Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-05, Vol.1021, p.114-121
• Main Authors: Davenport, Kaitlynn R., Smith, Christopher A., Hofstetter, Heike, Horn, James R., Hofstetter, Oliver
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antibodies, Immobilized - chemistry; Antibodies, Immobilized - genetics; Antibodies, Immobilized - metabolism; AviTag; Biotin; Biotin - chemistry; Biotin - metabolism; Camelid antibody; Camelus; Chromatography; Columns (structural); High performance liquid chromatography; Immobilization; Immunoaffinity chromatography; Immunochromatography - methods; Limit of Detection; Linear Models; Linearity; Methotrexate; Methotrexate - immunology; Methotrexate - metabolism; Oriented antibody immobilization; Peptides; Reproducibility of Results
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.01.021

•VHH-based immunochromatographic separation of methotrexate was demonstrated.•VHH variants contained a mutation that served as a pH-switch.•First use of AviTag technology for the production of affinity chromatography stationary phases. In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein’s surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R2>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04–12μM (R2>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first application of immunoaffinity chromatography for the analysis of MTX.