Giovannini R, Freitag R
Center of Biotechnology, Department of Chemistry, Swiss Federal Institute of Technology, 1015 Ecublens, Switzerland.
Biotechnol Bioeng. 2002 Feb 15;77(4):445-54. doi: 10.1002/bit.10149.
Continuous chromatographic separations, especially of multicomponent mixtures, constitute interesting options for biotechnological downstream processing. Taking the separation of plasmid DNA from clearified lysates on hydroxyapatite as a pertinent example, we discuss the potential of continuous annular chromatography (CAC) in comparison with conventional (preparative) batch chromatography. In CAC the column is realized in the form of a thin (5 mm, height 210 mm) slowly rotating annulus. The performance of such a CAC column is compared to that of an ("analytical") batch column of similar thickness (diameter) and length (4 x 250 mm) and that of a ("preparative") batch column of similar cross-sectional surface area and height (50 x 210 mm). The quality of the obtained plasmid as defined by the appearance of the corresponding agarose gels (native and linearized plasmid), the 260/280 ratio and the biological activity (transient transfection of HEK 293 cells) was found to be identical in all three cases. The yields are also shown to be equivalent. The loading factor is found to be the most decisive parameter for the transfer of a given separation method between the continuous and the batch columns. Under nonoptimized conditions, plate numbers tended to be lower in the continuous compared to the batch columns. This is shown to be largely due to an artifact created by the CAC design (collection of averaged fractions at the outlets) and can be overcome by optimizing the rotation speed. Surprisingly the large batch column consistently gave better plate numbers than either the small batch or the CAC column. Compared to the preparative batch column, wall effects are more pronounced in the CAC (respectively the small diameter batch column), which may translate into better bed stability but conceivably also contributes to an increase in plate height, due to the reduction in bed density usually observed in the proximity of the wall. The CAC is shown to be a powerful approach to continuous chromatography, which allows a direct and straightforward upscale of chromatographic bioseparation methods.
连续色谱分离，尤其是多组分混合物的分离，是生物技术下游加工中很有吸引力的选择。以在羟基磷灰石上从澄清裂解物中分离质粒DNA为例，我们将连续环形色谱法（CAC）与传统（制备性）间歇色谱法进行了比较，探讨了连续环形色谱法的潜力。在连续环形色谱法中，色谱柱以薄（5毫米，高210毫米）且缓慢旋转的环形形式实现。将这种连续环形色谱柱的性能与类似厚度（直径）和长度（4×250毫米）的“分析性”间歇色谱柱以及具有相似横截面积和高度（50×210毫米）的“制备性”间歇色谱柱的性能进行了比较。通过相应琼脂糖凝胶（天然和线性化质粒）的外观、260/280比值以及生物活性（HEK 293细胞的瞬时转染）来定义所获得质粒的质量，结果发现这三种情况下所获质粒质量相同。产率也显示是相当的。发现负载因子是给定分离方法在连续色谱柱和间歇色谱柱之间转换时最具决定性的参数。在未优化的条件下，连续色谱柱的塔板数往往比间歇色谱柱的低。结果表明，这在很大程度上是由连续环形色谱法的设计造成的假象（在出口处收集平均馏分），可以通过优化转速来克服。令人惊讶的是，大型间歇色谱柱的塔板数始终比小型间歇色谱柱或连续环形色谱柱的要好。与制备性间歇色谱柱相比，连续环形色谱柱（以及小直径间歇色谱柱）中的壁效应更为明显，这可能转化为更好的床稳定性，但也可能由于通常在壁附近观察到的床密度降低而导致塔板高度增加。连续环形色谱法被证明是一种强大的连续色谱方法，它能够直接且简便地扩大色谱生物分离方法的规模。
