Monoclonal antibodies (mAbs) are a major class of biopharmaceuticals. It is hypothesized that some concentrated mAb solutions exhibit formation of a solution phase consisting of reversibly self-associated aggregates (or reversible clusters), which is speculated to be responsible for their distinct solution properties. Here, we report direct observation of reversible clusters in concentrated solutions of mAbs using neutron spin echo. Specifically, a stable mAb solution is studied across a transition from dispersed monomers in dilute solution to clustered states at more concentrated conditions, where clusters of a preferred size are observed. Once mAb clusters have formed, their size, in contrast to that observed in typical globular protein solutions, is observed to remain nearly constant over a wide range of concentrations. Our results not only conclusively establish a clear relationship between the undesirable high viscosity of some mAb solutions and the formation of reversible clusters with extended open structures, but also directly observe self-assembled mAb protein clusters of preferred small finite size similar to that in micelle formation that dominate the properties of concentrated mAb solutions.
Recently, reversible cluster formation was identified as an underlying cause of anomalously large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations, which poses a major challenge to the use of subcutaneous injection for some mAbs. A fundamental understanding of the structural and dynamic origins of high viscosities in concentrated mAb solutions is thus of significant relevance to mAb applications in human health care, as well as being of scientific interest. Herein, we present a detailed investigation of an IgG1-based mAb to relate the short-time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. The combination of light scattering, small-angle neutron scattering, and neutron spin echo measurement techniques conclusively demonstrates that, upon addition of Na2SO4, these antibodies form strongly bound reversible dimers at dilute concentrations that interact with each other to form large, loosely bound, transient clusters when concentrated. This hierarchical structure formation in solution causes a significant increase in the solution viscosity.
Attractive protein–protein interactions (PPI) in concentrated monoclonal antibody (mAb) solutions may lead to reversible oligomers (clusters) that impact colloidal stability and viscosity. Herein, the PPI are tuned for two mAbs via the addition of arginine (Arg), NaCl, or ZnSO4 as characterized by the structure factor (S eff(q)) with small-angle X-ray scattering (SAXS). The SAXS data are fit with molecular dynamics simulations by placing a physically relevant short-range attractive interaction on selected beads in coarse-grained 12-bead models of the mAb shape. The optimized 12-bead models are then used to differentiate key microstructural properties, including center of mass radial distribution functions (g COM(r)), coordination numbers, and cluster size distributions (CSD). The addition of cosolutes results in more attractive S eff(q) relative to the no cosolute control for all systems tested, with the most attractive systems showing an upturn at low q. Only the All1 model with an attractive site in each Fab and Fc region (possessing Fab–Fab, Fab–Fc, and Fc–Fc interactions) can reproduce this upturn, and the corresponding CSDs show the presence of larger clusters compared to the control. In general, for models with similar net attractions, i.e., second osmotic virial coefficients, the size of the clusters increases as the attraction is concentrated on a smaller number of evenly distributed beads. The cluster size distributions from simulations are used to improve the understanding and prediction of experimental viscosities. The ability to discriminate between models with bead interactions at particular Fab and Fc bead sites from SAXS simulations, and to provide real-space properties (CSD and g COM(r)), will be of interest in engineering protein sequence and formulating protein solutions for weak PPI to minimize aggregation and viscosities.
Colloidal liquids interacting with short range attraction and long range repulsion, such as proposed for some protein solutions, have been found to exhibit novel states consisting of equilibrium particle clusters. Monte Carlo simulations are performed for two physically meaningful inter-particle potentials across a broad range of interaction parameters, temperatures and volume fractions to locate the conditions where clustered states are found. A corresponding states phase behavior is identified when normalized by the critical point of an appropriately selected reference attractive fluid. Clustered fluid states and cluster percolated states are found exclusively within the two phase region of the state diagram for a reference attractive fluid, confirming the underlying intrinsic relation between clustered states and bulk phase separation. Clustered and cluster percolated states consistently exhibit an intermediate range order peak in their structure factors with a magnitude above 2.7, leading to a semi-empirical rule for identifying clustered fluids in scattering experiments.
The ability to design and formulate mAbs to minimize attractive interactions at high concentrations is important for protein processing, stability, and administration, particularly in subcutaneous delivery, where high viscosities are often challenging. The strength of protein−protein interactions (PPIs) of an IgG1 and IgG4 monoclonal antibody (mAb) from low to high concentration was determined by static light scattering (SLS) and used to understand viscosity data. The PPI were tuned using NaCl and five organic ionic co-solutes. The PPI strength was quantified by the normalized structure factor S(0)/S(0) HS and Kirkwood−Buff integral G 22 /G 22,HS (HS = hard sphere) determined from the SLS data and also by fits with (1) a spherical Yukawa potential and (2) an interacting hard sphere (IHS) model, which describes attraction in terms of hypothetical oligomers. The IHS model was better able to capture the scattering behavior of the more strongly interacting systems (mAb and/or co-solute) than the spherical Yukawa potential. For each descriptor of PPI, linear correlations were obtained between the viscosity at high concentration (200 mg/mL) and the interaction strengths evaluated both at low (20 mg/mL) and high concentrations (200 mg/mL) for a given mAb. However, the only parameter that provided a correlation across both mAbs was the oligomer mass ratio (m oligomer /m monomer+dimer ) from the IHS model, indicating the importance of self-association (in addition to the direct influence of the attractive PPI) on the viscosity.
Colloidal dispersions with a short-range attraction and long-range repulsion can exhibit an intriguing intermediate range order, manifested in scattering experiments as a low-q peak in the structure factor. Monte Carlo simulations are performed on fluids that exhibit intermediate range order to explicitly determine its connection to a possible state of microphase separation, equilibrium clustering. This is accomplished by decomposing the structure factor into cluster-cluster, monomer-monomer, and cross-correlations that cannot be extracted from experimental scattering patterns. Our simulation results indicate that the intermediate range order arises from either monomeric or cluster species, depending on solution conditions, and reflects the presence of a preferred length scale that is not trivially related to the interparticle potential. Further, criteria are established to define monomer, cluster, and percolated states in these systems that facilitate further studies. Combining scattering techniques with simulations provides an effective method for identifying clustered states in complex fluids.
Although roughly 40% of pharmaceuticals being developed are poorly water soluble, this class of drugs lacks a formulation strategy capable of producing high loads, fast dissolution kinetics, and low energy input. In this work, a novel bottom-up approach is developed for producing and formulating nanocrystals of poorly water-soluble active pharmaceutical ingredients (APIs) using core-shell composite hydrogel beads. Organic phase nanoemulsion droplets stabilized by polyvinyl alcohol (PVA) and containing a model hydrophobic API (fenofibrate) are embedded in the alginate hydrogel matrix and subsequently act as crystallization reactors. Controlled evaporation of this composite material produces core-shell structured alginate-PVA hydrogels with drug nanocrystals (500-650 nm) embedded within the core. Adjustable loading of API nanocrystals up to 83% by weight is achieved with dissolution (of 80% of the drug) occurring in as little as 30 min. A quantitative model is also developed and experimentally validated that the drug release patterns of the fenofibrate nanocrystals can be modulated by controlling the thickness of the PVA shell and drug loading. Thus, these composite materials offer a "designer" drug delivery system. Overall, our approach enables a novel means of simultaneous controlled crystallization and formulation of hydrophobic drugs that circumvents energy intensive top-down processes in traditional manufacturing.
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