Accelerated Storage for Shelf-Life Prediction of Lyophiles: Temperature Dependence of Degradation of Amorphous Small Molecular Weight Drugs and Proteins.

Prediction of lyophilized product shelf-life using accelerated stability data requires understanding the temperature dependence of the degradation rate. Despite the abundance of published studies on stability of freeze-dried formulations and other amorphous materials, there are no definitive conclusions on the type of pattern one can expect for the temperature dependence of degradation. This lack of consensus represents a significant gap which may impact development and regulatory acceptance of freeze-dried pharmaceuticals and biopharmaceuticals. Review of the literature demonstrates that the temperature dependence of degradation rate constants in lyophiles can be represented by the Arrhenius equation in most cases. In some instances there is a break in the Arrhenius plot around the glass transition temperature or a related characteristic temperature. The majority of the activation energies (Ea), which are reported for various degradation pathways in lyophiles, falls in the range of 8 to 25 kcal/mol. The degradation Ea values for lyophiles are compared with the Ea for relaxation processes and diffusion in glasses, as wells as solution chemical reactions. Collectively, analysis of the literature demonstrates that the Arrhenius equation represents a reasonable empirical tool for analysis, presentation, and extrapolation of stability data for lyophiles, provided that specific conditions are met.

Water Distribution and Clustering on the Lyophilized IgG1 Surface: Insight from Molecular Dynamics Simulations.

Water has a critical role in the stability of the higher-order structure of proteins. In addition, it is considered to be a major destabilization factor for the physical and chemical stability of freeze-dried proteins and peptides. Physical and chemical aspects of protein/water relationships are commonly studied with the use of water vapor sorption isotherms for amorphous lyophilized proteins, which, in turn, are commonly analyzed using the Brunauer-Emmett-Teller (BET) equation to obtain the parameters, Wm and CB. The parameter Wm is generally referred to as the "monolayer limit of adsorption" and has a narrow range of 6-8% for most proteins. In this study, the water distribution on an IgG1 surface is investigated by molecular dynamics (MD) simulations at different water contents. The monolayer of water molecules was found to have limited coverage of the protein surface, and the true monolayer coverage of the protein globule actually occurs at a hydration level above 30%. The distribution of water molecules on the IgG1 surface is also highly heterogeneous, and the heterogeneity is not considered in the BET theory. In this study, a mechanistic model has been developed to describe the water vapor sorption isotherm. This model is based on the analysis of the hydrogen bonding network extracted from the MD simulations. The model is consistent with the experimental Type-II isotherm, which is usually observed for proteins. The physical meaning of the BET monolayer was redefined as the onset of water cluster formation. A simple model to calculate the onset water level, Wm, is proposed based on the hydration of different amino acids, as determined from the MD simulations.

Impact of Formulation and Suspension Properties on Redispersion of Aluminum-Adjuvanted Vaccines.

The adsorption of antigens to the surface of 2 commonly used insoluble adjuvants, aluminum phosphate and aluminum hydroxide, has been well characterized. In spite of the pharmaceutical benefits, alum-based vaccine formulations can present challenges in redispersion of the final product after storage. Inability to resuspend alum-based vaccines during administration results in inadequate dosing, thus rendering the product unusable. Here, the influence of formulation conditions on the resuspendability of aluminum adjuvant-containing vaccines was investigated. Particle size analysis by Micro-Flow Imaging (MFI™), zeta potential measurement, and sedimentation analysis by Turbiscan® were used to characterize suspension properties. Ionic strength, pH, and antigen concentration were found to significantly influence sedimentation behavior, particle size, and redispersion. Increasing ionic strength increased the sedimentation rate of adjuvants favoring resuspendability. The addition of bovine serum albumin to aluminum phosphate reduced resuspendability more significantly than the addition of lysozyme. Decreased resuspendability correlated with an increase in fine-to-large particle ratio and decrease in sedimentation rate. In summary, resuspendability of adjuvant drug product is favored by increased flocculation, decrease in fine-to-large particle ratio, and reduction in surface charge of antigen and adjuvant. A careful balance of these formulation conditions can therefore be an effective means to mitigate challenges of alum adjuvant redispersion.

Science and art of protein formulation development.

Protein pharmaceuticals have become a significant class of marketed drug products and are expected to grow steadily over the next decade. Development of a commercial protein product is, however, a rather complex process. A critical step in this process is formulation development, enabling the final product configuration. A number of challenges still exist in the formulation development process. This review is intended to discuss these challenges, to illustrate the basic formulation development processes, and to compare the options and strategies in practical formulation development.

Bulk Dynamic Spray Freeze-Drying Part 1: Modeling of Droplet Cooling and Phase Change.

In spray freeze-drying (SFD), the solution is typically dispersed into a gaseous cold environment producing frozen microparticles that are subsequently dried via sublimation. This technology can potentially manufacture bulk lyophilized drugs at higher rates compared with conventional freeze-drying in trays and vials because small frozen particles provide larger surface area available for sublimation. Although drying in SFD still has to meet the material collapse temperature requirements, the final characteristics of the respective products are mainly controlled by the spray-freezing dynamics. In this context, the main goal of this work is to present a single droplet spray-freezing model and validate it with previously published simulations and experimental data. For the investigated conditions, the droplet temperature evolutions predicted by the model agree with experiments within an error of ±10%. The proposed engineering-level modeling framework is intended to assist future development of efficient SFD processes and support scale up from laboratory to commercial scale equipment.

Bulk Dynamic Spray Freeze-Drying Part 2: Model-Based Parametric Study for Spray-Freezing Process Characterization.

Spray freeze-drying is an evolving technology that combines the benefits of spray-drying and conventional lyophilization techniques to produce drug substance and drug product as free-flowing powders. The high surface-to-volume ratio associated to the submillimeter spray-frozen particles contributes to shorter drying and reconstitution times. The formation of frozen particles is the most critical part of this dehydration technique because it defines the properties of final product. Based on a previously proposed and validated model, the current goal is to understand the role of various controllable parameters in the spray-freezing process. More specifically, given a set of spraying conditions, the model is used to predict the minimum distance required to cool and freeze the droplets below a temperature that prevents coalescence and product agglomeration. A parametric study is carried out to map the operational limit conditions of the actual spray-freezing column apparatus under consideration. For the spray freeze-drying conditions of interest, model simulations indicate that convection contributes to at least 80% of the total droplet heat transfer and, consequently, that freezing column gas temperature and droplet diameter are the most important process parameters affecting the freezing distance.

Freezing of Aqueous Solutions and Chemical Stability of Amorphous Pharmaceuticals: Water Clusters Hypothesis.

Molecular mobility has been traditionally invoked to explain physical and chemical stability of diverse pharmaceutical systems. Although the molecular mobility concept has been credited with creating a scientific basis for stabilization of amorphous pharmaceuticals and biopharmaceuticals, it has become increasingly clear that this approach represents only a partial description of the underlying fundamental principles. An additional mechanism is proposed herein to address 2 key questions: (1) the existence of unfrozen water (i.e., partial or complete freezing inhibition) in aqueous solutions at subzero temperatures and (2) the role of water in the chemical stability of amorphous pharmaceuticals. These apparently distant phenomena are linked via the concept of water clusters. In particular, freezing inhibition is associated with the confinement of water clusters in a solidified matrix of an amorphous solute, with nanoscaled water clusters being observed in aqueous glasses using wide-angle neutron scattering. The chemical instability is suggested to be directly related to the catalysis of proton transfer by water clusters, considering that proton transfer is the key elementary reaction in many chemical processes, including such common reactions as hydrolysis and deamidation.

Edetate Disodium as a Polysorbate Degradation and Monoclonal Antibody Oxidation Stabilizer.

Polysorbates are frequently used in biotherapeutic formulations. Interest in assessing their stability, in particular the impact of their degradation products on the stability of therapeutic proteins, has been steadily growing in the past decade. The work presented summarizes a case study of a monoclonal antibody formulation that demonstrated a simultaneous loss of polysorbate and an increase in methionine oxidation. Spiking studies were conducted to determine both the cause and a potential mitigation for the monoclonal antibody (mAb) oxidation and polysorbate 80 (PS80) loss. The results indicated that a different source material exhibited different rates of mAb oxidation and PS80 loss and that in all evaluated materials, the addition of edetate disodium to the formulation mitigated both observed issues. The mAb was assessed for the presence of lipases and lipoprotein lipase was detected at low levels. It is proposed that edetate disodium was effective in mitigating the mAb oxidation and PS80 loss by chelating calcium in the formulation and therefore decreasing the activity of the lipases.

Effect of Water on the Chemical Stability of Amorphous Pharmaceuticals: 2. Deamidation of Peptides and Proteins.

Role of water in chemical (in)stability is revisited, with focus on deamidation in freeze-dried amorphous proteins and peptides. Two distinct patterns for deamidation versus water have been reported, that is, a consistent increase in rate constant with water, and a "hockey stick"-type behavior. For the latter, deamidation is essentially independent of water at lower water contents and accelerates when water content increases above a threshold value. Two simple kinetic models are developed to analyze literature-reported relationships between water content and deamidation rate constants. One model is based on catalytic role of water clusters in enabling proton transfer, which is a critical reaction step. Water clusters are formed when water content increases above a threshold value, while unclustered (and less catalytically-active) water molecules are predominant at lower water levels. The second model considers the dual role of water, as both a destabilizer via catalysis and a stabilizer of protein native structure. Considering that both models emphasize the importance of local structure and that local structure is intrinsically related to fast (and non-cooperative) relaxation modes, it is plausible to expect correlations between local mobility, such as beta-relaxation, and amorphous chemical instability.

Stabilization of Live Attenuated Influenza Vaccines by Freeze Drying, Spray Drying, and Foam Drying.

PURPOSE: The goal of this research is to develop stable formulations for live attenuated influenza vaccines (LAIV) by employing the drying methods freeze drying, spray drying, and foam drying. METHODS: Formulated live attenuated Type-A H1N1 and B-strain influenza vaccines with a variety of excipient combinations were dried using one of the three drying methods. Process and storage stability at 4, 25 and 37°C of the LAIV in these formulations was monitored using a TCID50 potency assay. Their immunogenicity was also evaluated in a ferret model. RESULTS: The thermal stability of H1N1 vaccine was significantly enhanced through application of unique formulation combinations and drying processes. Foam dried formulations were as much as an order of magnitude more stable than either spray dried or freeze dried formulations, while exhibiting low process loss and full retention of immunogenicity. Based on long-term stability data, foam dried formulations exhibited a shelf life at 4, 25 and 37°C of >2, 1.5 years and 4.5 months, respectively. Foam dried LAIV Type-B manufactured using the same formulation and process parameters as H1N1 were imparted with a similar level of stability. CONCLUSION: Foam drying processing methods with appropriate selection of formulation components can produce an order of magnitude improvement in LAIV stability over other drying methods.

Investigation of the Sedimentation Behavior of Aluminum Phosphate: Influence of pH, Ionic Strength, and Model Antigens.

Evaluation of the physical characteristics of vaccines formulated in the presence of adjuvants, such as aluminum salts (Alum), is an important step in the development of vaccines. Depending on the formulation conditions and the associated electrostatic interactions of the adjuvant particles, the vaccine suspension may transition between flocculated and deflocculated states. The impact of practical formulation parameters, including pH, ionic strength, and the presence of model antigens, has been correlated to the sedimentation behavior of aluminum phosphate suspensions. A novel approach for the characterization of suspension properties of Alum has been developed to predict the flocculated state of the system using a sedimentation analysis-based tool (Turbiscan®). Two sedimentation parameters, the settling onset time (Sonset) and the sedimentation volume ratio (SVR) can be determined simultaneously in a single measurement. The results demonstrate the suspension characteristics to be significantly altered by solution conditions (pH and ionic strength) and the charge state of bound antigens. Formulation conditions that promote the flocculated state of the suspension are characterized by faster Sonset and higher SVR, and are generally easy to resuspend. The Turbiscan® method described herein is a useful tool for the characterization of aluminum-containing suspensions and may be adapted for screening and optimization of suspension-based vaccine formulations in general.

Next generation drying technologies for pharmaceutical applications.

Drying is a commonly used technique for improving the product stability of biotherapeutics. Typically, drying is accomplished through freeze-drying, as evidenced by the availability of several lyophilized products on the market. There are, however, a number of drawbacks to lyophilization, including the lengthy process time required for drying, low energy efficiency, high cost of purchasing and maintaining the equipment, and sensitivity of the product to freezing and various other processing-related stresses. These limitations have led to the search for next-generation drying methods that can be applied to biotherapeutics. Several alternative drying methods are reviewed herein, with particular emphasis on methods that are commonly employed outside of the biopharmaceutical industry including spray drying, convective drying, vacuum drying, microwave drying, and combinations thereof. Although some of the technologies have already been implemented for processing biotherapeutics, others are still at an early stage of feasibility assessment. An overview of each method is presented, detailing the comparison to lyophilization, examining the advantages and disadvantages of each technology, and evaluating the potential of each to be utilized for drying biotherapeutic products.

Structural characteristics of short peptides in solution.

Short peptides are important biopharmaceuticals as agonistic or antagonistic ligands, aggregation inhibitors, and vaccines, as well as in many other applications. They behave differently from globular proteins in solution. Many short peptides are unstructured and tend to aggregate and undergo structural transition in response to changes in solvent environment, including pH, temperature, ionic strength, presence of organic solvents or surfactants, and exposure to lipid membranes. Such structural transitions are often associated with fibril or ß-amyloid formation. These structural characteristics of short peptides have drastic impact on their function, immunogenicity, and storage stability.

Recombinant therapeutic protein vaccines.

Recombinant technology has ushered in a new era for the pharmaceutical industry. Protein therapeutics, including plasma-derived products and antibodies obtained from the serum of infected patients, have been successfully adopted and utilized to treat various indications. The development of recombinant technology and the subsequent improvement in expression, purification, and formulation technologies have enabled the generation of highly purified proteins in a scalable and cost-effective manner. The discovery and development of several recombinant proteins, such as growth factors and cytokines, will be described followed by a brief review of monoclonal antibodies and enzyme replacement therapy. Recombinant protein-based vaccine, which is the focus of the current review, is described in detail with particular emphasis on several viral and bacterial infections. Challenges and new approaches in their use as a replacement for the currently available vaccines are discussed.

Effect of water on the chemical stability of amorphous pharmaceuticals: I. Small molecules.

Amorphous states, ubiquitous in pharmaceutical products, possess higher tendency for chemical degradation in comparison to crystalline materials. This instability can be further enhanced by water, which is present even in nominally dry systems. It has been increasingly recognized that in addition to the plasticizing effect of lowering the glass transition temperature, water could influence the degradation rates through medium effects (e.g., through change in solvation of the reactants and the transition state) as well as by direct participation in solid-state hydrolytic degradation processes. In the current review, the impact of water on the chemical stability of small molecules is examined, with emphasis on hydrolysis reactions in freeze-dried materials remaining in the glassy state. Quantitative relationships between water content and stability are discussed, including molecular mobility (global and local) and solution-like mechanisms, using the medium effects concept that has been developed for liquid-state reactions. Further progress in this field requires the development of quantitative and mechanistic understanding of the relationship between local mobility and chemical reactivity in amorphous solids, as well as incorporating the learning from solution chemistry on the role of reaction media in chemical processes.

Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles.

The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs.

Interactions of formulation excipients with proteins in solution and in the dried state.

A variety of excipients are used to stabilize proteins, suppress protein aggregation, reduce surface adsorption, or to simply provide physiological osmolality. The stabilizers encompass a wide variety of molecules including sugars, salts, polymers, surfactants, and amino acids, in particular arginine. The effects of these excipients on protein stability in solution are mainly caused by their interaction with the protein and the container surface, and most importantly with water. Some excipients stabilize proteins in solution by direct binding, while others use a number of fundamentally different mechanisms that involve indirect interactions. In the dry state, any effects that the excipients confer to proteins through their interactions with water are irrelevant, as water is no longer present. Rather, the excipients stabilize proteins through direct binding and their effects on the physical properties of the dried powder. This review will describe a number of mechanisms by which the excipients interact with proteins in solution and with various interfaces, and their effects on the physical properties of the dried protein structure, and explain how the various interaction forces are related to their observed effects on protein stability.

Formulation and stabilization of Francisella tularensis live vaccine strain.

Francisella tularensis live vaccine strain (F. tularensis LVS), a promising vaccine candidate for protection against F. tularensis exposure, is a particularly thermolabile vaccine and difficult to stabilize sufficiently for storage under refrigerated conditions. Our preliminary data show that F. tularensis LVS can be stabilized in the dried state using foam drying, a modified freeze drying method, with sugar-based formulations. The process was conducted under mild drying conditions, which resulted in a good titer retention following processing. The inclusion of osmolytes in the growth media resulted in an acceleration of growth kinetics, although no change in osmotolerance was observed. The optimized F. tularensis formulation, which contained trehalose, gelatin, and Pluronic F68 demonstrated stability for approximately 1.5 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1 log(10) colony forming unit) and for 12 weeks at 25°C. At refrigerator storage condition (4°C), stabilized F. tularensis LVS vaccine exhibited no activity loss for at least 12 weeks. This stabilization method utilizes conventional freeze dryers and pharmaceutically approved stabilizers, and thus can be readily implemented at many manufacturing sites for large-scale production of stabilized vaccines. The improved heat stability of the F. tularensis LVS could mitigate risks of vaccine potency loss during long-term storage, shipping, and distribution.

Room temperature stabilization of oral, live attenuated Salmonella enterica serovar Typhi-vectored vaccines.

Foam drying, a modified freeze drying process, was utilized to produce a heat-stable, live attenuated Salmonella Typhi 'Ty21a' bacterial vaccine. Ty21a vaccine was formulated with pharmaceutically approved stabilizers, including sugars, plasticizers, amino acids, and proteins. Growth media and harvesting conditions of the bacteria were also studied to enhance resistance to desiccation stress encountered during processing as well as subsequent storage at elevated temperatures. The optimized Ty21a vaccine, formulated with trehalose, methionine, and gelatin, demonstrated stability for approximately 12 weeks at 37°C (i.e., time required for the vaccine to decrease in potency by 1log(10)CFU) and no loss in titer at 4 and 25°C following storage for the same duration. Furthermore, the foam dried Ty21a elicited a similar immunogenic response in mice as well as protection in challenge studies compared to Vivotif™, the commercial Ty21a vaccine. The enhanced heat stability of the Ty21a oral vaccine, or Ty21a derivatives expressing foreign antigens (e.g. anthrax), could mitigate risks of vaccine potency loss during long-term storage, shipping, delivery to geographical areas with warmer climates or during emergency distribution following a bioterrorist attack. Because the foam drying process is conducted using conventional freeze dryers and can be readily implemented at any freeze drying manufacturing facility, this technology appears ready and appropriate for large scale processing of foam dried vaccines.