Method for purification of bacteriophage particles
AssigneeAdaptive Phage Therapeutics, Inc.
InventorsJoseph Robert Fackler, Carl Merril, Jarrar Haider, Viet Dang
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Abstract
A method of recovering viable phage from, for example, a crude phage preparation such as a lysate resulting from amplification of phage in bacterial cell culture is disclosed. The method may be “universal”; that is, applicable to the purification of a broad range of phage species and strains. The phage product resulting from the method may have an acceptably low endotoxin titer (e.g. less than 500 EU/ml) and sufficiently high phage titer (e.g. >1×109 PFU/ml) for use in therapeutic applications.
Claims19
A method of recovering viable phage from a crude phage preparation, wherein the method comprises the steps of:
- a) adding the crude phage preparation to Tangential Flow filtration system comprising a size exclusion filter, wherein said filter comprises a pore size smaller than a size of the phage contained within the crude phage preparation;
- b) passing a series of buffers over the filter, wherein said series of buffers comprise, in any order:
- 1) A buffer that disrupts ionic protein interactions, wherein said buffer comprises sodium chloride and/or magnesium chloride at a concentration in the range of 250-950 mM;
- 2) A buffer comprising a detergent in a concentration sufficient to disrupt hydrophobic/hydrophilic interactions, wherein said detergent is a polyoxyethylene derivative and/or a zwitterionic surfactant;
- and
- 3) A buffer at a concentration that precipitates the phage without denaturing the phase;
- wherein said buffer comprises a salt selected from the group consisting of ammonium sulfate, sodium sulfate and magnesium sulfate;
- wherein one or both of steps 1) and 2) optionally may be repeated;
- and
- c) recovering said phage from the filter, wherein said recovered phage are viable.
A method of recovering viable phage from a crude phage preparation, comprising:
- a) adding the crude phage preparation to a tangential flow filtration system comprising a size exclusion filter, wherein said filter comprises a pore size smaller than the size of the phage in the crude phage preparation;
- b) washing the resulting lysate with a high ionic strength buffer that disrupts ionic protein interactions, wherein said buffer comprises 250-950 mM sodium chloride;
- c) washing the resulting lysate with a buffer comprising Triton X-100 in a concentration sufficient to disrupt hydrophobic/hydrophilic interactions;
- d) washing the resulting lysate with a buffer comprising ammonium sulfate at a concentration that precipitates the phage without denaturing the phage;
- and
- e) recovering said viable phage from the filter.
Dependencies are inferred from the claim text.