Method for purification of bacteriophage particles
Inventors
Fackler, Joseph Robert • Merril, Carl • Haider, Jarrar • Dang, Viet
Assignees
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Publication Number
US-12006517-B2
Publication Date
2024-06-11
Expiration Date
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.
Core Innovation
The invention relates to recovering viable phage from a crude phage preparation using tangential flow filtration with a size exclusion filter. The filter comprises a pore size smaller than the size of the phage contained within the crude phage preparation, and the phage are recovered from the filter as viable recovered phage. The approach uses a sequence of buffers passed over the filter to remove or disrupt contaminating components while maintaining phage viability.
A series of buffers is used in the method to disrupt different classes of interactions. A buffer that disrupts ionic protein interactions comprises sodium chloride and/or magnesium chloride at 250-950 mM. A buffer comprising a detergent and/or a zwitterionic surfactant is used at a concentration sufficient to disrupt hydrophobic/hydrophilic interactions, including a polyoxyethylene derivative and/or a zwitterionic surfactant such as Triton X-100.
The series further includes a buffer at a concentration that precipitates the phage without denaturing the phage. The precipitating buffer is a salt selected from ammonium sulfate, sodium sulfate, and magnesium sulfate, with ammonium sulfate identified in dependent claim language. The method allows one or both of the ionic-interaction and hydrophobic/hydrophilic-interaction disruptive steps to be optionally repeated, and the recovered phage from the filter are viable.
Claims Coverage
Two independent claims are provided. Their coverage centers on tangential flow filtration using a size exclusion filter with a pore size smaller than the phage, followed by sequential buffer washes that disrupt ionic protein interactions, disrupt hydrophobic/hydrophilic interactions, and precipitate phage without denaturing, then recovering viable phage from the filter.
Tangential flow filtration with size exclusion retaining viable phage
A method of recovering viable phage from a crude phage preparation using a tangential flow filtration system comprising a size exclusion filter, wherein the filter comprises a pore size smaller than the size of the phage contained within the crude phage preparation; recovering said phage from the filter, wherein said recovered phage are viable.
Disruption of ionic protein interactions using 250-950 mM salt
Passing a series of buffers over the filter, comprising a buffer that disrupts ionic protein interactions, wherein the buffer comprises sodium chloride and/or magnesium chloride at a concentration in the range of 250-950 mM.
Disruption of hydrophobic/hydrophilic interactions using detergent or zwitterionic surfactant
Passing a series of buffers over the filter, comprising a buffer comprising a detergent in a concentration sufficient to disrupt hydrophobic/hydrophilic interactions, wherein the detergent is a polyoxyethylene derivative and/or a zwitterionic surfactant.
Precipitation of phage without denaturing using selected sulfate salts
Passing a series of buffers over the filter, comprising a buffer at a concentration that precipitates the phage without denaturing the phage; wherein the buffer comprises a salt selected from the group consisting of ammonium sulfate, sodium sulfate and magnesium sulfate.
Sequential washing with Triton X-100 and ammonium sulfate
A method comprising washing with a buffer comprising Triton X-100 in a concentration sufficient to disrupt hydrophobic/hydrophilic interactions, and washing with a buffer comprising ammonium sulfate at a concentration that precipitates the phage without denaturing the phage.
The claim coverage centers on TFF using a size exclusion filter that retains phage, followed by sequential buffer passes that disrupt ionic protein interactions using 250-950 mM sodium chloride and/or magnesium chloride, disrupt hydrophobic/hydrophilic interactions using a polyoxyethylene detergent and/or zwitterionic surfactant such as Triton X-100, and precipitate phage without denaturing using sulfate salts including ammonium sulfate. Viable phage are then recovered from the filter.
Stated Advantages
Recovery of viable phage from a crude phage preparation.
Disruption of ionic protein interactions during phage recovery.
Disruption of hydrophobic/hydrophilic interactions during phage recovery.
Precipitation of phage without denaturing the phage.
Option to repeat one or both of the ionic-interaction and hydrophobic/hydrophilic-interaction buffer steps.
Lower endotoxin titer for recovered viable phage is explicitly described via endotoxin titer thresholds in dependent claim language.
Documented Applications
Phage therapy.
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