Search

CN-116348598-B - Improved removal of RNA and contaminants in DNA plasmid formulations by hydrophobic interaction chromatography

CN116348598BCN 116348598 BCN116348598 BCN 116348598BCN-116348598-B

Abstract

A method for preparing a purified pDNA formulation 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 co-liquid salt in a concentration capable of forcing adsorption of the pDNA and contaminants on the HIC material, after adsorption of the pDNA on the HIC material, diluting the concentration of the co-liquid salt in the presence of the 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 formulation. Furthermore, a method for preparing a sample to be subjected to the method of the invention or other purification method is disclosed, in particular by anion exchange chromatography exposing the sample to neutral salts in the presence of HIC material.

Inventors

  • GAGNON PETER S
  • R. Sikkernik
  • K. Bozk

Assignees

  • 赛多利斯比亚分离有限责任公司

Dates

Publication Date
20260508
Application Date
20211005
Priority Date
20201005

Claims (20)

  1. 1. A method for preparing a purified pDNA formulation from a sample comprising pDNA and contaminants, the method comprising the steps of: Contacting the sample with a hydrophobic interaction chromatography HIC material in a solution comprising a co-liquid salt and a neutral salt in a concentration capable of forcing pDNA and contaminants to adsorb on the HIC material, After adsorption of pDNA on HIC material, the concentration of the co-liquid salt is reduced in the presence of neutral salt, thereby Desorbing pDNA from HIC material while contaminants remain adsorbed by the continued presence of neutral salts, and Obtaining a pDNA preparation; wherein the concentration of the neutral salt is unchanged during the contacting step and the reducing step.
  2. 2. The method of claim 1, wherein the co-liquid salt is selected from the group consisting of ammonium sulfate, sodium sulfate, potassium dihydrogen phosphate, sodium citrate, potassium citrate, or a combination thereof.
  3. 3. The method of claim 1 or 2, wherein the concentration of the co-liquid salt is from 1.0M to 2.5M.
  4. 4. The method of claim 1 or 2, wherein the concentration of the co-liquid salt is from 1.25M to 2.25M.
  5. 5. The method of claim 1 or 2, wherein the concentration of the co-liquid salt is from 1.5M to 2.0M.
  6. 6. The method of claim 1 or 2, wherein the concentration of the co-liquid salt is from 1.7M to 1.9M.
  7. 7. The method of claim 1 or 2, wherein 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 a combination thereof.
  8. 8. The method of claim 1 or 2, wherein the concentration of the neutral salt is in the range of 0.5M to 5.0M.
  9. 9. The method of claim 1 or 2, wherein the concentration of the neutral salt is in the range of 0.75M to 4.0M.
  10. 10. The method of claim 1 or 2, wherein the concentration of the neutral salt is in the range of 1.0M to 3.0M.
  11. 11. The method of claim 1 or 2, wherein the concentration of the neutral salt is in the range of 1.25M to 2.5M.
  12. 12. The method of claim 1 or 2, wherein the concentration of the neutral salt is in the range of 1.5M to 2.0M.
  13. 13. The method of claim 1 or 2, wherein the sample is a lysate of prokaryotic cells containing plasmid DNA.
  14. 14. The method of claim 1 or 2, wherein the contaminant is selected from the group consisting of a protein, an RNA-protein aggregate, a DNA-protein aggregate, and a DNA-protein-RNA aggregate.
  15. 15. The method of claim 1 or 2, wherein the HIC material is a polymer with a hydrophobic ligand.
  16. 16. The method of claim 15, wherein the hydrophobic ligand has aromatic properties or has alkyl properties.
  17. 17. The method of claim 16, wherein the hydrophobic ligands having aromatic properties are phenyl and/or benzyl ligands.
  18. 18. The method of claim 16, wherein the hydrophobic ligand having alkyl properties is a butyl, hexyl, octyl ligand, or a combination thereof.
  19. 19. The method of claim 1 or 2, wherein the hydrophobic interaction chromatography HIC material is disposed in a chromatography column.
  20. 20. The method according to claim 1 or 2, characterized in that a further chromatography step is used before or after the hydrophobic interaction chromatography.

Description

Improved removal of RNA and contaminants in DNA plasmid formulations by hydrophobic interaction chromatography Technical Field The present invention relates to a method of preparing a purified pDNA preparation from a sample comprising pDNA and contaminants. Background DNA plasmids obtained by lysing producer cells are generally contaminated with proteins, RNA-protein aggregates and DNA-protein-RNA aggregates. Hydrophobic Interaction Chromatography (HIC) is one of the means known for purification of plasmid DNA (pDNA) [1]. The sample was added to a solution containing a high concentration of precipitated salt and applied to a HIC column. pDNA will bind to the column, but contaminants will also bind to the column. The column was eluted with a decreasing salt gradient. Good separation can be obtained between plasmid isomers, such as supercoiled (sc), open-loop (oc) and linear pDNA, but RNA co-elutes to a large extent with the desired scDNA. Good separation of pDNA from host cell proteins, RNA-protein aggregates and DNA-protein-RNA aggregates can also be achieved. HIC does not completely remove residual contaminants, which is typically compensated for by combining HIC with Anion Exchange Chromatography (AEC). Disclosure of Invention Surprisingly, it has been found that the removal of contaminants in a pDNA formulation by HIC can be enhanced by reducing the concentration of a co-liquid salt (first salt) when eluting the column in the presence of a neutral salt (second salt). In the case where neutral salts are continuously present, pDNA is recovered by reducing the concentration of the co-liquid salts. In the continued presence of the second salt, the RNA, protein, RNA-protein aggregates and DNA-protein-RNA aggregates remain bound to the chromatographic column. It is believed that the neutral salt forces the contaminants to remain tightly bound to the hydrophobic columns. This is in sharp contrast to conventional elution methods, which form a simple gradient by lowering the concentration of the co-liquid salt in the absence of neutral salts, where various contaminants co-elute with the pDNA. This method reduces the amount of contaminants in the pDNA fraction and alters the spectrum of contaminants remaining in the pDNA fraction, and therefore combining the method of the application with another chromatography can produce higher purity pDNA than would be the case if HIC were typically combined with another chromatography. The subject of the present invention is a method for preparing a purified pDNA preparation from a sample comprising pDNA and contaminants, said method comprising the steps of: Contacting the sample with a Hydrophobic Interaction Chromatography (HIC) material in a solution comprising a co-liquid salt in a concentration capable of forcing pDNA and contaminants to adsorb on the HIC material, After adsorption of pDNA on HIC material, the concentration of the co-liquid salt is diluted in the presence of neutral salt, thereby desorbing pDNA from HIC material while the contaminant remains adsorbed by the continued presence of neutral salt, and PDNA preparation was obtained. The term "precipitation salts" represents a continued concept in the field of protein chemistry, and certain salts are known to be effective in precipitating proteins. Ammonium sulfate is an example. Other species, such as guanidine hydrochloride, are strong dissolving agents that prevent precipitation. The ability of some salts (e.g., sodium chloride) to promote protein precipitation is greatly reduced. The effect of various salts on protein solubility is known to be related to their respective ranks in the Hofmeister series [2 ]. Salts that promote precipitation are generally classified as either co-liquid salts or lyotropic salts. Salts that promote solubility are generally classified as chaotropic salts. Salts with intermediate properties (e.g. sodium chloride) are generally classified as neutral salts. In this context, the term "neutral salt" is understood not to mean the pH of the aqueous solution in which the salt is dissolved. In contrast, the term neutral salt is understood to be any salt in which the effect resulting from the combination of cations and anions and the average contribution is neither chaotropic nor colotropic, in other words neutral. In general, monovalent metal halide salts and monovalent metal acetates are considered neutral salts. In one embodiment of the method of the present invention, the co-liquid salt may be a salt comprising a co-liquid anion, a cation or both, in particular 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 may be in the range of 1.0M to 2.5M, or 1.25M to 2.25M, or 1.5M to 2.0, or 1.7M to 1.9M. Many of the use of co-liquid salts is limited due to their low solubility in water. Sodium phosphate is an example. It saturates at