Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody

Tarja Nevanen (Corresponding Author), Marja-Leena Hellman, Nana Munck, Gerd Wohlfahrt, Anu Koivula, Hans Söderlund

Research output: Contribution to journalArticleScientificpeer-review

18 Citations (Scopus)

Abstract

The binding affinity and specificity of recombinant antibodies can be modified by site‐directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine‐tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen‐binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.
Original languageEnglish
Pages (from-to)1089-1097
Number of pages9
JournalProtein Engineering
Volume16
Issue number12
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

Fingerprint

Mutagenesis
Enantiomers
Fractionation
Antibodies
Immunoglobulin Fragments
Immunoglobulin Fab Fragments
Mutation
Antibody Affinity
Antibody Specificity
Haptens
Affinity Chromatography
Point Mutation
Pharmaceutical Preparations
Affinity chromatography
Molecular modeling

Keywords

  • antibodies
  • antibody Fab'-fragment
  • antibody fragments
  • mutagenesis
  • enantiomers
  • enantioselective antibody

Cite this

Nevanen, Tarja ; Hellman, Marja-Leena ; Munck, Nana ; Wohlfahrt, Gerd ; Koivula, Anu ; Söderlund, Hans. / Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody. In: Protein Engineering. 2003 ; Vol. 16, No. 12. pp. 1089-1097.
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Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody. / Nevanen, Tarja (Corresponding Author); Hellman, Marja-Leena; Munck, Nana; Wohlfahrt, Gerd; Koivula, Anu; Söderlund, Hans.

In: Protein Engineering, Vol. 16, No. 12, 2003, p. 1089-1097.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Model-based mutagenesis to improve the enantioselective fractionation properties of an antibody

AU - Nevanen, Tarja

AU - Hellman, Marja-Leena

AU - Munck, Nana

AU - Wohlfahrt, Gerd

AU - Koivula, Anu

AU - Söderlund, Hans

PY - 2003

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AB - The binding affinity and specificity of recombinant antibodies can be modified by site‐directed mutagenesis. Here we have used molecular modelling of the variable domains of an enantiospecific antibody fragment to fine‐tune its affinity so it is more suitable for the fractionation of the drug enantiomers. We have shown earlier that the Fab fragment of this antibody specifically recognizes one enantiomer from the racemic mixture of a medical drug and that it can be used for the fractionation of these enantiomers by affinity chromatography. However, the affinity was unnecessarily high, requiring harsh elution conditions to release the bound enantiomer. Thus, the continuous use of the antibody affinity columns was impossible. We made a homology model of the antibody and designed mutations to the antigen‐binding site to decrease the affinity. Four out of five point mutations showed decreased affinity for the hapten. Two of the mutations were also combined to construct a double mutant. The affinity columns made using one of the single mutants with lowered affinity and the double mutant were capable of multiple rounds of enantioseparation.

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