Extraction of monoclonal antibodies (IgG1) using anionic and anionic/nonionic reverse micelles

Abstract

Purification schemes for antibody production based on affinity chromatography are trying to keep pace with increases in cell culture expression levels and many current research initiatives are focused on finding alternatives to chromatography for the purification of Monoclonal antibodies (MAbs). In this article, we have investigated an alternative separation technique based on liquid-liquid extraction called the reverse micellar extraction. We extracted MAb (IgG1) using reverse micelles of an anionic surfactant, sodium bis 2-ethyl-hexyl sulfosuccinate (AOT) and a combination of anionic (AOT) and nonionic surfactants (Brij-30, Tween-85, Span-85) using isooctane as the solvent system. The extraction efficiency of IgG1 was studied by varying parameters, such as pH of the aqueous phase, cation concentration, and type and surfactant concentration. Using the AOT/Isooctane reverse micellar system, we could achieve good overall extraction of IgG1 (between 80 and 90%), but only 30% of the bioactivity of IgG1 could be recovered at the end of the extraction by using its binding to affinity chromatography columns as a surrogate measure of activity. As anionic surfactants were suspected as being one of the reasons for the reduced activity, we decided to combine a nonionic surfactant with an anionic surfactant and then study its effect on the extraction efficiency and bioactivity. The best results were obtained using an AOT/Brij-30/Isooctane reverse micellar system, which gave an overall extraction above 90 and 59% overall activity recovery. An AOT/Tween-85/Isooctane reverse micellar system gave an overall extraction of between 75 and 80% and overall activity recovery of around 40-45%. The results showed that the activity recovery of IgG1 can be significantly enhanced using different surfactant combination systems, and if the recovery of IgG1 can be further enhanced, the technique shows considerable promise for the downstream purification of MAbs.

Lactoperoxidase partitioning from whey using the reverse micelles of non‐ionic/ionic mixed surfactants: Improvement of back extraction

“…LP is a basic protein with a pI of about 9-9. Stuckey, 2010;Hilhorst et al, 1992). It was also observed that the size of the RM increases from 8.2 nm for empty RM without protein solubilization to a maximum size of 146.77 nm at pH 8 (Table 1).…”

“…The size of the reverse micelles also increased corresponding to the water content and LP extraction. The larger size protein extracted into the water core of RM requires the adaptation of the reverse micelle size to that of the protein (George & Stuckey, 2010; Hilhorst et al, 1992). It was also observed that the size of the RM increases from 8.2 nm for empty RM without protein solubilization to a maximum size of 146.77 nm at pH 8 (Table 1).…”