Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS
Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS
复制标题
DOI：
10.1039/c6an02041k
复制
发表时间：
2017-01-21
期刊：
影响因子：
4.2
通讯作者：
Kaltashov, I. A.
中科院分区：
文献类型：
--
作者：
Muneeruddin, K.;Bobst, C. E.;Kaltashov, I. A.
两种酶（例如，糖基化）和非酶（例如，氧化和脱酰胺）翻译后修饰（PTM）经常需要用于基于蛋白质的药物的质量评估。尽管如此，对于所谓的第二代生物药物来说，这一任务进一步复杂化，第二代生物药物还包含引入的“设计者PTM”，以增强其药代动力学特征（例如，PEG化蛋白质）或赋予它们治疗活性（例如，蛋白质-药物缀合物）。蛋白质共价结构的这种修饰可以显著增加结构异质性，使得“分子量”的概念毫无意义，因为代表PEG化蛋白质的不同糖型的离子可以具有几乎相同的离子电流分布作为m/z的函数，使得它们对质谱的贡献不可能区分。在这项工作中，我们证明了离子交换色谱（IXC）与在线检测电喷雾电离质谱（ESI MS）和离子操作的方法在气相中（有限的电荷减少和碰撞诱导解离）的组合允许有意义的结构信息，以获得结构异质性样品的聚乙二醇化干扰素β-1a。蛋白质样品的IXC分析产生了具有几个部分分离峰的卷积色谱图，这些峰可以代表蛋白质内的脱酰胺和不同糖基化模式以及不同程度的PEG化。因此，用在线IXC/ESI/MS/MS分析蛋白质允许通过提供关于单一蛋白质治疗剂内的三种不同类型的PTM（设计者、酶和非酶）的信息来表征蛋白质。
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 bio-pharmaceuticals, 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 beta-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.