Emerging NMR techniques as advanced tools for characterising biological parenteral products

An important step in the research and development of biopharmaceuticals is to identify molecules with favourable physical and chemical stability profiles. Yongchao Su, Wei Xu and Bruce Yu discuss how solution, solid-state and benchtop nuclear magnetic resonance (NMR) methods can provide advanced biophysical characterisations of biological products.

NMR technologies

Large molecules can exhibit complex structural behaviours due to their flexible molecular properties. An important step in the research and development of biopharmaceuticals is to identify molecules with favourable physical and chemical stability profiles, as these attributes are critical to the efficacy and safety of a drug product.In parenteral formulations, the conformational flexibility of proteins can result in different intra- and intermolecular contacts and sometimes trigger physical aggregation and chemical degradation. Thermal and mechanical stress encountered during the manufacturing processes, transport and storage of the finished products often introduce additional stability risk.

Biophysical characterisation plays an important role in the development and risk assessment of protein formulations. A wide array of analytical methods is necessary to probe the different aspects of the formulation, ranging from the molecular conformation to the higher order structure and the aggregated species. High-resolution characterisation tools are often required to determine structure, probe dynamics and study interaction in biological products. Particularly, nuclear magnetic resonance (NMR) methods have emerged as a powerful set of techniques for biophysical characterisation (Figure 1).

NMR methods

Figure 1: Advanced NMR techniques for characterising biologics formulation

High-field solution NMR as a versatile tool for in-depth biophysical characterisations

From a microscopic point of view, physical and chemical stability is governed by the structure of the molecular species in a biologics formulation. NMR characterises structures from Å to µm size and molecular dynamics from picoseconds to seconds, covering almost a full range of molecular behaviours in biologics products.2Compared to conventional tools, NMR is a versatile technique for investigating chemical and molecular structure, dynamics, and interaction at a high resolution and sensitivity.

In recent years, solution NMR has been explored by academia, biopharmaceutical industry and regulatory agencies to analyse biologics products. Successful examples include characterisations of physiochemical properties of biologics and mechanistic investigation of stabilising and destabilising interactions in parenteral products. For example, monoclonal antibodies (mAbs) represent the largest category of protein therapeutics on the market. One-dimensional (1D) NMR has been used extensively to identify and quantify the stability of mAbs.3-5It has facilitated investigation of structural and hydrodynamic profiles, the effects of excipients on self-association, and conformational change associated with aggregation in mAb formulations.

Solid-state NMR spectroscopy: predicting stability in lyophilised biological products…

Two-dimensional (2D) NMR methods enable examination of higher-order structure (HOS); describe the secondary, tertiary and quaternary structures; and probe the critical molecular properties of mAbs.6-8Fingerprint spectra can provide a sensitive spectroscopic identification and comparison on quality attributes of the drug substance over the time course of formulation development. Protein-excipient interactions have been studied to probe the mechanism of formulation instability at the molecular level9and screen stabilising excipients.10A significant number of NMR studies have focused on protein aggregation. For example, the particle size distribution is a critical quality attribute of drug products. Diffusion ordered spectroscopy (DOSY) NMR measures diffusion coefficients, which can derive the hydrodynamic radius for quantifying size distribution.11DOSY-NMR can also simultaneously report the particle size of the drug substance and excipients and identify protein-excipient complex in the formulation. Moreover, extensive efforts are made toward the mechanistic investigation of protein aggregation in studies of amyloid diseases.12The characterisations of prion protein oligomer, misfolding and effects of temperature, pH, metal ion and mechanical agitation in these studies offer opportunities for in-depth biophysical characterisations of large molecule formulations.

Solid-state NMR investigation of large biotherapeutic complexes and insoluble aggregates

In recent years, solid-state NMR (ssNMR) has been utilised to characterise a wide range of pharmaceutical materials including amorphous and crystalline dry powder, soft tissue specimen, fully-hydrated lipid systems, gel-like samples and insoluble protein complexes. In ssNMR experiments, samples are rotated at an angle (so-called magic angle spinning, MAS) with respect to the external magnetic field to average chemical shift anisotropies and dipolar interactions for narrower linewidth. As a cutting-edge technique, ultrafast MAS can spin up to 150kHz to generate 1H ssNMR spectra with significantly enhanced sensitivity and resolution. Another revolutionary technique, dynamic nuclear polarisation (DNP), can boost the ssNMR sensitivity by transferring high polarisation from electrons. Proton-detected techniques and DNP-enhanced ssNMR have opened new avenues in

NMR methods

Figure 2: Solution and solid-state NMR characterisations of biopharmaceutical interesting molecular behaviours including protein-protein and protein-excipient interaction, RNA structure, higher order structure (HOS), mAb stability, protein aggregation and crystalline proteins.

biopharmaceutical applications.13 Many large protein complexes and insoluble aggregates experience slower molecular dynamics and thus fall into the motional regime for MAS NMR to study. Existing applications include large protein assemblies, RNA structure, fibrils and crystalline proteins (Figure 2). Taking protein aggregations, for example, insoluble particles represent a significant risk for biological formulation developments. Glucagon or glucagon-like peptides exhibit a strong tendency of fibrilisation and consequently physical instability. MAS NMR has been demonstrated as a powder tool for structure determination of disease-related amyloid fibrils.14,15 This powerful tool has immediate applicability for investigating insoluble aggregation of biological drugs.The goal is to solve the structure of undesired aggregation and identify the mechanistic cause of fibrillation. This approach can guide the rational design of the molecular motifs to improve the inherent stability of the development candidates. Besides insoluble aggregates, multidimensional MAS NMR is a primary tool for studying large protein complexes16 and can be employed for characterising protein-excipient and protein-protein assemblies in the formulation. Moreover, MAS NMR has also been used to determine RNA structures17 and shows great potential for investigating RNA-excipient complexes. In addition to the structural events in the sterile formulation, biological crystals present a new category of large molecular drugs. Bulk crystallisation provides one strategy for purification and requires solid-state structural analysis. Ultrafast MAS applications of micro- or nanocrystalline proteins have demonstrated successful examples of characterising large molecule drugs in the crystalline form.18      

Time-domain benchtop NMR for probing biologics products

While high-field NMR techniques, whether solution or solid state, are apt to provide in-depth biophysical characterisations, they have limitations – such as expensive equipment with a large footprint,

NMR methods

Figure 3: Benchtop NMR as a nondestructive for drug product characterisation

sophisticated and time-consuming operations, and high skillset requirements. Benchtop NMR presents a nondestructive biophysical tool for accessible and quantitative analysis, particularly at the drug product level. In the solid state, benchtop NMR has been used to analyse content uniformity of tablets19 and powder weight in vials.20 In the solution state, the water proton NMR signal has been used to detect mAb aggregates,21,22 insulin23and aluminium adjuvant24 filling levels. As illustrated in Figure 3, benchtop NMR also exhibits a great potential on quantitative analysis of aluminium-adjuvanted vaccines. Sedimentation of heterogeneous micron-sized particles in vaccines poses challenges to manufacturing. Benchtop 1H2O NMR can potentially serve as an in-line tool to monitor aluminium adjuvant suspensions without need of optical transparency or physical contact.


Solution, solid-state and benchtop NMR methods provide advanced biophysical characterisations of biological products for quantitative assessment, structural characterisation and mechanistic investigation.


Yongchao Su, PhD is an Associate Principal Scientist in Pharmaceutical Sciences at Merck & Co, Inc., US. He received postdoctoral training at Massachusetts Institute of Technology (Cambridge, MA) in 2011-2014 after his PhD research at Iowa State University (Ames, IA) in 2011. He works as the analytical lead on physicochemical characterisations of solid dosages and biophysical and biochemical characterisation of biologics. His research focuses on developing and utilising NMR techniques for mechanistic investigations of membrane permeability in drug delivery and structural characterisations of small and large molecule formulations.

Wei Xu, PhD is a Director of Preformulation at Merck & Co, Inc., USA. She received her PhD in Medicinal Chemistry from Purdue University. She has over 20 years of drug development experience spanning from discovery support to late stage product development. She has led multiple cross-functional teams to advance new therapeutic molecules and pursue product value enhancement opportunities. Her research interests include materials and biophysical characterisation, predictive dissolution and novel formulation technologies.

Bruce Yu, PhD is a Professor of Pharmaceutical Sciences and Director of the Bio- and Nano-Technology Center at the University of Maryland School of Pharmacy. Dr Yu has conducted research on proteins, imaging agents and biomaterials. His current focus is on nondestructive analytics for biologics and complex drug products, including biomanufacturing. He received the 2004 Kimmel Scholar Award and the 2005 US Presidential Early Career Awards for Scientists and Engineers. He received a PhD in molecular biophysics from Johns Hopkins University.


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