Mapping of Methyl Epitopes of a Peptide-Drug with Its Receptor by 2D STDD-Methyl TROSY NMR Spectroscopy.

The current trend in the biopharmaceutical market has boosted the development and production of biological drugs with high efficacy and fidelity for receptor binding. While high-resolution structural insights into binding epitopes of the receptor are indispensable for better therapeutic design, it is tedious and costly. In this work, we develop a protocol by integrating two well-known NMR-based solution-state methods. Saturation transfer double-difference with methyl-TROSY (STDD-Methyl TROSY NMR) was used to probe methyl binding epitopes of the ligand in a label-free environment. This study was carried out with Human insulin as a model peptide drug, with the insulin growth factor receptor (IGFR), which is an off-target receptor for insulin. Methyl epitopes identified from STDD-Methyl TROSY NMR spectroscopy were validated through the HADDOCK platform to generate a drug-receptor model. Since this method can be applied at natural abundance, it has the potential to screen a large set of peptide-drug interactions for optimum receptor binding. Thus, we propose STDD-Methyl TROSY NMR spectroscopy as a technique for rapid screening of biologics for the development of optimized biopharmaceutics.

NMR based quality evaluation of mAb therapeutics: A proof of concept higher order structure biosimilarity assessment of trastuzumab biosimilars.

Higher-order structural (HOS) biosimilarity assessment is a regulatory requirement for intended biosimilars, and manufacturers are required to demonstrate the biosimilarity of their product in comparison to the reference product concerning both safety and efficacy. NMR has recently emerged as a powerful technique for complex biologics such as mAbs that offers holistic Higher-order structure (HOS) assessment. In this paper, a proof-of-concept for similarity assessment for marketed biosimilars of trastuzumab has been presented using both 1Dimensional (1H NMR) and 2Dimensional 1H-13C-methyl correlated NMR techniques. Samples were prepared without sample buffer exchange in the presence of excipients and assessed at natural abundance. Data were recorded using a 750 MHz NMR spectrometer equipped with a room temperature probe. Both visual and statistical assessment was performed to test biosimilarity. 1H- NMR was useful for the assessment of overall comparability with sensitivity towards changes in the formulation components, whereas 13C- 1H methyl correlation-based assessment indicated structural biosimilarity. All marketed biosimilars were found to be within the range established by originator batches used in this study. Using 1D and 2D NMR techniques, we aim to provide a perspective on the requirements and challenges of HOS biosimilarity assessments to be conducted for the drug product as a whole, inclusive of excipients as opposed to drug substance alone in buffer exchanged conditions.

Monitoring size and oligomeric-state distribution of therapeutic mAbs by NMR and DLS: Trastuzumab as a case study.

Monoclonal antibodies (mAbs) are the modalities of choice for immunotherapy. This class of products are known to exhibit considerable heterogeneity with respect to size, aggregation states, and charge. This makes it challenging for biopharmaceutical manufacturers, in particular biosimilar producers, to maintain consistency in product quality. In order to fingerprint these biotherapeutic products, multiple, high-resolution analytical tools are used to characterize the numerous critical quality attributes. Recently, there has been growing interest in enhancing adaptability of 1D and 2D NMR platforms for characterization of higher order structure with emphasis on 1D 1H, 2D 1H-15N and 1H-13C NMR experiments at natural abundance. In this communication, we report the applicability of 2D-DOSY NMR for quantification of colloidal diffusivities, namely diffusion coefficient (and associated hydrodynamic radius) for monomeric IgG1 mAb formulations at physiological conditions. Similarity assessment has been performed for trastuzumab originator (multiple batches) and marketed biosimilars to showcase the applicability of this approach. While dynamic light scattering measurements are known to be sensitive to presence of larger particles with a concentration dependence for estimation of colloidal diffusivities, size estimated by NMR experiments was found to be more in agreement with the computational hydrodynamic size estimations derived from the published crystal structures of intact mAb at formulation concentration.