Zhang Yuan, Wang Ying, Li Yifeng
Technology and Process Development (TPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Technology and Process Development (TPD), WuXi Biologics, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai, 200131, China.
Protein Expr Purif. 2019 Dec;164:105459. doi: 10.1016/j.pep.2019.105459. Epub 2019 Jul 23.
When monoclonal antibodies (mAbs) are analysed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), method-induced artifacts are a frequent phenomenon. Previous studies suggested that incomplete denaturation and disulfide-bond scrambling are two main causes of artifact bands. Thus, in practice samples are normally heated and treated with alkylating agent iodoacetamide (IAM) before loading to promote denaturation and block free sulfhydryl groups, respectively. In this work, we further studied the major cause of artifact bands on non-reducing SDS-PAGE and ways of eliminating artifacts with two purified mAbs. In both cases, it was found that artifact bands on non-gradient Tris-glycine gels are mainly caused by incomplete denaturation under typical gel conditions. In general, heating minimizes artifact bands due to incomplete denaturation but it also generates some extra bands. Combining heating with IAM treatment achieved slightly better results than heating alone. As an alternative to heating, treating the samples with 8 M urea also allows close to complete denaturation of samples and thus minimizes artifact bands. In addition, we learned that untreated samples (samples that are not heated or treated with urea) may look different on Bis-Tris gel depending on gel composition (non-gradient vs. gradient) and the buffer used (MES vs. MOPS). In certain case, the apparent lack of artifact bands on gradient Bis-Tris gel may be in fact due to insufficient resolution. In conclusion, this study further confirmed that full-denaturation of sample is critical for minimizing/avoiding artifact bands on non-reducing SDS-PAGE.
当通过非还原十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分析单克隆抗体（mAb）时，方法诱导的假象是一种常见现象。先前的研究表明，不完全变性和二硫键重排是假象条带的两个主要原因。因此，在实际操作中，样品通常在加样前进行加热并用烷基化剂碘乙酰胺（IAM）处理，分别以促进变性和封闭游离巯基。在这项工作中，我们进一步研究了非还原SDS-PAGE上假象条带的主要原因以及用两种纯化的单克隆抗体消除假象的方法。在这两种情况下，发现非梯度Tris-甘氨酸凝胶上的假象条带主要是由典型凝胶条件下的不完全变性引起的。一般来说，加热可减少由于不完全变性产生的假象条带，但也会产生一些额外的条带。加热与IAM处理相结合比单独加热取得了稍好的结果。作为加热的替代方法，用8 M尿素处理样品也能使样品接近完全变性，从而使假象条带最少。此外，我们了解到未处理的样品（未加热或未用尿素处理的样品）在Bis-Tris凝胶上的外观可能因凝胶组成（非梯度与梯度）和所用缓冲液（MES与MOPS）而有所不同。在某些情况下，梯度Bis-Tris凝胶上明显没有假象条带实际上可能是由于分辨率不足。总之，本研究进一步证实样品的完全变性对于最小化/避免非还原SDS-PAGE上的假象条带至关重要。
