Part 1: Physicochemical characterization of bevacizumab in undiluted 25 mg/mL drug product solutions: Comparison of originator with a biosimilar candidate.

The biosimilarity assessment of the physicochemical properties of high-concentration biopharmaceuticals is usually performed with measurements on diluted solutions, at concentrations below 1 mg/mL. In this study 13 orthogonal, spectroscopy and particle size determination methods were used to characterize the structure and aggregation of undiluted, 25 mg/mL bevacizumab drug products Avastin® manufactured in the USA and in Europe, and ABX-BEV, a bevacizumab biosimilar candidate produced by Apobiologix Inc. Secondary structure, conformation and the potential occurrence of chemical degradation of the monoclonal antibodies were characterized and compared using infrared spectroscopy, intrinsic fluorescence and ANS fluorescence spectroscopy. Protein aggregation and particulate matter in the monoclonal antibody solutions were compared using UV-Vis absorbance, 90° light-scattering, nanoparticle tracking analysis, Nile red fluorescence microscopy, particle flow imaging, ultrasound resonance technology and a new scanner-based method that visualizes protein aggregates inside unopened vials. A data wheel representation was used to plot in one figure the results from the multiple analytical methods and to highlight differences between samples. The 25 mg/mL Avastin® drug product is stored at 2-8 °C during its 2-year shelf life. After a thermal stress of 4 weeks at 40 °C the ABX-BEV solution was turbid, containing particles of 20-100 μm diameter, accompanied by strong changes in antibody structural properties. Characterization of unstressed samples stored at 2-8 °C showed that the physicochemical properties of bevacizumab in ABX-BEV and the two originator drug products were similar, the observed differences between the originators being in the same range as those between ABX-BEV and the originator. To investigate the similarity of the antibodies under stress conditions, a freeze-thaw study was performed. Although freeze-thawing of bevacizumab products is prohibited by the package insert, after two freeze-thaw cycles (24 °C to -80 °C) small changes in the structural and aggregation properties of bevacizumab were observed, changes that were similar for the originator and ABX-BEV. Our study showed a good similarity of the investigated physicochemical properties of bevacizumab in originator and ABX-BEV products. It also provides an analytical approach, based on orthogonal methods, to compare high-concentration formulations of monoclonal antibodies.