3-31: Aqueous two-phase micellar systems (ATPMS) as a new methodology to recover recombinant antibody fragment from Pichia pastoris fermented broth

Recombinant antibody fragments (scFv) are becoming one of the most popular tools in human therapies since they possess several advantageous features as easy manipulation, fast elimination of their inmunocomplex and the possibility of being produced in simple expression systems like bacteria and yeast. The objective of this work is to evaluate the application of aqueous two-phase micellar systems (ATPMS) of triton X-114 to recover an anti-LDL scFv produced extracellularly by Pichia pastoris. Cloud points were determined to construct the coexistence curves in buffered solutions of pH 7.4, with or without NaCl, Cibacrom Blue 3FGA (CB) and Fabsorbent^TM F1P HF (HF) ligand, and yeast fermented broth (60% of the total system mass). scFv partition was studied following a 2³-full factorial design in which NaCl, CB and HF concentrations were selected as independent variables, and scFv purification factor (PF) and percentage yield (Y%) as responses. Triton concentration (2% w/w) and temperature (17°C) were maintained constant. scFv partitioned  to the bottom, micelle-rich phase in the majority of conditions with values of Y% and FP of 88.15 and 3.46 respectively . The regression analysis pointed out that FH concentration had statistically significant effects on Y% as well as the interaction between the three variables evaluated.  Concerning to FP results, all independent variables studied resulted to have significant effect on the purification factor. These preliminary results present the ATPMS as a potential tool to purify scFv antibody from fermented broth. Further experiments with different combination of variable concentrations will be accomplished to maximize these responses.