By Subhabrata Majumder and Arun Alphonse Ignatius
Biologics can suffer from inherent long term storage stability problems. Thus optimal formulation of biologics is essential for maximizing its shelf life. Formulation development of biologics presents unique challenges which could be traced back to its inherent structural complexity.
For example, monoclonal antibodies (mAbs) and related modalities are multi-domain proteins and are susceptible to physical and chemical degradation leading to aggregation. Any or all of these could stem from local and global structure of the biologics, which can affect its function. These artifacts are even further amplified at very high concentrations typically used for monoclonal antibodies in the final formulation. Moreover, aggregation of biologics has been correlated with immunogenicity, which can adversely affect the clinical outcome of the product.
Biophysical studies to address aggregation have been focused either on the colloidal property of mAbs treating biologics as rigid spheres or on thermal shift assays to gauge thermal stability. Not only do these techniques probe macroscopic property of the biologics, the underlying assumptions are deceptively simple and at times, misleading. Moreover, the extrapolation of the protein behavior under dilute conditions (thermodynamically ideal) to the typically high formulation concentrations (non-ideal) may not be straightforward.
Nuclear magnetic resonance (NMR) spectroscopy is a technique that allows us to fill in the missing link between the structure and its molecular liabilities. It is a noninvasive technique and works well at close-to-formulation condition(s). Although it is widely used in small molecule drug development to assess the structure and purity, it has been scarcely applied to biologics, until now. The physical phenomenon underlying NMR is to treat the biologics or any molecule as a collection of nuclei. When placed in a spectrometer, a high field magnet, and in the presence of an oscillating radiofrequency field, these nuclei spin at various frequencies indicative of their local and overall environment (chemical shift). The readout of this exercise is an NMR spectrum, which contains the chemical shift dispersion of the chosen nuclei. In a more sophisticated version (2D NMR), one can investigate side chain methyl or backbone amide groups. Such molecular fingerprinting based on NMR is a valuable source of information for probing various formulation attributes of biologics. For example, one can examine hotspots of aggregation amongst different biologics or their respective predisposition toward a particular liability, which can significantly impact the time and resources of drug development.
Isotopically enriched biologics are required to realize the full potential of the technique. The advent of high field NMR spectrometers and cold probes allows us to assess biologics either in parts or as a whole, even without isotopic labeling.This to gauge(a) the intrinsic stability of the biologics, with respect to aggregation, can be probed at a residue specific level by molecular fingerprinting and (b) the extrinsic stability i.e effect of formulation of the biologics in different buffers. However, at this point NMR , is not a self-sustaining method and requires complimentary biophysical techniques to support/ validate the results. Finally, given the time and resources that the industry invests to discover and develop biologic drugs, a little deeper dive to look at the atomistic details of the molecule itself could provide invaluable insights leading to informed strategies for drug product development.