ArticlePharmaceutical research2011
A new approach to explore the impact of freeze-thaw cycling on protein structure: hydrogen/deuterium exchange mass spectrometry (HX-MS).
Article in Pharmaceutical research, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed.
- Comprehensive Analysis of Nivolumab, A Therapeutic Anti-Pd-1 Monoclonal Antibody: Impact of Handling and Stress.Pharmaceutics · 2022Article
- Fusion with heat-resistant obscure (Hero) proteins have the potential to improve the molecular property of recombinant proteins.PloS one · 2022Article
- Stability Studies of the Vaccine Adjuvant U-Omp19.Journal of pharmaceutical sciences · 2021Article
- Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines.Journal of the American Society for Mass Spectrometry · 2017Article
- Article
- Methods to study molecular mechanisms of the Neurospora circadian clock.Methods in enzymology · 2015Article
- Long-term stability of a vaccine formulated with the amphipol-trapped major outer membrane protein from Chlamydia trachomatis.The Journal of membrane biology · 2014Article
- Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies.FEBS letters · 2014Review
- Distinct aggregation mechanisms of monoclonal antibody under thermal and freeze-thaw stresses revealed by hydrogen exchange.Pharmaceutical research · 2012Article
Corrections and comments
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Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
purposeThe 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.
methodsA 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.
resultsIn 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.
conclusionsResults 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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.