Scalable purification of bacteriophages preparations

Area of Science:

• Biotechnology
• Microbiology
• Biopharmaceutical Manufacturing

Background:

• Phage therapy is a promising alternative to antibiotics for treating bacterial infections, especially those resistant to current drugs.
• Scalable and efficient purification methods are crucial for the clinical application of phage therapy, particularly for intravenous administration.
• Regulatory requirements necessitate the removal of impurities like endotoxins, proteins, and host cell DNA from phage preparations.

Purpose of the Study:

• To develop a scalable purification workflow for bacteriophages using anion-exchange chromatography (AEC).
• To investigate the efficacy of enzymatic pre-treatment with alkaline phosphatase (AP) to enhance endotoxin and phage separation via AEC.
• To assess the removal of endotoxins, proteins, and host cell DNA to meet regulatory standards for phage therapy.

Main Methods:

• A purification workflow centered on anion-exchange chromatography (AEC) was developed for lytic phage T4 and Escherichia coli K12.
• Alkaline phosphatase (AP) treatment at different concentrations (20 or 200 U/mL) was employed prior to AEC to reduce the negative charge of endotoxins and phages.
• Various AEC ligands, stationary phases, and H-bond chromatography were evaluated. Endotoxin, protein, and DNA removal were quantified, and phage recovery and titer were determined.

Main Results:

• A maximum phage titer of 1.26 × 10^11 PFU/mL and global recovery up to 45.1% were achieved.
• The highest endotoxin removal rate of 98.8% was observed after treatment with 20 U/mL of AP, followed by AEC using a quaternary amine column.
• All tested workflows effectively removed virtually all proteins and host cell DNA, with some preparations meeting criteria for intravenous administration based on endotoxin levels.

Conclusions:

• Enzymatic treatment with alkaline phosphatase combined with anion-exchange chromatography presents a promising and scalable purification strategy for bacteriophages.
• This purification workflow effectively removes critical impurities, making phage preparations suitable for clinical applications, including intravenous administration.
• The developed method offers a viable alternative to existing phage purification techniques, addressing key challenges in large-scale manufacturing for phage therapy.