Virus filtration is a critical step in the overall viral clearance strategy for the production of biotherapeutics. Virus filtration membranes provide a robust, primarily size-based, removal mechanism that compliments both adsorptive and inactivation steps. However, there is limited data suggesting that the actual degree of virus removal during virus filtration is also a function of solution conditions, suggesting that electrostatic interactions may also play a role in determining the effectiveness of virus capture. The objective of this work was to examine the effects of solution pH and ionic strength on virus retention by the Viresolve® NFP membrane.
Experiments were performed using the bacteriophage fX174 as a model virus, with the virus retention data complemented by the use of confocal microscopy to directly visualize the capture of fluorescently-labeled fX174 within the filter. Data were obtained during both constant pressure filtration and after a process disruption, in this case a short pressure release as might occur during typical process operations. Virus retention was highest at low pH and low ionic strength, conditions under which there was an attractive electrostatic interaction between the negatively-charged membrane and the positively-charged phage. In addition, the transient increase in virus transmission seen in response to a pressure disruption at pH 7.8 and 10 was completely absent at pH 4.9, suggesting that the trapped virus are unable to overcome the electrostatic attraction and diffuse out of the pores when the pressure is released.
This physical picture was further confirmed by the acquired confocal images. A two-fluorescent dye based technique was used to image and differentiate between virus particles captured before and after the pressure release. Images obtained at pH 10 showed the migration of previously captured phage; this phenomenon was absent at pH 4.9. These results provide important new insights into the factors governing virus retention using virus filtration membranes.