2.5. VLP purification
VLPs were purified in a single step by steric exclusion chromatography
(SXC). Chromatography runs were performed at 6 mL/min using an Äkta
Purifier system with software Unicorn 5.20 (Cytiva, Sweden). Two buffers
were used to obtain the desired PEG concentrations in each step: (i)
buffer A: PBS (137 mM NaCl, 2.7 mM KCl, 10 nM
Na2HPO4, 1.8 mM
KH2PO4, adjusted to pH 8.0); (ii) buffer
B: PBS (pH 8.0) containing 16% (m/v) PEG 6000. For equilibration and
wash steps, a 1:1 mixture of buffers A and B was fed to maintain PEG
concentration at 8% (m/v). During sample injection, cell culture
supernatant and buffer B were in-line mixed (1:1) in order to obtain the
desired PEG concentration (8% m/v) while reducing the risk of product
precipitation prior to injection (Lee et al., 2012). Elution was
accomplished by increasing buffer A proportion in order to decrease PEG
concentration and to release VLP.