US 11,426,679 B2 · Continuous sample purification systems and methods

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.

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.

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Continuous sample purification systems and methods

Life of this patent

Continuous sample purification systems and methods

Life of this patent

Abstract

Claims10