Utility of High Resolution 2D NMR Fingerprinting in Assessing Viscosity of Therapeutic Monoclonal Antibodies

Abstract

Purpose The viscosity of highly concentrated therapeutic monoclonal antibody (mAb) formulations at concentrations ≥ 100 mg/mL can significantly affect the stability, processing, and drug product development for subcutaneous delivery. An early identification of a viscosity prone mAb during candidate selection stages are often beneficial for downstream processes. Higher order structure of mAbs may often dictate their viscosity behavior at high concentration. Thus it is beneficial to gauge or rank-order their viscosity behavior using noninvasive structural fingerprinting methods and to potentially screen for suitable viscosity lowering excipients. Methods In this study, Dynamic Light Scattering (DLS) and 2D NMR based methyl fingerprinting were used to correlate viscosity behavior of a set of Pfizer mAbs. The viscosities of mAbs were determined. Respective Fab and Fc domains were generated for studies. Result Methyl fingerprinting of intact mAbs allows for differentiation of viscosity prone mAbs from well behaved ones even at 30-40 mg/ml, where bulk viscosity of the solutions are near identical. For viscosity prone mAbs, peak broadening and or distinct chemical shift changes were noted in intact and fragment fingerprints, unlike the well-behaved mAbs, indicative of protein protein interactions (PPI). Conclusion Fab-Fab or Fab-Fc interactions may lead to formation of protein networks at high concentration. The early transients to these network formation may be manifested through peak broadening or peak shift in the 2D NMR spectrum of mAb/mAb fragments. Such insights go beyond rank ordering mAbs based on viscosity behavior, which can be obtained by other methods as well..

Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy

“…For assigning the resonances of the Amadori products, the following 2D experiments were recorded: 1 H- 13 C HSQC, 1 H- 13 C HMBC (hmbcgpndqf), 1 H-1 H TOCSY with mixing times of 100 ms and 12 ms, 1 H-1 H COSY (cosygpppqf), 1 H- 13 C HSQC-TOCSY (hsqcdietgpsisp.2) with mixing times of 13 ms and 100 ms. More details of the experimental parameters are given in the Figure captions. The data was processed with Topspin 3.6.2 (Bruker) and analyzed with Sparky 1.470 [39].…”

“…Isolated signals were integrated using a Gaussian fit in Sparky (D. Goddard and D. G. Kneller, "SPARKY 3," University of California, San Francisco, 2000.). The volumes of isolated 1 H- 13 C correlations of the β-pyranose form were compared to isolated and well-integrable signals of the denatured protein. The latter could be either a typical random-coil signal resulting of a known amount of residues of an amino acid type in the protein sequence, e.g.…”

“…After dialysis against ddH 2 O and lyophilization, the glycation was monitored by 2D NMR spectra measured under denaturing conditions using 7 M urea-d 4 in D 2 O. In addition to the original protein signals and some remaining signals of free glucose, the 1 H- 13 C HSQC spectrum showed several sets of signals that pointed to Amadori products (Fig. 3).…”

“…Two recent developments enabled to overcome limitations of the large size of mAbs and the associated severe line broadening. 1 H- 13 C HSQC spectra are either used to detect methyl groups, which display sharper line widths due to favorable relaxation properties caused by fast methyl group rotation [13][14][15][16], or samples are measured under denaturing conditions using high concentrations of urea [17]. The methyl-HSQC spectra serve as fingerprint spectra, which are ideally suited to compare different batches of mAbs.…”

“…The second approach, which includes denaturing the protein with 7 M deuterated urea in D 2 O, is based on line-narrowing due to the significantly higher flexibility of denatured protein chains that is especially suited to detect PTMs. Under denaturing conditions, the 1 H- 13 C HSQC spectra are simplified dramatically resulting only in random coil chemical shift correlations of the 20 proteinogenic amino acids that are well known [18]. Any modification results either in additional signals, or lead to significant alterations of the amino acid signals to which the PTM is attached.…”