Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration

Abstract

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).

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

“…Similarly, H10 expressed in tobacco or N . benthamiana leaves was visualized as several protein bands following non-reducing SDS-PAGE and was purified as a mixture of full-size IgG and stable proteolytic fragments after protein A chromatography [16, 20]. In accordance with these reports, we here detected H10 as a multiple protein band pattern following non-reducing SDS-PAGE in protein extracts of both young and older leaves, encompassing the ~150-kDa full-size IgG and stable fragments of smaller size.…”

“…As expected given the reported stability of H10 light chain in N . benthamiana leaves [16], the stabilizing effect of Sl CYS8 was essentially associated with the heavy chain, notably in the CH2–CH3 constant region. From a quantitative standpoint, the cystatin had little impact on total amounts of IgG product purified from leaf extracts following protein A affinity chromatography.…”

“…benthamiana plant extracts by protein A affinity chromatography, essentially as described before [16]. Infiltrated leaves collected six days post-infiltration were pooled and homogenized mechanically in 80 ml of protein extraction buffer (1X PBS, pH 7.3) using an Ultra-Turrax homogenizer T25 (IKA, Staufen, Germany).…”

“…Endogenous proteases are involved in many biological processes, and hundreds of genes coding for these enzymes have been identified in plant genomes [12,13]. Protease activities in plant protein biofactories may lead to partial or complete hydrolysis of recombinant antibody chains in leaf cells or in the leaf apoplast [14, 15], typically leading to the concomitant isolation of full-size antibodies and stable fragments from crude protein extracts following purification [16]. Despite numerous reports on antibody degradation (e.g.…”

Tomato cystatin SlCYS8 as a stabilizing fusion partner for human serpin expression in plants

“…While plant cells are able to process, fold and assemble complex mammalian proteins, it is also likely that these processes have a considerable influence over expression levels. For example, the introduction or modification of post-translational modification sites can influence the stability and accumulation of secreted recombinant proteins in plants and plant cells (Lombardi et al, 2012;Meyers et al, 2008;Xu and Kieliszewski, 2011).…”