US 11,833,200 B2
Method of preparing plant-derived proteins
Louis-Philippe Vezina, Neuville (CA); Manon Couture, St. Augustin De Desmaures (CA); Dany Paquet, St. Jean Chrysostome (CA); Michele Dargis, Quebec (CA); and Marc-Andre D'Aoust, Quebec (CA)
Assigned to MEDICAGO INC., Quebec (CA)
Appl. No. 13/497,767
Filed by Louis-Philippe Vezina, Neuville (CA); Manon Couture, St. Augustin De Desmaures (CA); Dany Paquet, St. Jean Chrysostome (CA); Michele Dargis, Quebec (CA); and Marc-Andre D'Aoust, Quebec (CA)
PCT Filed Sep. 21, 2010, PCT No. PCT/CA2010/001489
§ 371(c)(1), (2), (4) Date Mar. 22, 2012,
PCT Pub. No. WO2011/035423, PCT Pub. Date Mar. 31, 2011.
Claims priority of provisional application 61/244,786, filed on Sep. 22, 2009.
Prior Publication US 2013/0067807 A1, Mar. 21, 2013
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 7/00 (2006.01); C12N 15/82 (2006.01); A61K 39/145 (2006.01); A61K 39/12 (2006.01); A61K 39/00 (2006.01)
CPC A61K 39/145 (2013.01) [A61K 39/12 (2013.01); C12N 7/00 (2013.01); C12N 15/8203 (2013.01); C12N 15/8257 (2013.01); C12N 15/8258 (2013.01); A61K 2039/517 (2013.01); A61K 2039/5258 (2013.01); C12N 2760/16123 (2013.01); C12N 2760/16134 (2013.01)] 11 Claims
 
1. A method of preparing plant derived antibodies, the method comprising:
a. obtaining a Nicotiana benthamiana plant or plant matter comprising apoplast-localized antibodies, wherein a nucleic acid comprising a nucleotide sequence encoding the antibodies has been introduced into the Nicotiana benthamiana plant in a transient manner, and wherein the Nicotiana benthamiana plant matter consists of plant leaves, leaf pieces, shredded leaves, or a combination thereof;
b. producing a digested fraction, wherein the digested fraction comprises an apoplast fraction, a protoplast fraction and plant debris, by extraction from the plant or plant matter, wherein the extraction consists of an enzymatic extraction, with a cell wall degrading enzyme mixture, wherein the cell wall degrading enzyme mixture comprises one or more than one cellulase and one or more than one pectinase, wherein a concentration of the one or more than one pectinase is about 1% (v/v) and a concentration of the one or more than one cellulase is about 1% (v/v), about 600 mM mannitol, about 75 mM citrate, and a buffer or buffer system which maintains a pH of about 6.9, at room temperature—with agitation for about 16 hours, wherein substantially all of the protoplasts in the protoplast fraction are disrupted protoplasts; and
c. separating the plant debris from the digested fraction using filtration with a mesh size of more than 250 μm, centrifuging at about 5,000 g for about 15 minutes at room temperature to remove organelles, and to produce a supernatant, and recovering the plant-derived antibodies from the supernatant.