基于MS的工作流程用于rAAV体蛋白的PTM分析,以支持生物过程监测和开发
Tomás M Mateus1, Sofia B Carvalho1, Bruno M Alexandre1
1iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Journal of biotechnology
|March 14, 2026
概括
我们开发了一种质谱工作流程,用于对复合腺相关病毒 (rAAV) 囊体的翻译后修饰 (PTM) 进行分析. 这种方法精确地监测净化和正在加工的rAAV中的PTM,帮助基因治疗的发展.
科学领域:
- 生物化学和分子生物学
- 基因治疗制造业 基因治疗制造业
- 分析化学 分析化学
背景情况:
- 在复合腺相关病毒 (rAAV) 体上,转化后修饰 (PTM) 影响载体结构,稳定性和功能.
- 对rAAV体蛋白质的全面PTM分析,特别是在工艺中间体中,提出了重大分析挑战.
研究的目的:
- 开发和验证基于质谱学的强大的工作流程,用于对rAAV体蛋白的PTM分析.
- 应用此工作流来分析不同阶段和血清型的净化和工艺中的rAAV样本.
- 评估该方法在应力条件和工艺变化下检测PTM变化的灵敏度.
主要方法:
- 为RAAV分析开发一个优化的样本准备协议.
- 基于质谱的基映射用于PTM识别和量化.
- 使用PTM的无标签,特定地点的相对量化.
- 在精制的rAAV8,工艺中的rAAV8和rAAV5样本上测试工作流程,包括压力研究.
主要成果:
- 获得了rAAV8的>80%病毒蛋白 (VP) 序列覆盖率,量化了N端乙化,脱化,氧化和酸化.
- 在无标签的PTM量化中证明了高的可重复性和可重复性 (内部测试5.5%,内部测试25.1%).
- 成功地将工作流应用于过程中的样本,揭示了动态PTM变化,并证实了rAAV血清型 (rAAV8和rAAV5) 的稳定性.
结论:
- 开发的分析平台为净化和加工中的RAAV样本提供可靠的PTM监测.
- 这种多功能方法支持在rAAV产品表征和基因治疗过程开发中的关键应用.
- 优化样品制备和消化对于准确的PTM分析至关重要,尽量减少人工修改.
相关概念视频
01:33Peptide Identification Using Tandem Mass Spectrometry
Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...
01:33Proteomics
A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term proteomics...
Proteomics is the study of proteomes' function. It involves the large-scale systematic study of the proteome to denote the protein complement expressed by a genome. Scientist Mark Wilkins coined the term proteomics...