Prediction of IgG1 aggregation in solution

Abstract

Interest in monoclonal antibody aggregation is increasing as aggregates of biopharmaceuticals can cause an immunogenic response when injected into the body. In this work, a stoichiometric reaction model from concentration-time data is developed to predict the dimer ratio in stored antibody solutions over time. IgG1 was incubated at pH from 4.5 to 5.5, salt concentrations from 100 to 600 mmol/kg and protein concentrations of 10.6-26.3 g/L; samples were taken at intervals of 20 min to 5 h over time periods from 4 h to 7.6 days, and analyzed with size-exclusion chromatography. The experiments showed the formation of dimers from monomers, but no higher order aggregates. Dilution of samples containing dimers led to the reversal of the dimerization reaction. Measurements of the concentrations of each component were made by fitting exponentially modified Gaussian peaks to the chromatograms used to measure the concentrations of the different forms of protein. This stoichiometric reaction model was able to predict the formation of dimers by the antibody studied. The equilibrium constant was found to be dependent on the salt concentration, and the kinetic constant showed a dependence on the pH of the solution. The prediction of the aggregation leads to a possibility of optimizing the conditions in order to prevent the dimer formation and to maximize the monomer concentration.

Model-Based Comparison of Antibody Dimerization in Continuous and Batch-Wise Downstream Processing

“…A fairly small increase considering that the concentration of antibodies is very high during the elution. This may be due to the fact that dimerization is very slow at pH 5, and the equilibrium is shifted towards monomers at high salt concentrations [9], which are conditions in which the antibodies are eluted. The interaction of antibodies with the stationary phase promotes the formation of aggregates [23][24][25], but the mechanism is unknown.…”

“…The elution conditions, low pH and high salt concentration, may also promote the formation of aggregates [9].…”

“…The reaction model, developed by Ojala et al [9], is based on a reversible reaction in which monomeric antibodies (M ) form dimers (D) according to:…”

“…These models, however, do not include the dimerization that occurs during the separation steps. Guo et al [8] showed that dimerization can occur due to interaction with the stationary phase, while Ojala et al [9] showed that dimers of the antibodies are formed in the mobile phase. Arosio et al [10] expanded the Lumry-Eyring model [11] to include the effects of reversibility.…”

“…Hold-up times of up to a couple of hours are common between process steps in the downstream processing [13]. Dimerization is a time-dependent reversible process [9], and it is therefore desirable to know how these hold-up times influence the yield of the process.…”