Brent S. Kendrick

A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody

The selection of the appropriate excipient and the amount of excipient required to achieve a 2-year shelf-life is often done by using iso-osmotic concentrations of excipients such as sugars (e.g., 275 mM sucrose or trehalose) and salts. Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage. Using a model recombinant humanized monoclonal antibody (rhuMAb HER2), we assessed the impact of lyoprotectants, sucrose, and trehalose, alone or in combination with mannitol, on the storage stability at 40 degrees C. Molar ratios of sugar to protein were used, and the stability of the resulting lyophilized formulations was determined by measuring aggregation, deamidation, and oxidation of the reconstituted protein and by infrared (IR) spectroscopy (secondary structure) of the dried protein. A 360:1 molar ratio of lyoprotectant to protein was required for storage stability of the protein, and the sugar concentration was 3-4-fold below the iso-osmotic concentration typically used in formulations. Formulations with combinations of sucrose (20 mM) or trehalose (20 mM) and mannitol (40 mM) had comparable stability to those with sucrose or trehalose alone at 60 mM concentration. A formulation with 60 mM mannitol alone provided slightly less protection during storage than 60 mM sucrose or trehalose. The disaccharide/mannitol formulations also inhibited deamidation during storage to a greater extent than the lyoprotectant formulations alone. The reduction in aggregation and deamidation during storage correlated directly with inhibition of unfolding during lyophilization, as assessed by IR spectroscopy. Thus, it appears that the protein must be retained in its native-like state during freeze-drying to assure storage stability in the dried solid. Long-term studies (23-54 months) performed at 40 degrees C revealed that the appropriate molar ratio of sugar to protein stabilized against aggregation and deamidation for up to 33 months. Therefore, long-term storage at room temperature or above may be achieved by proper selection of the molar ratio and sugar mixture. Overall, a specific sugar/protein molar ratio was sufficient to provide storage stability of rhuMAb HER2.

Roles of conformational stability and colloidal stability in the aggregation of recombinant human granulocyte colony‐stimulating factor

We studied the non‐native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (ΔGunf) and osmotic second virial coefficient (B22), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ΔGunf and negative B22), the first step is rate‐limiting, and increasing ΔGunf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B22 values) the second step is rate‐limiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate‐limiting.

Inhibition of stress-induced aggregation of protein therapeutics

Publisher Summary This chapter describes the types of stresses and conditions that are routinely found to cause aggregation of purified therapeutic proteins. Stresses encountered during processing include short-term exposure to high temperatures during pasteurization of aqueous solutions, freeze-thawing, freeze-drying, and exposure to denaturing interfaces because of agitation, filtration, air bubble entrainment during filling, and so on. It also considers the effects of long-term storage on protein stability in aqueous solutions and dried solids. Each section describes how the rational choice of stabilizing additives can be used to inhibit protein aggregation. These choices are based on a clear understanding of the mechanisms by which different additives succeed or fail as protein stabilizers under different conditions. The mechanisms are considered in detail and are illustrated in the chapter by selected examples. Finally, this chapter briefly describes a model that allows the calculation of the degree of expansion of the native state needed to form an aggregate-fostering species in aqueous solution and the utility of infrared spectroscopy to characterize the structure of proteins in precipitates.

Effects of sucrose on conformational equilibria and fluctuations within the native-state ensemble of proteins

Osmolytes increase the thermodynamic conformational stability of proteins, shifting the equilibrium between native and denatured states to favor the native state. However, their effects on conformational equilibria within native‐state ensembles of proteins remain controversial. We investigated the effects of sucrose, a model osmolyte, on conformational equilibria and fluctuations within the native‐state ensembles of bovine pancreatic ribonuclease A and S and horse heart cytochrome c. In the presence of sucrose, the far‐ and near‐UV circular dichroism spectra of all three native proteins were slightly altered and indicated that the sugar shifted the native‐state ensemble toward species with more ordered, compact conformations, without detectable changes in secondary structural contents. Thermodynamic stability of the proteins, as measured by guanidine HCl‐induced unfolding, increased in proportion to sucrose concentration. Native‐state hydrogen exchange (HX) studies monitored by infrared spectroscopy showed that addition of 1 M sucrose reduced average HX rate constants at all degrees of exchange of the proteins, for which comparison could be made in the presence and absence of sucrose. Sucrose also increased the exchange‐resistant core regions of the proteins. A coupling factor analysis relating the free energy of HX to the free energy of unfolding showed that sucrose had greater effects on large‐scale than on small‐scale fluctuations. These results indicate that the presence of sucrose shifts the conformational equilibria toward the most compact protein species within native‐state ensembles, which can be explained by preferential exclusion of sucrose from the protein surface.

Detection of Protein Aggregates by Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC): Sources of Variability and Their Relative Importance

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has found application in the biopharmaceutical industry as a method of detecting and quantifying protein aggregates. While the technique offers several advantages (i.e., matrix-free separation and minimal sample handling), its results exhibit a high degree of variability relative to orthogonal size-sensitive separation techniques such as size exclusion chromatography (SEC). The goal of this work is to characterize and quantify the sources of variability that affect SV-AUC results, particularly size distributions for a monoclonal antibody monomer/dimer system. Contributions of individual factors to the overall variability are examined. Results demonstrate that alignment of sample cells to the center of rotation is the most significant contributing factor to overall variability. The relative importance of other factors (e.g., temperature equilibration, time-invariant noise, meniscus misplacement, etc.) are quantified and discussed.

Common Excipients Impair Detection of Protein Aggregates During Sedimentation Velocity Analytical Ultracentrifugation

The final formulations of modern pharmaceutical protein products typically contain sugars or sugar alcohols as stabilizers. Migration of these sugars under the influence of an applied gravitational field during sedimentation velocity analytical ultracentrifugation (SV-AUC) produces dynamic density and viscosity gradients. If the formation of such gradients is not taken into account during data analysis, the capability of the SV-AUC technique to detect protein oligomers/aggregates may be dramatically impacted. In the example described here, the limit of quantitation (LOQ) of a simulated monoclonal antibody (mAb) dimer increases from 0.8% to 2.4% upon addition of 5% sorbitol to the formulation. This study uses simulated and experimental SV-AUC data to demonstrate the detrimental effect of dynamic gradients; it further explores how sophisticated data analysis techniques, including SEDFITs inhomogeneous solvent options, may be used to mitigate the detection problems caused by the sedimentation of excipients.

Physical Stabilization of Proteins in Aqueous Solution

The formulation scientist’s key goal is to achieve long-term stability of a drug compound. In the case of protein drugs, stabilization means not only maintaining the native chemical structure, but the native secondary and higher order structures necessary for biological activity (Cleland et al., 1993; Manning et al., 1989). Denaturation, as it is defined in this context, will be the process of forming any non-native physical or chemical state of the protein. Physical and chemical denat-urations are often accompanied by covalent and non-covalent aggregates that not only can destroy the activity of the drug, but also cause adverse side effects (Carpenter and Chang, 1996; Thornton and Ballow, 1993). Without the ability to stabilize native protein structures, even the most efficacious protein therapeutics will fail to make viable drug products.

Precision of protein aggregation measurements by sedimentation velocity analytical ultracentrifugation in biopharmaceutical applications.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is routinely applied in biopharmaceutical development to measure levels of protein aggregation in protein products. SV-AUC is free from many limitations intrinsic to size exclusion chromatography (SEC) such as mobile phase and column interaction effects on protein self-association. Despite these clear advantages, SV-AUC exhibits lower precision measurements than corresponding measurements by SEC. The precision of SV-AUC is influenced by numerous factors, including sample characteristics, cell alignment, centerpiece quality, and data analysis approaches. In this study, we evaluate the precision of SV-AUC in its current practice utilizing a multilaboratory, multiproduct intermediate precision study. We then explore experimental approaches to improve SV-AUC measurement precision, with emphasis on utilization of high quality centerpieces.

Measurement of the second osmotic virial coefficient for protein solutions exhibiting monomer-dimer equilibrium.

The second osmotic virial coefficient (B) is a measure of solution nonideality that is useful for predicting conditions favorable for protein crystallization and for inhibition of aggregation. Static light scattering is the technique most commonly used to determine B values, typically using protein concentrations less than 5 mg/mL. During static light scattering experiments at low protein concentrations, frequently the protein is assumed to exist either as a single nonassociating species or as a combination of assembly states independent of protein concentration. In the work described here, we examined the limit for ignoring weak reversible dimerization (Kd > or =1 mM) by comparing B values calculated with and without accounting for self-association. Light scattering effects for equilibrium dimer systems with Kd <20 mM and Kd <1 mM will significantly affect apparent B values measured for 20 and 150-kDa proteins, respectively. To interpret correctly light scattering data for monomer-dimer equilibrium systems, we use an expanded coefficient model to account for separate monomer-monomer (B(22)), monomer-dimer (B(23)), and dimer-dimer (B(33)) interactions.

Protein Misfolding and Aggregation

Interest in the problem of protein misfolding and aggregation has exploded in recent years for two reasons: ( 1 ) the sharp rise in the number and volume of therapeutic proteins produced commercially and ( 2 ) the recognition of the central role of protein aggregates in degenerative diseases. The systematic study of protein aggregation presents major challenges to both the experimentalist and the theoretician. Much of the work retains an empirical flavor due to the experimental complexities; the sensitivity of protein aggregation to the slightest change in protein amino acid composition, solvent properties, or protein concentration; and the lack of robust theoretical models of misfolding and aggregation. Novel experimental and computational approaches are being developed, and we anticipate substantial progress will be made in the near future. Several presentations describing the latest advances in protein misfolding and aggregation were given at the American Chemical Society meeting (BIOT division) held in September, 2006 in San Francisco.