Bacteriophage Purification via Affinity Chromatography

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Solution Overview

Problem

Current methods for producing bacteriophage preparations for bacterial infection therapy often fail to meet industrial purity requirements due to the presence of endotoxins and other contaminants, and existing phage display techniques require genetic modification of bacteriophage strains to express foreign proteins or peptides.

Innovation Solution

A method involving affinity chromatography using bacterial hosts expressing fusion proteins with affinity for chromatography resins, allowing for the purification and display of foreign peptides or proteins on bacteriophage capsids without modifying the phage genome, using strains like HisTag and GST, enabling effective purification and presentation of recombinant proteins on wild-type bacteriophages.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If multiple separate purification steps are used to remove endotoxins and contaminants from bacteriophage lysates, then the purity of the final preparation is improved, but the complexity and time of the manufacturing process increases

Engineering Contradiction:

Improvepurity of bacteriophage preparationVSAvoidcomplexity of purification process

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent combines multiple purification functions into a single affinity chromatography step by fusing the affinity tag to the capsid protein. This single step simultaneously purifies bacteriophages and removes endotoxins and other contaminants, replacing the traditional multi-step process that required separate purification and endotoxin removal steps.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The affinity-tagged capsid protein serves multiple functions: it enables specific binding to the chromatography resin for phage purification, and simultaneously provides a mechanism for co-purification of endotoxins and other contaminants through the affinity interaction, making the purification step universally effective for multiple contaminants.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If genetically modified bacteriophage strains are used to display foreign proteins on their surface, then the functionality and versatility of bacteriophages is improved, but the complexity of strain development and manufacturing increases

Engineering Contradiction:

Improveability to display foreign proteinsVSAvoidease of producing bacteriophage preparations

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The patent uses an affinity tag as an intermediary element fused to the capsid protein. This tag mediates the binding to the chromatography resin during purification, while the foreign protein is displayed on the capsid surface. The affinity tag acts as a bridge that enables both purification and foreign protein display without requiring complex genetic modifications of the entire phage genome.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

Instead of modifying the entire bacteriophage genome, the invention makes a localized modification by fusing the affinity tag and foreign protein coding sequences to the capsid protein gene. This local modification approach simplifies strain development while achieving the desired functionality of foreign protein display and purification capability.

Inventive Principle:

Principle #3Local quality

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 achieves single-stage purification of bacteriophages with maintained antibacterial activity, reducing the need for separate contaminant removal steps and facilitating the use of wild-type strains, thus enhancing the production of high-purity bacteriophage preparations for therapeutic and biotechnological applications.

Implementation Method 1

purification using affinity chromatography

Methodology Applied

Scientific EffectAffinity chromatography: Chromatography

Implementation Method 2

bacterial hosts expressing fusion proteins with affinity for chromatography resins

Methodology Applied

Scientific EffectAffinity interaction: Adsorption

Implementation Method 3

using affinity chromatography using bacterial hosts expressing fusion proteins with affinity for chromatography resins, allowing for the purification

Methodology Applied

Scientific EffectAffinity binding: Adsorption

Data Source

PatentUS10160956B2Method of producing bacteriophage preparations comprising purification using affinity chromatography

Publication Date: 2018.12.25 DABROWSKA KRYSTYNA

AI summary

The proposed method facilitates the single-stage and at the same time effective purification of phage preparations for therapeutic uses, and facilitates the maintenance of bacteriophage antibacterial activity both in the case of displacement of the bacteriophage from the resin and its proteolytic release. The protein modification of the phage capsid with appropriate binding motifs makes it possible to purify therapeutically bacteriophage strains using affinity chromatography. The proposed method is useful in the display of selected polypeptided on a bacteriophage capsid without the need to genetically modify the bacteriophage, and thus makes it possible to produce phage preparations for various uses using wild-type phages occurring naturally or others not additionally modified for phage-display purposes.