Journal of Proteomics ( IF 2.8 ) Pub Date : 2020-11-22 , DOI: 10.1016/j.jprot.2020.104053 Gauri Pradhan 1 , J M Sneha 1 , Babasaheb P Sonwane 2 , B Santhakumari 2 , Alka Rao 3 , Mahesh J Kulkarni 2
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Therapeutic monoclonal antibodies (mAbs) are structurally large and complex molecules. To be safe and efficacious, a biosimilar mAb must show high similarity to its reference product in Critical Quality Attributes (CQA). mAbs are highly sensitive to protein expression, production, manufacturing, supply chain, and storage conditions. All these factors make biosimilar mAbs intrinsically susceptible for variability during production. Accordingly, several lots of references and tests are required to establish the biosimilarity of a test mAb. The primary structure is a CQA of a mAb affecting its safety and efficacy. Here, we apply peptide mapping as an analytical method to decipher the primary structure and associated modifications for a quick quality assessment of TrastuzumAb and RituximAb innovator and biosimilar. A multiple-parallel-protease digestion strategy followed by high-resolution mass spectrometric analysis consistently achieved 100% sequence coverage along with reliable detection of post-translational modifications. Additionally, the use of supporting methods such as intact mass analysis and circular dichroism helped us to decipher the primary and higher order structures of these mAbs. We identify discernible variations in the profile of the innovator and biosimilar mAbs and validate the method for quick yet deep comparability analysis of the primary structure of biosimilar mAbs sold in the market.
Significance
Peptide mapping using bottom-up approach is one of the most common methods for the characterization of therapeutic monoclonal antibodies. Herein, we describe a multi-parallel-protease digestion strategy using a combination of five different proteases followed by high-resolution mass spectrometric analysis with TrastuzumAb and RituximAb as an example. This resulted in a comprehensive identification of peptides with increased reliability and identification of different PTMs. Additional supporting orthogonal methods like intact mass and higher-order structure analysis helped evaluate broader conformational properties.
中文翻译：
多重平行蛋白酶消化与高分辨率质谱联用：治疗性单克隆抗体的全面肽图分析方法
治疗性单克隆抗体（mAb）是结构大而复杂的分子。为了安全有效，生物仿制单克隆抗体必须在关键质量属性（CQA）中显示出与参考产品的高度相似性。单克隆抗体对蛋白质表达，生产，制造，供应链和储存条件高度敏感。所有这些因素使生物仿制药单抗本质上易于在生产过程中发生变异。因此，需要大量参考和测试来建立测试mAb的生物相似性。主要结构是影响其安全性和功效的mAb的CQA。在这里，我们将肽图分析作为一种分析方法，以解读曲妥珠单抗和利妥昔单抗创新药和生物仿制药的快速质量评估所需的主要结构和相关修饰。多重平行蛋白酶消化策略和高分辨率质谱分析一致地实现了100％的序列覆盖率以及对翻译后修饰的可靠检测。此外，使用完整的质量分析和圆二色性等支持方法有助于我们破译这些mAb的一级和高级结构。我们确定了创新药和生物仿制药单克隆抗体的概况中可辨别的变化，并验证了用于对市场上出售的生物仿制药单克隆抗体的主要结构进行快速而深入的可比性分析的方法。完整质量分析和圆二色性等支持方法的使用帮助我们破译了这些mAb的一级和高级结构。我们确定了创新药和生物仿制药单克隆抗体的概况中可辨别的变化，并验证了用于对市场上出售的生物仿制药单克隆抗体的主要结构进行快速而深入的可比性分析的方法。完整质量分析和圆二色性等支持方法的使用帮助我们破译了这些mAb的一级和高级结构。我们确定了创新药和生物仿制药单克隆抗体的概况中可辨别的变化，并验证了用于对市场上出售的生物仿制药单克隆抗体的主要结构进行快速而深入的可比性分析的方法。
意义
使用自下而上的方法进行肽图分析是表征治疗性单克隆抗体的最常用方法之一。在这里，我们描述了使用五个不同蛋白酶的组合，然后以曲妥珠单抗和利妥昔单抗为例的高分辨率质谱分析的多重平行蛋白酶消化策略。这导致了对肽的全面鉴定，具有更高的可靠性，并且鉴定了不同的PTM。其他完整的质量分析和高阶结构分析等正交方法也有助于评估更广泛的构象性质。
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