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

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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).

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Acknowledgments

We are grateful to Dr. Carla Marusic for manuscript revision and Philogen S.p.A. for the pilot-scale purification experiments. This work was partially funded by a grant of the Italian Government (‘Ministero per le Attività Produttive, Direzione Generale Produttivita’).

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Authors and Affiliations

  1. ENEA, Dipartimento BIOTEC, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, Via Anguillarese 301, 00123, Rome, Italy

    Raffaele Lombardi, Maria Elena Villani, Mariasole Di Carli, Patrizia Brunetti, Eugenio Benvenuto & Marcello Donini

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  1. Raffaele Lombardi
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  2. Maria Elena Villani
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  3. Mariasole Di Carli
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  4. Patrizia Brunetti
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  5. Eugenio Benvenuto
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  6. Marcello Donini
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Corresponding authors

Correspondence to Maria Elena Villani or Marcello Donini.

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Raffaele Lombardi and Maria Elena Villani share equal contribution to the work.

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Lombardi, R., Villani, M.E., Di Carli, M. et al. Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration. Transgenic Res 19, 1083–1097 (2010). https://doi.org/10.1007/s11248-010-9382-9

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  • DOI: https://doi.org/10.1007/s11248-010-9382-9

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