Method of preparing plant-derived VLPs

A method of preparing plant-derived virus-like particles (VLPs) comprising a plant-derived lipid envelope and an influenza hemagglutinin (HA) with reduced plant protein contaminants and increased yield, the method comprising:

• a. obtaining a plant, or plant matter comprising apoplast-localized VLPs, wherein the apoplast-localized VLPs comprising the influenza HA, wherein the plant matter consists of plant leaves, leaf pieces, shredded leaves or a combination thereof;
• b. digesting the plant or plant matter using a cell wall degrading enzyme mixture comprising one or more than one cellulase and one or more than one pectinase, a first salt consisting of about 0.01% to about 0.05% sodium bisulfite, about 200 mM to about 600 mM of a second salt, about 0.1 M to about 0.8 M osmoticum, and a buffer or buffer system which maintains the pH in the range of about 5.2 to about 6.2, at a temperature of about 18° C. to about 22° C., with agitation, to produce a digested fraction, the digested fraction comprising a protoplast fraction, an apoplast fraction comprising one or more than one apoplastic protein complex that comprises the VLPs and plant debris, wherein the digestion and agitation disrupts substantially all of the protoplasts in the protoplast fraction;
• and
• c. separating the plant debris from the digested fraction using filtration with a mesh size of more than 250 μm, centrifuging to remove organelles, and to produce a supernatant, recovering the one or more than one apoplastic protein complex from the supernatant,
• wherein the supernatant comprising the one or more than one apoplastic protein complex has reduced plant protein contaminants and increased yield compared to a supernatant of a corresponding digested fraction produced by mechanical extraction.