The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits.

This prototype metal-free SEC column was systematically compared with a conventional stainless-steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples.

It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were significantly improved for the different biopharmaceutical products on the metal-free SEC column. It has also been demonstrated that the largest differences between standard and metal-free SEC columns were observed for the most basic mAbs (high pI), which confirms that electrostatic interactions between the mAb and the metallic parts of the column (frits and inlet tube) could be responsible for the issues observed when performing SEC analysis with volatile mobile phase.

Finally, it was feasible to perform SEC-MS analysis for a wide range of biopharmaceutical products using volatile mobile phase. Our results also highlight that an inappropriate column could bias the quantification of size variants when using MS-compatible mobile phases. Therefore, metal-free column, such as the PEEK-lined column, should be preferentially selected for SEC-MS analysis.

该研究的目标是评估新一代无金属 SEC 色谱柱提供的可能性，以使用乙酸铵作为主要流动相添加剂对蛋白质生物药物进行直接 SEC-MS。在这项工作中使用的原型无金属 SEC 柱硬件是聚醚醚酮 (PEEK) 灌注不锈钢管，包括 PEEK 筛板。这种 PEEK 内衬色谱柱提供了完全生物惰性且不含金属的流体路径，同时保持了金属硬件的稳定性，并且可能是限制蛋白质与色谱柱壁/筛板之间可能发生的不良相互作用的良好解决方案。

将此原型无金属 SEC 柱与填充相同固定相材料的传统不锈钢 SEC 柱硬件进行了系统比较。四种不同的 mAb 产品，即曲妥珠单抗、帕利珠单抗、贝伐单抗和 NISTmAb，以及一种抗体药物偶联物 (ADC)，曲妥珠单抗 emtansine，被选为测试样品。

看来，在无金属 SEC 柱上，不同生物制药产品的峰对称性、低分子量物质 (LMWS) 的分离和高分子量物质 (HMWS) 的回收率都得到了显着改善。还已经证明，对于最基本的 mAb（高 p I），观察到标准和无金属 SEC 色谱柱之间的最大差异，这证实了 mAb 与色谱柱金属部分（筛板和入口管）之间的静电相互作用) 可能是使用挥发性流动相进行 SEC 分析时观察到的问题的原因。

最后，使用挥发性流动相对各种生物制药产品进行 SEC-MS 分析是可行的。我们的结果还强调，当使用兼容 MS 的流动相时，不合适的色谱柱可能会影响尺寸变异的量化。因此，应优先选择无金属色谱柱，例如 PEEK 内衬色谱柱进行 SEC-MS 分析。