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論文掲載:MSD社、治療用モノクローナル抗体の相補性決定領域でのアスパラギン酸残基におけるスクシンイミド形成・蓄積の検出と制御

Pharmaceutical Research
January 23, 2023

本研究では、インタクトならびに還元LC-MS質量測定によって、スクシンイミドの蓄積が確認されました。Merckの研究者は、生データのデコンボリューションと非標的/標的ペプチドマッピング実験のデータ解析においてExpressionistを使用しました。

In this study, accumulation of succinimide was identified through intact and reduced LC-MS mass measurements. The researchers at Merck used Genedata Expressionist for the deconvolution of raw data and for data analysis of both untargeted and targeted peptide mapping experiments.

Rationale

Succinimide formation and isomerization alter the chemical and physical properties of aspartic acid residues in a protein. Modification of aspartic acid residues within complementarity-determining regions (CDRs) of therapeutic monoclonal antibodies (mAbs) can be particularly detrimental to the efficacy of the molecule. The goal of this study was to characterize the site of succinimide accumulation in the CDR of a therapeutic mAb and understand its effects on potency. Furthermore, we aimed to mitigate succinimide accumulation through changes in formulation.

Methods

Nonreducing peptide mapping was applied for disulfide bridges assignment. This study presents an ad hoc method in which applying a neutral pH in the presence of an alkylating agent allowed to mitigate the formation of artifacts such as reshuffled disulfide bridges and permitted the detection of free cysteine. Ultra-high-performance liquid chromatography–MS analysis was performed on a Waters quadrupole time-of-flight Xevo G2-XS mass spectrometer acquiring data in MSE mode. MS data were processed using Expressionist MS Refiner 13.5 (Genedata).

Results

Succinimide accumulation in Formulation A was accelerated when stored at elevated temperatures. A strong correlation between succinimide accumulation in the CDR, an increase in basic charge variants, and a decrease in potency was observed. Statistical modeling suggest that a combination of ion exchange chromatography and potency measurements can be used to predict succinimide levels in a given sample. Reformulation of the mAb to Formulation B mitigates succinimide accumulation even after extended storage at elevated temperatures.

Conclusions

Succinimide formation in the CDR of a therapeutic mAb can have a strong negative impact on potency of the molecule. We demonstrate that thorough characterization of the molecule by LC-MS, ion exchange chromatography, and potency measurements can facilitate changes in formulation that mitigate succinimide formation and the corresponding detrimental changes in potency.