Characterization of therapeutic protein AvidinOX by an integrated analytical approach

• Published in: Analytical and bioanalytical chemistry 2018, Vol.410 (2), p.553-564
• Main Authors: Giannini, Giuseppe, Alekseeva, Anna, Naggi, Annamaria, Salvini, Laura, Tei, Lorenzo, De Santis, Rita
• Format: Article
• Language: English
• Subjects: Adducts; Aldehydes; Aldehydes - analysis; Amino acids; Amino groups; Analytical Chemistry; Avidin; Avidin - chemistry; Azo Compounds - chemistry; Backbone; Biochemistry; Biological activity; Biotin; Carbohydrates; Carboxylic acids; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Colorimetry; Food Science; Fractionation; Glycan; Glycoproteins; Half-life; Integrated approach; Laboratory Medicine; Mass spectrometry; Mass spectroscopy; Mathematical analysis; Microheterogeneity; Models, Molecular; Monitoring/Environmental Analysis; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular - methods; Oxidation; Oxidation-Reduction; Phenylacetates - chemistry; Polysaccharides - analysis; Protein Aggregates; Proteins; Radioactive half-life; Research Paper; Sodium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Stability analysis; Structural analysis; Sugar; Tumors
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-017-0754-3

AvidinOX, the oxidized derivative of Avidin, is a chemically modified glycoprotein, being currently under clinical investigation for targeted delivery of radioactive biotin to inoperable tumors. AvidinOX is produced by 4-hydroxyazobenzene-2-carboxylic acid (HABA)-assisted sodium periodate oxidation of Avidin. The peculiar property of the periodate-generated glycol-split carbohydrate moieties to form Schiff’s bases with amino groups of the tissue proteins allows to achieve a tissue half-life of 2 weeks compared to 2 h of native Avidin. Carbohydrate oxidation, along with possible minor amino acid modifications, introduces additional microheterogeneity in the glycoprotein structure, making its characterization even more demanding than for native glycoproteins. Aiming at the elucidation of the effects of oxidation conditions on the AvidinOX protein backbone and sugars, this microheterogeneous glycoprotein derivative was characterized for the first time using a combination of different analytical methods, including colorimetric methods, mass spectrometry, hollow-fiber flow field-flow fractionation with UV and multi-angle laser scattering detection (HF5-UV-MALS), and NMR. The proposed integrated approach reveals structural features of AvidinOX relevant for its biological activity, e.g., oxidized sites within both carbohydrate moieties and protein backbone and conformational stability, and will be considered as an analytical tool for AvidinOX industrial preparations. It is worth noting that this study enriches also the structural data of native Avidin published up-to-date (e.g., glycan structure and distribution, peptide fingerprint, etc.). Graphical abstract Scheme of phenylacetic hydrazide/MALDI-TOF approach for quantification of aldehydes in AvidinOX based on the determination of the number of hydrazone adducts between hydrazide reagent and aldehyde groups of protein