Derrick S. Katayama

Mixing properties of lyophilized protein systems: A spectroscopic and calorimetric study

The purpose of this study was to investigate the solid-state properties of lyophilized formulations of protein (ribonuclease A) containing sucrose or trehalose across a wide range of compositions, both in the presence or absence of hydroxyethylstarch (HES). Infrared spectroscopy reveals that the protein forms hydrogen bonds to sugars (sucrose or trehalose) as water is removed from the sample. The strength and/or number of hydrogen bonds in dried samples increase as the weight fraction of sugar increases. Significant deviations of glass transition temperatures (T(g)s) from those predicted by free volume theory are seen in both protein-sugar systems. The behavior can be explained by formation of protein-sugar hydrogen bonds at the expense of self-interactions between the sugars. Attractive interactions between lyophilized ribonuclease A and HES were detected spectroscopically and from thermodynamic analysis of T(g) values, contrary to the view that HES is sterically hindered from interacting with the protein surface. Sucrose-HES interactions were much less favorable than trehalose-HES interactions, suggesting that phase separation in sugar/HES/protein mixtures would be more likely in the presence of sucrose than trehalose. Finally, the thermodynamics of mixing were investigated using differential scanning calorimetry (DSC) providing some of the first data for such solid protein sugar formulations with and without HES. In nearly all samples, positive excess enthalpy, excess entropy and excess free energy were observed, with the excess free energy being greater for samples containing sucrose rather than trehalose. Analysis of Flory-Huggins chi parameters suggests that phase separation between protein and excipients may be thermodynamically favored in these dried solid preparations.

Role of Buffers in Protein Formulations

Buffers comprise an integral component of protein formulations. Not only do they function to regulate shifts in pH, they also can stabilize proteins by a variety of mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid formulations, frozen solutions, or the solid state is highlighted in this review. Addition of buffers can result in increased conformational stability of proteins, whether by ligand binding or by an excluded solute mechanism. In addition, they can alter the colloidal stability of proteins and modulate interfacial damage. Buffers can also lead to destabilization of proteins, and the stability of buffers themselves is presented. Furthermore, the potential safety and toxicity issues of buffers are discussed, with a special emphasis on the influence of buffers on the perceived pain upon injection. Finally, the interaction of buffers with other excipients is examined.

Solution behavior of a novel biopharmaceutical drug candidate : A gonadotropin-toxin conjugate

ABSTRACT There is little known about the solution structure and stability of peptide-protein conjugates, which comprise a new class of potential biopharmaceutical agents. This study describes the solution behavior of gonadotropins-releasing hormone (GnRH) chemically conjugated to pokeweed antiviral protein (PAP). The conjugate adopts a well-defined conformation across a pH range of 4 to 8. Even after heating to 80°C, the conjugate retains a significant amount of secondary and tertiary structure. Heating for 1 h at 60°C does lead to chemical damage, as determined by cation exchange chromatography. Using an experimental design approach, the optimal pH and salt concentration for limiting chemical damage was determined.

Denaturation and Aggregation of Interferon-τ in Aqueous Solution

PurposeTo evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride.MethodsAsymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system.ResultsThe two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1.ConclusionChemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.