What Is SEC Used For?
- Impurity testing (separation of monomer, dimer, aggregates)
- Molecular weight characterization
- Expression and binding studies
- Separation of reaction components and products (especially antibodies, fragments, and conjugates)
- Desalting and exchange of sample buffer
(e.g. coupled with ion exchange chromatography to allow the use of mass spectrometric detection)
- Collection of fractions under non-denaturing conditions
SEC is utilized at numerous stages throughout the production and release for biopharmaceutical products, primarily monitoring protein modifications (protein-drug conjugates, pegylated proteins etc.), aggregation, and quaternary structure. 5 SEC can be applied in different ways at each stage of the manufacturing process. 6
Monoclonal antibody (mAb) Manufacturing Process Development:
SEC can guide mAb cell-line selection. The data aids in selection of a cell-line which produces the lowest level of aggregates, or to discriminate between aggregate forms that may be more or less difficult to remove during purification. Furthermore, during cell-line development SEC can be used to ensure that the specific activity of the purified protein is not under- or over-reported.
Development of Purification Processes:
Protein A or G affinity purification is an effective technique for removing conditioned media impurities during mAb production. However, removal of aggregate impurities is often not achieved, and the elution conditions employed with affinity purification can potentially induce aggregate formation. 7 Typically subsequent polishing steps are required to remove aggregates; including, gel-filtration, ion exchange, hydrophobic interaction, or hydroxyapatite chromatography.
Some limitations of size exclusion chromatography include an inability to separate similar sized molecules, the need for adequate sample concentrations to provide meaningful data prior to SEC analysis as on-column analyte concentration is not possible, and the complication of the biopharmaceutical product eluting with excipients such as the non-ionic surfactant polysorbate 80. The limitations of concentration and co-elution can be addressed by the use of alternative UV absorbance wavelengths or fluorescence detection to provide greater specificity.