Pharmaceutical Perspective on Opalescence and Liquid-Liquid Phase Separation in Protein Solutions.

Opalescence in protein solutions reduces aesthetic appeal of a formulation and can be an indicator of the presence of aggregates or precursor to phase separation in solution signifying reduced product stability. Liquid-liquid phase separation of a protein solution into a protein-rich and a protein-poor phase has been well-documented for globular proteins and recently observed for monoclonal antibody solutions, resulting in physical instability of the formulation. The present review discusses opalescence and liquid-liquid phase separation (LLPS) for therapeutic protein formulations. A brief discussion on theoretical concepts based on thermodynamics, kinetics, and light scattering is presented. This review also discusses theoretical concepts behind intense light scattering in the vicinity of the critical point termed as "critical opalescence". Both opalescence and LLPS are affected by the formulation factors including pH, ionic strength, protein concentration, temperature, and excipients. Literature reports for the effect of these formulation factors on attractive protein-protein interactions in solution as assessed by the second virial coefficient (B2) and the cloud-point temperature (Tcloud) measurements are also presented. The review also highlights pharmaceutical implications of LLPS in protein solutions.

Effect of Excipients on Liquid-Liquid Phase Separation and Aggregation in Dual Variable Domain Immunoglobulin Protein Solutions.

Liquid-liquid phase separation (LLPS) and aggregation can reduce the physical stability of therapeutic protein formulations. On undergoing LLPS, the protein-rich phase can promote aggregation during storage due to high concentration of the protein. Effect of different excipients on aggregation in protein solution is well documented; however data on the effect of excipients on LLPS is scarce in the literature. In this study, the effect of four excipients (PEG 400, Tween 80, sucrose, and hydroxypropyl beta-cyclodextrin (HPßCD)) on liquid-liquid phase separation and aggregation in a dual variable domain immunoglobulin protein solution was investigated. Sucrose suppressed both LLPS and aggregation, Tween 80 had no effect on either, and PEG 400 increased LLPS and aggregation. Attractive protein-protein interactions and liquid-liquid phase separation decreased with increasing concentration of HPßCD, indicating its specific binding to the protein. However, HPßCD had no effect on the formation of soluble aggregates and fragments in this study. LLPS and aggregation are highly temperature dependent; at low temperature protein exhibits LLPS, at high temperature protein exhibits aggregation, and at an intermediate temperature both phenomena occur simultaneously depending on the solution conditions.

Viscosity Analysis of Dual Variable Domain Immunoglobulin Protein Solutions: Role of Size, Electroviscous Effect and Protein-Protein Interactions.

PURPOSE: Increased solution viscosity results in difficulties in manufacturing and delivery of therapeutic protein formulations, increasing both the time and production costs, and leading to patient inconvenience. The solution viscosity is affected by the molecular properties of both the solute and the solvent. The purpose of this work was to investigate the effect of size, charge and protein-protein interactions on the viscosity of Dual Variable Domain Immunoglobulin (DVD-Ig(TM)) protein solutions. METHODS: The effect of size of the protein molecule on solution viscosity was investigated by measuring intrinsic viscosity and excluded volume calculations for monoclonal antibody (mAb) and DVD-Ig(TM) protein solutions. The role of the electrostatic charge resulting in electroviscous effects for DVD-Ig(TM) protein was assessed by measuring zeta potential. Light scattering measurements were performed to detect protein-protein interactions affecting solution viscosity. RESULTS: DVD-Ig(TM) protein exhibited significantly higher viscosity compared to mAb. Intrinsic viscosity and excluded volume calculations indicated that the size of the molecule affects viscosity significantly at higher concentrations, while the effect was minimal at intermediate concentrations. Electroviscous contribution to the viscosity of DVD-Ig(TM) protein varied depending on the presence or absence of ions in the solution. In buffered solutions, negative k D and B 2 values indicated the presence of attractive interactions which resulted in high viscosity for DVD-Ig(TM) protein at certain pH and ionic strength conditions. CONCLUSIONS: Results show that more than one factor contributes to the increased viscosity of DVD-Ig(TM) protein and interplay of these factors modulates the overall viscosity behavior of the solution, especially at higher concentrations.

Liquid-Liquid Phase Separation in a Dual Variable Domain Immunoglobulin Protein Solution: Effect of Formulation Factors and Protein-Protein Interactions.

Dual variable domain immunoglobulin proteins (DVD-Ig proteins) are large molecules (MW ∼ 200 kDa) with increased asymmetry because of their extended Y-like shape, which results in increased formulation challenges. Liquid-liquid phase separation (LLPS) of protein solutions into protein-rich and protein-poor phases reduces solution stability at intermediate concentrations and lower temperatures, and is a serious concern in formulation development as therapeutic proteins are generally stored at refrigerated conditions. In the current work, LLPS was studied for a DVD-Ig protein molecule as a function of solution conditions by measuring solution opalescence. LLPS of the protein was confirmed by equilibrium studies and by visually observing under microscope. The protein does not undergo any structural change after phase separation. Protein-protein interactions were measured by light scattering (kD) and Tcloud (temperature that marks the onset of phase separation). There is a good agreement between kD measured in dilute solution with Tcloud measured in the critical concentration range. Results indicate that the increased complexity of the molecule (with respect to size, shape, and charge distribution on the molecule) increases contribution of specific and nonspecific interactions in solution, which are affected by formulation factors, resulting in LLPS for DVD-Ig protein.

Opalescence in monoclonal antibody solutions and its correlation with intermolecular interactions in dilute and concentrated solutions.

Opalescence indicates physical instability of a formulation because of the presence of aggregates or liquid-liquid phase separation in solution and has been reported for monoclonal antibody (mAb) formulations. Increased solution opalescence can be attributed to attractive protein-protein interactions (PPIs). Techniques including light scattering, AUC, or membrane osmometry are routinely employed to measure PPIs in dilute solutions, whereas opalescence is seen at relatively higher concentrations, where both long- and short-range forces contribute to overall PPIs. The mAb molecule studied here shows a unique property of high opalescence because of liquid-liquid phase separation. In this study, opalescence measurements are correlated to PPIs measured in diluted and concentrated solutions using light scattering (kD ) and high-frequency rheology (G'), respectively. Charges on the molecules were calculated using zeta potential measurements. Results indicate that high opalescence and phase separation are a result of the attractive interactions in solution; however, the presence of attractive interactions do not always imply phase separation. Temperature dependence of opalescence suggests that thermodynamic contribution to opalescence is significant and Tcloud can be utilized as a potential tool to assess attractive interactions in solution.

Psoriasis clinical implications and treatment: a review.

Psoriasis is a common skin disorder affecting the population worldwide. It is a T-cell mediated autoimmune disorder leading to keratinocyte hyperproliferation. Psoriasis has genetic predisposition that is further aggravated by certain stimulating factors. In spite of significant advances in understanding the pathogenesis of psoriasis, the exact etiology of the disease remains unknown. The clinical manifestations of this disease include various forms that affect different parts of the body. Treatment options vary according to the mode of application or severity of the disease. Earlier treatments have included application of emollients or keratolytic agents to hydrate the skin or shed off the skin. But later treatments have been modified to treat the underlying T-cell proliferation. Hence, topical treatments like coal tar, vitamin D, retinoids, topical calcineurin inhibitors for treating mild psoriasis, systemic treatments including methotrexate, cyclosporine, acitretin, hydroxyurea, as well as light therapy for severe psoriasis have become more prominent. Current treatment modalities are associated with the risk of serious side effects from prolonged treatment. Combinations of these therapies have provided effective and rapid modalities to suppress the disease and reduce the side effects of treatment. In addition, newer carrier systems for conventional drugs are being developed to improve the effectiveness of treatment and reduce the side effects. Development of biologics and gene therapy have revolutionized the treatment of this skin disease. Although an array of therapies to suppress the psoriatic condition exists, none are curative.