Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration.
It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15-20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).
ENEA, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, 00123, Rome, Italy.
Raffaele
R
Lombardi
Maria Elena
ME
Villani
Mariasole
M
Di Carli
Patrizia
P
Brunetti
Eugenio
E
Benvenuto
Marcello
M
Donini
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D010865:Pilot Projects; D018515:Plant Leaves / Q000276:immunology; D046549:Plantibodies / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D030821:Plants, Genetically Modified; D015202:Protein Engineering; D020349:Surface Plasmon Resonance; D014026:Nicotiana / Q000235:genetics* / Q000276:immunology*; D014618:Vacuum
D000912:Antibodies, Neoplasm; D004731:Endotoxins; D007074:Immunoglobulin G; D046549:Plantibodies