Trivalent Peptide Design for Bevacizumab Salt-Induced Affinity Precipitation ^†

Introduction: Bevacizumab, applied in cancer treatment, binds vascular endothelial growth factor (VEGF) inhibiting blood vessels growth, avoiding tumours expansion. During its production, its purification is performed using protein-A affinity chromatography, which highly increases its overall production cost. Although salt induced precipitation with ammonium sulphate (AMS) is a cheap alternative, it results in a low purity product. To achieve better purity, in this work, an affinity-based precipitation has been designed with a trivalent peptide corresponding to the VEGF epitope that interacts with bevacizumab: P⁸⁵HQGQHIG⁹². Methodology: Peptide (Ac-PHQGQHIG-Ahx₃)₂-K-Ahx₃-PHQGQHIG-NH₂ was synthesized by solid-phase peptide synthesis (SPPS). After incorporating PHQGQHIG, Fmoc-Lys(Fmoc)-OH was coupled to add two more branches. Between each epitope-branch, three molecules of 6-aminohexanoic acid (Ahx) were added as spacer arms, to ensure that each epitope of the trivalent peptide interacts with a different molecule of bevacizumab and the formation of cyclic complexes. Bevacizumab precipitation with the peptide, adding different concentrations of AMS, was assayed. Results: The SPPS allowed to obtain (Ac-PHQGQHIG-Ahx₃)₂-K-Ahx₃-PHQGQHIG-NH₂ with high purity and yield. Its identity was confirmed by ESI-MS (peaks 954, 763 and 636 corresponding to ions [M + 4H]⁴⁺, [M + 5H]⁵⁺ and [M + 6H]⁶⁺ respectively, were detected). The addition of the peptide to solution with increasing concentrations of AMS, demonstrated the specific precipitation of bevacizumab as was observed after centrifugation and measurement of absorbance at 280 nm. Conclusions: The branched peptide forms cyclic complexes with bevacizumab increasing its molecular mass thus decreasing its solubility. This will allow the design of an economic purification method avoiding the use of expensive protein-A affinity chromatography.