Continuous Sample Purification Systems and Methods

Patent metadata

Field	Value
Publication number	US-2019001237-A1
Application number	US-201616067509-A
Country	US
Kind code	A1
Filing date	Dec 21, 2016
Priority date	Dec 29, 2015
Publication date	Jan 3, 2019
Grant date	—

Abstract

Sample purification systems include a particle extraction assembly having a mixing compartment and a settling compartment. A biological sample is mixed with two liquid phases formulated to effectuate transfer of a biological molecule into a first phase and particulate contaminants into a second phase. The first phase includes a solubilizing salt, the second phase includes an organic molecule, and the mixture can have little or no monoatomic salt or dextran. The molecule-containing first phase can be optionally concentrated without also concentrating the particulate contaminants and introduced into a multi-stage liquid-liquid extractor, by which the biological molecule or molecular contaminants are extracted from the first phase into a third phase, thereby purifying the molecule away from contaminants. The extracted sample can be further purified through a series of processing steps. The system can be run in continuously mode to maintain sterility of the sample.

First claim

1 . A method of purifying a biological molecule, comprising:
mixing a biological sample with a first fluid phase and a second fluid phase to form a first mixture, the first fluid phase having a first density and the second fluid phase having a second density, the second density being different than the first density, the first fluid phase being immiscible with the second fluid phase, the biological sample comprising an amount of a biological molecule and an amount of a particulate contaminant; and allowing the first mixture to settle into a first phase body comprising the first fluid phase and a second phase body comprising the second fluid phase, the first phase body being separable from the second phase body, at least a portion of the amount of the biological molecule being disposed in the first phase body, and at least a portion of the amount of the particulate contaminant being disposed in the second phase body.

2 . The method of claim 1 , wherein:
(i) greater than about 75% of the amount of the biological molecule is disposed in the first phase body; (ii) greater than about 75% of the amount of the particulate contaminant is disposed in the second phase body; (iii) the first phase body having less than about 10% second fluid phase by volume disposed therein; and/or (iv) the second phase body having less than about 10% first fluid phase by volume disposed therein.

3 . The method of claim 1 , wherein:
(i) the biological molecule:
(a) has a concentration greater than about 100 mg/L in the first phase body;
(b) has a molecular weight, the molecular weight of the biological molecule being less than about 1000 kDa;
(c) is less than about 200 nm in size; and/or
(d) comprises a protein, monoclonal antibody, enzyme, nucleic acid, peptide, antibiotic, painkiller, or sedative; and/or
(ii) the particulate contaminant: (a) has a molecular weight greater than about 1000 kDa; (b) is greater than about 200 nm in size; (c) comprises cellular material; and/or (d) comprises one or more structural components of a cell.

4 . The method of claim 1 , wherein the first mixture:
(i) has a pH less than about 4.5; (ii) comprises, if any, less than about 5% by weight or by volume dextran; and/or (iii) comprises, if any, 0-10% by weight sodium chloride or potassium chloride.

5 . The method of claim 1 , wherein:
(i) one of the first fluid phase or the second fluid phase comprises an organic molecule; (ii) one of the first fluid phase or the second fluid phase comprises a hydrophilic polymer or polymeric glycol, the hydrophilic polymer or polymeric glycol; (iii) one of the first fluid phase or the second fluid phase comprises a salt; (iv) one of the first fluid phase or the second fluid phase comprises PEG having an average molecular weight of or between about 400 and/or 600 kDa at a concentration of about 15-20% by weight and the other of the first fluid phase or the second fluid phase comprises PO4, SO4 or Citrate at a concentration of about 12.5-17.5% by weight; (v) one of the first fluid phase or the second fluid phase comprises PEG having an average molecular weight of about 1450 kDa at a concentration of about 6-10% by weight and the other of the first fluid phase or the second fluid phase comprises PO4, SO4 or Citrate at a concentration of about 12-15% by weight; (vi) one of the first fluid phase or the second fluid phase comprises PEG having an average molecular weight of about 3350 kDa at a concentration of about 5-9% by weight and the other of the first fluid phase or the second fluid phase comprises PO4, SO4 or Citrate at a concentration of about 10-13% by weight; and/or (vii) one of the first fluid phase or the second fluid phase comprises PEG having an average molecular weight of about 6000 kDa at a concentration of about 4-8% by weight and the other of the first fluid phase or the second fluid phase comprises PO4, SO4 or Citrate at a concentration of about 9-12% by weight.

6 . The method of claim 1 , wherein the step of allowing the first mixture to settle comprises:
(i) positioning of a less-dense phase body of the first phase body and second phase body vertically above a more-dense phase body of the first phase body and second phase body; and/or (ii) formation of an at least partially horizontal interface between the first phase body and the second phase body.

7 . The method of claim 1 , wherein mixing comprises forming an emulsion of the first fluid phase and the second fluid phase, the amount of the biological molecule and the amount of the particulate contaminant being dispersed in the emulsion.

8 . The method of claim 1 , wherein the mixing is performed in a sample mixing compartment of a particulate removal assembly, the mixing compartment having means for mixing the biological sample with the first fluid phase and the second fluid phase, the particulate removal assembly further comprising:
a sample settle compartment in fluid communication with the mixing compartment such that the first mixture flows from the mixing compartment into the settling compartment, the first mixture being allowed to settle in the settling compartment; and a shielding element disposed between the mixing compartment and the settling compartment so as to at least partially separate the mixing compartment from the settling compartment.

9 . The method of claim 8 , wherein the particulate removal assembly is adapted for continuous flow operation wherein:
(i) the biological sample, first fluid phase, and second fluid phase are continuously introduced into the mixing compartment and mixed by the means for mixing, thereby continuously forming the first mixture, during a period of time; (ii) the first mixture continuously flows from the mixing compartment to the settling compartment and is allowed to continuously settle into the first phase body and the second phase body in the settling compartment during the period of time; (iii) the first phase body continuously fluidly exits the settling compartment through a first phase body outlet formed in a first end of the settling compartment during the period of time; and/or (iv) the second phase body continuously fluidly exits the settling compartment through a second phase body outlet formed in a second end of the settling compartment during the period of time, the first end being separated from the second end by a vertical distance.

10 . The method of claim 1 , further comprising:
(i) separating the first phase body from the second phase body; (ii) concentrating the portion of the amount of the biological molecule in the first phase body; and/or (iii) extracting at least part of the portion of the amount of the biological molecule from the first phase body, wherein extracting comprises:
(a) providing a third fluid phase having a third density, the third density being different than the first density, the first fluid phase and the third fluid phase being immiscible;
(b) mixing the first phase body with the third fluid phase to form a second mixture, the second mixture comprising the first fluid phase, the third fluid phase, and the portion of the amount of the biological molecule;
(c) allowing the second mixture to settle into a first phase portion and a third phase portion, the third phase portion comprising the third fluid phase and the at least part of the portion of the amount of the biological molecule;
(d) optionally applying an acoustic wave to a portion of the second mixture, thereby enhancing formation of the first phase portion and the third phase portion; and
(e) optionally separating the third phase portion from the first phase portion.

11 . The method of claim 10 , wherein the step of extracting is performed in

Assignees

• Life Technologies Corp

Inventors

• Collins Scott P
• Rodriguez Peralta Federico Carlos
• Jones Nephi D
• Brau Christopher D

Classifications

• B01D15/362

  Cation-exchange · CPC title

• B01D15/3847

  Multimodal interactions · CPC title

• B01D15/363

  Anion-exchange · CPC title

• B01D11/0488

  Flow sheets · CPC title

• B01D11/0465 Primary

  comprising vibrating mechanisms, radiations · CPC title