Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization, University of Salzburg, Hellbrunnerstrasse 34, 5020, Salzburg, Austria.
Department of Biosciences, University of Salzburg, Billrothstrasse 11, 5020, Salzburg, Austria.
J Biomol NMR. 2021 Jan;75(1):71-82. doi: 10.1007/s10858-020-00356-4. Epub 2021 Jan 21.
The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pK values of isoAsp and C-terminal Asp in short peptides. The characteristic H-C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4-5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.
非酶促翻译后修饰（PTMs）的监测对于确保药物安全性和疗效至关重要。与蛋氨酸和天冬酰胺一样，天冬氨酸（Asp）对自发变化非常敏感。特别是，Asp 残基可以发生异构化和肽键水解，尤其是当嵌入序列基序中时，这些基序容易形成琥珀酰亚胺，或者当 Asp 残基后面跟着脯氨酸（Pro）时。由于 Asp 和异Asp 具有相同的质量，并且 Asp-Pro 肽键的断裂可能导致在天然条件下或在形成二硫键的断裂产物的情况下，+18 Da 的非特异性质量差异，因此很难通过质谱（MS）直接检测和表征这种修饰。在这里，我们提出了一种基于 2D NMR 的方法，用于明确鉴定异 Asp 和 Asp-Pro 肽键断裂的产物，即 N 端 Pro 和 C 端 Asp，并证明其在包括治疗性单克隆抗体（mAb）在内的蛋白质中的适用性。为了选择 NMR 信号的理想 pH 条件，使异 Asp 和 C 端 Asp 的 NMR 信号与其他随机卷曲信号区分开来，我们确定了短肽中异 Asp 和 C 端 Asp 的 pK 值。在标准条件下，异 Asp、N 端 Pro 和 C 端 Asp 的特征 H-C 化学位移相关用于在酸性条件（pH 4-5）和 40°C 下长期储存后鉴定溶菌酶和治疗性 mAb 利妥昔单抗（MabThera）中的这些 PTMs。结果表明，我们的基于 2D NMR 的方案的应用非常简单，可以检测到其他分析方法可能无法察觉的蛋白质化学变化。
