Utility of Solution X-Ray Scattering for the Development of Antibody Biopharmaceuticals

Abstract

Characterization of immunoglobulin solutions at high concentrations represents a significant challenge. A current trend in the biopharmaceutical industry is to manufacture highly concentrated drug products, which can be used to deliver high doses in small volumes, via subcutaneous injections. Studying a molecule's structure and properties in its final drug product formulation is ideal, but characterization is typically performed under dilute solution conditions with critical stabilizing buffer components removed because of interference effects, which can result in an incomplete understanding of the molecule's properties. Direct study of protein conformation and protein-protein interactions in concentrated solutions is challenging for most biophysical and biochemical techniques; however, X-ray solution scattering offers opportunities. Combined with other biophysical techniques, X-ray scattering has the potential to provide relevant information on both structure and interactions in protein solutions over a broad concentration range. Here, we report X-ray solution scattering of 4 monoclonal antibodies, designated mAb1 (glycosylated and de-glycosylated), mAb2, and mAb3 at concentrations between 0.5 and >168 mg/mL. Data acquired from these measurements are combined with the results from other biophysical measurements to generate a comprehensive profile of their solution behaviors. Our results show that X-ray solution scattering can assess key parameters needed to aid in formulation development.

X-ray Scattering and Coarse-Grained Simulations for Clustering and Interactions of Monoclonal Antibodies at High Concentrations

“…The S eff (q) values approach 1 at q > 0.1 A −1 and have a small shoulder at q ≈ 0.06 A −1 (∼10 nm length scale as estimated by Bragg length = π l q Bragg 2 , typical of mAb hydrodynamic diameter 16,70,71 ), which has been seen for many other mAbs. 16,[49][50][51]54,61 The effect of adding cosolutes is most pronounced at lower q values, where the profile is more strongly dependent on the underlying PPI. 16,54,61 For mAb2 (Figure 1A), S eff (q) is the smallest (most repulsive) for the no cosolute control.…”

Dynamic Views of the Fc Region of Immunoglobulin G Provided by Experimental and Computational Observations

“…Dynamic views of antibody structures thus offer deep insights into their functional mechanisms. Dynamic structures of antibodies in solution have recently been characterized by sophisticated experimental techniques, including nuclear magnetic resonance (NMR) spectroscopy [13,14], solution scattering [15,16,17,18,19], cryo-electron microscopy [7,20], and high-speed atomic force microscopy [21]. In addition, computational approaches offer powerful tools for providing dynamic views of antibody structures [22].…”

Native peptide mapping – A simple method to routinely monitor higher order structure changes and relation to functional activity

“…[1][2][3] Therefore, the process of defining and controlling product critical quality attributes (CQA), which are defined by the quality by design approach (QbD), [4][5][6] is crucial, and ensures that the safety and efficacy of the product are maintained. 7 The higher order structure (HOS) of a product, which may be considered a CQA, can be studied by a wide range of techniques, such as fluorescence detection, 8 circular dichroism, [9][10][11][12][13] X-ray crystallography, [14][15][16] nuclear magnetic resonance (NMR), [17][18][19] epitope detection and Fourier-transform infrared spectroscopy. [20][21][22][23] This characterization approach is in line with the U.S. Food and Drug Administration's Quality by design (QbD) initiative 24 linking a desired product quality with physico-chemical or biological properties/characteristics.…”