Small-angle x-ray scattering screening complements conventional biophysical analysis: comparative structural and biophysical analysis of monoclonal antibodies IgG1, IgG2, and IgG4

Abstract

A crucial step in the development of therapeutic monoclonal antibodies is the selection of robust pharmaceutical candidates and screening of efficacious protein formulations to increase the resistance toward physicochemical degradation and aggregation during processing and storage. Here, we introduce small-angle X-ray scattering (SAXS) to characterize antibody solution behavior, which strongly complements conventional biophysical analysis. First, we apply a variety of conventional biophysical techniques for the evaluation of structural, conformational, and colloidal stability and report a systematic comparison between designed humanized IgG1, IgG2, and IgG4 with identical variable regions. Then, the high information content of SAXS data enables sensitive detection of structural differences between three IgG subclasses at neutral pH and rapid formation of dimers of IgG2 and IgG4 at low pH. We reveal subclass-specific variation in intermolecular repulsion already at low and medium protein concentrations, which explains the observed improved stability of IgG1 with respect to aggregation. We show how excipients dramatically influence such repulsive effects, hence demonstrating the potential application of extensive SAXS screening in antibody selection, eventual engineering, and formulation development.

Keywords: IgG antibody; analysis; physicochemical properties; protein aggregation; protein formulation; small-angle X-ray scattering (SAXS); stability.

Figure 1

(a) Stability of antibodies investigated by SE-HPLC under accelerated storage conditions (40°C for 8 weeks). LMWS indicate the low-molecular-weight species. Blue, red, green, and pink trace lines indicate the samples at pH 5.0, 6.5, 7.4, and 8.5, respectively, containing 100 mM NaCl. The black trace line indicates the nonstressed sample at pH 7.4. (b) Stability of antibodies investigated by SE-HPLC under normal storage conditions (5°C or 25°C for 8 weeks). Trace lines indicate the samples in Formulation A at 5°C (cyan), Formulation A at 25°C (pink), Formulation B at 5°C (blue), and Formulation B at 25°C (green). Formulation A: 50 mM histidine, pH 6.5, 250 mM sucrose. Formulation B: 50 mM Na-phosphate, pH 7.4, 100 mM NaCl.

Figure 2

(a) Primary structure of IgG1, IgG2, and IgG4 hinge regions (Kabat numbering14). (b) T_m measured by DSF and (c) R_h measured by DLS at various pH values and by inclusion of various excipients. IgG1 (blue), IgG2 (red), and IgG4 (green). DLS measurements were performed on stressed samples that were stored at 40°C for 4 days.

Figure 3

(a) Comparison of the SAXS curves at different pH. The SAXS curves of the samples at pH 3.3, pH 5.0, pH 6.5, pH 7.4, and pH 8.5 are translated for comparison and ordered sequentially from top to bottom for each IgG (i.e., within each color). (b and c) The P(r) of the samples at pH 6.5 and pH 3.3 from the indirect Fourier transformation of the scattering intensity. (d–f) Kratky plots based on the SAXS data of IgG1, IgG2, IgG4, respectively, at pH 6.5 and 3.3.

Figure 4

(a) R_g of three IgG subclasses in different formulation buffers and (b) the corresponding I(0) values. 1–5 on x-axis indicate the samples at pH 3.3, 5.0, 6.5, 7.4, and 8.5, respectively, containing 100 mM NaCl. 6–10 indicate the samples at pH 3.3, 5.0, 6.5, 7.4, and 8.5, respectively, containing 250 mM sucrose. 11 indicate the samples at pH 7.4 containing 0.05% Tween 80. (c and d) Concentration dependence of the three antibodies in Na-citrate buffer (pH 3.3). The data curves of the individual IgGs have been transposed for clarity. (e and f) SAXS curves comparing the effect of sucrose and NaCl at pH 6.5 and 3.3. Dimerization is observed at pH 3.3 for IgG2 and IgG4. The data curves of the individual IgGs have been transposed for clarity.