Introduction
Monoclonal antibodies (mAbs) are powerful detection tools in science [1, 2] and valuable therapeutic agents in medicine [3, 4]. Their significant contribution to human health derives from their strong binding affinity and high specificity towards various antigens, including proteins, carbohydrates, lipids and nucleotides, and , as such, allow their use in personalized medicine [5] and cancer therapy [6, 7]. In 2021 alone, 16 new mAbs were expected to be approved by the American Food and Drug Administration [8]. Clearly, as the global demand for diverse mAbs grows, increase in large scale production and expression levels of mAbs (i.e. upstream processing) combined with their purification (i.e. downstream processing), is becoming ever more challenging [9].
The majority of mAbs are purified from cell culture via Protein A chromatography due to the high binding affinity [10] and specificity [11] of the Protein A ligand to a wide range of antibody (Ab) isotypes [12]. The unique properties of Protein A translate into excellent recovery yields (generally >90%) and high purity (>98%), both accomplished within a single chromatographic step [13], making Protein A chromatography the gold standard technology in Ab purification [14]. In general, mAbs are eluted very efficiently from Protein A resins under acidic conditions (e.g. 0.1 M sodium citrate, pH 3.3 [15]). Low pH is required for (a) weakening the interactions between the immobilized Protein A and the Fc domain of the target antibody; and for (b) inactivating any viruses which may be present as impurities in the system [16, 17]. However, low pH may also promote disruption of antibody secondary structure which can lead to aggregate formation [16-20]. Therefore, suppression or elimination of antibody aggregation , during both upstream and downstream processing, has become a subject of on-going investigation in bioprocess development [20]. The high antibody concentration present in the cell culture medium increases the frequency of protein self-adsorption events [16, 21, 22], which, of course, can also lead to aggregation. Several strategies and/or small molecule additives, aimed at minimizing antibody aggregation during column elution, have been tested. They have included for example: (a) addition of 0.5-1M urea [16] or (b) of arginine monomers [21] to the running buffer. Suppressing the formation of antibody oligomers is vital: studies have shown that such aggregates may reduce (i) therapeutic antibody potency; (ii) batch to batch reproducibility; as well as (iii) promoting undesirable immunogenic response [18, 23-25].
We have studied an alternative purification method that would avoid exposing polyclonal human IgG’s to acidic conditions. This approach relies on a recently described non-chromatographic, ligand-free strategy that uses nonionic detergent micelles conjugated via the amphiphilic complex [(bathophenanthroline)3:Fe2+] [26-28] as the purification platform. Such conjugated detergent micelles were found to quantitatively capture human and mouse IgG’s at neutral pH, exclude hydrophilic protein background impurities and allow extraction of relatively pure IgG’s without parallel co-extraction of background impurities or dissolution of the conjugated micelles [26-28]. The approach was found to function efficiently with three commercially available surfactant families characterized by polyethylene oxide (PEO) headgroups: Tween; Brij and Triton X-100. We note that PEO-based surfactants are approved for use in pharmaceutical formulations. However, it was found to be necessary to lower the pH to 3.8 during IgG extraction in order to achieve overall satisfactory yield of pure antibodies (generally >80%, [28]). Here, we investigated the possibility of weakening the interactions between captured antibodies and the detergent matrix to such an extent that antibody capture would not be affected while efficient extraction could be performed at close to neutral pH.