1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. [2] Netherlands Proteomics Centre, Utrecht, The Netherlands. [3].
1] Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands. [2] Netherlands Proteomics Centre, Utrecht, The Netherlands.
Nat Protoc. 2014 Apr;9(4):967-76. doi: 10.1038/nprot.2014.057. Epub 2014 Mar 27.
The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation.
生物制药的分子复杂性对其全面结构特征所需的生物分析技术提出了严格的要求。质谱（MS）在生物制药分析中变得越来越重要，采用了从基于肽的测序到直接分析完整蛋白质和蛋白质组装等不同的互补方法。在本方案中，我们描述了优化的程序，用于在伪天然条件下使用天然 MS 对完整蛋白质水平的单克隆抗体（mAb）进行分析。天然 MS 在生物制药分析中的一些优势在于其分析速度、灵敏度和特异性：对于大多数实验，整个方案需要一个工作日的时间，其中可以以多路复用的方式分析数十个样本，使其适合高通量分析。该方法可用于不同的应用，例如 mAb 混合物、抗体药物偶联物的分析以及 mAb 翻译后修饰的分析，包括 mAb 糖基化的定性和定量分析。
