2 research outputs found
Kinetic Analysis of the Multistep Aggregation Mechanism of Monoclonal Antibodies
We investigate by kinetic analysis
the aggregation mechanism of
two monoclonal antibodies belonging to the IgG1 and IgG2 subclass
under thermal stress. For each IgG, we apply a combination of size
exclusion chromatography and light scattering techniques to resolve
the time evolution of the monomer, dimer, and trimer concentrations,
as well as the average molecular weight and the average hydrodynamic
radius of the aggregate distribution. By combining the detailed experimental
characterization with a theoretical kinetic model based on population
balance equations, we extract relevant information on the contribution
of the individual elementary steps on the global aggregation process.
The analysis shows that the two molecules follow different aggregation
pathways under the same operating conditions. In particular, while
the monomer depletion of the IgG1 is found to be rate-limited by monomeric
conformational changes, bimolecular collision is identified as the
rate-limiting step in the IgG2 aggregation process. The measurement
of the microscopic rate constants by kinetic analysis allows the quantification
of the protein–protein interaction potentials expressed in
terms of the Fuchs stability ratio (<i>W</i>). It is found
that the antibody solutions exhibit large <i>W</i> values,
which are several orders of magnitude larger than the values computed
in the frame of the DLVO theory. This indicates that, besides net
electrostatic repulsion, additional effects delay the aggregation
kinetics of the antibody solutions with respect to diffusion-limited
conditions. These effects likely include the limited efficiency of
the collision events due to the presence of a limited number of specific
aggregation-prone patches on the heterogeneous protein surface, and
the contribution of additional repulsive non-DLVO forces to the protein–protein
interaction potential, such as hydration forces
Role of Cosolutes in the Aggregation Kinetics of Monoclonal Antibodies
We propose a general strategy based
on kinetic analysis to investigate
how cosolutes affect the aggregation behavior of therapeutic proteins.
We apply this approach to study the impact of NaCl and sorbitol on
the aggregation kinetics of two monoclonal antibodies, an IgG1 and
an IgG2. By using a combination of size exclusion chromatography and
light scattering techniques, we study the impact of the cosolutes
on the monomer depletion, as well as on the formation of dimers, trimers,
and larger aggregates. We analyze these macroscopic effects in the
frame of a kinetic model based on Smoluchowski’s population
balance equations modified to account for nucleation events. By comparing
experimental data with model simulations, we discriminate the effect
of cosolutes on the elementary steps which contribute to the global
aggregation process. In the case of the IgG1, it is found that NaCl
accelerates the kinetics of aggregation by promoting specifically
aggregation events, while sorbitol delays the kinetics of aggregation
by specifically inhibiting protein unfolding. In the case of the IgG2,
whose monomer depletion kinetics is limited by dimer formation, NaCl
and sorbitol are found respectively to accelerate and inhibit conformational
changes and aggregation events to the same extent