Background

Therapeutic monoclonal antibodies (mAbs) have become essential biopharmaceuticals for clinical targeted therapies due to their high specificity, affinity and low side effects. The specificity and affinity of mAb to targeting antigen are mainly dependent on the three complementarity determining regions (CDRs) with high variations in amino acid sequences. Therefore, mAb CDR sequencing is crucial for the characterization of therapeutic mAbs. Here, we developed a 193-nm ultraviolet photodissociation (UVPD) based multiple charge integration middle-down mass spectrometry (MCI-MDMS) strategy for mAb sequencing.

Results

We demonstrate that the UVPD spectra of mAb subunit ions with different charge states exhibit high complementarity, and integration can result in higher sequence coverage compared to single charge states. Finally, over 95% sequence coverage of two different mAbs has been achieved with full sequence coverage of CDRs, underscoring the great potential of this strategy in accurate sequencing of mAb variable regions. Compared with the conventional higher energy collisional dissociation (HCD) strategy of mAb subunit sequencing, the sequence coverage of CDRs at single UVPD subunit charge state has increased by an average of 30%. In addition, almost complete sequence coverage of mAb ensures the accurate localization of mAb post-translational modifications (PTMs), including glycosylation of two different sites, C-terminal lysine truncation, and N-terminal cyclization of glutamine.

Significance

The integration of MCI-MDMS and UVPD realizes high sequence coverage and reliable PTM determination of mAbs. This integrated strategy holds significant promise for accurate analysis of antibody-drug conjugates, polyclonal antibodies and unknown mAbs including sequences and PTMs, and providing a crucial tool for the discovery and development of therapeutic mAbs.

中文翻译：

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使用多重电荷整合 Middle-Down 策略和紫外光解离对单克隆抗体可变区进行测序

 背景

治疗性单克隆抗体 （mAb） 因其高特异性、亲和力和低副作用而成为临床靶向治疗的重要生物制药。mAb 对靶向抗原的特异性和亲和力主要取决于氨基酸序列高度变化的三个互补决定区 （CDR）。因此，mAb CDR 测序对于治疗性 mAb 的表征至关重要。在这里，我们开发了一种基于 193 nm 紫外光解离 （UVPD） 的多电荷积分自中而下质谱 （MCI-MDMS） 策略，用于 mAb 测序。

 结果

我们证明，具有不同电荷态的 mAb 亚基离子的 UVPD 谱图表现出高度互补性，与单一电荷态相比，积分可以产生更高的序列覆盖率。最后，在 CDR 完全序列覆盖的情况下，两种不同 mAb 的序列覆盖率超过 95%，这突显了该策略在 mAb 可变区准确测序方面的巨大潜力。与单克隆抗体亚基测序的常规高能量碰撞解离 （HCD） 策略相比，单个 UVPD 亚基电荷状态下 CDR 的序列覆盖率平均提高了 30%。此外，mAb 几乎完全的序列覆盖确保了 mAb 翻译后修饰 （PTM） 的准确定位，包括两个不同位点的糖基化、C 端赖氨酸截断和谷氨酰胺的 N 端环化。

 意义

MCI-MDMS 和 UVPD 的集成实现了高序列覆盖率和可靠的 mAb PTM 测定。这种综合策略对于抗体-药物偶联物、多克隆抗体和未知 mAb（包括序列和 PTM）的准确分析具有重要前景，并为治疗性 mAb 的发现和开发提供了关键工具。