School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva 4, Switzerland.
Anal Bioanal Chem. 2013 Apr;405(10):3137-51. doi: 10.1007/s00216-013-6759-7. Epub 2013 Jan 29.
Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ~250 Å and macropores of ~ 1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ~300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.
最近研究了各种宽孔径反相固定相，用于分析大小在 25 至 50 kDa 之间的完整单克隆抗体 (mAb) 及其片段。评估了不同类型的柱技术，即含有约 250 Å 中孔和 1.1 μm 大孔的原型硅胶基无机整体、填充有 3.6 µm 宽孔径核壳颗粒的柱，该颗粒具有平均孔径约 200 Å 的宽孔径分布和填充有全多孔 1.7 µm 颗粒的柱，其孔径约为 300 Å。将这些宽孔径材料的性能与具有约 1 µm 大孔尺寸但无中孔（停滞区）的大孔聚苯乙烯-二乙烯基苯有机整体柱的性能进行了比较。使用模型 IgG1 和 IgG2 mAb（利妥昔单抗、帕尼单抗和贝伐单抗）进行了系统研究。首先，比较了两种整体相中完整和还原 mAb 的回收率，结果表明，有机整体相中吸附程度较低，这可能是由于化学性质（C18 与苯基）和中孔缺失（停滞区）的差异所致。其次，在梯度洗脱模式下研究了所有柱子的动力学性能。为此，在各种流速下计算了每米的峰容量以及每时间单位和每压力单位的峰容量 (PPT)，以比较不同尺寸柱子的性能。就每米的峰容量而言，在 60°C 时，核壳 3.6 µm 和全多孔 1.7 µm 柱优于两种整体相。然而，当考虑 PPT 值时，核壳 3.6 µm 柱仍然是最佳相，而原型硅胶整体相变得非常有趣，主要是因为与有机整体相相比，其渗透性非常高。因此，这些核壳和硅胶整体相提供了最快的可实现分离。最后，在每个柱子的最大工作温度下，核壳 3.6 µm 柱远远优于其他柱子，因为它是唯一稳定在 90°C 以上的柱子。最后，还在这四种不同的相上测量了载量。由于缺乏中孔，有机整体相是最不有趣和最快过载的。另一方面，原型硅胶整体相的载量确实合理。
