Improving RNA and contaminant removal from DNA plasmid preparations by hydrophobic interaction chromatography

Abstract

A method for preparing a purified pDNA preparation from a sample comprising pDNA and contaminants is disclosed, the method comprising the steps of contacting the sample with a hydrophobic interaction chromatography (HIC) material in a solution comprising a kosmotropic salt at a concentration capable of forcing the pDNA and contaminants to adsorb on the HIC material, after adsorption of the pDNA on the HIC material, diluting the concentration of the kosmotropic salt in the presence of a neutral salt, thereby desorbing the pDNA from the HIC material while the contaminants remain adsorbed by the continued presence of the neutral salt, and obtaining the pDNA preparation. Furthermore, a method for preparing a sample to be subjected to the method of the invention or other purification methods is disclosed, in particular anion exchange chromatography by exposing the sample to a neutral salt in the presence of a HIC material.

Description

Technical Field

[0001] This invention relates to a method for preparing a purified pDNA formulation from a sample containing pDNA and contaminants. Background Technology

[0002] DNA plasmids obtained by lysing production cells are commonly contaminated with proteins, RNA, RNA-protein aggregates, and DNA-protein-RNA aggregates. Hydrophobic interaction chromatography (HIC) is one of the known methods for purifying plasmid DNA (pDNA) [1]. The sample is added to a solution containing a high concentration of precipitating salts and applied to an HIC column. pDNA binds to the column, but contaminants also bind to the column. The column is eluted with a descending salt gradient. Good separation can be achieved between plasmid isoforms, such as supercoiled (sc), open-circular (oc), and linear pDNA, but RNA is largely co-eluted with the desired scDNA. Good separation of pDNA from host cell proteins, RNA-protein aggregates, and DNA-protein-RNA aggregates can also be achieved. However, HIC cannot completely remove residual contaminants, which is usually compensated for by combining HIC with anion exchange chromatography (AEC). Summary of the Invention

[0003] Surprisingly, it has been found that reducing the concentration of the kosmotropic salt (first salt) during column elution enhances the removal of contaminants from pDNA formulations by HIC in the presence of a neutral salt (second salt). pDNA is recovered by reducing the concentration of the kosmotropic salt in the persistent presence of the neutral salt. In the persistent presence of the second salt, RNA, proteins, RNA-protein aggregates, and DNA-protein-RNA aggregates remain bound to the column. It is believed that the neutral salt forces contaminants to remain tightly bound to the hydrophobic column. This contrasts sharply with conventional elution methods, which create a simple gradient by reducing the concentration of the kosmotropic salt in the absence of a neutral salt, where various contaminants co-elute with pDNA. This method reduces the amount of contaminants in the pDNA fraction and alters the spectrum of residual contaminants in the pDNA fraction. Therefore, combining the method of this application with another chromatographic method can produce pDNA with higher purity compared to the usual combination of HIC with another chromatographic method.

[0004] The subject of this invention is a method for preparing a purified pDNA formulation from a sample containing pDNA and contaminants, the method comprising the following steps:

[0005] The sample is contacted with hydrophobic interaction chromatography (HIC) material in a solution containing a co-liquid salt at a concentration sufficient to force pDNA and contaminants to adsorb onto the HIC material.

[0006] After pDNA is adsorbed onto the HIC material, the concentration of the co-solution salt is diluted in the presence of a neutral salt, thereby desorbing the pDNA from the HIC material. Meanwhile, the pollutants remain adsorbed due to the continued presence of the neutral salt.

[0007] Obtain pDNA formulation.

[0008] The term “precipitating salt” represents a continuation of the concept in the field of protein chemistry, where certain salts are known to effectively precipitate proteins. Ammonium sulfate is one example. Other types, such as guanidine hydrochloride, are strong solubilizers that prevent precipitation. Some salts (such as sodium chloride) have a significantly reduced ability to promote protein precipitation. The effects of various salts on protein solubility are known to be related to their respective ranking in the Hofmeister series [2].

[0009] Salts that promote precipitation are typically classified as co-liquid salts or colloidal ion salts. Salts that promote solubility are typically classified as ionizing salts. Salts with intermediate properties (e.g., sodium chloride) are typically classified as neutral salts.

[0010] In this article, the term "neutral salt" should be understood not to refer to the pH value of the aqueous solution in which the salt is dissolved. Rather, the term neutral salt is understood to be any salt in which the combined and average contribution of the cations and anions produces an effect that is neither iono-liquid nor co-liquid; in other words, it is neutral. Generally, monovalent metal halide salts and monovalent metal acetates are considered neutral salts.

[0011] In one embodiment of the method of the present invention, the co-liquid salt may be a salt containing a co-liquid anion, a cation, or both, particularly a salt selected from the group consisting of ammonium sulfate, sodium sulfate, potassium phosphate, sodium citrate, potassium citrate, and combinations thereof. Typically, the concentration of the co-liquid salt can range from 1.0 M to 2.5 M, or 1.25 M to 2.25 M, or 1.5 M to 2.0 M, or 1.7 M to 1.9 M. The use of many co-liquid salts is limited due to their low solubility in water. Sodium phosphate is one example. It saturates at approximately 0.8 M, which is too low for it to be used in the method of the present invention or any precipitation-based technique. Potassium phosphate is practical because it remains soluble at higher concentrations.

[0012] In another embodiment of the method of the present invention, the neutral salt may be selected from the group consisting of sodium chloride, potassium chloride, lithium chloride, ammonium chloride, sodium acetate, potassium acetate, lithium acetate, ammonium acetate, and combinations thereof. Typically, the concentration of the neutral salt may be in the range of 0.5M to 5.0M, or 0.75M to 4.0M, or 1.0M to 3.0M, or 1.25M to 2.5M, or 1.5M to 2.0M.

[0013] In another embodiment of the method of the present invention, the sample may be a lysate of prokaryotic cells containing plasmid DNA.

[0014] In another embodiment of the method of the present invention, the contaminant may be selected from the group consisting of: proteins, RNA, RNA-protein aggregates, DNA-protein aggregates, and DNA-protein-RNA aggregates.

[0015] In another embodiment of the method of the present invention, the HIC material may be a polymer with hydrophobic ligands.

[0016] In another embodiment of the method of the present invention, the hydrophobic ligand may have aromatic properties, such as phenyl and / or benzyl ligands; or have alkyl properties, such as butyl, hexyl and / or octyl ligands or combinations thereof.

[0017] In another embodiment of the method of the present invention, the HIC material can be arranged in a chromatographic column.

[0018] In one embodiment, the method of the present invention is a single method for removing contaminants from pDNA. Another embodiment of the invention is a pre-purification method for preparing samples to be subjected to other purification methods, particularly anion exchange chromatography. Specifically, if a pre-purification method is used, the neutral salt may be lithium chloride or calcium chloride. In yet another embodiment, the method of the present invention is a post-purification method for improving the purity of pDNA prepared by other methods (particularly anion exchange chromatography).

[0019] In one embodiment of the pre-purification method of the present invention, the HIC material may be a hydrophobic depth filtration material or a column filled with porous hydrophobic particles or nanofibers.

[0020] In another embodiment of the pre-purification method of the present invention, a co-liquid salt may be added to the sample. In a closely related variant, the co-liquid salt may be added as a liquid concentrate. In one such embodiment, the addition may be regulated by a mixing device to rapidly obtain a homogeneous mixture. In one such embodiment, the mixing may be performed immediately before the mixture contacts the HIC column to minimize the formation of precipitates before the sample contacts the column.

[0021] In another embodiment of the pre-purification method of the present invention, the sample can be diluted with water or a low-conductivity buffer to prepare a sample for anion exchange chromatography.

[0022] In one embodiment of the post-purification method of the present invention, pDNA eluted from the anion exchanger and still containing the neutral salt used for elution from the anion exchanger can be combined with a co-solution salt to regulate the binding of the pDNA to the HIC column. Generally, this method is less complex and has a smoother overall process flow compared to the pre-purification method of the present invention. Attached Figure Description

[0023] Figure 1 The various stages of the method of the present invention are described.

[0024] Figure 2 The elution of DNA in a co-liquid salt gradient is described.

[0025] Figure 3 The elution of plasmid DNA in a co-liquid salt gradient is described with the neutral salt concentration kept constant.

[0026] Figure 4 The elution behavior of RNA is described, which maintains binding while the concentration of co-solvent salt decreases and the concentration of neutral salt remains constant, and elutes when the concentration of neutral salt decreases.

[0027] Figure 5 The elution behavior of plasmid DNA and RNA was described, wherein pDNA was eluted under a decreasing gradient of cosoluble salt while the concentration of neutral salt was kept constant, and RNA was eluted as the concentration of neutral salt decreased. Detailed Implementation

[0028] In some embodiments, the sample to be processed by the method of the present invention is a filtered bacterial lysate containing plasmid DNA (pDNA). In some such embodiments, the bacterial host is *Escherichia coli*. In another embodiment, the sample to be processed by the method of the present invention is a filtered bacterial lysate containing pDNA, which has been treated with calcium chloride to precipitate a portion of the RNA. In another embodiment, the sample to be processed by the method of the present invention is prepared for chromatography using another method. In still other embodiments, the sample to be processed by the method of the present invention is partially purified. In still other embodiments, the sample is substantially purified supercoiled plasmid DNA, still contaminated with any amount or relative proportion of protein, RNA, and / or DNA-protein-RNA aggregates. In still other embodiments, the partially purified pDNA to be processed by the method of the present invention is partially purified by anion exchange chromatography.

[0029] In some embodiments, the HIC material used to implement the methods of the present invention is a polymer with hydrophobic ligands. The HIC ligands can be aromatic, alkyl, or a mixture thereof. Examples of aromatic HIC media include phenyl and benzyl media. Examples of alkyl HIC media include butyl, hexyl, and octyl media. Both types are used for pDNA purification and are widely available globally in various physical forms. These forms include columns packed with porous particles, bulk materials, membranes, and nanofibers, etc., which are used in chromatographic apparatuses to facilitate practical chromatography and are commonly referred to as chromatographic columns.

[0030] In some embodiments, the co-liquid salt is ammonium sulfate, or sodium sulfate, or potassium phosphate, or sodium citrate, or potassium citrate, or other co-liquid salts. In one such embodiment, the co-liquid salt is ammonium sulfate, and the concentration of ammonium sulfate used to bind DNA to the HIC column can range from 1.0 M to 2.5 M, or 1.25 M to 2.25 M, or 1.5 M to 2.0 M, or 1.7 M to 1.9 M, depending on the hydrophobicity of the HIC column. It is well known in the art how to determine the concentration of the co-liquid salt to achieve pDNA binding to the HIC column. Generally, the more hydrophobic the column, the lower the salt concentration required to achieve the desired effect.

[0031] In some embodiments, the neutral salt is sodium chloride, or potassium chloride, or lithium chloride, or ammonium chloride, or sodium acetate, or potassium acetate, or lithium acetate, or ammonium acetate, or other neutral salts. In one such embodiment, the neutral salt is sodium chloride, and the concentration of sodium chloride used to maintain the binding of RNA and host cell DNA-protein-RNA aggregates to the HIC column can be in the range of 0.5M to 5.0M, or 0.75M to 4.0M, or 1.0M to 3.0M, or 1.25M to 2.5M, or 1.5M to 2.0M, depending on the hydrophobicity of the HIC column. As with co-liquid salts, determining the appropriate salt concentration is well known in the art.

[0032] Those skilled in the art will recognize the benefit of maintaining ionic continuity between salt buffer solutions, which can prevent spontaneous precipitation of salts due to the low solubility constants of some combinations. For example, mixing potassium dihydrogen phosphate with sodium chloride carries the risk of sodium phosphate precipitation, as sodium phosphate is much less soluble than potassium dihydrogen phosphate. Mixing potassium dihydrogen phosphate with potassium chloride mitigates this risk. In similar embodiments, mixing high concentrations of ammonium sulfate and sodium chloride may result in precipitation of less soluble sodium sulfate. This problem does not occur if ammonium sulfate is mixed with ammonium chloride or sodium sulfate with sodium chloride. Such a reminder does not mean to eliminate all combinations that may produce salt solubility problems. Rather, it is important to note that if a combination proves problematic, it needs to be consciously managed.

[0033] In some embodiments, the method of the present invention can be carried out at a near-neutral pH, wherein the term "near-neutral pH" is understood to mean a range from about pH 6.5 to about pH 7.5. In other embodiments, the entire method or different segments of the method or buffer solutions can be carried out at a wider range of pH values, such as from pH 6.0 to pH 8.0, or from pH 5.5 to pH 8.5, or from pH 5.0 to pH 9.0, or from pH 4.0 to pH 9.0. Those skilled in the art will recognize that the use of ammonium salts is discouraged at pH values ​​greater than 7.0, because ammonium ions spontaneously convert to ammonia gas, which makes the buffer unstable, potentially posing safety hazards and possibly damaging the desired DNA plasmid.

[0034] In some implementations, a neutral salt is not required during the initial binding of pDNA to the HIC column. The key requirement is that it must be present with the co-liquid salt during DNA elution. In one implementation, the pDNA may bind only in the first co-liquid salt, and the neutral salt may be introduced in a subsequent step to ensure its presence during the elution of the DNA plasmid.

[0035] In some embodiments, the concentration of the co-liquid salt is gradually reduced while maintaining a constant neutral salt concentration. This gradual reduction of the first salt results in a so-called linear gradient, more specifically, a descending linear gradient. In some embodiments, the concentration of the co-liquid salt is incrementally reduced while maintaining a constant neutral salt concentration. This incremental reduction results in a so-called step gradient, more specifically, a descending step gradient. In some embodiments, the elution gradient may include step segments and linear segments.

[0036] In some implementations, the concentration of the neutral salt may vary during the operation of the method.

[0037] In some embodiments, the method can be configured such that pDNA binds in the presence of a buffer containing only co-solution salts, and the gradient endpoint buffer contains only neutral salts. In this configuration, simple mathematical calculations emphasize that the concentration of the second salt in the gradient endpoint buffer must be sufficiently high to reach the threshold concentration of the second salt required to maintain the binding of RNA, protein, and DNA-protein-RNA aggregates during pDNA elution. In one such embodiment, the concentration of the first salt in the pDNA binding buffer is 2M, and the concentration of the second salt in the gradient endpoint buffer is 4M, with the concentration of the second salt increasing to 2M as the concentration of the first salt decreases to 1M. This gradient-forming method is known in the art as a cross-gradient, where one component is concentrated in the starting buffer and a different component is concentrated in the endpoint buffer.

[0038] In one embodiment using a cross-gradient method, the binding buffer containing the co-liquid salt may contain a neutral salt, wherein the concentration of the neutral salt in the binding buffer differs from the concentration of the neutral salt in the gradient endpoint buffer. In one such embodiment, the concentration of the second salt in the gradient initiation buffer is lower than its concentration in the gradient endpoint buffer. In other embodiments, the concentration of the co-liquid salt in the gradient endpoint buffer is lower than the concentration of the co-liquid salt in the gradient initiation buffer.

[0039] In some implementations, once the desired pDNA has eluted, the column can be regenerated via a cleaning step. In some such implementations, the cleaning step formulation may be 1M NaOH, or lower or higher concentrations of NaOH, or 1M NaOH plus 2M NaCl, or 500mM NaOH plus 3M NaCl, or some other combination of NaOH and NaCl, or some combination of KOH and KCl. The cleaning solution may also include a chelating agent, such as ethylenediaminetetraacetic acid (EDTA) in concentrations ranging from 1mM to 100mM.

[0040] In some embodiments, a second elution may be performed after the DNA plasmid elution, wherein the concentration of the neutral salt is reduced to recover the contaminants. In one such embodiment, the second elution may be performed in a single step, such that the contaminants are concentrated for analysis. In another such embodiment, the second elution may be performed as a multi-step or linear gradient to assess the relative retention of various contaminants.

[0041] In some implementations, the aim is to concentrate pDNA while removing small molecule contaminants, RNA, host cell DNA-protein-RNA, and protein contaminants, and to intentionally sacrifice the ability of the first salt to separate supercoiled pDNA from open-loop pDNA. pDNA can be eluted in the step to reduce or eliminate co-liquid salts while maintaining the presence of neutral salts.

[0042] In some embodiments, to precipitate a portion of the contaminants, the sample can be prepared for application to the HIC column by first exposing the sample to a neutral salt. In the case of such precipitate formation, it can be removed before adding a first co-liquid salt to the sample, prior to binding the sample to the HIC column.

[0043] In some embodiments, the sample can be prepared for use on a HIC column by first exposing it to a neutral salt in the presence of hydrophobic particles, allowing protein, RNA, and DNA-protein-RNA contaminants to bind to the particles to aid in their sedimentation. In one such embodiment, the neutral salt can be lithium chloride or calcium chloride. Those skilled in the art of plasmid DNA purification will recognize that this can replace the common practice of precipitation with calcium chloride to reduce RNA contamination, or be used in combination with calcium precipitation, to more effectively remove protein, RNA, and DNA-protein-RNA aggregates prior to the first chromatographic purification step.

[0044] In one embodiment, the sample preparation method is performed on a hydrophobic surface, rather than on loose particles. In such an embodiment, the sample preparation method is performed using a hydrophobic depth filtration device or another device or surface accommodating the hydrophobic surface, possibly comprising a column filled with porous hydrophobic particles or nanofibers. In one embodiment, in the presence of a hydrophobic surface, after preparing the sample by treatment with a neutral salt, a co-liquid salt may be subsequently added to the treated sample to prepare the sample according to the method of the present invention. In another embodiment, in the presence of a hydrophobic surface, after preparing the sample by treatment with a neutral salt, the sample may be diluted with water or a low-conductivity buffer to prepare a sample for anion exchange chromatography. In yet another embodiment, in the presence of a hydrophobic surface, after preparing the sample by treatment with a neutral salt, the sample may be equilibrated for another type of treatment.

[0045] In some embodiments, the method of the present invention is performed as a first chromatographic step during the purification of plasmid DNA. In other embodiments, the method of the present invention is performed as a second or subsequent chromatographic step during the purification of plasmid DNA.

[0046] In some embodiments, the method of the present invention is performed prior to an anion exchange chromatography step. In other embodiments, the method of the present invention is performed after an anion exchange chromatography step. In another embodiment, the method of the present invention is combined with a metal affinity chromatography step. In another embodiment, the method of the present invention is combined with another chromatographic step. In another embodiment, the method of the present invention is combined with more than one additional chromatographic step. In one such embodiment, the method of the present invention is combined with an anion exchange chromatography step and a metal affinity chromatography step. In another such embodiment, the method of the present invention is combined with an anion exchange or metal affinity chromatography step and another chromatographic step. In any of the above embodiments, the additional chromatographic step may be a size exclusion chromatography step. In other embodiments, the method of the present invention may be a single chromatographic step.

[0047] Those skilled in the art will recognize that RNA contamination in pDNA formulations is diverse. It may contain RNA of various sizes, and RNA is often tightly bound to proteins, forming complexes with chromatographic behavior different from that of purified RNA. The method of the present invention will particularly enhance the removal of large RNA, RNA-protein complexes, and RNA-protein-DNA complexes. Very small RNA populations may be eluted during the reduction of co-liquid salts to elute DNA, but such RNA populations will be reduced more effectively by anion exchange chromatography compared to RNA contaminating pDNA when eluting HIC with a simple co-liquid salt gradient under conventional conditions.

[0048] References

[0049] All references cited in this article are incorporated by way of citation where they do not conflict with the explicit teachings of this article.

[0050] [1] C Schafer-Nielsen, C Rose, Separation of nucleic acids and chromatin proteins by hydrophobic interaction chromatography, Biochim Biophys Acta 696 (1982) 323-331.

[0051] [2] W Melander, C Horvath, Effects of salt on protein precipitation and hydrophobic interactions in chromatography: an interpretation of anisotropic series, Arch Biochem Biophys 183 (1977) 200-215.

[0052] The invention is further explained by the following non-limiting examples.

[0053] Example

[0054] Example 1

[0055] Removal of proteins, RNA, DNA-protein-RNA aggregates, and low molecular weight contaminants from plasmid DNA preparations.

[0056] The butyl stock for HIC was equilibrated with a combination of 50 mM Hepes, 1.8 M ammonium sulfate, 1.8 M NaCl, 10 mM EDTA, and pH 7.0. A sample containing *E. coli* lysate precipitated with calcium chloride and filtered through supernatant was titrated to pH 7.0, and ammonium sulfate was added to a final concentration of 1.8 M. The sample was loaded onto the column, and the column was washed with equilibration buffer to displace unbound low-molecular-weight contaminants. Plasmid DNA was then eluted with a linear gradient decreasing in ammonium sulfate concentration while maintaining a constant sodium chloride concentration. See [link to relevant documentation] Figure 1 (ft: flow through, sc: superspiral, oc: open-circular).

[0057] Example 2

[0058] Binding and elution of double-stranded DNA

[0059] Samples of double-stranded DNA of varying sizes, ranging from 80 to 10,000 base pairs, were loaded into a high-density butyl (HIC) stock solution at 50 mM Tris pH, 10 mM EDTA, 2.5 M ammonium sulfate, and pH 7.2. The loaded column was reequilibrated to 50 mM Tris, 10 mM EDTA, 2.5 M ammonium sulfate, 1.2 M sodium chloride, and pH 7.2. The column was then eluted with a linear gradient to 50 mM Tris pH, 10 mM EDTA, 1.2 M sodium chloride, and pH 7.2. Double-stranded DNA was eluted in the gradient. See [link to relevant documentation]. Figure 2 .

[0060] Example 3

[0061] Binding and non-elution of single-stranded RNA

[0062] The conditions of Example 1 were repeated, except that the sample contained single-stranded RNA of varying sizes, ranging from 200 to 6,000 bases. The RNA failed to elute in a decreasing ammonium sulfate gradient and remained bound to the column in NaCl.

[0063] Example 4

[0064] pDNA was eluted from the HIC column by reducing the concentration of the co-solution salt while maintaining a constant neutral salt concentration. A binding buffer concentrate (Buffer A) containing 50 mM Hepes, 1.25 M sodium sulfate, 3.0 M sodium chloride, and pH 7.2 was prepared. A pDNA elution buffer (Buffer B) containing 50 mM Hepes, 3.0 M sodium chloride, and pH 7.2 was prepared. An RNA elution buffer (Buffer C) containing 50 mM Hepes and pH 7.2 was prepared. 100 μL of butylated stock was equilibrated with a mixture of 90% Buffer A and 10% Buffer B at a flow rate of 0.5 mL / min. Approximately 4.7 kbp of purified DNA plasmid was loaded onto the column and washed with the equilibration buffer for 10 min. pDNA was then eluted to Buffer B using a linear gradient for 4 min. Buffer B was then passed through the column for another 4 min. The buffer composition was then switched to Buffer C and continued for an additional 7 min. Results are shown below. Figure 3 As shown. pDNA binds to the column under equilibration conditions and remains bound during washing. It elutes in a gradient to buffer B. The thin solid line represents the buffer baseline.

[0065] Example 5

[0066] The HIC column retained RNA while the concentration of the co-solution salt was decreased and the concentration of the neutral salt was kept constant. RNA was then eluted by decreasing the concentration of the neutral salt. The conditions of Example 4 were repeated, except that the sample used consisted of mRNA of 4400 nucleotides in length. Results were as follows: Figure 4 As shown. mRNA bound to the column under equilibration conditions remains bound during washing. It remains bound during the gradient to buffer B and subsequent washes in buffer B. Eluting is then performed to buffer C in this step. The thin solid line represents the buffer baseline.

[0067] Example 6

[0068] While maintaining a constant neutral salt concentration, pDNA was eluted from the HIC column by decreasing the concentration of the co-solution salt, followed by elution of RNA by decreasing the concentration of the neutral salt. The conditions of Example 3 were repeated, except that the sample used consisted of a mixture of 4.7 kbp pDNA and 4400 nucleotides mRNA. Results were as follows... Figure 5 As shown. pDNA was eluted as in Example 4. mRNA was eluted as in Example 5. The thin solid line represents the buffer baseline.

[0069] Given that the purpose of the method of the present invention is to produce pDNA components of higher purity, it should be understood that the second elution step, which reduces the concentration of neutral salt, is included in the above embodiments primarily to illustrate that the concentrations of co-solvent salt and neutral salt can be manipulated independently to control different types of nucleic acids and their derivatives. In a manufacturing environment, obtaining eluted contaminants by reducing the concentration of neutral salt is of little value. This shortens and simplifies the process of directly proceeding to the sodium hydroxide washing step.

Claims

1. A method for preparing a purified pDNA formulation from a sample containing pDNA and contaminants, the method comprising the following steps: The sample is contacted with a hydrophobic interaction chromatographic HIC material in a solution containing co-liquid salts and neutral salts at concentrations sufficient to force pDNA and contaminants to adsorb onto the HIC material. After adsorbing pDNA onto the HIC material, the concentration of the co-solution salt is reduced in the presence of neutral salt, thereby... pDNA is desorbed from the HIC material, while pollutants remain adsorbed through the continued presence of neutral salt. Obtain pDNA formulation; in, During the contact step and the reduction step, the concentration of the neutral salt remains unchanged.

2. The method as described in claim 1, characterized in that, The co-liquid salt is selected from the group consisting of ammonium sulfate, sodium sulfate, potassium dihydrogen phosphate, sodium citrate, potassium citrate, or combinations thereof.

3. The method as described in claim 1 or 2, characterized in that, The concentration of the co-liquid salt is from 1.0 M to 2.5 M.

4. The method as described in claim 1 or 2, characterized in that, The concentration of the co-liquid salt is from 1.25 M to 2.25 M.

5. The method as described in claim 1 or 2, characterized in that, The concentration of the co-liquid salt is 1.5 M to 2.0 M.

6. The method as described in claim 1 or 2, characterized in that, The concentration of the co-liquid salt is 1.7 M to 1.9 M.

7. The method as described in claim 1 or 2, characterized in that, The neutral salt is selected from the group consisting of sodium chloride, potassium chloride, lithium chloride, ammonium chloride, sodium acetate, potassium acetate, lithium acetate, ammonium acetate, or combinations thereof.

8. The method as described in claim 1 or 2, characterized in that, The concentration of the neutral salt is in the range of 0.5 M to 5.0 M.

9. The method as described in claim 1 or 2, characterized in that, The concentration of the neutral salt is in the range of 0.75 M to 4.0 M.

10. The method as described in claim 1 or 2, characterized in that, The concentration of the neutral salt is in the range of 1.0 M to 3.0 M.

11. The method as described in claim 1 or 2, characterized in that, The concentration of the neutral salt is in the range of 1.25 M to 2.5 M.

12. The method as described in claim 1 or 2, characterized in that, The concentration of the neutral salt is in the range of 1.5 M to 2.0 M.

13. The method as described in claim 1 or 2, characterized in that, The sample is a lysate of prokaryotic cells containing plasmid DNA.

14. The method as described in claim 1 or 2, characterized in that, The contaminants are selected from the group consisting of: proteins, RNA-protein aggregates, DNA-protein aggregates, and DNA-protein-RNA aggregates.

15. The method as described in claim 1 or 2, characterized in that, The HIC material is a polymer with hydrophobic ligands.

16. The method as described in claim 15, characterized in that, The hydrophobic ligand has aromatic properties; or has alkyl properties.

17. The method as described in claim 16, characterized in that, The aromatic hydrophobic ligands are phenyl and / or benzyl ligands.

18. The method as described in claim 16, characterized in that, The hydrophobic ligand with alkyl properties is a butyl, hexyl, or octyl ligand, or a combination thereof.

19. The method as described in claim 1 or 2, characterized in that, The hydrophobic interaction chromatographic HIC material is configured in the chromatographic column.

20. The method as described in claim 1 or 2, characterized in that, Further chromatographic steps may be used before or after the hydrophobic interaction chromatography.

21. The method as described in claim 20, characterized in that, The further chromatographic step is anion exchange chromatography.

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