Purification of the baculovirus Autographa californica M nucleopolyhedrovirus by tangential flow ultrafiltration

Membrane-based virus filtration is used in biotechnological processes due to advantages such as easy scaleup but is currently lacking standardized validation protocols. Thus, ultrafiltration (UF) of a rod-shaped virus was assessed to contribute to the body of knowledge regarding the characterization of virus filtration. A recombinant baculovirus of Autographa californica M nucleopolyhedrovirus (AcMNPV), vHSGFP, expressing egfp was filtered using polyethersulfone membranes ranging from 30 to 1000 kDa molecular weight cut-off (MWCO) and filtration parameters were previously assessed for their potential to affect virus stability. AcMNPV was concentrated 20-fold in the retentate using 100–1000 kDa membranes. However, evidence for a superposition of cake formation and pore plugging through viruses and other solution components (likely proteins) causing partially irreversible fouling of membranes dependent on the MWCO was observed. Results support UF using a 300 kDa MWCO to concentrate AcMNPV with minimized membrane fouling.

AcMNPV is a baculovirus with broad applications [10,11] that require its purification and concentration and validation of virus membrane filtration is vital in industry [1,3,6]. Viruses of similar shape and size are commercially attractive, e.g., poxviruses [18]. In this study, a 20-fold concentration of AcMNPV (BV) in a SFM was achieved using UF with commercial flat-sheet PES membranes (100–1000 kDa MWCO) at an average of 0.15 bar TMP and about 25°C. UF appears to be a useful unit operation in downstream processing of BV. However, flux loss through fouling, discussed as a superposition of various physical mechanisms, cannot be avoided [17]. An “intermediate” 300 kDa membrane showed minimum fouling. This might be rationalized through the interplay of large (virus) and small (likely protein) solution components with the pore structure of the range of membranes investigated here. Acknowledgments The authors thank Kent E. Hampton and Chris Lehiy for technical assistance. This work was funded in part by the DAAD (German Academic Exchange Service) and by the Innovation Funds of the University of Applied Sciences GiessenFriedberg.