McCarthy E, Vella G, Mhatre R, Lim Y P
PerSeptive Biosystems, Inc., Framingham, MA 01701, USA.
J Chromatogr A. 1996 Aug 30;743(1):163-70. doi: 10.1016/0021-9673(96)00358-5.
A purification and on-line monitoring procedure for IgM was developed. Perfusion ion-exchange chromatography was used for rapid purification of IgM from ascites fluid and hybridoma supernatant. Crude ascites was directly loaded onto an ion exchanger. Due to the complexity of IgM, a two-step ion-exchange procedure had to be developed. This procedure involved a rapid cation-exchange chromatography capture step followed by further purification using anion-exchange chromatography. High linear velocities, in excess of 3500 cm/h, enabled separations to be performed under 5 min. Purity of the final product by SDS-PAGE was shown to be greater than 95%. Furthermore, the antibodies retained biological activity as measured by indirect immunofluorescence (IIF) and ELISA. The IgM peak was also monitored on-line using a novel peak tracking approach. This involved placing an antibody column (specific to the IgM) prior to the ion-exchange column and operating the ion-exchange column with and without the antibody column in-line. The missing peak that is identified by comparing the two chromatograms indicates where the IgM elutes.
开发了一种IgM的纯化及在线监测程序。采用灌注离子交换色谱法从腹水和杂交瘤上清液中快速纯化IgM。粗腹水直接加载到离子交换剂上。由于IgM的复杂性，必须开发两步离子交换程序。该程序包括快速阳离子交换色谱捕获步骤，随后使用阴离子交换色谱进一步纯化。超过3500 cm/h的高线速度使得分离可在5分钟内完成。SDS-PAGE显示最终产物的纯度大于95%。此外，通过间接免疫荧光法（IIF）和酶联免疫吸附测定（ELISA）测定，抗体保留了生物活性。还使用一种新颖的峰跟踪方法对IgM峰进行在线监测。这包括在离子交换柱之前放置一个（针对IgM的）抗体柱，并分别在有和没有抗体柱在线的情况下操作离子交换柱。通过比较两张色谱图识别出的缺失峰表明IgM的洗脱位置。
