Modulation of the Hydration Water Around Monoclonal Antibodies on Addition of Excipients Detected by Terahertz Time-Domain Spectroscopy

Abstract

Terahertz time-domain spectroscopy (THz-TDS) has been shown to detect overlapping extended hydration layers around proteins. Here, we used THz-TDS to detect modulation of the extended hydration layer around monoclonal antibodies (mAbs) by the introduction of commonly used excipients. Proline and sucrose altered the hydration layer around a mAb (mAb1), which was observed as a negative shift in the plateau in absorbance above ~100 mg/mL mAb1 (~70,000 water molecules per mAb); arginine had no effect. At lower concentrations of ~10 mg/mL mAb1 (~700,000 water molecules per mAb) proline and arginine modulated the hydration layer, which was observed as a negative shift in the relative absorbance, whereas sucrose had no effect. The changes in the extended hydration layer were not translated to shifts in the thermal stability or protein:protein interaction parameter. The hydration layer of a second mAb (mAb2) was further shown to be modulated by more complex formulations composed of two or more excipients; although the differences in terahertz absorbance were not predictive of viscosity or long-term stability. THz-TDS promises to be a useful tool for understanding a protein's interaction with excipients in solution and the challenge will be to determine how to apply this knowledge to protein formulation.

The interaction between electromagnetic fields at megahertz, gigahertz and terahertz frequencies with cells, tissues and organisms: risks and potential

“…The nonlinear relationship between the protein–water solution absorption coefficient and concentration of the tested protein suggests an intense interaction of the water molecules with protein surface and enveloping layer could be as thick as tens of angstroms [ 196 , 197 ]. Studies with monoclonal antibodies (mAbs) conducted by Wallace et al [ 198 ] demonstrated a steady increase in absorbance along terahertz range 0.25–1.5 THz. Notably, the technique was sensitive enough to detect submillimolar changes in protein concentration [ 198 ].…”

“…Studies with monoclonal antibodies (mAbs) conducted by Wallace et al [ 198 ] demonstrated a steady increase in absorbance along terahertz range 0.25–1.5 THz. Notably, the technique was sensitive enough to detect submillimolar changes in protein concentration [ 198 ]. Also, the plot of the change in absorption coefficient versus protein concentration, measured at 1 THz, indicated the characteristic shape of gradual decrease with a subsequent plateau at some critical concentration (approx.…”

“…In some experiments, the addition of extra components into solution was done to test the extended hydration layer hypothesis, in other words, whether modification of protein hydration layer may affect its interaction with excipients. The plateau mentioned above was attenuated by the addition of 200 mM of uncharged sucrose or zwitterionic proline, whereas addition of charged arginine (same concentration) did not affect the absorption curve [ 198 ].…”

Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015

“…There is evidence that a protein's hydration layer is more extensive and complex than previously believed . It has also been shown that cosolutes can modify the hydration layer . There is scope for future work using ITC in conjunction with low‐frequency analysis of water.…”

“…Cosolutes interact with macromolecules both by direct binding and indirectly by modifying their hydration layers . Macromolecules similarly interact with each other or compete with each other for water for their hydration layers . The issue of cosolutes altering the thermodynamics of ligand binding is unavoidable, and the researcher has to accept that the thermodynamic constants derived from their research are for the solution conditions used and will change if different buffer, pH or temperatures are used.…”