caa a22 4500 445313935 CHVBK 20180317142639.0 cr unu---uuuuu 170323e20110501xx s 000 0 eng 10.1007/s11095-011-0383-z doi (NATIONALLICENCE)springer-10.1007/s11095-011-0383-z A New Approach to Explore the Impact of Freeze-Thaw Cycling on Protein Structure: Hydrogen/Deuterium Exchange Mass Spectrometry (HX-MS) [Elektronische Daten] [Aming Zhang, Wei Qi, Satish Singh, Erik Fernandez] ABSTRACT: Purpose: The impact of freeze-thaw (F/T) on structure integrity of protein therapeutics is poorly understood, partially due to lack of methods to detect protein structural perturbations during F/T processing in the frozen state. Methods: A new approach of hydrogen/deuterium exchange was developed to separate and distinguish the specific impact of single freezing and F/T cycling on protein structure, using lactate dehydrogenase (LDH) as model system. Results: In the freezing process, a fraction of LDH molecules that was inversely dependent on protein concentration was observed to partially denature its structure. Local structural perturbations were localized by peptide level HX analysis to the surface residues in segments 91-132, 170-237 and 288-331. In contrast, F/T cycling led to irreversible LDH aggregation with global structural unfolding. Residual solvent-protected structure was only detected in the aggregates for three segments, 13-31, 109-117 and 133-143, that were coincident with the consensus aggregation hotspots predicted by four different algorithms. Conclusions: Results indicate freezing preferentially disturbs local structure at the surface residues, consistent with ice-solution interface-mediated denaturation mechanism. F/T-induced aggregation begins as partial denaturation during freezing, but is accompanied by more comprehensive structural rearrangement during F/T cycling. Springer Science+Business Media, LLC, 2011 aggregation nationallicence freeze-thaw nationallicence hydrogen/deuterium exchange nationallicence lactate dehydrogenase nationallicence structural denaturation nationallicence DLS : dynamic light scattering nationallicence F/T : freeze-thaw nationallicence HX : hydrogen/deuterium exchange nationallicence HX-MS : hydrogen/deuterium exchange mass spectrometry nationallicence LDH : lactate dehydrogenase nationallicence Zhang Aming Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, 22904, Charlottesville, Virginia, USA aut Qi Wei Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, 22904, Charlottesville, Virginia, USA aut Singh Satish BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Fernandez Erik Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, 22904, Charlottesville, Virginia, USA aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/5(2011-05-01), 1179-1193 0724-8741 28:5<1179 2011 28 11095 https://doi.org/10.1007/s11095-011-0383-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-011-0383-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zhang Aming Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, 22904, Charlottesville, Virginia, USA aut NATIONALLICENCE 700 1- Qi Wei Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, 22904, Charlottesville, Virginia, USA aut NATIONALLICENCE 700 1- Singh Satish BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 700 1- Fernandez Erik Department of Chemical Engineering, University of Virginia, 102 Engineer's Way, 22904, Charlottesville, Virginia, USA aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/5(2011-05-01), 1179-1193 0724-8741 28:5<1179 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer