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.