Two-Step Purification of Antibody from Tobacco Plants for Vaccine Manufacturing: Aqueous Two-Phase Extraction and Affinity Chromatography

by Williams Ferro, Tatiana Álvarez, Déborah Geada, Yenisley Medina, Yarysel Guevara, José Montero, Andrés Tamayo, Ariadna López, Daily Hernández, Mayra Wood, Tatiana González, Regla Somoza, and Rodolfo Valdés
Volume 14, Issue 1 (Spring 2015)

The purification of PHB-01 plantibody derived from tobacco leaves imposed difficulties when the plantibody solid-liquid extraction design was performed. Thus, our study focused on assessing a combination of an aqueous two-phase extraction (ATPE) procedure and affinity chromatography for solving some of the issues in plantibody purification. This was done using a complete factorial redesign, different polyethylene glycol (PEG)/K2PO4 proportions, and pH values in each partitioning variant. Out of the results of 27 variants, ten were selected for the subsequent purification step, considering an antibody recovery of ≥ 80% and a leaf-soluble protein removal capacity of ≥ 30%. Regarding this, the best ATPE combination was PEG 6000 (20%)/K2PO4 (10%), pH 5.5, showing 95.44 ± 7.89% of antibody recovery and 32.86 ± 17.02% of leaf-soluble protein removal capacity. Besides, the organoleptic properties of ATPE preparation were similar to those observed in solid-liquid extraction preparation, but a reduction in operation unit number and bioprocessing time was demonstrated. Next, the antibody extracted in the three of ten selected variants was submitted to affinity chromatography for increasing molecule recovery and purity. The highest recovery was measured in PEG 4000 (10%)/K2PO4 (15%), pH 5.5, (92.69 ± 4.81%). Conversely, a significant decrease in antibody recovery (p = 0.000) was observed when antibody was purified after ATPE with PEG 6000 (ranging from 54.81 ± 17.05% to 65.37 ± 13.47%). The antibody electrophoretic mobility was unmodified by the addition of PEG in ATPE, and antibody purity measured by SDS-PAGE did not show significant differences (> 97%). These preliminary results allowed us to predict that a combination of ATPE and affinity chromatography could be useful for solving issues in industrial-scale PHB-01 purification...

Ferro W et al. Two-step purification of antibody from tobacco plants for vaccine manufacturing: aqueous two-phase extraction and affinity chromatography. BioProcess J, 2015; 14(1): 43–8.

Posted online May 1, 2015.