Monoclonal Antibody NMR and DLS

1D ¹H NMR Analyses

In addition to the amide finger print region of 1D 1H NMR spectra (6 – 11 ppm, see here for more details),  the spectral region of the protons of the amino acid side chains (0 – 3 ppm, shown in Figures 1 and 2) are also sensitive to primary, secondary, and tertiary structure. 

Figure 1: Full 1D 1H NMR Spectrum of NIST mAb #8671 buffer exchanged to 25mM bis-tris-d19 with 10% D2O at pH = 6.1 measured at 37ºC. Blue box indicates the region where the methyl protons of the protein methyl reside.

Figure 2: Zoom of protein methyl region of the 1D 1H NMR Spectrum of NIST mAb #8671 buffer exchanged to 25mM bis-tris-d19 with 10% D2O at pH = 6.1 measured at 37ºC.

2D ¹H-¹³C HMQC NMR Analyses

Due to the broadness of the 1D NMR spectra, the use of 2D NMR can provide an additional glimpse into the HOS. Following the findings of the NIST Round Robin study that provided a method for analyzing the unenriched isolated FAB portion of the mAb, a similar method was used to characterize the intact NIST mAb # 8671 by 2D 1H-13C HMQC NMR highlighting the region between 0-4 ppm x 10-50 ppm (Figure 3 below). 

Figure 3: 2D 1H-13C NMR Spectrum of NIST mAb #8671 buffer exchanged to 25mM bis-tris-d19 with 10% D2O at pH = 6.1 measured at 37ºC for 54 hours.

Notably, upon digesting the NIST mAb # 8671 with Genovis FABricator, additional sharper signals became visible above the noise (Figure 4). These findings are consistent with the results of the study by Chen et al in 2016, where upon digestion the spectral resolution improved from a SNR of 3.85 to 5.00 for peak 2.04 x 14.72 ppm and a SNR of 5.31 to 11.51 for peak 2.1 x 22.6 ppm as indicated by the yellow boxes in Figure 5.   

Figure 4: 2D 1H-13C NMR Spectrum of NIST mAb #8671 digested with 167 units of FABricator in 25mM bis-tris-d19 with 10% D2O at pH = 6.1 measured at 37ºC.

Figure 5: Overlay of 2D 1H-13C NMR Spectra of NIST mAb #8671 (red) and FABricator digested NIST mAb #8671 (blue) in 25mM bis-tris-d19 with 10% D2O at pH = 6.1 measured at 37ºC. Yellow boxes indicate peaks where signal to noise increased by 2 fold after digestion.

The use of Dynamic Light Scattering which has a higher sensitivity for aggregates that would be considered immunogenic, would be an appropriate method for determining whether aggregates may be present. The regularization autocorrelation function for the NIST mAb and FABricator digested NIST mAb are shown in Figures 6 and 7 below. A diffusion coefficient (D_t), hydrodynamic radius (R_h), and percent polydispersity (%Pd) of 4.91 x 10^-7 cm²/s, 4.89 nm, and 12.16%, respectively, are consistent with a 140-150 kDa mAb that doesn’t have additional oligomeric states such as dimers or trimers (Figure 6). Upon digestion, the hydrodynamic radius decreases from 4.89 nm to 3.81 nm, and the %Pd  increases to > 15% which is indicative of more than one species being present at that radius. This is consistent with both the FAB and FC portions of the mAb being cleaved and present together in solution. However, due to the reduced sensitivity of the DLS for characterizing species with less than 3 – 5 fold difference in size, the FAB and FC portions cannot be differentiated from each other. 

Figure 6: DLS Autocorrelation function using a regularization fit of NIST mAb #8671.

Figure 7: DLS Autocorrelation function using a regularization fit of FABricator digested NIST mAb #8671.

Although DOSY is not as sensitive to larger sized molecules compared to DLS, the use of DOSY would also be a suitable method to characterize a mAb or other peptides/proteins. The DOSY spectrum for the  FABricator digested NIST mAb is shown in Figure 8 below. The corrected diffusion coefficient (D_t) for the methyls of the digested mAb (highlighted in Figure 8) was 7.33 x 10^-7 cm²/s which is similar to the value determined by DLS. 

Figure 8: 2D 1H DOSY Spectrum of FABricator digested NIST mAb #8671.