Cao Mingyan, Mo Wenjun David, Shannon Anthony, Wei Ziping, Washabaugh Michael, Cash Patricia
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD.
Analytical Biotechnology Department, MedImmune Inc, Gaithersburg, MD
PDA J Pharm Sci Technol. 2016;70(6):490-507. doi: 10.5731/pdajpst.2015.006239. Epub 2016 Apr 18.
Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing.
Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality.
天冬氨酸（Asp）异构化是重组治疗性蛋白质常见的翻译后修饰，可发生在生产、储存或给药过程中。单克隆抗体互补决定区中的Asp异构化可能会影响靶点结合，因此需要一种足够稳健的常规监测质量控制方法。在这项工作中，我们采用基于液相色谱 - 质谱（LC/MS）的方法来鉴定治疗性单克隆抗体互补决定区中的Asp异构化。为了定量单克隆抗体位点特异性的Asp异构化，开发了一种利用相同LC平台基于紫外检测的定量分析方法。该分析方法经过验证并用于该产品特异性修饰的常规监测。与现有方法相比，这种分析模式适用于鉴定Asp异构化（或其他修饰），随后开发一种快速、足够稳健的质量控制方法，用于常规位点特异性监测和定量，以确保产品质量。这种方法首先识别并定位由异构化、脱酰胺、氧化或其他翻译后修饰引起的与产品相关的杂质（关键质量属性），然后利用合成肽和质谱辅助开发基于LC - UV的色谱方法，通过紫外峰分离和定量与产品相关的杂质。所建立的LC - UV方法具有可接受的峰特异性、精密度、线性和准确度；它可以经过验证并用于良好生产规范环境中的批次放行和稳定性测试。
天冬氨酸异构化是重组蛋白在生产过程和储存期间常见的翻译后修饰。已检测到单克隆抗体A（mAb - A）互补决定区（CDR）中的异构化，并且已证明其对抗原结合亲和力有影响。在这项工作中，我们采用基于质谱的肽图谱分析方法来检测和定量mAb - A的CDR中的Asp异构化。为了常规监测mAb - A的CDR异构化，开发并验证了一种利用反相色谱分离和紫外检测的聚焦肽图谱分析方法。这种方法通常适用于以特定且高通量的模式监测蛋白质的异构化和其他翻译后修饰，以确保产品质量。
