None of these treatments eliminated the size or size heterogeneity artifacts

None of these treatments eliminated the size or size heterogeneity artifacts. condition. Analyzing our human anti-cocaine monoclonal antibody, we noted unexpectedly larger apparent molecular weights and apparent protein size heterogeneities using non-reducing SDS-PAGE. These apparent molecular excess weight heterogeneities are not consistent with other analysis techniques. Heterogeneities were noted using several heating and pre-electrophoretic sample preparation protocols, and are altered by the inclusion of small concentrations of certain alcohols such as propanol and butanol. All of these unexpected results Gboxin were also observed for any commercial human IgG1 antibody, suggesting that these observations are applicable to IgGs in general. Thus, careful attention must be paid to the interpretation of non-reducing SDS-PAGE results for IgGs. It is hypothesized that differential thermal unfolding of the Gboxin Fab, CH2 and CH3 domains of the IgGs in SDS give rise to the stable, discreet bands observed using different heating protocols prior to non-reducing SDS-PAGE. Keywords: Monoclonal antibody, non-reducing SDS-PAGE, alcohol additives, antibody analysis, electrophoretic migration, antibody domain name unfolding Graphical Abstract Introduction Monoclonal antibodies (mAbs) are important therapeutic agents, and both monoclonal and polyclonal antibodies are widely used in basic and translational research. However, it has been reported that a large percentage of commercially available antibodies for basic research are unable to accurately and selectively identify their intended targets [1,2], and there are substantial ongoing efforts to systematically validate antibodies for their intended usages and targets. There are very well-developed protocols and methodologies used to characterize protein therapeutics, e.g., monoclonal antibodies, as well as fragments and derivatives of monoclonal and polyclonal antibodies. Our laboratory has developed a humanized mAb (h2E2) that has high affinity for cocaine and selectivity for cocaine over its inactive metabolites [3], which is intended for treatment of cocaine dependency. This recombinant mAb Gboxin protein can be produced in gram quantities in Chinese hamster ovary (CHO) cells [4]. SDS-PAGE is one of the simplest, least expensive, and most commonly used techniques to analyze antibodies for purity. Reduced samples of the IgG class of ITGAM antibodies Gboxin give rise to glycosylated heavy chains of approximately 50 kDa and light chains of approximately 25 kDa on SDS-PAGE. When analyzed without reduction of disulfide bonds, these antibodies should give rise to a single band on SDS-PAGE, i.e., the intact antibody consisting of 2 heavy and 2 light chains, with a combined size of approximately 150 kDa. In this work, we notice apparent overestimated molecular weights and size heterogeneities for any recombinant humanized monoclonal anti-cocaine mAb (h2E2), as evidenced by multiple bands on non-reducing gels, some of which migrate with artificially low electrophoretic mobilities (high apparent molecular weights). These anomalies are dependent on the sample pre-electrophoretic heating heat and period. In addition, we noted that under some conditions, both the apparent band heterogeneity and the anomalously high molecular weights observed are modulated by the inclusion of a small concentration of certain short chain alcohols (e.g., propanol and butanol) in the sample buffer. These unexpected effects of heating and the addition of alcohols were similar for both the humanized h2E2 monoclonal antibody and a commercial human IgG1 polyclonal antibody, suggesting that these properties may be shared by many polyclonal and monoclonal antibodies in use for both research and clinical applications. It is well known that, Gboxin in the absence of SDS, IgGs have structural domains that unfold relatively independently, and exhibit differential thermal stabilities, as measured by the multiple transitions and melting temperatures observed for the native proteins in non-denaturing buffers, as assessed by differential scanning calorimetry (DSC, [5,6,7]). In this study, we hypothesize that this observed changes in electrophoretic mobilities and apparent size heterogeneities of these IgG antibodies in non-reducing SDS-PAGE are due to differential unfolding of the Fab, CH2, and CH3 domains of the immunoglobulins in the SDS sample buffer prior to non-reducing electrophoresis. Using such analyses could add to the existing therapeutic antibody characterization techniques, as well as possibly represent a simple means to analyze the effects of mutations around the stability of IgG protein domains in therapeutic antibodies. Materials and Methods Materials The generation and production of the h2E2 anti-cocaine monoclonal antibody was previously explained [8]. The recombinant h2E2 mAb was purified by protein A affinity chromatography and used as supplied by the manufacturer, Catalent. The purity, structure, and function of the resultant mAb protein has been well characterized in our laboratory [4,9,10,11]. The human polyclonal IgG1 protein used for comparative purposes was purchased from Sigma-Aldrich (catalogue # I5029,.