Separation and quantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography.

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 micrograms 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.