Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches

Abstract

Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (K_D) and nonspecific binding constants (N_s). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.

Keywords: Excipient ranking; Monte carlo; STD NMR; mab stability; mab–excipient interaction; molecular dynamics.

Figure 1.

Structures and ¹H 1D solution NMR spectra of sucrose, trehalose, mannitol, sorbitol, succinic acid, and glycine. For each excipient, the peaks under study are numbered in the structure and assigned in the spectra. ¹H spectra in display were acquired at 283 K using bruker 700 MHz spectrometer.

Figure 2.

Experimental data points and fitting plots for sucrose at 283 K (black) and 293 K (blue). Site-specific binding curves fitted by use of Eq (2) are shown in dashed lines. Fitting curves with orange marks are outliers based on P test.

Figure 3.

Experimental data points and fitting plots for a) trehalose, b) sorbitol, c) mannitol, d) succinic acid and e) glycine at 283 K (black) and 293 K (blue). No site-specific binding is observed, and the nonspecific binding curves fitted by use of Eq (3) are shown in solid lines. Fitting curves with orange marks are outliers (Trehalose H10 was rejected because of peak overlapping).

Figure 4.

Ranking of excipients of nonspecific binding constant by STDNMR (a), thermal stability measurement (b), B₂₂ measurement (c) and excipient count by MD (within 2 Å) (d) of MD simulation with 3D illustration (e). (a) the nonspecific binding constant of each proton (outliers excluded) at 283 K (blue) and 293 K (yellow) were ranked with error bar depicted. Ranking: Sucrose > Trehalose > Mannitol ≈ Sorbitol ≈ Succinic acid ≈ Glycine. (b) the thermal stability measurement (Tm) of each excipient at 293 K were ranked with error bar depicted. Control: 20 mM His buffer with BMSmAb (bar 1). Sucrose, trehalose, mannitol, and sorbitol increases the thermal stability of BMSmAb, whereas succinic acid and glycine decrease it (bar 2–7). Ranking: Sucrose ≈ Trehalose > Mannitol ≈ Sorbitol > Succinic acid > Glycine. (c) the colloidal stability measurement (B₂₂) of each excipient at 293 K were ranked with error bar depicted. Control: 20 mM His buffer with BMSmAb (bar 1). Ranking: Sucrose ≈ Trehalose > Mannitol ≈ Sorbitol > Glycine > Succinic acid. (d) the block average (using 8 ns blocks) for the last 80 ns of the simulation of each excipient with an atom within 2 Å of any atom on the BMSfAb was counted (normalized by SASA) and ranked with error bar depicted. Ranking: Sucrose > Trehalose > Mannitol > Sorbitol > Glycine ≈ Succinic acid. (e) the number of binding sites as a function of LGFE value for different excipients. Ranking: Trehalose ≈ Sucrose > Sorbitol ≈ Mannitol > Succinic acid > Glycine.

Figure 5.

3D maps of the screened excipient clusters where dense clusters (>85% occupancy) are shown as space filling models and less dense cluster (>20% occupancy) are shown as ball and stick models along a blue-green-red color gradient by occupancy. These 3D illustrations are generated by VMD.

Figure 6.

Binding site of sucrose (left) and mannitol (right) with BMSmAb characterized by STD effect analysis and MD simulation. First row: structure of each excipient with the affected proton highlighted in circle (ranking from red for the strongest effect to yellow). Second (283 K) and third (293 K) row: STD effect plot of different proton of each excipient at different concentration. Different protons show different amplitude of STD effect. Fourth row: the 3D illustration of most probable binding position of each excipient with BMSmAb calculated by MD simulation. The closest two distances of each proton which are less than 3 Å are shown. These 3D illustrations are generated by pymol.