Purposes

The main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon.

Methods

A series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques.

Results

The formation of the impurity as observed in CE-SDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LC-MS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS.

Conclusions

Our study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.

中文翻译：

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CE-SDS分析重组蛋白过程中史无前例的杂质形成

目的

本文的主要目的是描述一种前所未有的现象，其中在重组融合蛋白X的常规非还原毛细管电泳-十二烷基硫酸钠（CE-SDS）分析中观察到大量的肩峰杂质，并进行评估造成这种现象的根本原因。

方法

进行了一系列实验以研究这种降解的性质。使用多种表征技术评估了碘乙酰胺（IAM），加热温度，持续时间和SDS对这种特定杂质形成的影响。

结果

CE-SDS中观察到的杂质形成实际上是由于赖氨酸和丝氨酸残基被IAM烷基化所致，如肽图分析和LC-MS / MS所证实的那样，这增​​加了分子量，因此降低了电泳迁移率。杂质的数量也强烈取决于样品制备条件，包括是否存在SDS。

结论

我们的研究清楚地表明，即使IAM在单克隆抗体和其他蛋白质的传统非还原CE-SDS分析中已被广泛用作烷基化试剂，但用IAM烷基化可能会导致其他杂质峰，从而使分析变得复杂。因此，在执行CE-SDS和其他分析之前，必须评估样品制备程序对分析结果的影响。对于蛋白质X，应排除IAM用于CE-SDS分析。