Dyes with High Affinity for Diphtheria Toxoid as Promising Affinity Ligands for its Industrial Purification

Background: Usually, diphteria toxin concentration and purification are performed after detoxification due to its high danger. In this work, an alternative diphteria toxoid (DTx) concentration and purification method by affinity chromatography is proposed. Methods: Screening of 19 triazinic dyes for selecting the best ligand for DTx concentration and purification by affinity chromatography was performed. Results: Cibacron Blue 3GA and Reactive Green 10 showed the highest affinity for DTx. The adsorption isotherms of DTx on Cibacron Blue 3GA-Sepharose and Reactive Green 19-Sepharose showed a good fit of experimental data to a Langmuir-type isotherm and allowed the calculation of a maximum capacity (qm) of 1.21 ± 0.12 μmol DTx/mL matrix and 1.44 ± 0.14 μmol DTx/mL matrix, respectively and a dissociation constant (Kd) of 9.75 ± 2.90 μM and 1.58 ± 0.38 μM, respectively. Blue R-HE-Sepharose isotherm did not show a good fit of experimental data to a Langmuirtype isotherm due to its high Kd. Conclusion: Cibacron Blue 3GA and Reactive Green 10 are promising ligands for concentration and purification of DTx by affinity chromatography.