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Using different proteolytic enzymes to digest antibody and its impact on stability of antibody mimetics
Journal of Immunological Methods ( IF 1.6 ) Pub Date : 2020-11-21 , DOI: 10.1016/j.jim.2020.112933
Hanieh Khalili 1
Affiliation  

There are opportunities to formulate antibodies as solid-state depots for local therapy, which would minimise large systemic doses that are typically required. We have developed antibody mimetics known as Fab-PEG-Fab (FpF) that display similar binding affinity and functional activity as IgG antibodies. For head-to-head comparison between FpF and IgG, FpF is prepared from the Fabs obtained by enzymatic digestion of IgGs. Here, we report for the first time that using different enzymes to proteolytically digest IgG plays an important role in stability profile of the obtained Fabs leading in different stability profiles of the final conjugated product such as FpF. We prepared an anti-vascular endothelial growth factor (VEGF) FpF from either clinical Fabrani (ranibizumab) or Fabs obtained by enzymatic digestion of bevacizumab (IgG) using immobilised papain and gingisKHANTM (KGP) enzyme. The stability of FpFs was then studied after being lyophilised in comparison with both ranibizumab and bevacizumab. Lyophilisation is being evaluated to produce solid material that can be used for depot fabrication. We observed that using immobilised papain to digest IgG resulted in the heterogenous isomers Fab leading to the preparation of heterogenous FpFbeva-papain mimetic that underwent aggregation during lyophilisation. However, using KGP enzyme generated a homogenous intact Fabbeva-KGP as determined by mass spectral analysis. Interestingly, the FpF mimetics prepared from the homogenous Fabs (Fabrani and Fabbeva-KGP), displayed greater stability compared to their starting bevacizumab and ranibizumab after being lyophilised as determined by DLS analysis. There is a potential to lyophilize FpFs to be used to fabricate solid-state depots.



中文翻译:

使用不同的蛋白水解酶消化抗体及其对抗体模拟物稳定性的影响

有机会将抗体配制为用于局部治疗的固态贮库,这将最大限度地减少通常需要的大全身剂量。我们开发了称为 Fab-PEG-Fab (FpF) 的抗体模拟物,其显示出与 IgG 抗体相似的结合亲和力和功能活性。对于 FpF 和 IgG 之间的头对头比较,FpF 是从通过酶消化 IgG 获得的 Fab 制备的。在这里,我们首次报告使用不同的酶来蛋白水解消化 IgG 在获得的 Fabs 的稳定性曲线中起着重要作用,导致最终缀合产物(如 FpF)的稳定性曲线不同。我们从任何临床的Fab制备的抗血管内皮生长因子(VEGF)FPF拉尼(ranibizumab) 或通过使用固定化木瓜蛋白酶和 gingisKHANTM (KGP) 酶对贝伐单抗 (IgG) 进行酶消化获得的 Fab。然后与雷珠单抗和贝伐单抗相比,在冻干后研究 FpF 的稳定性。正在评估冻干以生产可用于仓库制造的固体材料。我们观察到,使用固定化木瓜蛋白酶消化 IgG 会产生异质异构体 Fab,从而导致制备异质 FpF贝伐木瓜蛋白酶模拟物,该模拟物在冻干过程中发生聚集。然而,如通过质谱分析所确定的,使用KGP酶产生了均质完整的Fab beva-KGP。有趣的是,从同质 Fab(Fab rani和 Fabbeva-KGP ),与它们的起始贝伐单抗和雷珠单抗相比,在冻干后显示出更高的稳定性,如通过 DLS 分析确定的。有可能冻干 FpFs 以用于制造固态仓库。

更新日期:2020-11-22
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