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.