Nuclear Magnetic Resonance (NMR) Spectroscopy Laboratory
Therapeutic Protein Structure by Nuclear Magnetic Resonance Spectroscopy
At the Centre for Biologics Evaluation, we apply the latest technology to study the structure of proteins used in therapeutics and to develop new tools for evaluating biologics. We focus on the use of nuclear magnetic resonance (NMR) spectroscopy to extract detailed molecular information on proteins of therapeutic interest.
- Why we study protein structure
- How we are using nuclear magnetic resonance spectroscopy
- Concepts and tools we use to study protein structure
- Research highlight 1: Nuclear magnetic resonance fingerprinting - A tool to analyse proteins and identify biologics
- Research highlight 2: Proteins are dynamics: their motions influence their functions
Why we study protein structure
Proteins consist of long chains of amino acids—the building blocks of life. These chains fold into complex three-dimensional scaffolds or structures (conformations) that directly determine the ability of an active pharmaceutical ingredient (API) or targeted molecule to function properly. As such, proteins are complex biomolecules that form the APIs of biologics such as hormones, antibodies, and vaccine antigens. These therapeutic proteins target and interact with other proteins in the body.
Changes in protein structure can affect how a protein functions. By studying protein structure, we can obtain information on what structural characteristics make an API work well, identify what normal and abnormal proteins look like, and discover how protein function is affected. In addition, biologics products do not only contain the API, they are also formulated with excipients. These are additives that stabilize the API in the product and facilitate its administration to patients. Our studies allow us to develop analytical tools to identify and assess the structure of the API and to characterize the possible interactions with excipients.
How we are using nuclear magnetic resonance spectroscopy
At the Centre for Biologics Evaluation, our team studies protein structure. We focus on the close relationship between biological function, protein conformation and dynamics. As we obtain a description in structural terms of biologics, proteins or polypeptides, we lay the foundation for building a complete understanding of these complex molecules. We use this knowledge of the relationship between protein structure and function to identify and develop bioanalytical tools for characterizing or assessing biologics.
To study protein structure, our research team has developed expertise in the use of a powerful technique called nuclear magnetic resonance (NMR) spectroscopy. We also use the latest biotechnology methods to produce protein samples necessary for applying NMR techniques. We use recombinant protein and bioprocess technologies to incorporate stable isotopes such as nitrogen-15 and carbon-13 that let us apply multi-dimensional NMR techniques. These techniques greatly increase resolution, so that we can determine and study the structure and dynamics of proteins at atomic level. Methods for the analysis of biologics are usually developed and tested with labelled proteins first, before they can be applied to test the proteins in biologic formulations.
Research in this area has applications such as
- Characterizing the structure of recombinant protein therapeutics.
- Developing new methods to assess biosimilars and other biologics.
- Studying the effects of formulation excipients on protein motions.
Concepts and tools we use to study protein structure
Nuclear magnetic resonance (NMR) spectroscopy measures the precession frequency of atomic nuclei, such as the hydrogen atom, when subjected to strong magnetic fields. When a molecule (for example, water, protein or drug) is exposed to a very strong magnetic field, the nuclei move like spinning tops that are under the effect of the earth’s gravitational field. This movement, as illustrated in Figure 1, is called precession. The precession frequency of a given nucleus is directly related to the surrounding magnetic field, also referred to its surrounding magnetic environment. NMR spectroscopy measures all individual frequencies of all nuclei (1H 13C and 15N) in a biomolecule. Using appropriate techniques, all frequencies can be attributed to each amino acid of a biomolecule and inter-nuclear distances can be measured in order to derive three-dimensional structures. Other parameters can be measured to inform on the various internal motions that are experienced by the protein.
1.1: Spinning top
1.3: Field (Gravitational or Magnetic)
© Her Majesty the Queen in Right of Canada, represented by the Minister of Health (2009).
Research highlight 1: Nuclear magnetic resonance fingerprinting - A new tool to analyse proteins and identify biologics
Recombinant protein therapeutics form a class of biologics in which proteins, such as hormones and antibodies, are the key active pharmaceutical ingredients (APIs). These large molecules can vary greatly in shape, composition and structure depending on their environment and how they are produced. The structure of the protein (that is, its conformation) determines its ability to work as a therapeutic agent. A change in conformation may lead to an ineffective therapeutic that may generate unwanted effects.
Delivery of a therapeutic protein into the human body requires that the protein be mixed (formulated) with appropriate additives, called excipients. As added ingredients, excipients allow proper delivery of the APIs, maintain protein conformation, and provide biochemical stability over long periods of time (shelf-life). To assess the quality attributes of these therapeutic products, researchers must develop the ability to analyse the conformation of the protein from formulated products.
The nuclear magnetic resonance (NMR) spectra of a protein measure the frequencies from most nuclei (proton, nitrogen and carbon). These frequencies are directly influenced by the local magnetic field surrounding the nuclei. Therefore, the conformation of a protein will have a unique NMR signature that can be seen as a fingerprint, analogous to the unique fingerprints of a human. Researchers use a protein NMR fingerprint to identify and assess the conformation of the API. They compare it with the matching fingerprint recorded as a reference standard.
Research in this area provides new tools to
- assess if changes in formulations of biotechnology derived products affect the structure of the protein and possibly the activity of the biologic
- help evaluate the quality of biosimilars or other recombinant protein therapeutics.
Research highlight 2: Proteins are dynamics: their motions influence their functions
Proteins are large biomolecules with fascinating three-dimensional structures. These, like all molecules, can experience motions that can span a wide range of frequencies and amplitudes. Therefore, protein dynamics often play a central role in biological function and the intrinsic stability of the structure.
We are working to
- Learn about how excipients influence the stability of recombinant protein therapeutics by measuring their effects on the structure dynamics. These studies will help develop a rigorous knowledge on how the various excipients can enhance the stability and functions of biological products, as well as guide decision making in cases where modification of a formulation is proposed by a sponsor.
For information about the lead scientist of this laboratory, please visit their Directory of Scientists and Professionals profile.