Department of Chemistry, University of Massachusetts Amherst, Amherst, MA, USA.
Analytical Development, Biogen, Cambridge, MA, USA.
Analyst. 2017 Jan 16;142(2):336-344. doi: 10.1039/c6an02041k.
Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.
详细分析蛋白质药物质量评估所需的酶促（例如糖基化）和非酶促（例如氧化和脱酰胺）翻译后修饰（PTMs）。对于所谓的第二代生物制药来说，这是一项具有挑战性的任务，因为它们还包含旨在增强其药代动力学特征（例如聚乙二醇化蛋白质）或赋予其治疗活性（例如蛋白质 - 药物偶联物）的“设计 PTM”。这些蛋白质共价结构的修饰可以显著增加结构异质性，使得“分子量”的概念变得毫无意义，因为代表聚乙二醇化蛋白质不同糖型的离子可能具有几乎相同的离子流分布作为 m/z 的函数，使得它们对质谱的贡献无法区分。在这项工作中，我们证明了离子交换色谱（IXC）与电喷雾电离质谱（ESI MS）在线检测以及气相中离子操纵方法（有限电荷还原和碰撞诱导解离）的组合可以对聚乙二醇化干扰素β-1a 的结构异质样品获得有意义的结构信息。蛋白质样品的 IXC 分析产生了一个复杂的色谱图，其中有几个部分解析的峰，这些峰可以代表蛋白质内的脱酰胺和不同的糖基化模式，以及聚乙二醇化的不同程度。因此，通过在线 IXC/ESI/MS/MS 对蛋白质进行分析，可以提供有关单个蛋白质治疗中三种不同类型的 PTM（设计、酶促和非酶促）的信息，从而对其进行表征。
