Associate Professor Rutgers University, United States
Introduction: Monoclonal antibodies (mAbs) are therapeutic proteins with proven efficacy against a host of diseases ranging from arthritis to cancer [1]. Therapeutic mAbs are physically unstable and prone to irreversible aggregation at room temperature and at high dosage, which is required for their subcutaneous administration in humans. Aggregation is one of the degradation pathways that triggers immunogenicity, lower potency and affects product’s safety, quality and efficacy. We here present our results related to the aggregation behavior of one of the mAbs, bovine immunoglobulin G (bIgG), that could advance our understanding of the effect of concentration on the structure of the bIgG. Despite detailed investigations, the sample conditions that drive IgGs towards aggregation is not clearly understood. Our results are expected to enable designing strategies, i.e., design appropriate polymeric excipients, to inhibit this aggregation.
Materials and
Methods: bIgG was obtained as a lyophilized powder from MP Biomedicals and was solubilized at 50 mg/mL in DI water. These solutions were then concentrated using spin concentrators with 10kDa MW cutoff at 6000xg and 15 °C until 200 mg/mL concentration was obtained. bIgG concentrations were measured with NanoDropTM One. The bIgG solution was then diluted for obtaining 10, 50, 100, 150 mg/mL concentrations. Similar procedure was employed to obtain bIgG solutions in 25mM pH 6 Histidine buffer, 30mM pH 5.5 Acetate buffer, and 30 mM pH 4.5 Acetate buffer. bIgG solutions were also prepared with addition of polysorbate 20 at polysorbate:bIgG molar ratios of 0.5:1, 1:1, and 2:1 in 30mM pH 4.5 buffer. Dynamic light scattering (DLS) measurements were carried out to on DynaPro DLS Plate Reader III (Wyatt Technologies) to follow the aggregation behavior with concentration and temperature. The data were collected at a wavelength of 830 nm and a scattering angle of 173°. Regularization analysis was performed using Rayleigh spheres model for hydrodynamic size measurement. Small-angle X-ray scattering (SAXS) measurements were made at 16-ID-C LIXS beam line (National Synchrotron Light Source II (NSLS-II), Brookhaven National Laboratory; 15.14 keV X-rays (λ= 0.8189 Å) and three Pilatus 1 M detectors). Data over a q range of 0.005–0.25 Å-1 was analyzed. Background subtraction was done using the sodium acetate buffer scattering obtained after every three samples. The data was analyzed in BioXTAS RAW 2.1 with ATSAS 3.0.4 to quantify radius of gyration (Rg) by Guinier analysis; pair-distance distribution functions, P(r).
Results, Conclusions, and Discussions: Figure 1a shows an example of SAXS intensity curves at increasing concentrations. The weak peaks at q values of 0.04, 0.08 an 0.16 Å-1 arise from the multidomain structure in IgG. The shape of the scattering curve, and hence the conformation of the IgG, does not change with commonly used preservatives. The data at 10 mg/ mL gives a radius of gyration (Rg) of 48 Å, in agreement with the value reported in the literature [2]. This curve was used to generate a pair distribution function, P(r), which was used to generate a bead model for the IgG molecule (Figure 1b). This model is similar to that published in the literature [2]. As the concentration is increased, the Rg decreases, to ~ 31 Å at 150 mg/mL. This decrease in Rg at higher concentrations is too large to be explained by interparticle interference, and is suggestive of the structural changes occurring at higher concentrations. There was also clear evidence of aggregation at higher concentrations. Bead models were generated from the P(r) functions derived from the SAXS data at higher concentrations. This models support the hypothesis that three domains that linked by flexible linkers in mAbs could move in response to the close proximity of the IgG molecules at higher concentrations. These changes in the disposition of the domains is a likely explanation for the decrease in Rg and the concomitant propensity of IgG, and mAbs in general, to aggregate at higher concentrations. DLS data (Figure 2) show that at 10mg/mL, bIgG does not aggregate in DI water until 85 °C whereas it starts aggregating (Tonset) at 45 °C in pH 5.5 and 60 °C in pH 6. However, at 50 mg/mL, bIgG aggregates at 55 °C in DI water, 40 °C in pH 5.5, and 35 °C in pH 6. This suggests that increasing concentrations lowers the aggregation onset temperatures.
Conclusions: The large changes seen at higher concentrations, including large decrease in Rg suggests that conformational changes occur due to crowding the molecules. It is likely that these conformational changes are precursor to the observed aggregation behavior of mAbs
Acknowledgements (Optional): This was work was funded by the National Science Foundation (DMREF-2118860). The SAXS experiments were performed at NSLS-II beamline 16-ID for life science x-ray scattering (LiX), which is supported by NIH through Grant S10 OD01233, and NIGMS grant P30GM133893, and by the DOE Office of Biological and Environmental Research (grant KP1605010) and by the U.S. Department of Energy, Office of Basic Energy Sciences (contract number DE-SC0012704). We thank Dr. James Byrnes for his assistance with the SAXS measurements
