Local Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies

“…The detailed methodology for these measurements is described elsewhere. 19 Briefly, intrinsic Trp fluorescence spectra were acquired using a peltier controlled PTI Quanta Master Spectrophotometer (Lawrenceville, NJ) using 0.1 mg/mL (6.7 3 10 24 mM) of mAb (IgG1 and IgG2) over a temperature range of 10-40 C (10 C intervals) and 40-90 C (5 C intervals). An equilibration time of 3 min was used at each temperature.…”

“…Any inner filter effects due to the presence of acrylamide were corrected as described earlier. 19,38 Static light scattering, extrinsic (1-anilino naphthalene-8-sulphonate) fluorescence, far-UV circular dichroism and differential scanning calorimetry. Static light scattering intensity (SLS) and extrinsic (1-anilino naphthalene-8-sulphonate; ANS) fluorescence were both performed as a function of temperature C at 2.5 C intervals) using 0.1 mg/mL of mAb using a PTI Quanta Master Spectrophotometer.…”

“…Such regions in immunoglobulins can manifest differences in their conformational stability [12][13][14] and dynamic [15][16][17][18][19] properties in response to changes in temperature and the presence of stabilizing or destabilizing cosolutes. 19 These variables have the potential to induce partially folded intermediate structures, which are less stable than the native state and more prone to aggregation. For many proteins, such partially folded intermediates are known to proceed via competing pathways to retain elements of the native state and can lead to the formation of native/non-native aggregates.…”

“…[34][35][36] Recently, the role of intramolecular dynamics of regions with distinct solvent exposures in modulating global conformational stability was investigated using an IgG1 monoclonal antibody. 19 A fluorescence spectroscopy based approach employing red-edge excitation (REE) and fluorescence quenching was employed in these studies. REE, an approach in which the excitation wavelength was increased towards the red edge of the absorption band, was used to sample and monitor a fraction of tryptophan (Trp) residues in environments that were increasingly exposed to solvent.…”

“…Fluorescence quenching studies, used to probe protein matrix dynamics, were performed at selected temperatures including a pre-unfolding temperature range, an onset temperature (T onset ), and at the apparent thermal melting temperature (T MA ) to obtain a snapshot of local dynamics of the antibody as a function of temperature. 19 The effect of sucrose or arginine as representative stabilizing and destabilizing excipients, 18,37 respectively, were also evaluated under these conditions. 19 The current study is aimed at (1) characterizing and comparing the conformational stability and dynamic properties of regions with distinct solvent exposure at various temperatures using two additional monoclonal antibodies (an IgG1 and IgG2) and (2) understanding the relevance of local conformational stability and dynamics to the aggregation propensity of representative IgG1 and IgG2 monoclonal antibodies.…”