Chromatographic tools for plant-derived recombinant antibodies purification and characterization.

In the last two decades, plants became an interesting alternative for the production of recombinant proteins for human therapy and several antibodies expressed in plants have reached the clinical development stage. Plants are capable of post-translational modifications (PTMs) necessary for protein activity and pharmacokinetics, such as glycosylation. However, there are important kingdom-specific modifications that have to be considered when expressing recombinant proteins. Therefore, there is a need for efficient analytical methods for deep protein characterization starting from the expression platform design until the product approval to guarantee product authenticity, quality and efficacy. Literature lacks of reviews dealing with plant-derived proteins purification and characterization by chromatographic methods, thus the focus of the present review is on this topic for the most representative biotechnological drugs i.e. monoclonal antibodies (mAbs). In the first part, a comprehensive discussion of the methods applied in dowstream processes (extraction and clarification) and a detailed overview of the chromatographic techniques useful for the purification of plant-made mAbs are reported. Among purification techniques, Protein A affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, hydrophobic charge induction chromatography or mixed mode chromatography are described. In the second part, we will discuss analytical platforms based on chromatographic techniques (reverse phase, size exclusion chromatography, ion-exchange chromatography, hydrophilic interaction liquid chromatography) coupled with different detection systems (UV, Fluorescence, MS) used at protein, peptide and glycan level to characterize plant-made mAbs with their unique features.

Investigating recombinant protein exudation from roots of transgenic tobacco.

It is widely acknowledged that plant-made pharmaceuticals (PMPs) offer numerous benefits, including inexpensive production, biological safety and the facility for production at agricultural scale. At the same time, it is important to minimize any potential risk associated with this new technology, including the potential release of bioactive proteins into the environment. To address this issue, we studied transgenic Nicotiana benthamiana and Nicotiana tabacum plants expressing two recombinant single-chain variable fragment (scFv) antibodies, respectively scFvB9 and scFvH10. ScFvB9 was raised against glycoprotein G1 of Tomato spotted wilt virus (TSWV), and scFvH10 was raised against human tumor-associated antigen tenascin-C. Both antibodies were targeted to the secretory pathway using the N-terminal signal peptide from Phaseolus vulgaris polygalacturonase-inhibiting protein (PGIP), and scFvH10 carried in addition a C-terminal KDEL tetrapeptide for retention in the endoplasmic reticulum (ER). Sterile hydroponic cultures were established, allowing us to investigate whether scFvB9 and scFvH10 were present in root exudates. Intercellular fluids extracted from different plant tissues were analyzed by western blotting revealing the presence of scFvB9. Successful secretion of scFvB9 in hydroponic medium was also demonstrated, whereas no scFvH10 could be detected in the leaf, stem or root apoplast, nor secreted into the hydroponic medium. Our results show that scFvH10 release or diffusion from the roots of transgenic plants was not occurring, suggesting that the KDEL signal might contribute to the environmental biosafety of crops producing PMPs.

A rapid and sensitive ELISA to quantify an HBsAg specific monoclonal antibody and a plant-derived antibody during their downstream purification process.

An enzyme-linked immunosorbent assay to quantify the mAb CB.Hep-1 during downstream purification process was standardized and validated. This assay is characterized by a short time of incubation at high temperature, allowing the detection of this antibody with high specificity and sensitivity. Detection of antigen-antibody reaction was achieved using a horseradish peroxidase conjugated anti-mouse IgG whose enzyme activity was revealed with o-phenylenediamine substrate. The immunoassay is linear in a range between 3.12 and 50 ng/mL, with a recovery of 98.55-107.62%. According to results, it is possible to estimate the mAb CB.Hep-1 concentration with high precision and reproducibility. The intra- and interassay coefficient of variation ranged from 0.25 to 8.64% and 1.84 to 9.43%, respectively. Significant differences were not observed in the plant-derived antibody quantification by HRP-ELISA and PhoA-ELISA (n=18), demonstrating that plant endogenous peroxidases do not produce interferences in the quantification of this molecule. Therefore, both antibodies can be tested with the same immunoassay with high precision, specificity and accuracy during their respective purification processes without interference of the buffers and sample characteristics.