Development of Salmonella phage SE40 production technology: from cultivation to obtaining purified phage lysate for phage therapy

Bacteriophages are considered as a promising alternative to antibiotics, especially in cases where standard therapy is ineffective due to antibiotic resistance of pathogens. Bacteriophages obtained in production require effective purification from residual proteins, bacterial cell DNA, endotoxins and other contaminants to ensure their safety, quality and compliance with regulatory requirements. Thus, the purpose of this study is to develop a technology for obtaining phage SE40 in high titer, develop and optimize a multi-stage process for purification and concentration of phage lysate aimed at obtaining highly purified and stable concentrates with an increased titer of viral particles suitable for further use in phage therapy. In the course of the work, the optimal nutrient medium for cultivation, the kinetics of host strain growth and the ratio of multiplicity of infection for a high yield of phage particles were determined. Multi-stage filtration was carried out to purify and concentrate the phage lysate. At the first stage, the phage lysate was passed through glass fiber filters with pores of 1,2 and 0,65 μm, then through polyethersulfone membranes of 0,45/0,22 μm at a constant flow and a pressure of up to 1,0 bar. Then, a Sartoflow Smart tangential filtration system with a polysulfone recirculation cup (1 L) was used to remove residual proteins, DNA and endotoxins. The solution was supplied by a membrane piston pump, and the pressure was controlled by sensors on the feed, permeate and retentate lines. As a result of phage lysate purification by the proposed method, it was possible to reduce the endotoxin content by 420 times, residual protein – by 2,5 times and residual DNA of host bacteria – by 15 times. The concentration of bacteriophages was 1011–1012 PFU/ml.