skilled person is capable of routinely optimizing the first and second concentrations depending on the binding and eluting behavior of the target biomolecules and the binding and eluting behavior of other components of the sample such as impurities and contaminants.

[0312] For example, the first concentration may be selected to allow the target biomolecules to bind to the chromatography support while less hydrophobic impurities / contaminants pass through the chromatography support without binding to it (bind-elute mode). In this case, the chromatography support may subsequently be flushed with a washing solution comprising the at least one multivalent halide salt at approx, the first concentration (same concentration as the sample) or a higher concentration to remove any unbound impurities from the support. Subsequently, the target biomolecules can be eluted from the support by passing through the first solution which contains the multivalent halide salt at a second concentration which is lower than the first concentration, e.g. a constantly lower concentration or a decreasing salt gradient.

[0313] Before passing the sample through the chromatography support, the support may be flushed with a preconditioning buffer comprising the at least one multivalent halide salt, preferably at approx, the first (higher) concentration.

[0314] In some embodiments, the method comprises steps and embodiments described herein for the first aspect of the invention. In this context, it is understood that the first and second aspect of the invention can be combined.

[0315] In some embodiments, the method comprises:

[0316] (a) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities;

[0317] (b) passing the sample comprising the biomolecules through a chromatography support, wherein the sample further comprises the multivalent halide salt at a first concentration; and (c) passing a first solution through the chromatography support, wherein the first solution comprises the multivalent halide salt at a second concentration, wherein the second concentration is lower than the first concentration.

[0318] In this context it is understood that the step of passing the sample comprising the biomolecules through a chromatography support results in an incubation of the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with the support.

[0319] In some embodiments, the method comprises:

[0320] (al) providing a sample comprising cells, wherein the cells comprise the biomolecules which should be purified;

[0321] (a2) lysing the cells to obtain a sample comprising the biomolecules and one or more impurities;

[0322] (a3) mixing the sample comprising the biomolecules and the impurities with CaC12 to precipitate one or more of the impurities; (b) passing the sample comprising the biomolecules through a chromatography support, wherein the sample further comprises the CaC12 at a first concentration; and

[0323] (c) passing a first solution through the chromatography support, wherein the first solution comprises the CaC12 at a second concentration, wherein the second concentration is lower than the first concentration.

[0324] In some embodiments, the sample comprises a buffer to maintain the pH in the range of 2.5 - 11. It is understood that the pH is measured at room temperature (20 °C). Therein, the compound and concentration of the buffer compound is not crucial. It is, however, preferred that the buffering compound does not include a kosmotropic anion such as sulfate or phosphate as these tend to precipitate with the multivalent halide salts.

[0325] In some embodiments, the multivalent halide salt is present in the sample at a total concentration of 0.25 - 5.0 M.

[0326] In some embodiments, the at least one multivalent halide salt is present in the sample at a total concentration of 0.25 - 5.0 M, wherein preferably, passing the first solution through the chromatography support comprises applying a decreasing salt gradient of the at least one halide salt, wherein preferably the concentration of the multivalent halide salt is decreased from an initial concentration of 0.25 - 5 M to a final concentration of 0 - 1 M, wherein preferably, the decrease is carried out in a continuous or step-wise fashion.

[0327] In some embodiments, the chromatography support is suitable for hydrophobic interaction chromatography or reverse-phase chromatography, preferably wherein the chromatography support comprises neutral ligands selected from the group consisting of alkyl, aryl, cycloalkyl, hydroxy, epoxy, ether, amide and ester , and / or multimodal hydrophobichydrogen bonding ligands such as pyridine, further preferably wherein the chromatography support is a porous particle support, a membrane support, a monolith support or a nanofiber support.

[0328] In some embodiments, the hydrophobic interaction support is suitable for hydrophobic interaction chromatography, preferably wherein the chromatography support comprises neutral ligands selected from the group consisting of alkyl, aryl, cycloalkyl, hydroxy, epoxy, ether, amide and ester, and / or multimodal hydrophobic-hydrogen bonding ligands such as pyridine,

[0329] In some embodiments, in step (a) the multivalent halide salt is present at a concentration of at least about 0.25 M, optionally from about 0.5 M to about 3 M, preferably from about 0.75 M to about 3 M. In some embodiments, in step (a) the multivalent halide salt is present at a concentration of about 0.25 M, 0.5 M, or 0.75 M. In some embodiments, the concentration may be about 1.0 M, 1.5 M, or 2.0 M. In some embodiments, the concentration is about 2.5 M or about 3.0 M. In some embodiments, the concentration may range from about 0.25 M to about 1.0 M. In some embodiments, the concentration may range from about 1.0 M to about 2.0 M. In some embodiments, the concentration may range from about 2.0 M to about 3.0 M. In some embodiments, the concentration is selected based on the support, desired ionic strength, solubility, and compatibility with other components in the sample.

[0330] In some embodiments, in step (b) the multivalent halide salt is present at a concentration of at least about 0.25 M, optionally from about 0.5 M to about 3 M, preferably from about 0.75 M to about 3 M. In some embodiments, in step (a) the multivalent halide salt is present at a concentration of about 0.25 M, 0.5 M, or 0.75 M. In some embodiments, the concentration may be about 1.0 M, 1.5 M, or 2.0 M. In some embodiments, the concentration is about 2.5 M or about 3.0 M. In some embodiments, the concentration may range from about 0.25 M to about 1.0 M. In some embodiments, the concentration may range from about 1.0 M to about 2.0 M. In some embodiments, the concentration may range from about 2.0 M to about 3.0 M. In some embodiments, the concentration is selected based on the support, desired ionic strength, solubility, and compatibility with other components in the sample.

[0331] In some embodiments, in step (a) the sample has a pH value in the range from about 4 to about 9, preferably pH is about 4.5-5.5, preferably about 5. In some embodiments, in step (a) the sample has a pH value in the range from about 4 to about 9. In some embodiments, the pH is about 4.0, 4.5, or 5.0. In some embodiments, the pH is about 5.5, 6.0, or 6.5. In some embodiments, the pH is about 7.0, 7.5, or 8.0. In some embodiments, the pH is about 8.5 or about 9.0. In some embodiments, the pH is preferably in the range of about 4.5 to about 5.5. In some embodiments, the pH is most preferably about 5.0. It is understood that the pH is measured at room temperature (20 °C).

[0332] In some embodiments, in step (b) the sample has a pH value in the range from about 4 to about 9, preferably pH is about 4.5-5.5, preferably about 5. In some embodiments, in step (a) the sample has a pH value in the range from about 4 to about 9. In some embodiments, the pH is about 4.0, 4.5, or 5.0. In some embodiments, the pH is about 5.5, 6.0, or 6.5. In some embodiments, the pH is about 7.0, 7.5, or 8.0. In some embodiments, the pH is about 8.5 or about 9.0. In some embodiments, the pH is preferably in the range of about 4.5 to about 5.5. In some embodiments, the pH is most preferably about 5.0. It is understood that the pH is measured at room temperature (20 °C).

[0333] In some embodiments, step (b) is not preceded by an ionic exchange chromatography, optionally wherein the method does not comprise an ionic exchange chromatography.

[0334] As can be seen from figure 10, the method described herein beneficially leads to an improved purification process of the target biomolecule. In particular, the inventive method requires fewer steps since an additional purification step prior incubating the biomolecule with the hydrophobic interaction support is not needed. Also adjusting the sample to optimize binding condition is not required as the incubation step to bind impurities to the support (e.g., flow-through) can directly follow the precipitation step. Prior to incubating the biomolecule with the support (bind-and-elute), solely the multivalent halide salt concentration may be increased. This has the further advantage of reducing the process volume several-fold. The method described herein results in high purity target biomolecule, e.g., with a purity of >95% for the SC pDNA as target biomolecule. With this method, endotoxins as cell-derived impurities are removed below the limit of quantification even if using only a single chromatographic step (being the bind-and-elute of the target biomolecule). Importantly, there is no need to use environmentally problematic salts such as ammonium sulfate.

[0335] In some embodiments, the impurities comprise cellular components, optionally wherein the cellular components comprise RNA, endotoxins, proteins, open-circular plasmid DNA, linear pDNA, or genomic DNA or any combination thereof. In some embodiments, the impurities comprise impurities derived from lysed cells producing the biomolecule.

[0336] In some embodiments, the biomolecules are selected from the group consisting of nucleic acids, optionally DNA, polypeptides, viruses, and virus-like particles or combinations thereof, optionally wherein the biomolecules comprise nucleic acids, optionally plasmid DNA, optionally supercoiled plasmid DNA (SC pDNA). In some embodiments, the biomolecule to be purified is a pDNA, optionally a SC pDNA. The DNA can be a modified or unmodified DNA.

[0337] In some embodiments, the sample comprises less than 0.5 mol / L ammonium sulfate, optionally less than 0.05 mol / L ammonium sulfate, more optionally wherein the sample is essentially free from ammonium sulfate.

[0338] The invention of the second aspect further provides a use of a multivalent halide salt for purifying biomolecules in a hydrophobic interaction chromatography, optionally according to a method disclosed herein. In some embodiments, the multivalent halide salt is used for precipitation of impurities, followed by binding of impurities to the hydrophobic interaction support. In some embodiments, the multivalent halide salt is used for precipitation of impurities, followed by binding of the biomolecules to the hydrophobic interaction support.

[0339] The invention of the second aspect further provides a use of a support for purifying biomolecules in a method as described herein according to the second aspect. In some embodiments, the support is used for binding of impurities to the hydrophobic interaction support following the precipitation of impurities. In some embodiments, the support is used for binding of the biomolecules to the hydrophobic interaction support following the precipitation of impurities.

[0340] In some embodiments, the chromatography support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl, preferably butyl ligand. The invention of the second aspect further provides a use of a kit for purifying biomolecules optionally in a method described herein, the kit comprising:

[0341] a hydrophobic interaction support

[0342] a multivalent halide salt and

[0343] a chromatography support comprising ligands for hydrophobic interaction chromatography, ion (anion or cation) exchange chromatography, or mixed mode chromatography.

[0344] In some embodiments, the multivalent halide salt is present in a buffer solution, preferably in the form of a stock solution. The stock solution can be understood to refer to a solution in need of dilution prior use.

[0345] In this respect, any chromatography support described herein that is suitable for hydrophobic interaction may be part of the kit. Moreover, it is generally appreciated that the multivalent halide salt is present in the kit in a sufficient amount or concentration that allows for carrying out a method disclosed herein using the kit, in particular a method where the at least one multivalent halide salt is for precipitation and in a hydrophobic interaction workflow.

[0346] In some embodiments, the hydrophobic interaction support comprises ligands selected from the group consisting of polymers, polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins, polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene- AB S, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, or polyisobutylene-PIB or any combination thereof.

[0347] Further features and advantages of the aspects and embodiments of the invention emerge from the following description of experimental data with reference to the attached figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0348] Fig. 1 shows a preparative chromatogram for binding of pDNA (4.7 kbp) containing a mixture of supercoiled (SC) and open circular (OC) isoforms, onto CIM® C4 HLD monolith chromatographic column in presence of 1.75 M CaC12 at pH 7.2 used as a mobile phase modifier.

[0349] Fig. 2 shows an analytical HPLC chromatogram using the in-process samples from purification shown in Fig. 1.

[0350] Fig. 3 shows an agarose gel comprising the chromatography fractions shown in Fig. 2.

[0351] Fig. 4 shows the binding capacity for various concentrations of CaC12, ammonium sulfate and MgC12 used as mobile phase modifiers enabling binding of 4.7 kbp pDNA to a CIM® C4 HLD monolithic chromatographic column.

[0352] Fig. 5 shows the binding capacity for binding of pDNA (4.7 kbp) onto a CIM® C4 HLD monolithic chromatographic column, while 1.5 M ammonium sulfate or 1.5 M CaC12 are present as mobile phase modifiers at different pH values.

[0353] Fig. 6 shows a preparative chromatogram for binding of pDNA (4.7 kbp) containing a mixture of supercoiled (SC) and open circular (OC) isoforms to CIM® C4 HLD monolith chromatographic column in the presence of 2.25 M MgC12 at pH 7.2 used as a mobile phase modifier.

[0354] Fig. 7 shows an analytical HPLC chromatogram using the in-process samples from the purification shown in Fig. 6.

[0355] Fig. 8 shows a preparative chromatogram for the protein bovine serum albumin (BSA) binding to CIMmultus® C4 A monolith in the presence of 2.25 M CaC12 at pH 7.2 as mobile phase modifier, and elution in 0 M CaC12. Fig. 9 shows a preparative chromatogram for binding of 4.7 kbp pDNA containing a mixture of SC and OC isoforms to CIMmultus® C4 HLD 40 mL (2 pm channel diameter) monolith in the presence of 2 M CaC12 at pH 7.2 as a mobile phase modifier.

[0356] Fig. 10 shows schematic representation of the number of unit operations according to a classical pDNA process (fig. 10 A) compared to an exemplary process according to the second aspect of the invention (fig. 10 B).

[0357] Fig. 11 shows a preparative chromatogram for purification of pDNA from E. coli cell lysate with a single chromatographic purification step using CIMmultus C4 HLD.

[0358] Fig. 12 shows an overlay of CIMac pDNA analytical UV chromatograms showing high SC pDNA purity of eluate (solid line) compared to load (clarified lysate, dotted line).

[0359] Comparison of fluorescence signals (tryptophan fluorescence kex 280 nm em 348 nm) for eluate (dashed line) and load (dotted-dashed line) indicated significant reduction of hostcell proteins.

[0360] Fig. 13 shows a scheme of a purification process, where CaC12-promoted binding to hydrophobic resin resuspended in CaC12-containing clarified lysate serves as an impurity removal step prior to capture chromatography step.

[0361] Fig. 14 shows a preparative chromatogram for a sample containing pDNA with 16% open circular (OC) and linear (LIN) and 84% supercoiled (SC) isoforms which was directly loaded onto a CIM C4 HLD chromatography column.

[0362] Fig. 15 shows CIMac pDNA analytical UV chromatograms demonstrating high SC pDNA purity of eluate (solid line) compared to load (dotted line). Comparison of tryptophan fluorescence signals for eluate (dashed line) and load (dotted-dashed) also indicates removal of residual host-cell proteins after C4 HLD demonstrating the high efficiency of the chromatographic separation.

[0363] Fig. 16 shows how multiple lysate-PES iterations promote ETX clearance (bars). Starting ETX concentration (before addition of PES) was 962 EU / mL and was decreased to 112 EU / mL after two PES iterations, 9.55 EU / mL after three PES iterations, and 3.89 EU / mL after four PES iterations. pDNA concentration remained unchanged through PES iterations (dots and line).

[0364] Fig. 17 shows CIMac pDNA analytical UV chromatograms demonstrating no significant differences in composition of pDNA isoforms between multiple PES iterations.

[0365] Fig. 18 shows significant decrease in FLD peak at retention time of 4.4 min already after 2 PES iterations (dashed line) in comparison to untreated lysate (dotted line), indicating high degree of clearance of host-cell proteins. With additional PES iterations, the peak at this retention time was <LOD (dashed-dotted and solid lines) and FLD peak at retention time 1 min decreased by 35%.

[0366] Fig. 19 shows how two lysate-PES iterations promote ETX clearance (bars). Starting ETX concentration (before addition of PES) was 1061 EU / mL and decreased to 45.9 EU / mL after one addition of PES particles, it was further reduced to <5 EU / mL (<LOQ) after second addition of PES particles. pDNA concentration decreased by -10% between PES iterations (dots).

[0367] Fig. 20 shows CIMac pDNA analytical UV chromatograms showing partial removal of RNA at retention time 4.5-5 min and no significant differences in composition of pDNA isoforms between multiple samples from PES iterations.

[0368] Fig. 21 shows significant decrease in FLD peak at retention time of 4.4 min already after first PES iteration (dashed line) in comparison to untreated lysate (dotted line). With second PES iteration, this peak was <LOD (dashed-dotted line). The FLD peak at retention 1 min decreased by 30%.

[0369] Fig. 22 shows preparative chromatograms. Results obtained in run 5 were highly reproducible compared to run 1 : the homogeneity of the SC pDNA recovered from the elution process was 96%. Fig. 23 shows significant enrichment of SC pDNA between load and elution and highly comparable quality of pDNA in two eluates is observed by CIMac pDNA analytical UV chromatograms.

[0370] Fig. 24 shows CIMac pDNA analytical fluorescence (FLD) signals demonstrating complete depletion of FLD peaks in eluates from CIM C4 HLD (solid and dashed lines) in comparison to load sample (dotted line), indicating - complete removal of residual proteins. Eluate contained 12% of residual host-cell RNA. Concentration of endotoxins in eluate was <3.5 EU / mL (<LOQ).

[0371] Fig. 25 and 26 show CIMac pDNA analytical chromatograms revealing that residual RNA was successfully removed from pDNA sample (fig. 25), which is confirmed by agarose gel showing removal of hcRNA throughout the purification process: TFF retentate is RNA-free (fig- 26).

[0372] Exemplary, non-limiting working examples of the invention are disclosed in the following. These examples are intended to illustrate that the invention leads to beneficial effects over a broad range of conditions and in combination with a large variety of chromatography paradigms, biomolecules, and chromatography supports.

[0373] EXAMPLES

[0374] Materials and Preparations

[0375] Buffer solutions were freshly prepared with double-distilled water (ddEEO) and analytical grade reagents. They were filtered through a 0.2 pm PES filter (Thermo Fisher Scientific, Nalgene Rapid-Flow). Trizma-base (TRIS), MES hydrate, CaC12 x 2H2O, MgC12 x 6H2O, hydrochloric acid (HC1), (NH4)2SO4 and agarose were from Sigma-Aldrich, NaCl and NaOH were from Honeywell, disodium salt of ethylenediaminetetraacetic acid (EDTA) was from Kemika and glycine was from Merck. The ddH2O used for all experiments was prepared with Adrona. Plasmid-containing E. coli pastes were prepared as described previously (Kos et al., 2021) and purified by clarification and DEAE chromatography as described elsewhere (Cernigoj & Strancar, 2021; Smrekar et al., 2010). Pre-purified plasmid pUCBS4.7 (4.7 kbp, 25% OC, 75% SC) was stored in 10 mM TRIS, 1 mM EDTA, pH 8.0 or in 50 mM TRIS, 10 mM EDTA, 0.75 M NaCl, pH 7.2 (used for scale-up run - see example 6 below).

[0376] Chromatographic separations were performed on PATfix™ (Sartorius BIA Separations), equipped with UV detection (260 and 280 nm wavelength, 10 mm optical path length), conductivity and pH probes, or were performed on Akta Pure 150 (Cytiva), equipped with UV detection (260 and 280 nm, 2 mm optical path length), conductivity and pH probes. PATfix (Sartorius BIA Separations) or UNICORN (Cytiva) software were used for instrument control, data acquisition and data analysis. Flowthrough fractions were analyzed by UV-Vis spectrophotometry at wavelengths of 260 and 280 nm (NanoDrop One, Thermo Fischer Scientific) to determine the concentration of pDNA. Isoform purity for each of the fractions was determined by CIMac® pDNA AEX HPLC analysis (PATfix, Sartorius BIA Separations) as described previously (Pavlin et al., 2023). Mupid®-One (Advance Co., Ltd.) was used for gel electrophoresis. QuantStudio™ 5 (Thermo Fisher Scientific) was used for qPCR. Endosafe® nexgen-PTS™ (Charles River Laboratories) was used for endotoxin testing according to the manufacturer’s instructions.

[0377] Wash solution (free from multivalent halide salt) was 50 mM Tris, pH 7.2. Loading solutions contained 50 mM Tris, pH 7.2 with different (first) concentrations of CaC12, MgC12 or ammonium sulfate (AS) equal to the concentration present in the sample to be analyzed. In experiments with AS, buffers further comprised 10 mM EDTA, which acts as a chelator for divalent metal ions and was therefore avoided when CaC12 or MgC12 was used. In experiments at pH 5.5, 50 mM MES was used as a buffering compound instead of Tris. At pH 3.5 and pH 10, 50 mM glycine was used as a buffering compound instead of Tris. At pH 9, 50 mM Tris was used as buffering compound.

[0378] Before each run, columns were sanitized, regenerated and equilibrated with 10 column volumes (CV) of cleaning-in-place sanitizing solution (1 M NaOH, contact time 15 min), followed by 10 CV of ddH2O, >3 CV of wash solution and >3 CV of loading solution.

[0379] All chromatographic experiments were performed at least in triplicates. Example 1 : Dynamic binding capacity of plasmid DNA when using multivalent halide salts as mobile phase modifier

[0380] The example was carried out to determine the binding capacity and the selectivity of the chromatographic support when multivalent halide salt CaC12 at neutral / physiological pH values (exemplary pH: 7.2) is used as a mobile phase modifier in hydrophobic interaction chromatography. As a chromatography support, a hydrophobic monolithic column (CIMultus® C4 HLD 1 mL, 2 pm channel diameter, Sartorius BIA Separations) comprising high density butyl ligands was used. To a sample comprising 0.2 mg / mL of plasmid DNA, of which 75 % was present in supercoiled form (sc pDNA; biomolecule of interest) and 25 % was present in open circular form (oc pDNA; impurity), CaC12 was added to the sample from a stock solution to achieve multiple total concentrations in the range of 1.25 to 2.5 M. Additionally, samples comprising 1.25 to 2.5 M ammonium sulfate at pH 7.2 were generated.

[0381] The samples comprising CaC12 or ammonium sulfate, respectively, were passed through the chromatography support at a flow rate of 2.5 column volumes per minute (CV / min). After a respective sample had been loaded onto the chromatography support, the support was flushed with a solution containing the same total concentration of CaC12 or ammonium sulfate at 2.5 CV / min, and thereafter a first solution comprising 0.9 M CaC12 or ammonium sulfate was passed through the support at 1 CV / min, and thereafter the support was flushed with a wash solution (“STRIP”) free from multivalent halide salt (0 M CaC12) at 1 CV / min. In each case, the flowthrough fractions were collected for further analysis.

[0382] Parameters such as the flow rate of liquid passing through the chromatography support, as well as the salt concentration and conductivity were also tracked. Fig. 1 shows an exemplary chromatogram depicting the UV absorbance value (continuous line), the salt concentration (dotted line) and the conductivity (dashed line) over the course of the chromatography run, where 1.75 M CaC12 at pH 7.2 was used as a mobile phase modifier enabling binding of pDNA having a length of 4.7 kilobasepairs (kbp) to the CIM® C4 HLD monolith. As documented in Fig. 1, the sample was loaded onto the support between the 1-minute and 5-minute timestamps, the support was washed approx, between the 5-minute and 9-minute timestamps, the first solution was passed through the column approx, between the 9-minute and 14-minute timestamps, and the wash solution was applied approx, between the 14-minute and 17-minute timestamps.

[0383] Fig. 1 documents that a breakthrough of biomolecules occurred, which is indicated by an increase of absorbance starting between the 1 -minute and 2-minute timestamps. Plasmid DNA was eluted from the support by applying the first solution, which is indicated by the large increase in the absorbance between approx, the 10-minute and 12-minute timestamps. More pDNA was removed from the support by applying the wash solution, which is indicated by a smaller absorbance peak around the 15-minute timestamp. This indicates that under binding conditions, the SC isoform was retained on the column while the OC isoform did not bind and was recovered in flow-through (“FT1”), and that SC was eluted upon reducing the concentration of CaC12 to 0.9 M (“E”).

[0384] The binding capacity for pDNA was determined as the amount of biomolecule that can be loaded onto the chromatography support in certain conditions. The binding capacity may be interpreted as the amount of biomolecule that can bind to a chromatography support until a breakthrough is observed. For example, the binding capacity can be determined by calculating the amount of biomolecule that is eluted from the chromatography support. Through the present disclosure, the binding capacity is expressed as the amount (in mg) of biomolecule that is retained by 1 mL of chromatography support, the unit is mg / mL.

[0385] Fig. 4 shows the binding capacity obtained when using different concentrations of CaC12 (crosses), MgC12 (circles) and ammonium sulfate (squares). For CaC12, Fig. 4 shows that the binding capacity for pDNA at neutral pH 7.2 increases between 1.25 and 2 M while remaining constant above 2 M. For ammonium sulfate, the binding capacity for pDNA at pH 7.2 increases up until a concentration of 2.5 M, which is a concentration typically used in hydrophobic interaction chromatography for pDNA. Moreover, the binding capacity of C4 HLD monolith for pDNA in a CaC12 solution is approx. 25 % lower than in an ammonium sulfate solution. The box shown in Fig. 4 indicates the operating range of salt concentrations in the mobile phase at which a successful separation of sc pDNA from oc pDNA can be achieved (also termed “OC clearance”) and the binding capacity increases approx, linearly with the concentration of multivalent halide salt. Fig. 2 shows HPLC chromatograms for four process samples: the loaded sample containing the biomolecule of interest and impurities (“Load”; dotted line), the flowthrough after passing the sample through the support (“flow-through 1” or “FT1”; dashed line), the flowthrough after passing the first solution through the support (“Elution” or “E”; continuous line), and the flowthrough after passing the wash solution through the support (“Strip”; dotted-dashed line). 1.75 M CaC12 at pH 7.2 was used as a mobile phase modifier enabling binding of 4.7 kbp pDNA to the CIM® C4 HLD monolith.

[0386] The HPLC analysis revealed two distinct peaks. In a previous experiment not shown here, the right peak according to Fig. 2 was attributed to the biomolecule of interest, sc pDNA, and the left peak was attributed to the impurity, oc pDNA. Fig. 2 shows that the load fraction contained both sc pDNA and oc pDNA, whereas the FT1 fraction contained >99 % oc pDNA (impurity), and the E fraction contained >95 % sc pDNA (biomolecule of interest). Thereby, the HPLC analysis indicates that biomolecules of interest can be successfully separated from impurities and / or contaminants by the method according to the invention.

[0387] This result was confirmed by agarose gel electrophoresis using a gel comprising 1 % agarose and SYBRgold in lx Tris-Acetate-EDTA (TAE) buffer. The Load, flow-through 1 (FT1) and / or flow-through 2 (FT2 - corresponding to first break-through of SC isoform through the column) and elution (E) fractions from purification when OC / SC pDNA containing samples further containing a total concentration of 1.25 to 2 M CaC12 at pH 7.2 were subjected to standard gel electrophoresis (100 V, 60 min) together with a DNA ladder (SC ladder, NEB). The results are shown in Fig. 3. In a previous experiment not shown here, the lower band according to Fig. 3 had been attributed to the biomolecule of interest (sc pDNA, “SC”), and the higher band to the impurity / contaminant (oc pDNA, “OC”).

[0388] Fig. 3 shows that the oc pDNA predominantly did not bind to the support at all, which is indicated by the respective bands in the FT1 (dashed-frame boxes) and FT2 samples. Moreover, the sc pDNA was completely bound by the chromatography support, which is indicated by the fact that a sc pDNA band was only visible in the FT2 fractions, i.e. when the support was overloaded with sample, i.e. when the binding capacity of the support was not sufficient to bind the entirety of sc pDNA biomolecules contained in the sample. Fig. 3 further confirms that in the range between 1.25 and 2 M CaC12, independently from the specific CaC12 concentration in the sample, predominantly sc pDNA and almost no oc pDNA was eluted by the first solution, which is indicated by the strong sc pDNA band and very weak oc pDNA band in the E samples (continuous-frame boxes).

[0389] The experiment shows that the multivalent halide salt CaC12 can be successfully used for separating a biomolecule of interest from an impurity in hydrophobic interaction chromatography, e.g. when using a CIM® HLD monolith. At pH 7.2, CaC12 provides selective binding conditions for sc pDNA at least in the concentration range between 1.25 M and 2 M, and the binding capacity for pDNA is comparable to the one obtained with known methods based on ammonium sulfate as the mobile phase modifier. In a solution comprising CaC12 and a mixture of sc pDNA and oc pDNA, the hydrophobic chromatography support retained predominantly sc pDNA, and the sc pDNA could be further isolated from oc pDNA impurities by elution with a first solution containing a lower second concentration of the multivalent halide salt, in this case 0.9 M CaC12. Thereby, the present invention provides a workflow for hydrophobic interaction chromatography which is based on environmentally friendly and easy-to-handle multivalent halide salts.

[0390] Example 2: Hydrophobic interaction chromatography using magnesium chloride as mobile phase modifier

[0391] Preparative runs (loading and wash steps) and analytics for experiments in this example were performed the same way as experiments in Example 1, except that CaC12 was replaced with MgCh as a mobile phase modifier.

[0392] The example was carried out to determine the binding capacity and the selectivity of the chromatographic support when multivalent halide salt MgCh at neutral pH 7.2 is used as a mobile phase modifier in hydrophobic interaction chromatography. As a chromatography support, a hydrophobic monolithic column (CIMultus® C4 HLD 1 mL, 2 pm channel diameter, Sartorius BIA Separations) comprising high density butyl ligands was used. To a sample comprising 0.2 mg / mL of plasmid DNA, of which 75 % was present in supercoiled (sc pDNA) form (biomolecule of interest) and 25 % was present in open circular (oc pDNA) form (impurity), MgCh was added as a stock solution to achieve multiple total concentrations in the range of 1.5 to 2.25 M. Fig. 6 shows an exemplary chromatogram depicting the UV absorbance value (continuous line), the salt concentration (dotted line) and conductivity (dashed line) over the course of the chromatography run, where 2.25 M MgCh at pH 7.2 was used as a mobile phase modifier enabling binding of 4.7 kbp pDNA to the CIM® C4 HLD monolith. Under binding conditions, the SC isoform was retained on the column while OC isoform did not bind and was recovered in flow-through (FT1 / FT2). SC pDNA was eluted upon reducing the concentration of MgCh to 0.9 M (first solution: 0.9 M MgCh, 50 mM Tris, pH 7.2).

[0393] According to Fig. 6, the sample was loaded onto the support between the 1 -minute and 5-minute timestamps, the support was washed approx, between the 5-minute and 7.5-minute timestamps, the first solution was passed through the column approx, between the 7.5-minute and 16-minute timestamps, and the wash solution was applied approx, between the 16-minute and 27-minute timestamps.

[0394] Fig. 6 shows that two breakthroughs of biomolecules occurred, which is indicated by increases of absorbance starting between the 1 -minute and 2-minute timestamps and at the 4-minute timestamp. Without wishing to be bound by theory, it is currently assumed that the first breakthrough is of oc pDNA, which is bound to a lesser degree in this setup, and the second breakthrough is of sc pDNA due to the column capacity being exceeded.

[0395] Plasmid DNA was eluted from the support by applying the first solution, which is indicated by the large increase in the absorbance between approx, the 10-minute and 15.5-minute timestamps. More pDNA was removed from the support by applying the wash solution, which is indicated by a broader feature around the 18-minute timestamp.

[0396] Fig. 4 shows binding capacity values obtained by using different concentrations of MgCh (circles) besides results for CaC12 (crosses), and ammonium sulfate (squares). For MgCh, Fig. 4 shows that the binding capacity for pDNA at pH 7.2 increases between 1.5 and 2 M while remaining constant above 2 M. Moreover, the binding capacity of CIM® C4 HLD monolith for pDNA in a MgCh solution is approx. 50 % lower than in an ammonium sulfate solution.

[0397] Fig. 7 shows an analytical HPLC chromatogram of the following in-process samples: load (dotted line), the flowthrough after passing the sample through the support (“flow-through 1” or “FT1”; dashed line), the flowthrough after passing the first solution through the support (“Elution” or “E”; continuous line), and the flowthrough after passing the wash solution through the support (“Strip”; dotted-dashed line). In the chromatography preceding the HPLC analysis shown in Fig. 7, 2.25 M MgCh at pH 7.2 was used as a mobile phase modifier enabling binding of 4.7 kbp pDNA to the CIM® C4 HLD monolith.

[0398] The HPLC analysis revealed two distinct peaks. In a previous experiment not shown here, the right peak according to Fig. 7 had been attributed to the biomolecule of interest, sc pDNA, and the left peak was attributed to the impurity, oc pDNA. Fig. 7 shows that the load fraction contained both sc pDNA and oc pDNA, whereas the FT1 fraction contained >99 % oc pDNA and E fraction contained >95 % sc pDNA.

[0399] The experiment shows that the multivalent halide salt MgCh can be successfully used for separating a biomolecule of interest from an impurity in hydrophobic interaction chromatography on CIM® HLD monolith. At pH 7.2, MgCh provides selective binding conditions for sc pDNA in a concentration window between 1.5 M and 2.25 M, and the binding capacity for pDNA is approximately 50 % lower than the one with ammonium sulfate. In a solution comprising MgCh and a mixture of sc pDNA and oc pDNA, the hydrophobic chromatography support retained predominantly sc pDNA, and the sc pDNA could be further isolated from oc pDNA impurities by elution with a first solution containing a lower concentration of 0.9 M MgCh.

[0400] Example 3 : Dynamic binding capacity at different pH values

[0401] The aim of this example was to determine the binding capacity of biomolecules in hydrophobic interaction chromatography with multivalent halide salts as mobile phase modifiers at different pH values. Preparative runs (loading and wash steps) and analytics for experiments in this example were performed the same way as experiments in Example 1, except for different total concentrations of CaC12 and different pH of solutions. An additional set of experiments was performed to check if a similar trend for pH dependency of binding capacity may be observed also with the known mobile phase modifier ammonium sulfate. Fig. 5 shows results for binding capacity from experiments where 1.5 M ammonium sulfate or 1.5 M CaC12 at different pH values were used as loading conditions for 4.7 kbp pDNA to CIM® C4 HLD monolith. The binding capacity was observed to be higher at more alkaline than at neutral pH using same concentration of CaC12. Thus, by increasing the pH value, a desired binding capacity may be achieved at a lower concentration of CaC12. This was confirmed in an experiment at pH 10 (not shown in Fig. 5, where only 0.5 M CaC12 was used), and the resulting binding capacity (1.2 mg / mL) was comparable to the one achieved with 1.5 M CaC12 at pH 7.2. On the other hand, the binding capacity was reduced at a pH value of 3.5 in comparison to neutral pH using same concentration of CaC12.

[0402] With respect to the binding capacity as a function of the pH value, a different trend than in case of CaC12 was observed with ammonium sulfate: both at pH 5.5 and pH 9, the binding capacity was approximately 20 % lower (1.0 mg / mL) than at neutral pH (1.2 mg / mL), indicating that the maximum binding capacity value can be achieved at neutral pH and that the pH dependence of the binding capacity in ammonium sulfate does not follow the same pattern as CaC12. This strengthens the hypothesis that the mechanism enabling binding of pDNA in case of CaC12 (chaotrope) may be significantly different from the mechanism in case of ammonium sulfate (kosmotrope). Also, the experiment shows that when using a method according to the invention, HIC can be performed over a larger range of pH values than with known mobile phase modifiers such as ammonium sulfate.

[0403] Example 4: Removal of endotoxins by methods of the invention

[0404] Selected in-process samples from Examples 1 and 3 were analyzed for content of other important contaminants: wt.-% of genomic DNA (gDNA) and concentration of endotoxins (ETX) was tested as described elsewhere herein. Results in Table 1 show that a biomolecule of interest (here: pDNA) can be efficiently separated from impurities and contaminants (here: gDNA and ETX) using CaC12 instead of the well-established ammonium sulfate. In all samples, the percentage of gDNA was reduced by at least a factor of 5.5, and the endotoxin content was reduced by more than 99.5 %.

[0405] The following table shows results for content of gDNA and endotoxins in the load and elution fractions from a few representative experiments from Examples 1 and 3. Loading conditions ETX in load ETX in eluate gDNA in load gDNA in [EU / mL] [EU / mL] [w / w] eluate [w / w] 2 M CaC12, pH 7.2 2156 <10 1.7 % 0.3 %

[0406] 2 M CaC12, pH 8 n.d. n.d. 2 % 0.03 %

[0407] 1.5 M CaC12, pH n.d. n.d. 2 % 0.15 %

[0408] 8.5

[0409] 1.5 M CaC12, pH 9 n.d. n.d. 2 % 0.19 %

[0410]

[0411] n.d.: not determined

[0412] Example 5: Purification of proteins by methods of the invention

[0413] In this experiment, divalent chloride salts (CaC12) were used as mobile phase modifiers in hydrophobic interaction chromatography for protein purification. Bovine serum albumin (BSA) was used as a model protein. As proteins are known to be very hydrophobic in comparison to nucleic acids, a chromatographic support with lower hydrophobicity (CIMmultus® C4 A, 2 pm channel diameter, Sartorius BIA Separations) was used to prevent potential irreversible binding of proteins, which is a known issue in hydrophobic interaction chromatography. A BSA sample comprising 2.25 M CaC12 at pH 7.2 was prepared and loaded onto a chromatographic support until a breakthrough, representing unbound BSA, was observed. Fig. 8 shows a representative preparative chromatogram for the following chromatographic run (similar to the one described in Example 1 above): the sample was loaded onto a CIMmultus® C4 A monolith chromatographic support between the 37-minute and 48-minute timestamps, the chromatographic support was washed between the 48-minute and 52.5-minute timestamps (wash solution: 2.25 M CaC12, 50 mM Tris, pH 7.2), and the first (elution) solution was passed through the column between the 52.5-minute and 60-minute timestamps (first solution: 0 M CaC12, 50 mM Tris, pH 7.2). Plotted are the UV absorbance value (continuous line), the salt concentration (dotted line) and the conductivity (dashed line).

[0414] Fig. 8 shows an increase of absorbance starting between the 42-minute and 43-minute timestamps, indicating that a breakthrough of BSA occurred (meaning that the binding capacity of the chromatographic support was exceeded). BSA was fully eluted from the support by applying the first solution, which is indicated by the increase in the absorbance between approx, the 53.5-minute and 56-minute timestamps. This example shows that CaC12 can be used also as a mobile phase modifier in hydrophobic interaction chromatography for proteins.

[0415] Example 6: Purification of biomolecules at a larger scale

[0416] This example represents a scale-up experiment, performed the same way and with the same buffers as experiments in Example 1 but on a larger chromatography support. Plasmid DNA sample comprising 2 M CaC12 at pH 7.2 was loaded onto CIMmultus® C4 HLD (40 mL, 2 pm channel diameter, Sartorius BIA Separations) until the second pDNA breakthrough event occurred (which indicates that the column capacity was exceeded). The chromatography support had a 40 times larger volume than the one used in Example 1. A preparative chromatogram is shown in Fig. 9. Plotted are the UV absorbance value (continuous line), the salt concentration (dotted line) and the conductivity (dashed line).

[0417] Looking to Fig. 9, the sample was loaded onto the support between the 2-minute and 13.5-minute timestamps, the support was washed approx, between the 13.5-minute and 17-minute timestamps, the first solution was passed through the column approx, between the 17-minute and 23-minute timestamps, and the wash solution was applied approx, between the 23-minute and 29-minute timestamps.

[0418] Fig. 9 shows that two breakthroughs occurred, which is indicated by the rapid increase of absorbance starting between the 2-minute and 2.5-minute timestamps (corresponding to OC pDNA which did not bind to the column) and at approx, the 11 -minute timestamp (when the column capacity for SC pDNA was likely exceeded). Plasmid DNA was eluted from the support by applying the first solution, which is indicated by the large increase in the absorbance between approx, the 17.5-minute and 22.5-minute timestamps. More pDNA was removed from the support by applying the wash solution, which is indicated by a smaller absorbance peak around the 24-minute timestamp.

[0419] HPLC analytics (data not shown) indicated that the load fraction contained both sc pDNA (82 %) and oc pDNA, whereas the FT1 fraction contained >99 % oc pDNA and the E fraction contained >95 % sc pDNA. The binding capacity observed in this experiment (1.95 mg / mL) was comparable to the one obtained with 1 mL column (1.85 mg / mL). With this example it was confirmed that using CaC12 as a mobile phase modifier in hydrophobic interaction chromatography for pDNA is a scalable approach.

[0420] Example 7: Purification of biomolecules from lysate following CaC12-precipitation and hydrophobic interaction purification

[0421] Cell paste containing 4.7 kbp pDNA was resuspended in TE buffer and homogenized. Alkaline lysis was performed by addition of 0.2 M NaOH and 1 % SDS in 1 : 1 ratio (final cone. 0.1 M NaOH, 0.5 % SDS). After 5 min reaction was neutralized with 3 M CH3COOK to 1 M final concentration.

[0422] Precipitation of RNA was achieved by addition of 5 M CaC12 solution to final concentration of 1.25 M. Lysate was then filtered through 0.45 pm PES filter to obtain fine filtrate.

[0423] Lysate sample contained a mixture of 7% open circular (OC), 9% linear (LEST) and 84% supercoiled (SC) isoforms and was directly loaded onto a CIM C4 HLD monolithic chromatography column preconditioned in 1.25 M CaC12. The OC and LEST isoforms of the pDNA did not bind to the CIM C4 HLD column and were subsequently recovered in the flow-through, while SC isoform (the target isoform) bound to the column and was eluted with 0.9 M CaC12 (83%) and in strip (0 M CaC12) fraction (17%).

[0424] This indicated that 1.25 M can selectively bind SC isoform to C4 HLD, and can be eluted by reducing the concentration of CaC12. Small amount of residual RNA was removed from pDNA sample in TFF step after C4. Endotoxin in C4 HLD elution was <5 EU / mL by Limulus Amebocyte Lysate (LAL) test. Figure 11 corresponds to preparative chromatogram for purification of pDNA from E. coli cell lysate with a single chromatographic purification step using CIMmultus C4 HLD. Figure 12 is overlay of CIMac pDNA analytical UV chromatograms showing high SC pDNA purity of eluate (solid line) compared to load (clarified lysate, dotted line). Comparison of fluorescence signals (tryptophan fluorescence kex280 nm

[0425]

[0426] 348 nm) for eluate (dashed line) and load (dotted-dashed line) indicated significant reduction of host-cell proteins.

[0427] The purity of the SC pDNA recovered from the elution process was found to be greater than 95% (99%), including clearance of residual endotoxin < 5 EU / mg, undetectable tryptophan fluorescence indicating complete protein clearance and clearance of residual RNA by CIMac pDNA, demonstrating the high efficiency of the chromatographic separation under these conditions.

[0428] Example 8: Purification of biomolecules from lysate following CaC12-precipitation and hydrophobic interaction purification

[0429] E. coli cell paste containing 4.7 kbp pDNA was resuspended in TE buffer and homogenized. Alkaline lysis was performed by addition of 0.2 M NaOH and 1 % SDS in 1:1 ratio (final cone. 0.1 MNaOH, 0.5 % SDS). After 5 min, alkaline lysis was neutralized with 3 M CH3COOK to 1 M final concentration. Precipitation of RNA was achieved by addition of 5 M CaC12 solution to final concentration of 1.25 M.

[0430] Figure 13 presents a scheme of a purification process, where CaC12-promoted binding to hydrophobic resin resuspended in CaC12-containing clarified lysate serves as an impurity removal step prior to capture chromatography step.

[0431] CIMasphere C4 HLD particles (diameter 200 - 500 nm) were pre-conditioned with TE buffer pH 7.2, containing 1.25 M CaC12, then the liquid was removed to obtain almost dry CIMasphere particles. Particles were added to lysate (5 % w / w) to bind highly hydrophobic impurities (proteins, ETX, RNA), stirred in lysate for 30 min, then filtered through 0.45 pm PES filter to remove CIMasphere particles and obtain fine filtrate. This sample, containing pDNA with 16% open circular (OC) and linear (LIN) and 84% supercoiled (SC) isoforms was directly loaded onto a CIM C4 HLD chromatography column, preconditioned in 1.25 M CaC12 (Figure 14). The OC and LIN isoforms of the pDNA did not bind to the CIM C4 HLD column and were subsequently recovered in the flow-through. No SC pDNA was detected in ‘flow-through L, demonstrating that SC pDNA bound to the column. Second break-through of UV trace indicated that capacity for SC pDNA was exceeded, which was confirmed with detecting SC isoform in ‘flow-through 2’ (FT2) sample. CIM C4 HLD column was washed with higher cone, of CaC12 (1.5 M) to prevent conductivity drop and unwanted elution of pDNA. pDNA was then eluted from the column in reduced cone, of CaC12 (0.9 M). The homogeneity of the SC pDNA recovered from the elution process was 98%, it contained 5% of RNA. CIMac pDNA analytical UV chromatograms in Figure 15 show high SC pDNA purity of eluate (solid line) compared to load (dotted line). Comparison of tryptophan fluorescence signals for eluate (dashed line) and load (dotted-dashed) also indicates removal of residual host-cell proteins after C4 HLD demonstrating the high efficiency of the chromatographic separation under these conditions.

[0432] Example 9: Pre-treatment of lysate with PES particles to remove impurities

[0433] pDNA was purified from E. coli cell lysate with CaC12-promoted removal of impurities by PES material as follows.

[0434] E. coli cell paste containing 4.7 kbp pDNA was resuspended in TE buffer and homogenized in 5 mL plastic tubes. Alkaline lysis was performed by addition of 0.2 M NaOH and 1 % SDS in 1:1 ratio (final cone. 0.1 M NaOH, 0.5 % SDS). After 5 min reaction was neutralized with 3 M CH3COOK, pH 5.5 to 1 M final concentration. Precipitation of RNA was achieved by addition of 4 M CaC12 solution to a final concentration of 0.75 M.

[0435] PES (polyethersulfone) particles (diameter 380 - 550 pm) without any pretreatment were added to the lysate (20 % w / V) to bind host-cell impurities (e.g. ETX, proteins, RNA), mixed on a block shaker for 30 min and then filtered through 0.45 pm PES syringe filter to remove PES particles. Fine filtrate was a subject of the next PES iteration, where 20 % (w / V) of PES was added, mixed on a block shaker for 30 min and then filtered through 0.45 pm PES syringe filter. In total 4 consecutive PES iterations were performed.

[0436] Samples of lysate from all iterations were analyzed by CIMac pDNA analytics to determine nucleic acid content (pDNA and RNA), residual proteins (FLD trace) and by LAL test to determine ETX concentration.

[0437] Figure 16 shows how multiple lysate-PES iterations promoted ETX clearance (bars).

[0438] Starting ETX concentration (before addition of PES) was 962 EU / mL and was decreased to 112 EU / mL after two PES iterations, 9.55 EU / mL after three PES iterations, and 3.89 EU / mL after four PES iterations. pDNA concentration remained unchanged through PES iterations (dots and line). CIMac pDNA analytical UV chromatograms showing no significant differences in composition of pDNA isoforms (retention time of OC isoform is 7.1 min, retention time of SC isoform is 8.8 min) between multiple PES iterations are shown in Figure 17, whereas CIMac pDNA analytical fluorescence (FLD) signals in Figure 18 show significant decrease in FLD peak at retention time of 4.4 min already after 2 PES iterations (dashed line) in comparison to untreated lysate (dotted line), indicating high degree of clearance of host-cell proteins. With additional PES iterations, the peak at this retention time was <LOD (dashed-dotted and solid lines) and FLD peak at retention time 1 min decreased by 35%.

[0439] Example 10: Pre-treatment of E, coli lysate containing CaC12 with PES particles to remove contaminants - Scale-up

[0440] pDNA was purified from E. coli cell lysate with CaC12-promoted removal of impurities by PES material as follows.

[0441] E. coli cell paste containing 4.7 kbp pDNA was resuspended in TE buffer and homogenized in glass beaker with magnetic stirrer. Alkaline lysis was performed in the same container by addition of 0.2 M NaOH and 1 % SDS in 1 : 1 ratio (final cone. 0.1 M NaOH, 0.5 % SDS). After 5 min, alkaline lysis was neutralized with 3 M CH3COOK, pH 5.5 to a 1 M final concentration. Precipitation of RNA was achieved by addition of 5 M CaC12 solution to a final concentration of 0.75 M.

[0442] PES particles (diameter 380 - 550 pm) without pretreatment were added to the lysate (30 % w / V) to bind host-cell impurities (e.g. ETX, proteins, RNA), stirred in lysate with magnetic stirrer for 3.5 hours and then filtered to remove larger PES particles. In the next iteration that followed directly, PES (30 % w / V) was added to filtrate from previous cycle, stirred with a magnetic stirrer for 1 h and filtered to remove PES particles and to obtain fine filtrate, which was loaded onto CIM C4HLD column to capture SC pDNA.

[0443] Samples from iterations were analyzed by CIMac pDNA analytics to determine nucleic acid and protein content and by LAL test to determine ETX concentration.

[0444] Figure 19 shows how two lysate-PES iterations promoted ETX clearance (bars). Starting ETX concentration (before addition of PES) was 1061 EU / mL and decreased to 45.9 EU / mL after one addition of PES particles, it was further reduced to <5 EU / mL (<LOQ) after second addition of PES particles. pDNA concentration decreased by -10% between PES iterations (dots). CIMac pDNA analytical UV chromatograms showing partial removal of RNA at retention time 4.5-5 min and no significant differences in composition of pDNA isoforms between multiple samples from PES iterations are shown in Figure 20, whereas CIMac pDNA analytical fluorescence (FLD) signals in Figure 21 show significant decrease in FLD peak at retention time of 4.4 min already after first PES iteration (dashed line) in comparison to untreated lysate (dotted line). With second PES iteration, this peak was <LOD (dashed-dotted line). The FLD peak at retention 1 min decreased by 30%.

[0445] Example 11 : Loading of PES -pre-treated E. coli lysate containing CaC12 onto HIC chromatography (CIMmultus C4 HLD column)

[0446] Fine filtrate, obtained after second PES iteration in Example 4, containing a mixture of 17% open circular (OC) 7 % linear (LIN) and 76% supercoiled (SC) and multimer isoforms was spiked with concentrated CaC12 solution (50 mM TRIS, 4 M CaC12, pH 7.2) to increase CaC12 concentration to 2 M. Sample containing 2 M CaC12 was loaded onto a CIM C4 HLD chromatography column, preconditioned in 50 mM TRIS, 2 M CaC12, pH 7.2. The OC and LIN isoforms of the pDNA did not bind to the CIM C4 HLD column and were subsequently recovered in the flow-through. No SC pDNA was detected in ‘flow-through L, demonstrating that pDNA bound to the column. Second break-through of UV signal indicated that capacity for SC pDNA was exceeded. C4 HLD column was washed with 2 M CaC12 and eluted with 0.9 M CaC12. After elution, column was washed with 50 mM Tris, pH 7.2 (STRIP), purified water, then a cleaning step (CIP) with 1 M NaOH was applied (40 min contact time). After CIP, column was washed again with purified water, 50 mM TRIS, pH 7.2, and loading buffer (50 mM TRIS, 2 M CaC12, pH 7.2).

[0447] The homogeneity of the SC pDNA recovered from the elution process was 97%, demonstrating the high efficiency of the chromatographic separation under these conditions.

[0448] A series of four subsequent C4 HLD chromatographic runs was performed to demonstrate the robustness of the purification process. Preparative chromatograms are shown in Figure 22. Results obtained in run 5 were highly reproducible compared to run 1: the homogeneity of the SC pDNA recovered from the elution process was 96%.

[0449] Significant enrichment of SC pDNA between load and elution and highly comparable quality of pDNA in two eluates is observed by CIMac pDNA analytical UV chromatograms (Figure 23).

[0450] CIMac pDNA analytical fluorescence (FLD) signals in Figure 24 show complete depletion of FLD peaks in eluates from CIM C4 HLD (solid and dashed lines) in comparison to load sample (dotted line), indicating - complete removal of residual proteins. Eluate contained 12% of residual host-cell RNA. Concentration of endotoxins in eluate was <3.5 EU / mL (<LOQ).

[0451] Example 12: Tangential flow filtration (TFF) post-HIC formulates biomolecule into target formulation and removes residual host-cell RNA,

[0452] Residual host cell RNA was removed by tangential flow-filtration (TFF) Sartocon Slice cassette PES 50 cm2with 100 kDa cut-off was used, which also formulated pDNA in desired formulation matrix (ddFEO). Eluates after C4 HLD polishing, containing 12 % of residual hcRNA, were pooled together and used as a feed material for TFF. Concentration of sample was followed by diafiltration in purified water (10 diafiltration volumes). CIMac pDNA analytical chromatograms in Figure 25 reveal that residual RNA was successfully removed from pDNA sample, which is confirmed by agarose gel showing removal of hcRNA throughout the purification process: TFF retentate is RNA-free (Figure 26).

Claims

C l a i m s1. Method for purifying biomolecules, the method comprising:(a) mixing a sample comprising the biomolecules and one or more impurities with at least one multivalent halide salt to precipitate one or more of the impurities; and(b) incubating the sample comprising the biomolecules, any remaining impurities, and the multivalent halide salt with a hydrophobic interaction support.

2. Method according to claim 1, wherein in step (b) impurities bind to the hydrophobic interaction support, optionally by using a solid phase adsorption or powder assisted adsorption.

3. Method according to claim 1 or 2, wherein the hydrophobic interaction support comprises a particle support, a nanoparticle support, a fiber support, a nanofiber support, a gel support, a sheet support, a film support, a membrane support, a monolith support, or any combination thereof.

4. Method according to any of the claims 2-3, wherein the hydrophobic interaction support comprises a nanoparticle or particle support or any combination thereof, optionally selected from powders, granules, or particles or any combination thereof.

5. Method according to any of the claims 2-4, wherein the hydrophobic interaction support comprises porous, non-porous, or semi-porous material or any combination thereof.

6. Method according to any of the claims 2-5, wherein the hydrophobic interaction support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl or any combinations thereof; optionally comprising butyl ligands.

7. Method according to any of the claims 2-6, wherein the hydrophobic interaction support comprises ligands selected from the group consisting of polymers, polysthersulfone-PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins, poly vinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene- AB S, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene,poly chi orotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, or polyisobutylene-PIB or any combination thereof.

8. Method according to any of the claims 2-7, wherein in step (b) the sample and the support are agitated to promote binding of the impurities to the hydrophobic interaction support, optionally by aeration, stirring, shaking, swirling, churning, rocking, or any combination thereof.

9. Method according to any of the claims 2-8, further comprising a removal step of the hydrophobic interaction support, optionally by filtration, centrifugation, sedimentation, or the like.

10. Method according to any of the claims 2-9, wherein the hydrophobic interaction support is washed with a washing solution containing the multivalent halide salt, optionally wherein the multivalent halide salt has the same concentration as in the sample from step (b).

11. Method according to any of the claims 2-10, further comprising(d) passing the sample comprising the biomolecules through a chromatography support to bind the biomolecules to the support.

12. Method according to claim 11, wherein the chromatography is a mixed-mode chromatography, a hydrophobic interaction chromatography, an ion exchange chromatography, a size exclusion chromatography, an affinity chromatography, an immobilized metal affinity chromatography or the like.

13. Method according to claim 11 or 12, further comprising eluting the biomolecules to obtain purified biomolecules.

14. Method according to any of the claims 2-13, wherein step (b) comprises or is a flow- through step.

15. Method according to claim 1, wherein in step (b) the biomolecules bind to the hydrophobic interaction support.

16. Method according to claim 15, wherein the concentration of the multivalent halide salt is adjusted prior the incubation, optionally wherein the concentration of the multivalent halide salt is increased; optionally wherein the concentration of the multivalent halide salt is increased following the flow-through step.

17. Method according to claim 15 or 16, further comprising in step (b):(bl) passing the sample through the hydrophobic interaction support, wherein the sample further comprises the multivalent halide salt at a first concentration, and (b2) passing a first solution through the support, wherein the first solution comprises the multivalent halide salt at a second concentration,wherein the second concentration is lower than the first concentration.

18. Method according to any of claims 15-17, wherein the hydrophobic interaction support is suitable for hydrophobic interaction chromatography, preferably wherein the chromatography support comprises neutral ligands selected from the group consisting of alkyl, aryl, cycloalkyl, hydroxy, epoxy, ether, amide and ester, and / or multimodal hydrophobic-hydrogen bonding ligands such as pyridine.

19. Method according to any of the preceding claims, wherein the multivalent halide salt comprises divalent and / or tri valent metal cations, preferably Mg, Ca, Sr, Al, Fe, and / or Ba, more preferably Mg and / or Ca cations.

20. Method according to any of the preceding claims, wherein the multivalent halide salt comprises F anions, Cl anions, Br anions, I anions, or At anions, or any combination thereof, preferably F and / or Cl anions.

21. Method according to any of the preceding claims, wherein the multivalent halide salt is MgC12 and / or CaC12.

22. Method according to any of the preceding claims, wherein in step (a) the multivalent halide salt is present at a concentration of at least about 0.25 M, optionally from about 0.5 M to about 3 M, preferably from about 0.75 M to about 3 M.

23. Method according to any of the preceding claims, wherein in step (b) the multivalent halide salt is present at a concentration of at least about 0.25 M, optionally from about 0.5 M to about 3 M, preferably from about 0.75 M to about 3 M.

24. Method according to any of the preceding claims, wherein in step (a) the sample has a pH value in the range from about 4 to about 9, preferably pH is about 4.5-5.5, preferably about 5.

25. Method according to any of the preceding claims, wherein in step (b) the sample has a pH value in the range from about 4 to about 9, preferably pH is about 4.5-5.5, preferably about 5.

26. Method according to any of the preceding claims, wherein step (b) is not preceded by an anionic exchange chromatography, optionally wherein the method does not comprise an anionic exchange chromatography.

27. Method according to any of the preceding claims, wherein the impurities comprise cellular components, optionally wherein the cellular components comprise RNA,endotoxins, proteins, open-circular plasmid DNA, linear pDNA, or genomic DNA or any combination thereof.

28. Method according to any of the preceding claims, wherein the impurities are derived from lysed cells producing the biomolecule.

29. Method according to any of the preceding claims, wherein the biomolecules are selected from the group consisting of nucleic acids, optionally DNA, polypeptides, viruses, and virus-like particles or combinations thereof, optionally wherein the biomolecules comprise plasmid DNA, optionally supercoiled plasmid DNA.

30. Method according to any of the preceding claims, wherein the sample comprises less than 0.5 mol / L ammonium sulfate, optionally less than 0.05 mol / L ammonium sulfate, more optionally wherein the mobile phase is essentially free from ammonium sulfate.

31. Use of a multivalent halide salt for purifying biomolecules in a hydrophobic interaction chromatography, optionally according to a method of any one of claims 1 to 30.

32. Use of a chromatography support for purifying biomolecules in a method according to any one of claims 1 to 30.

33. Use according to claim 32, wherein the chromatography support comprises hydrophobic ligands selected from the group consisting of alkyl, aryl and cycloalkyl, preferably butyl ligand.

34. Use of a Kit for purifying biomolecules optionally in a method according to any one of claims 1 to 30, the kit comprising:a hydrophobic interaction support;at least one multivalent halide salt; anda chromatography support comprising ligands for hydrophobic interaction chromatography, ion exchange chromatography or mixed modechromatography.

35. Use of a kit according to claim 34, wherein the multivalent halide salt is present in a buffer solution, preferably in the form of a stock solution.

36. Use of a kit according to claim 34 or 35, wherein the hydrophobic interaction support comprises ligands selected from the group consisting of polymers, polysthersulfone- PES, polystyrene-PS, polyethylene-PE, polypropylene-PP, polyvinyl chloride-PVC, polyvinyl fluoride-PVF, poly(methyl methacrylate)-PMMA, polyoxymethylene-POM, polycarbonate-PC, nylon, polyethylene terephthalate-PET, epoxy resins, polyvinylidene chloride (PVDC), polyvinylidene fluoride-PVDF, polyphenylene sulfide-PPS, acrylonitrile butadiene styrene-ABS, poly n-butyl methacrylate-PnBMA, polytrifluoroethylene, polybutadiene, polychlorotrifluoroethylene-PCTFE, polydimethylsiloxane-PDMS, fluorinated ethylene propylene-FEP, hexatriacontane, paraffin, polytetrafluoroethylene-PTFE, hexafluoropropylene, or polyisobutylene-PIB or any combination thereof.

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