SEPARATION AND QUANTITATION OF MONOCLONAL-ANTIBODY AGGREGATES BY ASYMMETRICAL FLOW FIELD-FLOW FRACTIONATION AND COMPARISON TO GEL-PERMEATION CHROMATOGRAPHY
SEPARATION AND QUANTITATION OF MONOCLONAL-ANTIBODY AGGREGATES BY ASYMMETRICAL FLOW FIELD-FLOW FRACTIONATION AND COMPARISON TO GEL-PERMEATION CHROMATOGRAPHY
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DOI：
10.1006/abio.1993.1356
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发表时间：
1993-08-01
影响因子：
2.9
通讯作者：
WAHLUND, KG
中科院分区：
文献类型：
--
作者：
LITZEN, A;WALTER, JK;WAHLUND, KG
在单抗的下游处理过程中，可能会形成二聚体和/或寡聚体。目前，测定土壤团聚体含量最常用的方法是凝胶渗透色谱(GPC)，但其分辨率和分离速度有限。低聚物尤其难以分解。非对称流场-流动分馏是一种分离单抗聚集体的新方法。单体和二聚体的峰被很好地分辨出来，另外三个峰被认为是三聚体、四聚体和五聚体。分离的总时间可以保持在最短6分钟。凝胶渗透色谱分离时间约为15min，分辨率较低。对这两种技术的重复性研究表明，GPC的精密度略好于GPC。对样品浓度的依赖关系研究表明，单抗的载量极限约为4μg。GPC技术需要预过滤的样品，以避免柱堵塞。对于FFF，可以在不进行任何前处理的情况下注射含有沉淀物质的样品。可调整流动条件，使沉淀物随峰淋洗，与单体峰分离良好。
During the downstream processing of monoclonal antibodies (Mab) the formation of dimers and/or oligomers may occur. Today, the most common technique for determination of aggregate content is gel permeation chromatography (GPC) but it has limited resolution and separation speed. Oligomers are particularly difficult to resolve. Asymmetrical flow field-flow fractionation (FFF) is a new analytical method for the separation of Mab aggregates. The monomer and dimer peaks were well resolved and three additional peaks, thought to be trimers, tetramers, and pentamers were partially resolved. The total time for a separation can be kept as short as 6 min. The GPC separation takes about 15 min with less resolution. A study of the reproducibility for the two techniques revealed that the precision was slightly better for GPC. Investigation of the dependence of sample concentration showed that the load limit for FFF was about 4 μg of Mab. The GPC technique requires prefiltered samples in order to avoid clogging of the column. For FFF it is possible to inject samples containing precipitated material without any pretreatment. The flow conditions can be adjusted so that the precipitated material elutes with the front, well separated from the monomer peak.