Role of NMR in High Ordered Structure Characterization of Monoclonal Antibodies

Abstract

Obtaining high ordered structure (HOS) information is of importance to guarantee the efficacy and safety of monoclonal antibodies (mAbs) in clinical application. Assessment of HOS should ideally be performed in a non-invasive manner under their formulated storage conditions, as any perturbation can introduce unexpected detritions. However, most of the currently available techniques only indirectly report HOS of mAbs and/or require a certain condition to conduct the analyses. Besides, the flexible multidomain architecture of mAbs has hampered atomic-resolution structural analyses using X-ray crystallography and cryo-electron microscopy. In contrast, the ability of nuclear magnetic resonance (NMR) spectroscopy to structurally analyze biomolecules in various conditions in a non-invasive and quantitative manner is suitable to meet the needs. However, the application of NMR to mAbs is not straightforward due to the high molecular weight of the system. In this review, we will discuss how NMR techniques have been applied to HOS analysis of mAbs, along with the recent advances of the novel 15N direct detection NMR strategy that allows for obtaining the structural fingerprint of mAbs at lower temperatures under multiple formulation conditions. The potential application of these NMR strategies will benefit next-generation mAbs, such as antibody-drug conjugates and bispecific antibodies.

Analytical Techniques for the Characterization and Quantification of Monoclonal Antibodies

“…On the other hand, 1D and 2D Nuclear Magnetic Resonance (NMR) has been utilized by several researchers to obtain highly specific High Ordered Structures (HOS) of mAbs. Two- dimensional NMR can indicate molecular fingerprints of proteins at atomic resolution level providing detailed structural information [ 49 ]. These analytical processes facilitate the separation of the required mAbs isoform and deliver a thorough characterization, illustrating the organization of the protein.…”

“…To elucidate the specific high ordered structures (HOS) of mAbs, spectroscopic methods were also employed by different researchers during the last two decades [ 49 , 164 ]. Although several previously studied analytical methods, such as liquid chromatography, bioassays, diverse forms of electrophoresis, and peptide mapping have illustrated the covalent structure of the proteins (sequences of amino acid and post translational modifications), they all lacked the information about the HOS of mAbs [ 165 ].…”

“…In recent years, the application of numerous advanced spectroscopic strategies, for example circular dichroism (CD) and Raman and Fourier transform infrared spectroscopy (FTIR) were utilized to determine the tertiary structures of mAbs. These techniques involve specific conditions to perform the chemical analysis and reported the HOS of mAbs [ 49 , 66 ]. Consequently, NMR played a crucial role in elucidating the three-dimensional (3D) structure by providing the HOS of mAbs and is possibly the best method for the structural characterization of proteins especially the therapeutic mAbs.…”

“…The reversible self-association of mAbs has also been reported to be monitored by 1D 1 H NMR, which ultimately reveal the dimension of the transient protein clusters and viscosity of the solution [ 49 , 69 , 70 ]. In another study, the combined approach of fast protein liquid chromatography (FPLC)-NMR, CD, SEC, and MALS (multi-angle light scattering), was suitable for yielding an overall outline of HOS and different quinary structure of mAb therapeutics [ 71 ].…”

“…On the contrary, owing to the short transverse relaxation times along with the slow molecular tumbling, multidimensional NMR method has resulted in its lesser adaptability for structure analysis of intact mAbs. Therefore, to prevail over such hindrance (rapid transverse relaxation), different approaches were adopted, such as deuteration of non-labile 1 H sites and utilization of the TROSY (transverse relaxation optimized spectroscopy) or CRINEPT (cross-correlated relaxation enhanced polarization transfer) [ 49 , 170 ]. Subsequently, an innovative 15 N-direct revealing NMR analysis i.e., 15 N-detected CRINEPT has also been reported that works on the same concept of coherence transfer as in the case of 1 H-revealing CRINEPT and supports high-sensitive dimensions of non-deuterated high molecular weight proteins [ 75 ].…”