Printed monolith adsorption as an alternative to expanded bed adsorption for purifying M13 bacteriophage

Abstract

An ordered 3D printed chromatography stationary phase was used to purify M13 bacteriophage (M13) directly from crude cell culture. This new approach, which offers the same advantages as expanded bed adsorption (EBA) with regard to tolerating solids-laden feed streams but without the corresponding issues associated with fluidized bed stability that affect the latter, can be described as “printed monolith adsorption (PMA)”. PMA columns (5, 10 and 15 cm length by 1 cm diameter) were made via a wax templating method from cross-linked cellulose hydrogel and functionalized with a quaternary amine ligand. The recovery of M13 was found to be strongly linked to load flow rate, with the highest recovery 89.7% ± 6% for 1.4 × 1011 pfu/mL of resin occurring at 76 cm/h with a 10 cm column length. A recovery of 87.7% ± 5% for 1.49 × 1011 pfu/mL of media was achieved with a 15 cm column length under conditions comparable to a reported EBA process. The PMA process was completed three times faster than EBA because PMA flow rates can readily be adjusted during operation, with high flow rates and low back pressure, which is unique to the ordered monolithic media geometry used. Equilibration, wash, and cleaning steps were carried out at high flow rates (611 cm/h), minimizing process time and were limited only by the volumetric flow rate capacity of the pumps used, rather than column back pressure (<0.1 MPa at 611 cm/hr). Initial capture of M13 appears to occur on the surface of the monolith solid phase (i.e. the mobile phase channel walls) and subsequently, at a slower rate, within the internal pores of the solid phase media. The difference in binding rate between these two sites is likely caused by slow pore diffusion of the large M13 particles into the pores, with similar slow diffusion out of the pores resulting in tailing of the elution peak. The results indicate that PMA is a promising technology for the efficient purification of viruses directly from crude cell culture.