HIGH-THROUGHPUT SCREENING OF MEMBRANE ADSORBERS FOR BACTERIOPHAGE PURIFICATION: ADDRESSING VARIABILITY IN STRAIN-SPECIFIC PROCESS DEVELOPMENT

Abstract

Bacteriophages, a promising antimicrobial alternative to modern antibiotics, can rapidly overcome bacterial resistance. However, the propagation of therapeutic phages in gram-negative bacteria leads to proinflammatory contaminants, notably endotoxins (lipopolysaccharides). To meet strict regulations on endotoxin levels, the widely accepted method for phage purification is based on polyethylene glycol precipitation followed by cesium chloride density gradient ultracentrifugation. This method, while effective, is labor-intensive and produces only small quantities of purified phage products. Moreover, the current studies on using membrane adsorbers in this application have primarily focused on removing endotoxins or recovering phages, without addressing the variability in physical properties of bacteriophages that necessitates strain-specific process development. In contrast, this thesis introduces a novel high-throughput flat sheet membrane screening approach. This approach not only aims to evaluate the effect of varying solution conditions on bacteriophage purification but also provides a comprehensive solution to the limitations of the current method, guiding larger-scale purification work. Using two clinically derived bacteriophage, this work demonstrates that the multi well device used in this work is appropriate for this application, with little background binding of bacteriophage to the device itself. Bacteriophage binding to the membrane was found to be both sensitive to the dilution of the preparation and contact time with a given membrane. The device was used to evaluate the overall binding productivity of the membranes, finding that the Natrix and Sartobind Q membranes possess superior productivity to that of Mustang Q. Furthermore, this work demonstrated that the method could be used to rapidly screen the effects of varying sodium chloride molarity on both bacteriophage and endotoxin binding, with the effect of greater NaCl molarity on both bacteriophage and endotoxin binding being generally comparable to expectations.Finally, this work demonstrates that improvements in the translatability of screened conditions to larger-scale techniques (e.g., Syringe Filtration) require a better understanding of the underlying interactions occurring between the biomolecules and membranes.