Biopharmaceutical powders: particle formation and formulation considerations

It is well known that protein/peptide-based drug formulations are more stable in the solid state than in the liquid state, thereby offering stability advantages in ambient temperature storage, product shipping/distribution, and long-term shelf life. Novel powder-based drug delivery systems recently emerging for applications in sustained release, inhalation, intradermal delivery, etc, add more value to protein solid dosage forms. Despite great research interests in understanding the drying effects on protein stability and a large collection of publications focusing on this area, systematic accounts of powder formation techniques are lacking. This review is to summarize a number of methods currently available for protein powder preparation. Some are common methods such as lyophilization, spray drying, pulverization, and precipitation, and some methods are more recently developed such as supercritical fluid precipitation, spray-freeze drying, fluidized-bed spray coating and emulsion precipitation. In addition to examining the individual process effect on protein stability that is always the focus of formulation scientists, this review also likes to evaluate each method from a more practical sense in terms of process versatility and scalability. The conclusion is that each method has its own advantages and the use of a method is formulation and application specific. With the understanding of the principles and advantages of these methods, it can benefit our choice on selecting appropriate techniques for preparing a desired protein powder formulation for specific applications.     

Development of a Highly Stable,Nonaqueous Glucagon Formulation for Delivery via Infusion Pump Systems

Background:Despite a vigorous research effort, to date, the development of systems that achieve glucagon stability in aqueous formulations (without reconstitution) has failed to produce any clinical candidates. We have developed a novel, nonaqueous glucagon formulation based on a biocompatible pharmaceutical solvent, dimethyl sulfoxide, which demonstrates excellent physical and chemical stability at relatively high concentrations and at high temperatures.Methods:This article reports the development of a novel, biocompatible, nonaqueous native human glucagon formulation for potential use in subcutaneous infusion pump systems.Results:Data are presented that demonstrate physical and chemical stability under presumed storage conditions (>2 years at room temperature) as well as “in use” stability and compatibility in an Insulet’s OmniPod^® infusion pump. Also presented are results of a skin irritation study in a rabbit model and pharmacokinetics/pharmacodynamics data following pump administration of glucagon in a diabetic swine model.Conclusions:This nonaqueous glucagon formulation is suitable for further clinical development in pump systems.     

Moisture effects on protein-excipient interactions in spray-dried powders. Nature of destabilizing effects of sucrose

The preparation of stable solid protein formulations presents significant challenges. Ultimately, the interactions between incorporated excipients and the pharmaceutical protein determine the formulation stability. In this study, moisture was utilized to probe the interactions between a model protein, trypsinogen, and sucrose in the solid state, following spray drying. Through investigation of the physical properties of the spray-dried formulations, we attempted to elucidate the mechanisms underlying the previously observed stabilizing and destabilizing effects of the carbohydrate during spray drying. Both dynamic and equilibrium moisture uptake studies indicated the presence of an optimal protein-sugar hydrogen bonding network. At low sucrose contents, a preferential protein-sucrose hydrogen bonding interaction was dominant, resulting in protein stabilization. However, at high carbohydrate concentrations, preferential sugar-sugar interactions prevailed, resulting in a phase separation within the formulation matrix. The preferential incorporation of the sucrose molecules in a sugar-rich phase reduced the actual amount of the carbohydrate available to interact with the protein and thereby decreased the number of effective protein-sucrose contacts. As a consequence, the protein could not be effectively protected during spray drying. We hypothesize that the observed phase separation at this sucrose concentration regime originates from its exclusion from the protein in solution before spray drying, further accompanied by preferential clustering of the sucrose molecules.     

The Effect of the Reconstitution Medium on Aggregation of Lyophilized Recombinant Interleukin-2 and Ribonuclease A

Pharmaceutical Research -     

Activity-stability considerations of trypsinogen during spray drying: effects of sucrose

The preparation and processing of protein pharmaceuticals into powders may impose significant stresses that could perturb and ultimately denature them. In many cases their stabilization through added excipients is necessary to yield native and active proteins. In this study, the effect of spray drying on the structure and activity of a model protein (trypsinogen) was investigated. In the absence of excipients, spray drying resulted in small losses of its enzymatic activity. Protein conformational rearrangements in the solid state (observed via FTIR) and irreversible aggregation (upon reconstitution) constituted the major degradation pathways. The irreversible unfolding in the solid state was also confirmed by solution calorimetric studies that indicated a decreased thermal stability of the spray-dried protein after reconstitution. The presence of sucrose, a thermal and dehydration stress stabilizer, induced a concentration-dependent protective effect. Protein protection was afforded even at low carbohydrate concentrations, while at specific mass ratios (sucrose-to-protein = 1:1) complete activity preservation was achieved. However, at the high end of sucrose concentrations, a small destabilization was evident, indicating that excluded volume effects may be undesirable during preparation of protein microparticles via spray drying. The profile of both the protein conformational changes and thermal stability in the solid state closely followed that of the incurred activity losses, indicating that protein stabilization during dehydration is crucial during processing of these polypeptides.     

Some Factors Associated with the Ultrasonic Nebulization of Proteins

Ultrasonic nebulization of lactate dehydrogenase (LDH) was investigated using a DeVilbiss Aerosonic nebulizer. The enzyme (8ml, 0.025mg/ml Na₂HPO₄, pH 7.0) was completely inactivated after 20 minutes of operation. However, the inactivation profile observed during ultrasonic nebulization was different from that previously observed using air-jet nebulization. At least two mechanisms are involved, one associated with heating and the other with aerosol production. By preventing heating of the nebulizer fluid during operation, the denaturation profile was dramatically altered. By additionally including 0.01% w/v Tween 80 or l%w/v PEG 8000, almost all activity was retained. Similar results were obtained by preventing aerosol production and heating. However, 100% of activity was lost when heating was allowed to occur without aerosol formation. The results demonstrate that cooling in conjunction with a surfactant is one approach that could be used to stabilize proteins to ultrasonic nebulization. However, cooling also significantly reduced solute output from the nebulizer. When operated at 10°C output was negligible. At 50°C the output was 5× greater than that found at room temperature. The median droplet size (µm) was not significantly influenced by the operating temperature of the nebulizer fluid (3.6 ± 0.4, 21°C; 3.9 ± 0.2, 50°C, p = NS (n = 6)) although the size distribution was noted to increase at the higher temperature.     

Factors affecting short-term and long-term stabilities of proteins

Proteins are marginally stable and, hence, are readily denatured by various stresses encountered in solution, or in the frozen or dried states. Various additives are known to minimize damage and enhance the stability of proteins. This review discusses the current knowledge of the mechanisms by which these additives stabilize proteins against acute stresses, and also the various factors to be considered for long-term storage of proteins in solution.     

Effects of Excipients on the Chemical and Physical Stability of Glucagon during Freeze-Drying and Storage in Dried Formulations

Purpose

To evaluate the effects of several buffers and excipients on the stability of glucagon during freeze-drying and storage as dried powder formulations.

Methods

The chemical and physical stability of glucagon in freeze-dried solid formulations was evaluated by a variety of techniques including mass spectrometry (MS), reversed phase HPLC (RP-HPLC), size exclusion HPLC (SE-HPLC), infrared (IR) spectroscopy, differential scanning calorimetry (DSC) and turbidity.

Results

Similar to protein drugs, maintaining the solid amorphous phase by incorporating carbohydrates as well as addition of surfactant protected lyophilized glucagon from degradation during long-term storage. However, different from proteins, maintaining/stabilizing the secondary structure of glucagon was not a prerequisite for its stability.

Conclusions

The formulation lessons learned from studies of freeze-dried formulations of proteins can be applied successfully to development of stable formulations of glucagon. However, peptides may behave differently than proteins due to their small molecule size and less ordered structure.     

A New Strategy for Enhancing the Stability of Lyophilized Protein: The Effect of the Reconstitution Medium on Keratinocyte Growth Factor

Purpose. Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF). Methods. The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer. Results. Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein. Conclusions. These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.     

Optimization of Lyophilization Conditions for Recombinant Human Interleukin-2 by Dried-State Conformational Analysis Using Fourier-Transform Infrared Spectroscopy

Purpose. Examination of the dried-state conformation of interleukin-2 (IL-2) was used to determine the pH conditions and stabilizers that provide optimal storage stability for the lyophilized product. Methods. Fourier-transform infrared spectroscopy and accelerated stability studies which examined solubility, aggregate formation, and covalent cross-linking were used. Results. Varying the pH in the absence of excipients resulted in dramatic differences in the dried state conformation of IL-2. At pH 7, IL-2 unfolds extensively upon lyophilization while at pH below 5 it remains essentially native. Additional unfolding was observed upon incubation at elevated temperatures. A strong direct correlation between the retention of the native (aqueous) structure during freeze-drying and enhanced stability is demonstrated. IL-2 prepared at pH 5 is approximately an order of magnitude more stable than at pH 7 with regard to formation of soluble and insoluble aggregates. A similar pH profile was observed in the presence of excipients, although the excipients alter the overall stability profile. Additional accelerated stability studies examined the stabilizers necessary for optimal stability. Conclusions. Excipients with the capacity to substitute for water upon dehydration better preserve the native structure resulting in enhanced stability. Those that have high glass transition temperatures provide the highest level of stability during storage, although they do not prevent dehydration induced unfolding.