Continuous sample purification systems and methods

Patent metadata

Field	Value
Publication number	US-11426679-B2
Application number	US-201616067509-A
Country	US
Kind code	B2
Filing date	Dec 21, 2016
Priority date	Dec 29, 2015
Publication date	Aug 30, 2022
Grant date	Aug 30, 2022

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

What is claimed is:

1. A method of purifying a biological molecule in a continuous flow sample purification system, the method comprising:
(i) providing a biological sample comprising a population of a biological molecule of interest and a population of particulate contaminants;
(ii) mixing the biological sample with a first fluid phase and a second fluid phase to form an emulsion of the first fluid phase and the second fluid phase with the population of the biological molecule and the population of particulate contaminants dispersed in the emulsion as a colloidal suspension, 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 wherein one of the first fluid phase or the second fluid phase comprises an organic molecule, hydrophilic polymer or polymeric glycol, and the other of the first fluid phase or the second fluid phase comprises a salt, the emulsion comprising, if any, less than about 5% by weight or by volume dextran;
(iii) allowing the emulsion 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, wherein (i) greater than about 75% of the population of the biological molecule from the emulsion is disposed in a first one of the first phase body or the second phase body, and greater than about 75% of the population of particulate contaminants from the emulsion are disposed in a second one of the first phase body and the second phase body, and (ii) the first phase body has less than about 10% second fluid phase by volume disposed therein, and the second phase body has less than about 10% first fluid phase by volume disposed therein;
(iv) separating the first phase body from the second phase body;
(v) concentrating the population of the biological molecule disposed in the first phase body;
(vi) extracting at least a portion of the population 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 mixture, the mixture comprising the first fluid phase, the third fluid phase, and the portion of the population of the biological molecule;
(c) allowing the mixture to settle into a first phase portion and a third phase portion, the third phase portion comprising the third fluid phase and the portion of the population of the biological molecule;
(d) optionally applying an acoustic wave to a portion of the mixture, thereby enhancing formation of the first phase portion and the third phase portion; and
(e) separating the third phase portion from the first phase portion;

(vii) optionally performing sterile filtration of the third phase portion;
(viii) performing a liquid buffer exchange on the third phase portion to produce a chromatography sample, the chromatography sample comprising the portion of the population of the biological molecule disposed in a chromatography buffer;
(ix) performing a first ion exchange chromatographic purification step on the chromatography sample, the first ion exchange chromatographic purification step comprising multi-modal cation exchange or multi-modal anion exchange;
(x) optionally performing a second ion exchange chromatographic purification step on the chromatography sample, the second ion exchange chromatographic purification step comprising multi-modal cation exchange or multi-modal anion exchange;
(xi) performing bacterial filtration on the chromatography sample; and
(xii) performing viral filtration on the chromatography sample.

2. The method of claim 1 , wherein:
(i) the biological molecule:
(a) has a molecular weight of less than about 1000 kDa; and/or
(b) is less than about 200 nm in size; and

(ii) the particulate contaminants:
(a) have a molecular weight greater than about 1000 kDa; and/or
(b) are greater than about 200 nm in size.

3. The method of claim 1 , wherein the emulsion:
(i) has a pH less than about 4.5; and/or
(ii) comprises, if any, less than or equal to 10% by weight sodium chloride or potassium chloride.

4. The method of claim 1 , wherein:
(i) 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;
(ii) 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;
(iii) 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
(iv) 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.

5. The method of claim 1 , wherein the step of mixing the biological sample with the first fluid phase and the second fluid phase to form the emulsion is performed in a 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 settling compartment in fluid communication with the mixing compartment such that the emulsion flows from the mixing compartment into the settling compartment, the emulsion 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.

6. The method of claim 5 , wherein the particulate removal assembly is adapted for continuous flow operation wherein:
(i) the biological sample, the first fluid phase, and the second fluid phase are continuously introduced into the mixing compartment and mixed by the means for mixing, thereby continuously forming the emulsion, during a period of time;
(ii) the emulsion 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.

7. The method of claim 1 , wherein the step of extracting at least the portion of the population of the biological molecule from the first phase body is performed in a multi-stage liquid-liqu

Assignees

• Life Technologies Corp

Inventors

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

Classifications

• B01D11/02 Primary

  of solids · CPC title

• B01D15/362

  Cation-exchange · CPC title

• B01D11/0465 Primary

  comprising vibrating mechanisms, radiations · CPC title

• B01D11/0492

  Applications, solvents used · CPC title

• B01D11/0488

  Flow sheets · CPC title