Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.

IF 3.5 3区医学 Q2 CHEMISTRY, MULTIDISCIPLINARY Pharmaceutical Research Pub Date : 2024-10-01 Epub Date: 2024-09-25 DOI:10.1007/s11095-024-03773-4

Meng Li, Weiyu Li, Xin Wang, Gang Wu, Jialiang Du, Gangling Xu, Maoqin Duan, Xiaojuan Yu, Chunbo Cui, Chunyu Liu, Zhihao Fu, Chuanfei Yu, Lan Wang

{"title":"Identification and Activity Study of an Impurity Band Observed in the nrSDS-PAGE of Aflibercept.","authors":"Meng Li, Weiyu Li, Xin Wang, Gang Wu, Jialiang Du, Gangling Xu, Maoqin Duan, Xiaojuan Yu, Chunbo Cui, Chunyu Liu, Zhihao Fu, Chuanfei Yu, Lan Wang","doi":"10.1007/s11095-024-03773-4","DOIUrl":null,"url":null,"abstract":"Background: Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required.Methods: In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity.Results: The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected.Conclusions: The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":"2031-2042"},"PeriodicalIF":3.5000,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Pharmaceutical Research","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s11095-024-03773-4","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/9/25 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}

引用次数: 0

Abstract

Background: Aflibercept is a biopharmaceutical targeting vascular endothelial growth factor (VEGF) that has shown promise in the treatment of neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DME) in adults. Quality control studies of aflibercept employing non-reduced SDS-PAGE (nrSDS-PAGE) have shown that a significant variant band (IM1) is consistently present below the main band. Considering the quality control strategy of biopharmaceuticals, structural elucidation and functional studies are required.

Methods: In this study, the variant bands in nrSDS-PAGE were collected through electroelution and identified by peptide mass fingerprinting based on liquid chromatography-tandem MS (LC-MS/MS). This variant was expressed using knob-into-hole (KIH) design transient transfection for the detection of ligand affinity, binding activity and biological activity.

Results: The variant band was formed by C-terminal truncation at position N99 of one chain in the aflibercept homodimer. Then, this variant was successfully expressed using KIH design transient transfection. The ligand affinity of the IM1 truncated variant was reduced by 18-fold, and neither binding activity nor biological activity were detected.

Conclusions: The efficacy of aflibercept is influenced by the loss of biological activity of the variant. Therefore, this study supports the development of a quality control strategy for aflibercept.

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

阿弗利贝赛在 nrSDS-PAGE 中观察到的杂质带的鉴定和活性研究

背景：阿弗利百普是一种靶向血管内皮生长因子（VEGF）的生物制药，在治疗成人新生血管性老年黄斑变性（nAMD）和糖尿病性黄斑水肿（DME）方面前景看好。采用非还原SDS-PAGE（nrSDS-PAGE）对阿弗利百普进行的质量控制研究表明，在主带下方始终存在一个重要的变异带（IM1）。考虑到生物制药的质量控制策略，需要进行结构阐明和功能研究：本研究通过电洗脱收集了 nrSDS-PAGE 中的变异条带，并通过基于液相色谱-串联质谱（LC-MS/MS）的肽段质量指纹图谱进行了鉴定。利用KIH设计瞬时转染法表达该变体，检测配体亲和力、结合活性和生物活性：结果：阿弗利贝赛普同源二聚体中一条链的N99位C端截短形成了变异带。然后，利用 KIH 设计瞬时转染成功表达了该变体。IM1截短变体的配体亲和力降低了18倍，且未检测到结合活性和生物活性：结论：阿弗利百普的疗效受变体生物活性丧失的影响。因此，本研究支持制定阿弗利百普的质量控制策略。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文去求助

来源期刊

Pharmaceutical Research 医学-化学综合

CiteScore

6.60

自引率

5.40%

发文量

276

审稿时长

3.4 months

期刊介绍： Pharmaceutical Research, an official journal of the American Association of Pharmaceutical Scientists, is committed to publishing novel research that is mechanism-based, hypothesis-driven and addresses significant issues in drug discovery, development and regulation. Current areas of interest include, but are not limited to: -(pre)formulation engineering and processing- computational biopharmaceutics- drug delivery and targeting- molecular biopharmaceutics and drug disposition (including cellular and molecular pharmacology)- pharmacokinetics, pharmacodynamics and pharmacogenetics. Research may involve nonclinical and clinical studies, and utilize both in vitro and in vivo approaches. Studies on small drug molecules, pharmaceutical solid materials (including biomaterials, polymers and nanoparticles) biotechnology products (including genes, peptides, proteins and vaccines), and genetically engineered cells are welcome.