Methods for purifying bacteriophage and products of manufacture containing endotoxin-free bacteriophage preparations

A method for purifying a bacteriophage from a sample comprising unpurified bacteriophages comprising or contaminated with a bacterial product or contaminant and generating a bacterial product- or contaminant-free preparation of the bacteriophage, comprising:

• (a) providing a sample comprising an unpurified bacteriophage,
• wherein the sample comprises a bacteriophage lysate or culture, and to provide a sample, the lysate or culture is cooled to between 4° C. to 37° C., at the point where bacteriophage-insensitive or bacteriophage-resistant bacteria appear in the lysate or the culture;
• and
• (b)
• (i)
• (1) amplifying bacteriophage in the unpurified bacteriophage sample by cultivating in a culture or an enrichment medium for between 18 to 24 hours, between 20 to 22 hours, or between 15 to 30 hours, and then
• (2) removing bacterial contaminants by a process comprising high-speed centrifugation at 8,000×g, or at least 8,000×g, to pellet bacterial contaminants;
• or
• (ii)
• (1) removing bacterial contaminants by a process comprising high-speed centrifugation at 8,000×g, or at least 8,000×g to pellet bacterial contaminants, and then
• (2) amplifying bacteriophage in the unpurified bacteriophage sample by cultivating in a culture or an enrichment medium for between 18 to 24 hours, between 20 to 22 hours, or between 15 to 30 hours;
• and
• (c) filtering the sample with:
• (i) a dead-end microfiltration process comprising inline stepwise 0.8, 0.45, 0.40, and 0.22 μm membrane filtration, or
• (ii) a pressure-driven cross-flow ultrafiltration (CFF) comprising a molecular weight cut-off (MWCO) at 100 kDa,
• wherein a membrane pore size of 100 kDa provides the ability to filter out an equivalent of a spherical particle with a 3 nm diameter, thereby having the ability to retain most bacteriophages,
• wherein any material smaller than a cross-flow membrane pore passes through the membrane, while bacteriophage particles that do not pass through the membrane remain in the retentate stream, and the CFF with a 100 kDa membrane pore size removes both free endotoxins 10 kDa in size, and exotoxins smaller than 30 kDa, and
• a membrane pore size of 100 kDa retains an equivalent to a spherical particle with a 3 nm diameter, thereby retaining bacteriophages,
• thereby generating a bacterial product- or contaminant-free preparation of bacteriophage.