Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity
| Autor: | Chelsey Bennett, Mcclure Megan J, Georgia D. Tomaras, Michael S. Seaman, Clark Rutilio H, Randal R. Ketchem, J. Alaina Floyd, Kelly E. Seaton, Christine C. Siska, Yan Brodsky, Jeremy M. Shaver, Bryan T. Mayer, Nicole L. Yates, Bruce A. Kerwin, Alison J. Gillespie |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
analytical biochemistry
HP-SEC High performance size exclusion chromatography Protein Conformation Pharmaceutical Science CDR Complementary determining region 02 engineering and technology Protein aggregation HIV Antibodies 030226 pharmacology & pharmacy Neutralization Protein Structure Secondary high throughput technology(s) PK Pharmacokinetic chemistry.chemical_compound HC Heavy chain Mice 0302 clinical medicine Protein structure protein folding antibody(s) Gdn-HCl Guanidine hydrochloride Chemistry Protein Stability fluorescence spectroscopy 021001 nanoscience & nanotechnology stabilization bnAbs broadly neutralizing antibodies Monomer PEG Polyethylene glycol DSF Differential scanning fluorimetry CDCA Stability design by covariance analysis HIV/AIDS Protein folding 0210 nano-technology pharmacokinetics COGs Cost of goods PBS Phosphate buffered saline HMW High molecular weight Article physical stability protein aggregation 03 medical and health sciences Residue (chemistry) LC Light chain developability Molecule Animals Humans protein structure mAb monoclonal antibody biopharmaceutical characterization stability HEK293 Cells Mutation Biophysics Salt bridge Broadly Neutralizing Antibodies |
| Zdroj: | Journal of Pharmaceutical Sciences |
| ISSN: | 1520-6017 0022-3549 |
| Popis: | The broadly neutralizing anti-HIV antibody, 10-1074, is a highly somatically hypermutated IgG1 being developed for prophylaxis in sub-Saharan Africa. A series of algorithms were applied to identify potentially destabilizing residues in the framework of the Fv region. Of 17 residues defined, a variant was identified encompassing 1 light and 3 heavy chain residues, with significantly increased conformational stability while maintaining full neutralization activity. Central to the stabilization was the replacement of the heavy chain residue T108 with R108 at the base of the CDR3 loop which allowed for the formation of a nascent salt bridge with heavy chain residue D137. Three additional mutations were necessary to confer increased conformational stability as evidenced by differential scanning fluorimetry and isothermal chemical unfolding. In addition, we observed increased stability during low pH incubation in which 40% of the parental monomer aggregated while the combinatorial variant showed no increase in aggregation. Incubation of the variant at 100 mg/mL for 6 weeks at 40°C showed a 9-fold decrease in subvisible particles ≥2 μm relative to the parental molecule. Stability-based designs have also translated to improved pharmacokinetics. Together, these data show that increasing conformational stability of the Fab can have profound effects on the manufacturability and long-term stability of a monoclonal antibody. |
| Databáze: | OpenAIRE |
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