Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants

Abstract

SummaryMonoclonal antibody production from transgenic tobacco plants offers many advantages over other heterologous production systems, creating the prospect of production at a scale that will allow new prophylactic and therapeutic applications in global human and animal health. However, information on the major processing factors to consider for large-scale purification of antibodies from transgenic plants is currently limited, and is in urgent need of attention. The purpose of this project was to investigate methods for the initial extraction of recombinant immunoglobulin G (IgG) antibodies from transgenic tobacco leaf tissue.Three different transgenic plant lines were studied in order to establish the parameters for optimal extraction of monoclonal antibodies that accumulate in the apoplasm, at the plasma membrane or within the endoplasmic reticulum. For each transgenic line, seven techniques for physical extraction were compared. The factors that determine the optimal extraction of antibodies from plants have a direct influence on the initial choice of expression strategy, and so must be considered at an early stage. The use of small-scale techniques that are applicable to large-scale purification was a particularly important consideration. The optimal extraction technique varied with the target location of IgG in the plant cell, and the dependence of antibody yield on the physical extraction methodology employed, the pH of the extraction buffer and the extraction temperature was demonstrated in each case. The addition of detergent to the extraction buffer may improve the yield, but this was found to be dependent on the site of accumulation of IgG within the plant cell.

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

“…Apigenin-7-glycosides have similar maxima but contain a more rounded trough compared to that of apigenin and vitexin. A double absorbance band at 255 and 270 nm with a stronger absorption band at about 350 nm is characteristic of luteolin (5,7,3 0 ,4 0 -tetrahydroxyflavone) and related glycosides. Although we were not able to definitively assign names to all of the different peaks in our chromatographic profile, the predominant peaks were categorized as apigenin-7-glycoside, ferulic acid-form, vitexin-form, depending on their UV-VIS spectra.…”

“…A preliminary determination of the phenolic peaks (and the classes the majority of them belong to) was done by comparison to spectra of reference compounds or by comparison to published spectra. 24 For example, absorption maxima at 269 and 337 nm is characteristic of apigenin (5,7,4 0 -trihydroxyflavone) and C-glycosides of apigenin, such as vitexin (8-C-glucosylapigenin). Apigenin-7-glycosides have similar maxima but contain a more rounded trough compared to that of apigenin and vitexin.…”

“…5 The extracts of Lemna, tobacco, and other leafy plants can be complex and contain a number of water-soluble nonprotein impurities including phenolics, amino acids, organic acids, carbohydrates, pectin, and nucleic acids. [6][7][8] Phenolic compounds are a potential concern in the production of protein therapeutics such as mAb for two reasons. First, they can interact with proteins through a number of mechanisms including hydrogen bonding, oxidative coupling, and ionic and/or hydrophobic interactions.…”

Application of a PEG/salt aqueous two‐phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

“…Future progress in utilizing transgenic plants for biopharmaceuticals production will depend on the efficiency of the purification methods, since purification represents most of the cost of biopharmaceutical production in transgenic plants [14,15].…”

“…These include cation exchange chromatography [16], anion exchange chromatography [17] Protein isolation and purification from plants is a difficult task, owing to the complexity of the plant system and the presence of phenolic compounds, pigments, and mucilages [14,31]. The presence of such compounds is potential interfering factor in downstream processing and has to be addressed early in the isolation process.…”