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Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity.

Publication ,  Journal Article
Kerwin, BA; Bennett, C; Brodsky, Y; Clark, R; Floyd, JA; Gillespie, A; Mayer, BT; McClure, M; Siska, C; Seaman, MS; Seaton, KE; Shaver, J ...
Published in: J Pharm Sci
January 2020

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.

Duke Scholars

Published In

J Pharm Sci

DOI

EISSN

1520-6017

Publication Date

January 2020

Volume

109

Issue

1

Start / End Page

233 / 246

Location

United States

Related Subject Headings

  • Protein Structure, Secondary
  • Protein Stability
  • Protein Conformation
  • Pharmacology & Pharmacy
  • Mutation
  • Mice
  • Humans
  • HIV Antibodies
  • HEK293 Cells
  • Broadly Neutralizing Antibodies
 

Citation

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Kerwin, B. A., Bennett, C., Brodsky, Y., Clark, R., Floyd, J. A., Gillespie, A., … Ketchem, R. R. (2020). Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity. J Pharm Sci, 109(1), 233–246. https://doi.org/10.1016/j.xphs.2019.07.009
Kerwin, Bruce A., Chelsey Bennett, Yan Brodsky, Rutilio Clark, J Alaina Floyd, Alison Gillespie, Bryan T. Mayer, et al. “Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity.J Pharm Sci 109, no. 1 (January 2020): 233–46. https://doi.org/10.1016/j.xphs.2019.07.009.
Kerwin BA, Bennett C, Brodsky Y, Clark R, Floyd JA, Gillespie A, et al. Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity. J Pharm Sci. 2020 Jan;109(1):233–46.
Kerwin, Bruce A., et al. “Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity.J Pharm Sci, vol. 109, no. 1, Jan. 2020, pp. 233–46. Pubmed, doi:10.1016/j.xphs.2019.07.009.
Kerwin BA, Bennett C, Brodsky Y, Clark R, Floyd JA, Gillespie A, Mayer BT, McClure M, Siska C, Seaman MS, Seaton KE, Shaver J, Tomaras GD, Yates NL, Ketchem RR. Framework Mutations of the 10-1074 bnAb Increase Conformational Stability, Manufacturability, and Stability While Preserving Full Neutralization Activity. J Pharm Sci. 2020 Jan;109(1):233–246.
Journal cover image

Published In

J Pharm Sci

DOI

EISSN

1520-6017

Publication Date

January 2020

Volume

109

Issue

1

Start / End Page

233 / 246

Location

United States

Related Subject Headings

  • Protein Structure, Secondary
  • Protein Stability
  • Protein Conformation
  • Pharmacology & Pharmacy
  • Mutation
  • Mice
  • Humans
  • HIV Antibodies
  • HEK293 Cells
  • Broadly Neutralizing Antibodies