Barredo Gabriela R, Giudicessi Silvana L, Martínez Ceron María C, Saavedra Soledad L, Rodriguez Santiago, Filgueira Risso Lucas, Erra-Balsells Rosa, Mahler Gustavo, Albericio Fernando, Cascone Osvaldo, Camperi Silvia A
Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de Biotecnología, Junín 956, 1113, Buenos Aires, Argentina; CONICET-Universidad de Buenos Aires, Instituto de Nanobiotecnología (NANOBIOTEC), Facultad de Farmacia y Bioquímica, Junín 956, 1113, Buenos Aires, Argentina.
MAbxience SAU, Carlos Villate 5148, 1605, Munro, Buenos Aires, Argentina.
Protein Expr Purif. 2020 Jan;165:105500. doi: 10.1016/j.pep.2019.105500. Epub 2019 Sep 19.
Bevacizumab is a vascular endothelial growth factor (VEGF)-directed monoclonal antibody (mAb) used for the treatment of several human cancers. Given that bevacizumab is administered intravenously, it must have extremely high purity, which is achieved by purification with protein A affinity chromatography (AC). However, protein A is a very expensive ligand, thereby increasing the cost of purification. Furthermore, the harsh elution conditions required to recover bevacizumab from the AC column can damage both the mAb and protein A. In contrast, short peptides show higher stability, easier synthesis and lower cost and are therefore ideal ligands for AC. In the present study, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment that binds bevacizumab, was synthesized and immobilized on agarose. The peptidyl-agarose showed affinity for bevacizumab, with an equilibrium dissociation constant value of 2.2±0.5 x 10 M under optimal conditions. Samples of CHO cell filtrate producing bevacizumab were loaded on the peptidyl-agarose chromatography column. Bevacizumab was recovered from the elution fraction with a yield of 94% and a purity of 98%. The maximum capacity (qm) 38±2 mg of bevacizumab per mL of matrix was comparable to that of commercial protein A matrices. Moreover, the peptide ligand showed greater stability and a lower cost than protein A. Unlike peptides previously reported for IgG purification, the ligand described herein allows mAb elution under mild conditions, thereby favoring the integrity of bevacizumab. The lack of Trp, Met or Cys in the peptide prevents its oxidation and extends the useful life of the chromatographic matrix.
贝伐单抗是一种靶向血管内皮生长因子（VEGF）的单克隆抗体（mAb），用于治疗多种人类癌症。鉴于贝伐单抗通过静脉给药，它必须具有极高的纯度，这可通过蛋白A亲和色谱法（AC）纯化来实现。然而，蛋白A是一种非常昂贵的配体，从而增加了纯化成本。此外，从AC柱上回收贝伐单抗所需的苛刻洗脱条件可能会损坏mAb和蛋白A。相比之下，短肽显示出更高的稳定性、更易合成且成本更低，因此是AC的理想配体。在本研究中，合成了与贝伐单抗结合的VEGF片段中包含的肽Ac-PHQGQHIGVSK，并将其固定在琼脂糖上。肽基琼脂糖对贝伐单抗表现出亲和力，在最佳条件下平衡解离常数为2.2±0.5×10⁻⁶M。将产生贝伐单抗的CHO细胞滤液样品加载到肽基琼脂糖色谱柱上。从洗脱级分中回收贝伐单抗，产率为94%，纯度为98%。每毫升基质中贝伐单抗的最大容量（qm）为38±2mg，与市售蛋白A基质相当。此外，该肽配体比蛋白A表现出更高的稳定性和更低的成本。与先前报道的用于IgG纯化的肽不同，本文所述的配体允许在温和条件下洗脱mAb，从而有利于贝伐单抗的完整性。该肽中缺乏色氨酸、甲硫氨酸或半胱氨酸可防止其氧化，并延长色谱基质的使用寿命。
