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Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions

Sakhnini, Laila I LU ; Greisen, Per J ; Wiberg, Charlotte LU ; Bozoky, Zoltan ; Lund, Søren ; Wolf Perez, Adriana-Michelle ; Karkov, Hanne S ; Huus, Kasper ; Hansen, Jens-Jacob and Bülow, Leif LU , et al. (2019) In Biochemistry 58(24). p.2750-2759
Abstract

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human... (More)

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.

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published
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Biochemistry
volume
58
issue
24
pages
2750 - 2759
publisher
The American Chemical Society (ACS)
external identifiers
  • pmid:31117388
  • scopus:85067417278
ISSN
0006-2960
DOI
10.1021/acs.biochem.9b00251
language
English
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yes
id
6eb42767-a3a8-46f7-b9e3-5b36a41d7665
date added to LUP
2019-06-17 16:52:24
date last changed
2020-01-16 03:59:11
@article{6eb42767-a3a8-46f7-b9e3-5b36a41d7665,
  abstract     = {<p>Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.</p>},
  author       = {Sakhnini, Laila I and Greisen, Per J and Wiberg, Charlotte and Bozoky, Zoltan and Lund, Søren and Wolf Perez, Adriana-Michelle and Karkov, Hanne S and Huus, Kasper and Hansen, Jens-Jacob and Bülow, Leif and Lorenzen, Nikolai and Dainiak, Maria B and Pedersen, Anja K},
  issn         = {0006-2960},
  language     = {eng},
  number       = {24},
  pages        = {2750--2759},
  publisher    = {The American Chemical Society (ACS)},
  series       = {Biochemistry},
  title        = {Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions},
  url          = {http://dx.doi.org/10.1021/acs.biochem.9b00251},
  doi          = {10.1021/acs.biochem.9b00251},
  volume       = {58},
  year         = {2019},
}