Introduction
Antibodies (Abs) are routinely used as detection and quantitation agents in scientific research and as vehicles for delivering radioisotopes, drugs and enzymes to cells of interest in medicine.1-2 It is the inherent high specificity combined with the strong binding affinity towards diverse antigens (proteins, carbohydrates, nucleotides and haptens) that explains why personalized medicine relies to a great extent on monoclonal antibodies (mAbs),3 many of which find their use in cancer therapy.4-5 The striking success of mAbs as efficient therapeutic agents resulted in 61 FDA- approved mAbs by 2017 6 and ~ 570 mAbs are currently in advanced clinical trials.7 The increasing global demand for diverse mAbs pressures pharmaceutical companies to improve the expression levels of mAbs (upstream processing) and their purification (downstream processing).
Antibody purification is most commonly achieved viachromatography using Protein A (ProA) as the ligand of choice for capturing Abs. ProA binds strongly8 and specifically9 to diverse Ab subclasses, 10 making it possible to efficiently capture Abs from very complex media and to reach high purity (>98%) within a single chromatographic step. 11 These features have thus made ProA chromatography the gold standard technology in Ab purification. 12However, the necessity to purify ton amounts13 of therapeutic mAbs has led pharmaceutical companies to search for a more economical route to meet the above challenge. The ideal purification technology would thus rely on a non-chromatographic step capable of efficiently capturing Abs. This challenging objective was termed the: ”ABC philosophy”,i.e., Anything But Chromatography.12, 14 We sought to take this philosophy an additional step forward by entirely excluding ProA from the process.
Development of a non-chromatographic, ligand-free Ab purification platform has the potential of overcoming major problems related to ProA chromatography. These include: (i) the high cost of ProA resins and the potential leaching of ProA (or its fragments) into the purified Ab;12 (ii) the limited binding capacities of ProA columns which may pose a problem at high Ab concentrations; 15(iii) deamidation of ProA asparagines during column sanitation,16 leading to lower binding efficiency; and (iv) the high maintenance costs associated with the use of HPLC\FPLC instrumentation.
Great efforts have been directed toward replacing ProA chromatography with a non-chromatographic approach. These included: (a) diverse precipitation methods;17 (b) extraction systems; 18-19 and (c) ultrafiltration with charged membranes;20 but none of these are widely used.
Recently, we described an alternative Ab purification platform that complies with such challenging criteria.21-22 Our approach relied on literature reports describing purification of mAbs viahydrophobic interaction chromatography (HIC)23-25 or hydrophobic membrane interaction chromatography.26 Those studies demonstrated the tendency of immunoglobulins (IgGs) to bind more strongly to non-polar surfaces than do non-IgG water-soluble proteins. We exploited this greater binding affinity of IgGs to hydrophobic resins by demonstrating that Tween-20 detergent micellar aggregates can be used for purification of human, mouse, rabbit and sheep IgGs.21-22
Tween-20 aggregates were formed in two-steps: first, the amphiphilic chelator, bathophenanthroline (batho), was added to a dispersion of Tween-20 micelles and transformed the latter into what we have termed: ‘engineered-micelles’,27-29 in which the chelator is presumably positioned at the micelle/water interface. This was followed by specific conjugation of engineered-micelles with Fe2+ ions (known to bind with high affinity up to three batho molecules in parallel14, 30) leading to Tween-20 aggregates as a distinct amphiphilic phase. The latter were shown to efficiently capture IgG’s from hybridoma serum-free media, or from E. coli lysate, and allow extraction of pure target IgG’s without concomitant extraction of impurities or aggregate dissolution.21-22
In the present report, we demonstrate the generality of our non-chromatographic, ligand-free approach, as well as providing evidence which would further justify efforts at up-scaling. Achieving these challenging objectives is expected to form the basis for considering integration of our purification platform into standard downstream processing of therapeutic grade mAbs on an industrial scale.