Size exclusion chromatography (SEC) is a standard technique for analysing monoclonal antibodies (MAbs) such as Bevacizumab (Avastin®).
It is also a standard separation mode used in quality control to obtain information about aggregation and/or fragmentation of the MAb. Detection by light scattering, e.g. multi-angle laser light scattering (MALLS), can provide additional information, as the signal intensity is also influenced by the molar mass in contrast to usual concentration detectors such as UV or RI.
In this application note YMC’s dedicated SEC column for antibodies, YMC-SEC MAB, has been used to develop a method for Bevacizumab using MALLS detection. Four different buffers, a phosphate buffered saline (PBS; containing 0.138 M NaCl, 0.027 M KCl) pH 7.4 and phosphate buffers pH 6.6 (Sigma-Aldrich, 0.034 M) with varying concentrations of NaCl (0.1, 0.3 and 0.5 M), were used. A defined minimum ionic strength is necessary to achieve a robust method with good resolution, which is not fulfilled by the use of phosphate buffer with 0.1 M NaCl.
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