(384b) Providing a Molecular Basis to Modeling Protein Adsorption to Multimodal Carbohydrate-Based Ligands
- Conference: AIChE Annual Meeting
- Year: 2016
- Proceeding: 2016 AIChE Annual Meeting
- Group: Pharmaceutical Discovery, Development and Manufacturing Forum
- Time: Tuesday, November 15, 2016 - 12:52pm-1:14pm
Here we study the adsorption of carbohydrate-binding proteins to two distinct model polysaccharide substrates with identical molecular composition but varying surface hydrophilicity using complementary bulk and single-molecule based adsorption techniques. We have modified a recently developed optical tweezers force spectroscopy technique  to characterize the single molecule protein binding-unbinding force dynamics to polysaccharide surfaces and probed the protein-ligand bond lifetimes at the single molecule level. Analysis of our records was used to identify trends in the force-binding lifetime datasets to find that the unbinding force and total binding lifetimes reflects the underlying heterogeneity of the available binding sites for carbohydrate-derived ligands. This distribution reflects the spectrum of varying affinity sites present on the polysaccharide surface and ultimately provides a molecular basis for using various multi-site Langmuir-type adsorption models to characterize protein adsorption to polysaccharide surfaces. Interestingly, simple regression analysis of bulk adsorption data using multi-site Langmuir-type models reveals that traditional protein bulk adsorption measurements are unable to provide data with sufficiently high resolution to discriminate between the low and high affinity protein binding sites for polysaccharides if the difference in the protein-carbohydrate ligand unbinding force is relatively small. In summary, we now have the necessary tools to systematically characterize protein adsorption to multimodal carbohydrate-derived ligands for optimizing industrial bioseparations using a first-principles approach.
 Pinto, I. F., Aires-Barros, M. R. & Azevedo, A. M. Multimodal chromatography: debottlenecking the downstream processing of monoclonal antibodies. Pharm. Bioprocess. 3, 263â??279 (2015).
 Brady, S. K., Sreelatha, S., Feng, Y., Chundawat, S. P. S. & Lang, M. J. Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps. Nature Communications. 6, 10149 (2015).