Purification of Virus Particles
Virus particles are a difficult challenge for purification with columns of packed porous particles because such columns rely on diffusion for the virus particles to be transported into the pores where most of the binding surface area resides. The large size of these particles translates to extremely slow diffusion constants. The low efficiency of diffusion reduces binding capacity and resolution.
Besides that, many species of virus are so large that they cannot fit into the pores at all, so binding capacity is further limited to the external surface of the particles. Capacity is frequently 1010 particles or less per mL of chromatography media. Figure 2 shows a chromatogram of bacteriophage M13 purified from E. coli broth by SXC on a hydroxyl monolith.
Dynamic binding capacity was 1013 particles per mL, and the technique achieved 99.8% reduction of E. coli host proteins in a single step. DNA was reduced 93%. Despite the elevated viscosity due to the presence of PEG, more than 90% of the virus was captured during the 6 seconds transit time of a given unit of sample through the monolith. This corresponds to a flow rate of 10 bed volumes per minute that supports very short process times.
Two additional benefits of the method are that PEG is virus-stabilizing, and flow-through monolith channels is laminar. The latter feature avoids the destructive shear forces that occur in the void volume of packed particle columns. As a result, full infectivity of the purified virus was conserved.
The combination of these results makes SXC on monoliths thousands of times more effective and productive than chromatography on columns packed with porous particles. Similar results have since been achieved with several other bacteriophage species as well as influenza and Dengue virus. These results suggest that the technique will prove to be a universal method for virus purification.
This brings up the issue of how to purify virus particles using SXC. This requires a binary gradient chromatography system with pressure capabilities of at least 2 MPa.
Hydroxyl monoliths ranging from 1 mL to 8 L are available from BIA Separations. Purification of the M13 phage was done by equilibrating the A-pump inlet line with 12% PEG-6000, 500 mM NaCl, 50 mM MES, pH 6.0, and the B-pump inlet with 500 mM NaCl with, 50 mM MES, pH 6.0.
The monolith was equilibrated with 50% A/50% B at 10-bed volumes per minute. The system was paused and the B-pump inlet transferred to the sample to be purified.
When the sample was fully loaded, the system was paused and the pump-B inlet transferred back to the original buffer. The monolith was washed to baseline, and the virus eluted with a 10-bed volume linear gradient to 100% B.