Modeling Antibody Aggregation in Downstream Processing

Abstract: Antibodies are part of the immune system where their purpose is to recognize and neutralize foreign species such as viruses and bacteria, thus preventing and curing infections. The trend of using antibodies in the treatment of, for example, cancer is increasing. These antibodies are produced in bioreactors, but the production stream from the bioreactors also includes impurities such as viruses and host cell proteins. Antibody aggregates are also found in this stream, and can result in an immune response when they are injected into the body. A purification process is thus essential to ensure a safe product. It has been shown that aggregation is not confined to the bioreactors, but also occurs during the purification process. The work described in this thesis deals with the aggregation of antibodies in the downstream process, the influence of the buffer solution on aggregation, and how the purification process influences the degree of aggregation. A facilitated method of measuring aggregation over time has been developed, including incubation of antibodies in various solutions, analysis of the degree of aggregation using size-exclusion chromatography, and calculation of antibody concentrations utilizing Gaussian curves. The effect of pH and salt content on aggregation in the mobile phase of a chromatography step and in solution has been studied. The results showed that only dimers are formed within the studied time frame (up to 13 days) and that the aggregation is reversible. The dimer fraction increased with decreasing salt concentration, and the rate of dimerization increased with decreasing pH. Modeling of chromatographic separation has been produced for monomers and dimers. The diversity of this kind of modeling was demonstrated by successfully utilizing similar models to predict separation of rare earth elements. The model of size exclusion separation of monomers and dimers showed that, given sufficiently long retention times, the dilution occurring in the mobile phase causes the reversible aggregation to reform monomers from dimers. The batch-wise and continuous separation of monomers and dimers including dimer formation was modeled, and the change in dimer fraction occurring in the mobile phase during downstream processing was analyzed. The conclusion was that dimers are primarily formed during the virus inactivation step in the batch-wise process, when the pH and salt concentration are low. Use of continuous downstream processing, performing the virus inactivation in a size exclusion column instead of in a tank, greatly diminished the formation of dimers. This makes it a preferred process compared to batch-wise, from an aggregation point of view.