Tim J. Kamerzell

Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development

The purpose of this review is to demonstrate the critical importance of understanding protein-excipient interactions as a key step in the rational design of formulations to stabilize and deliver protein-based therapeutic drugs and vaccines. Biophysical methods used to examine various molecular interactions between solutes and protein molecules are discussed with an emphasis on applications to pharmaceutical excipients in terms of their effects on protein stability. Key mechanisms of protein-excipient interactions such as electrostatic and cation-pi interactions, preferential hydration, dispersive forces, and hydrogen bonding are presented in the context of different physical states of the formulation such as frozen liquids, solutions, gels, freeze-dried solids and interfacial phenomenon. An overview of the different classes of pharmaceutical excipients used to formulate and stabilize protein therapeutic drugs is also presented along with the rationale for use in different dosage forms including practical pharmaceutical considerations. The utility of high throughput analytical methodologies to examine protein-excipient interactions is presented in terms of expanding formulation design space and accelerating experimental timelines.

The Complex Inter-Relationships Between Protein Flexibility and Stability

The ability to successfully formulate and manufacture therapeutic protein dosage forms requires a thorough understanding of their physico-chemical properties. Proteins are inherently dynamic molecules of marginal stability. These properties present unique challenges to the pharmaceutical scientist attempting to develop protein based therapeutics. The physicochemical stability and biological functions of proteins are thought to be intimately related to their global flexibility, intramolecular fluctuations and various other dynamic processes. Our understanding of these relationships, however, is incomplete but undeniably necessary for the development of efficacious therapies. Therefore, a better understanding of the complex inter-relationships between protein flexibility and stability should enable the rational design and optimization of protein formulation conditions based on protein dynamics. This review attempts to define protein dynamics and flexibility while summarizing a select number of studies of potential pharmaceutical interest that evaluate these relationships.

Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability

Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic EPDs. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed.

Increasing IgG Concentration Modulates the Conformational Heterogeneity and Bonding Network that Influence Solution Properties

Multiple molecular driving forces mediate protein stability, association, and recognition in concentrated solutions. Here we investigate the interactions that modulate the nonideal solution behavior of two immunoglobulins (IgG1s) in highly concentrated solutions using two-dimensional vibrational correlation spectroscopy (2D-COS) and principal components analysis (PCA). A specific sequence of changes is observed in the concentration-dependent vibrational spectra of the highly viscous IgG solution that deviates from ideality, whereas that sequence is reversed for all other conditions examined. The asynchronous spectra reveal variation in beta-sheet and turn regions occur before intensity variations in disordered and alpha-helical regions as the concentration is increased for the highly viscous regime. This is in contrast to the sequence observed for all other conditions studied and to the idea that beta-sheet regions are resistant to concentration-dependent affects. Finally, we show that increased hydrogen bonding and electrostatics primarily modulate the intermolecular association and nonideal behavior. Specifically, 2D-COS and PCA analysis of the amide II region suggests that Glu and Asp residues trigger the change resulting in increased viscosity and association of one IgG.

Immunoglobulin Dynamics, Conformational Fluctuations, and Nonlinear Elasticity and Their Effects on Stability

The relationships between protein dynamics, function, and stability are incompletely understood. Two external perturbations (temperature and pH) were used to modulate the flexibility and stability of an IgG1kappa monoclonal antibody (mAb) in an attempt to better understand the possible correlations between flexibility and stability. Ultrasonic velocimetry, densitometry, differential scanning calorimetry (DSC), and pressure perturbation calorimetry (PPC) were used to experimentally determine the adiabatic and isothermal compressibility, expansibility, fractional volumes of unfolding, and various nonlinear thermoacoustical parameters as a function of pH and temperature. By combining these results, state parameter fluctuations were calculated from fundamental statistical mechanical relationships. The most dynamic and rigid mAb ensemble is measured at pH 4 and 6, respectively, based on state parameter fluctuations and compressibility. The effect of pH appears to couple mAb dynamics to solvent fluctuations, which control its dynamics and stability. A nonlinear response to mechanical perturbation, comparable to that seen with many polymers, is observed for this monoclonal antibody at pH 4-8. This behavior is characterized as strongly anisotropic and anharmonic, especially at pH 4. The midpoint of thermal unfolding as measured by DSC does not necessarily correlate with flexibility.

Characteristics of rhVEGF Release from Topical Hydrogel Formulations

PurposeTo study recombinant human vascular endothelial growth factor (rhVEGF), the release characteristics from topical gel formulations, and its interaction with the gelling agents.MethodsThe release kinetics were followed by quantifying rhVEGF that diffused into the receptor chamber of Franz cells. Analytical ultracentrifuge (AUC) was used to characterize the sedimentation velocity of rhVEGF experienced in the gel. The interactions were characterized by isothermal calorimetry (ITC), and rhVEGF conformation was assessed by circular dichroism (CD).ResultsThe fraction of protein released was linear with the square root of time. The release rate constants did not show significant change within a wide range of bulk viscosities created by different concentrations of hydroxypropyl methylcellulose (HPMC) or MC gels. Sedimentation velocity determined by AUC generated comparable sedimentation coefficients of protein in these gels. AUC and ITC revealed no significant interaction between rhVEGF and HPMC and some change on secondary structure of the protein by Far UV CD, which was not the case with carboxymethyl cellulose (CMC).ConclusionsMicroviscosity, not bulk viscosity, was the key factor for the release of rhVEGF from cellulosic gels such as HPMC. Interaction between rhVEGF and CMC resulted in slower, and reduced amount of, release from the gel.

Parathyroid hormone is a heparin/polyanion binding protein: binding energetics and structure modification.

The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1–84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1–84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce α‐helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.