Barredo Gabriela R, Giudicessi Silvana L, Martínez Ceron María C, Saavedra Soledad L, Rodríguez 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.
MethodsX. 2019 Dec 16;7:100769. doi: 10.1016/j.mex.2019.12.010. eCollection 2020.
Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture. •Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.
贝伐单抗是一种在CHO细胞中产生的单克隆抗体，用于治疗多种人类癌症。它是一种抗血管内皮生长因子（抗VEGF），可阻断肿瘤血管的生长。如今，其纯化是通过使用蛋白A的亲和色谱法（AC）实现的，而蛋白A是一种非常昂贵的配体。另一方面，VEGF片段中包含的肽Ac-PHQGQHIGVSK与贝伐单抗具有高亲和力。这种短肽配体比蛋白A具有更高的稳定性和更低的成本，并且可以通过固相肽合成非常容易地制备。本方案描述了Ac-PHQGQHIGVSK-琼脂糖的合成及其用于从澄清的CHO细胞培养物中亲和色谱纯化贝伐单抗的方法。•Ac-PHQGQHIGVSK-琼脂糖的容量和选择性与蛋白A基质相当。•肽配体显示出更高的稳定性和更低的成本。肽配体中缺乏色氨酸、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。•色谱过程中使用的温和条件保持了贝伐单抗的完整性。
