Bioprocessing of bacteriophage as dry powders

This thesis describes an alternative bioprocess for bacteriophages involving room temperature co-precipitation of an aqueous mixture of phage (Siphoviridae) and a crystallisable carrier (glutamine or glycine) in excess water miscible organic solvent (isopropanol or isobutanol). The resultant suspension of phage-coated microcrystals can be harvested by filtration and the residual solvent removed rapidly by air-drying at a relative humidity of 52 or 75%. Albumin or trehalose added at 5% w/w of the crystalline carrier provide for better stabilization of the phage during co-precipitation. Free-flowing dry powders generated from an aqueous solution of phage (~14 log¹⁰ pfu/ml) can be reconstituted in the same aqueous volume to a phage titre of almost 11 log¹⁰ pfu/ml; high enough to permit subsequent formulation steps following bioprocessing. The phage-coated microcrystals remain partially stable at room temperature for at least one month. We anticipate that this bioprocessing technique will have application to other phage families as required for the development of phage therapies. In the final part of this thesis we describe unfinished studies aiming to investigate the potential uses of other excipients for bacteriophage formulation, including polyethylene glycol and a series of salts selected from a Hofmeister series.This thesis describes an alternative bioprocess for bacteriophages involving room temperature co-precipitation of an aqueous mixture of phage (Siphoviridae) and a crystallisable carrier (glutamine or glycine) in excess water miscible organic solvent (isopropanol or isobutanol). The resultant suspension of phage-coated microcrystals can be harvested by filtration and the residual solvent removed rapidly by air-drying at a relative humidity of 52 or 75%. Albumin or trehalose added at 5% w/w of the crystalline carrier provide for better stabilization of the phage during co-precipitation. Free-flowing dry powders generated from an aqueous solution of phage (~14 log¹⁰ pfu/ml) can be reconstituted in the same aqueous volume to a phage titre of almost 11 log¹⁰ pfu/ml; high enough to permit subsequent formulation steps following bioprocessing. The phage-coated microcrystals remain partially stable at room temperature for at least one month. We anticipate that this bioprocessing technique will have application to other phage families as required for the development of phage therapies. In the final part of this thesis we describe unfinished studies aiming to investigate the potential uses of other excipients for bacteriophage formulation, including polyethylene glycol and a series of salts selected from a Hofmeister series