Scientists find a way to ‘fingerprint’ the structure of a monoclonal antibody
Stephanie Sutton |
Precisely measuring the structural configuration of monoclonal antibodies (mAbs) is important when it comes to safety and efficacy. To that end, Robert Brinson, a chemist at the National Institute of Standards and Technology (NIST), and his team have used a technique called near-magnetic resonance (NMR) spectroscopy to measure the fingerprint of mAbs (1). Brinson explains NMR fingerprinting in more detail.
What are the challenges to fingerprinting mAbs?
The intact mAb we looked at is around 150,000 Daltons, and the Fc and Fab fragments are 50,000 Daltons. For comparison, aspirin, the classic small-molecule drug, is 180 Daltons. Neupogen, a biologic, is 18,800 Daltons. A small-molecule drug can be easily characterized, but this is not the case for a complex biotherapeutic protein. While the primary amino acid sequence may be known, one protein batch can be safe and another toxic. This is due to higher-order protein folding – the primary sequence folds back on itself into a secondary and tertiary structure and quaternary structures.
The general purpose of our method was to develop and apply NMR spectroscopy as a higher-order structure assessment tool to mAbs. Our goal was to show that this technique could deliver data that demonstrate highly similar or fingerprint-like similarity between protein lots, or between an innovator and a biosimilar.
How did you make the method work?
Our goal as a lab is to push the practical limits of NMR spectroscopy. Within that framework, it was natural for us to attempt this type of characterization. We were pleasantly surprised, however, that we successfully collected data with such high quality on the intact mAb.
We demonstrated that collecting the 2D 13C,1H NMR methyl fingerprint is feasible on the intact mAb. The methyl group has greater rotation than other functional groups, which leads to sharper peaks and therefore higher spectral quality. These groups are dispersed throughout the protein and directly report on how well the protein is folded.
Since NMR systems with lower magnetic field strength are more commonly found in analytical research labs, we divided the mAb enzymatically into its two constituent Fc and Fab fragments, so that the NMR mapping approach could be employed using more commonly available NMR systems. Importantly, we demonstrated that the two fragments generated from the full mAb showed no loss of structural information and that the sum of the fingerprint patterns of the fragments could be matched to the intact mAb.
What are the implications for drug development?
This measurement technique provides a robust means for a company to characterize the higher-order structure of a protein drug product. This is useful in pre-clinical and clinical settings as it shows where the drug is acting and why. In a QC environment, NMR could potentially be used to evaluate multiple lots through statistical comparability methods and compare biosimilars to innovator products.
- L.W. Abrogast, R.G. Brinson and J.P. Marino, “Mapping Monoclonal Antibody Structure by 2D 13C NMR at Natural Abundance,” Analytical Chemistry 87, 3556-3561 (2015). DOI: 10.1021/ac504804m