Application of maximum bubble pressure surface tensiometer to study protein-surfactant interactions.

Binding of a surfactant to proteins can affect their physicochemical stability and solubility in a formulation. The extent of the effect depends on the binding stoichiometry. In this study, we have utilized the technique of maximum bubble pressure surface tensiometry to characterize the binding between human serum albumin (HSA) and surfactants (sodium dodecyl sulfate (SDS) and polysorbate 80) by dynamic surface tension measurements. Results show that two classes of binding sites are present in HSA for SDS, 5 primary binding sites with high binding affinity (K(a)=5.38×10(5) M(-1)) and 12 secondary binding sites with low affinity (K(a)=6.7×10(4) M(-1)). The binding is high affinity and limited capacity due to both, ionic and hydrophobic interactions between HSA and SDS. For polysorbate 80, the binding does not follow the Scatchard plot, and is low affinity and high capacity, indicating that polysorbate 80 interacts with HSA through hydrophobic interactions. The results show that maximal bubble pressure surface tensiometry is a fast and convenient technique to determine the concentration of free and bound surfactants in the presence of proteins.

Ultrasonic rheology of a monoclonal antibody (IgG2) solution: implications for physical stability of proteins in high concentration formulations.

The purpose of this work was to investigate if physical stability of a model monoclonal antibody (IgG(2)), as determined by extent of aggregation, was related to rheology of its solutions. Storage stability of the model protein was assessed at 25 degrees C and 37 degrees C for three months in solutions ranging from pH 4.0 to 9.0 and ionic strengths of 4 mM and 300 mM. The rheology of IgG(2) solutions has been characterized at 25 degrees C in our previous work and correlation of solution storage modulus (G') with protein-protein interactions established. The extent of aggregation was consistent with solution rheology as understood in terms of changes in G' with protein concentration. Thermodynamic stability of native IgG(2) conformation increased with increasing pH. The correlation between rheology and aggregation was also assessed at increased ionic strengths. The decrease in aggregation was consistent with change in solution rheology profile at pH 7.4 and 9.0. The results provide evidence of a relationship between solution rheology and extent of aggregation for the model protein studied. The implications of this relationship for formulation and physical stability assessment in high concentration protein solutions are discussed.

Ultrasonic storage modulus as a novel parameter for analyzing protein-protein interactions in high protein concentration solutions: correlation with static and dynamic light scattering measurements.

The purpose of this work was to establish ultrasonic storage modulus (G') as a novel parameter for characterizing protein-protein interactions (PPI) in high concentration protein solutions. Using an indigenously developed ultrasonic shear rheometer, G' for 20-120 mg/ml solutions of a monoclonal antibody (IgG(2)), between pH 3.0 and 9.0 at 4 mM ionic strength, was measured at frequency of 10 MHz. Our understanding of ultrasonic rheology indicated decrease in repulsive and increase in attractive PPI with increasing solution pH. To confirm this behavior, dynamic (DLS) and static (SLS) light scattering measurements were conducted in dilute solutions. Due to technical limitations, light scattering measurements could not be conducted in concentrated solutions. Mutual-diffusion coefficient, measured by DLS, increased with IgG(2) concentration at pH 4.0 and this trend reversed as pH was increased to 9.0. Second virial coefficient, measured by SLS, decreased with increasing pH. These observations were consistent with the nature of PPI understood from G' measurements. Ultrasonic rheology, DLS, and SLS measurements were also conducted under conditions of increased ionic strength. The consistency between rheology and light scattering analysis under various solution conditions established the utility of ultrasonic G' measurements as a novel tool for analyzing PPI in high protein concentration systems.

Antibody structure, instability, and formulation.

The number of therapeutic monoclonal antibody in development has increased tremendously over the last several years and this trend continues. At present there are more than 23 approved antibodies on the US market and an estimated 200 or more are in development. Although antibodies share certain structural similarities, development of commercially viable antibody pharmaceuticals has not been straightforward because of their unique and somewhat unpredictable solution behavior. This article reviews the structure and function of antibodies and the mechanisms of physical and chemical instabilities. Various aspects of formulation development have been examined to identify the critical attributes for the stabilization of antibodies.

Application of high-frequency rheology measurements for analyzing protein-protein interactions in high protein concentration solutions using a model monoclonal antibody (IgG2).

The purpose of this work was to explore the utilization of high-frequency rheology analysis for assessing protein-protein interactions in high protein concentration solutions. Rheology analysis of a model monoclonal immunoglobulin G2 solutions was conducted on indigenously developed ultrasonic shear rheometer at frequency of 10 MHz. Solutions at pH 9.0 behaved as most viscous and viscoelastic whereas those at pH 4.0 and 5.4 exhibited lower viscosity and viscoelasticity, respectively. Intrinsic viscosity, hydrophobicity, and conformational analysis could not account for the rheological behavior of IgG2 solutions. Zeta potential and light scattering measurements showed the significance of electroviscous and specific protein-protein interactions in governing rheology of IgG2 solutions. Specific protein-protein interactions resulted in formation of reversible higher order species of monomer. Solution storage modulus (G'), and not loss modulus or complex viscosity, was the more reliable parameter for predicting protein-protein interactions. Predictions about the nature of protein-protein interactions made on the basis of solution G' were found to be consistent with observed effect of pH and ionic strength on zeta potential and scattered intensity of IgG2 solutions. Results demonstrated the potential of high-frequency storage modulus measurements for understanding behavior of proteins in solutions and predicting the nature of protein-protein interactions.

Retrospective statistical analysis of lyophilized protein formulations of progenipoietin using PLS: determination of the critical parameters for long-term storage stability.

Although certain criteria have become recognized as being essential for a stable lyophilized formulation, the relative importance of different stability criteria has not been demonstrated quantitatively. This study uses multivariate statistical methods to determine the relative importance of certain formulation variables that affect long-term storage stability of a therapeutic protein. Using the projection to latent structures (PLS) method, a retrospective analysis was conducted of 18 formulations of progenipoietin (ProGP), a potential protein therapeutic agent. The relative importance of composition, pH, maintenance of protein structure (as determined by infrared (IR) spectroscopy), and thermochemical properties of the glassy state (as measured by differential scanning calorimetry (DSC)) were evaluated. Various stability endpoints were assessed and validated models constructed for each using the PLS method. Retention of parent protein and the appearance of degradation products could be adequately modeled using PLS. The models demonstrate the importance of retention of native structure in the solid state and controlling the pH. The relative importance of T(g) in affecting storage stability was low, as all of the samples had T(g) values above the highest storage temperature (40 degrees C). However, other indicators of molecular mobility in the solid state, such as change in DeltaC(p) upon annealing, appear to be important, even for storage below T(g). For the first time, the relative importance of certain properties in controlling long-term storage stability could be assessed quantitatively. In general, the most important parameters appear to be pH and retention of native structure in the solid state. However, for some stability endpoints, the composition (concentration of protein or various excipients), as well as some DSC parameters, were found to be significant in predicting long-term stability.

Purification and characterization of progenipoietins produced in Escherichia. coli.

The progenipoietins (ProGPs) are a family of genetically engineered chimeric proteins that contain receptor agonist activity for both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. These unique proteins have previously been shown to induce the proliferation of multiple cell lineages. The characterization of two progenipoietins, ProGP-1 and ProGP-4, refolded and purified from an Escherichia coli expression system is described. These ProGP molecules differ in the orientation of the two receptor agonists and, in addition, ProGP-4 contains a fetal liver tyrosine kinase-3 receptor agonist that has been circularly permuted to modulate its activity. Static light scattering analyses demonstrated that both ProGP molecules exist as dimers, most likely through non-covalent interaction of the fetal liver tyrosine kinase-3 receptor agonist domains. ProGP-1 and ProGP-4 have comparable secondary structures, as analyzed by circular dichroism; however, their tertiary structures, as measured by intrinsic fluorescence, were demonstrated to be different. Differential scanning calorimetry demonstrated that the thermal stability of these two proteins was indistinguishable. Interestingly, these dual agonist proteins yielded only a single melting temperature value that was intermediate between that of their individual receptor agonist components, indicating that these chimeric molecules behave as a single domain protein during thermal denaturation. This study describes the purification and physico-chemical properties of this class of proteins generated using an E. coli expression system.