Population Balance Modeling of Antibodies Aggregation Kinetics

The aggregates morphology and the aggregation kinetics of a model monoclonal antibody under acidic conditions have been investigated. Growth occurs via irreversible cluster–cluster coagulation forming compact, fractal aggregates with fractal dimension of 2.6. We measured the time evolution of the average radius of gyration, ⟨<i>R</i>_<i>g</i>⟩, and the average hydrodynamic radius, ⟨<i>R</i>_<i>h</i>⟩, by in situ light scattering, and simulated the aggregation kinetics by a modified Smoluchowski‘s population balance equations. The analysis indicates that aggregation does not occur under diffusive control, and allows quantification of effective intermolecular interactions, expressed in terms of the Fuchs stability ratio (<i>W</i>). In particular, by introducing a dimensionless time weighed on <i>W</i>, the time evolutions of ⟨<i>R</i>_<i>h</i>⟩ measured under various operating conditions (temperature, pH, type and concentration of salt) collapse on a single master curve. The analysis applies also to data reported in the literature when growth by cluster–cluster coagulation dominates, showing a certain level of generality in the antibodies aggregation behavior. The quantification of the stability ratio gives important physical insights into the process, including the Arrhenius dependence of the aggregation rate constant and the relationship between monomer–monomer and cluster–cluster interactions. Particularly, it is found that the reactivity of non-native monomers is larger than that of non-native aggregates, likely due to the reduction of the number of available hydrophobic patches during aggregation

Density-Gradient-Free Microfluidic Centrifugation for Analytical and Preparative Separation of Nanoparticles

Sedimentation and centrifugation techniques are widely applied for the separation of biomolecules and colloids but require the presence of controlled density gradients for stable operation. Here we present an approach for separating nanoparticles in free solution without gradients. We use microfluidics to generate a convective flow perpendicular to the sedimentation direction. We show that the hydrodynamic Rayleigh–Taylor-like instability, which, in traditional methods, requires the presence of a density gradient, can be suppressed by the Poiseuille flow in the microchannel. We illustrate the power of this approach by demonstrating the separation of mixtures of particles on the nanometer scale, orders of magnitude smaller than the micrometer-sized objects separated by conventional inertial microfluidic approaches. This technique exhibits a series of favorable features including short analysis time, small sample volume, limited dilution of the analyte, limited interactions with surfaces as well as the possibility to tune easily the separation range by adjusting the geometry of the system. These features highlight the potential of gradient-free microfluidic centrifugation as an attractive route toward a broad range of nanoscale applications

Microfluidic Diffusion Analysis of the Size Distribution and Microrheological Properties of Antibody Solutions at High Concentrations

The size distribution and the rheological properties of dispersions of biological colloids are relevant quality attributes for a variety of industrial applications, including pharmaceutical, food, and cosmetic products. For instance, the biophysical properties of monoclonal antibodies and therapeutic proteins, which represent an important class of drugs in the pharmaceutical market, are important for their safety and efficacy. In this work, we apply a microfluidic diffusion platform to analyze protein sizes and interactions in high-concentration antibody solutions directly in the liquid state with minimal perturbation of the sample. We show that this method provides size distributions in a size range scaling from a few angstroms to hundreds of nanometers. The detection sensitivity of the technique is independent of the particle size, and the method provides number-average distributions, enabling the simultaneous detection of both monomeric species and soluble aggregates. We further show that the same platform can be applied to measure viscosity–scaling effects in crowded environments by probing the Brownian motion of several tracers with different sizes. Such tracers experience a shift from the microviscosity to the macroviscosity of the sample at a critical probe size that is equal to the characteristic dimension of the main components of the dispersions. The technique simultaneously provides quantitative measurement of the microrheological properties and the macroviscosity of the sample, as well as information on the characteristic size of the components of the solution. Overall, these methods represent attractive tools in the context of the analysis of sizes and interactions of proteins in both diluted and high-concentration solutions during development, manufacturing, and formulation

Contribution of Electrostatics in the Fibril Stability of a Model Ionic-Complementary Peptide

In this work we quantified the role of electrostatic interactions in the self-assembly of a model amphiphilic peptide (RADA 16-I) into fibrillar structures by a combination of size exclusion chromatography and molecular simulations. For the peptide under investigation, it is found that a net charge of +0.75 represents the ideal condition to promote the formation of regular amyloid fibrils. Lower net charges favor the formation of amorphous precipitates, while larger net charges destabilize the fibrillar aggregates and promote a reversible dissociation of monomers from the ends of the fibrils. By quantifying the dependence of the equilibrium constant of this reversible reaction on the pH value and the peptide net charge, we show that electrostatic interactions contribute largely to the free energy of fibril formation. The addition of both salt and a charged destabilizer (guanidinium hydrochloride) at moderate concentration (0.3–1 M) shifts the monomer-fibril equilibrium toward the fibrillar state. Whereas the first effect can be explained by charge screening of electrostatic repulsion only, the promotion of fibril formation in the presence of guanidinium hydrochloride is also attributed to modifications of the peptide conformation. The results of this work indicate that the global peptide net charge is a key property that correlates well with the fibril stability, although the peptide conformation and the surface charge distribution also contribute to the aggregation propensity

Quantification of the Concentration of Aβ42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay

The aggregation of the amyloid beta peptide, Aβ42, implicated in Alzheimer’s disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar Aβ42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase

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

Evaluation of exogenous human FTL mRNA in the indicated areas of the brain of 8 month-old Tg-mice.

<p>Solid circles for males and the empty ones for females. The horizontal lines are the means. Data obtained by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The transcript of the transgene was higher in males than females, and higher in the hippocampus > cortex > cerebellum > midbrain.</p

Ferritin and iron accumulation in the brain of Tg-mice.

<p>(A) Paraffin-embedded brain sections (40 <i>μ</i>m thick) of Tg and control mice were stained with a specific antibody for human L-ferritin chain. (B) Slices stained with DAB-enhanced Perl’s reaction showed a strong accumulation of ferritin/iron bodies in Tg-mouse brains. The quantitative evaluation of the percentage of brown staining in various fields showed the increase of the number and size of the granules with aging. At the optical microscope these granules seemed to be present mostly in neurons, and in the center of the cells in a nuclear/paranuclear position. (C) Mice were treated for 3 weeks with oral iron chelator Deferiprone (DFR) 1 mg/ml in drinking water ad libitum to reduce iron burden. The treatment reduced inclusion bodies in the brain both in number and in size.</p

Assessment of spontaneous locomotor activity 24 h following administration of PQ+MB in old mice (21 month-old).

<p>After treatment Tg-mice showed increased locomotor (A, B) and exploratory activities (C, D), compared to wild type subjects of the same age. Results are presented as mean + S.E.M. * <i>P</i> < 0.05.</p