Bacteriophage Purification via Size Exclusion Filtration
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Solution Overview
Problem
Current methods for purifying bacteriophages for therapeutic use are cumbersome, time-consuming, and not scalable, often resulting in loss of viable phage due to binding with host cell materials like endotoxins and proteins, and are not universally applicable across different phage species and strains.
Innovation Solution
A method involving selective filtration using a size exclusion filter with a series of buffers that disrupt ionic and hydrophobic interactions, followed by phage precipitation with ammonium sulfate, to retain phages while removing unwanted host cell materials, suitable for continuous filtration systems like TFF.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ion-exchange chromatography is used for phage purification, then separation based on affinity is achieved, but each unique phage requires optimization of chromatography conditions making the process cumbersome and time-consuming
Solution Approach 1:
The patent applies universality by developing a single purification protocol using Amicon filter devices that can be applied to all phage species and strains without requiring optimization for each unique phage. This universal approach eliminates the time-consuming condition optimization required by ion-exchange chromatography while maintaining effective separation.
Solution Approach 2:
The patent extracts the phage from the crude lysate using size-based filtration through Amicon filters with specific molecular weight cut-offs. This extraction method separates phage particles from host cell materials based on size differences, achieving purification without the complex optimization procedures required by chromatographic methods.
2Ease of manufacture
If isopycnic centrifugation on CsCl gradient is used for phage purification, then no optimization is required, but the process is time-consuming and not readily scalable
Solution Approach 1:
The patent replaces the mechanical centrifugation system with a filtration system using Amicon filter devices. This substitution eliminates the need for time-consuming centrifugation while enabling scalability through simple filtration operations that can be performed rapidly and scaled up by increasing filter surface area or number of filters.
Solution Approach 2:
The patent changes the purification parameter from density-based separation (centrifugation) to size-based filtration. By using filters with specific molecular weight cut-offs, the method achieves rapid separation without requiring optimization of centrifugation conditions, thereby improving both ease of use and productivity.
3Measurement precision
If conventional purification methods are used, then phage can be separated from host cell materials, but endotoxins and HCPs bind to phage resulting in multiple bands and loss in titer
Solution Approach 1:
The patent applies local quality by using filters with specific molecular weight cut-offs tailored to the size of phage particles. This localized approach ensures that only phage particles of the appropriate size are retained on the filter, while smaller contaminants like endotoxins and HCPs pass through, achieving precise separation without binding losses.
Solution Approach 2:
The patent extracts phage particles from the crude lysate through size-based filtration, physically separating them from host cell materials. This extraction method prevents binding interactions between phage and contaminants that occur in chromatographic methods, thereby maintaining phage titer while achieving purification.
4Measurement precision
If conventional purification methods are used, then phage can be purified, but the process is not universally applicable across different phage species and strains
Solution Approach 1:
The patent achieves universality by developing a purification protocol using Amicon filter devices that can be applied to all phage species and strains. The method relies on size-based filtration rather than species-specific interactions, making it universally applicable while maintaining purification precision through appropriate selection of filter molecular weight cut-offs.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method effectively reduces endotoxin titers to acceptable levels and maintains high phage titers, making the purified phage suitable for therapeutic use across a broad range of phage species and strains without substantial loss of viability.
Implementation Method 1
Adding the crude phage preparation to an active 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
Implementation Method 2
a buffer that disrupts ionic protein interactions and which comprises a high ionic strength buffer
Implementation Method 3
a buffer that disrupts hydrophobic/hydrophobic interactions and which comprises a detergent and/or non-denaturing organic solvent
Implementation Method 4
a buffer that precipitates the phage which is an ammonium sulfate buffer, a sodium sulfate buffer, a magnesium sulfate buffer or a mixture thereof
Data Source
AI summary
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 x 109 PFU/ml) for use in therapeutic applications.