Cycle Development in a Mini-Freeze Dryer: Evaluation of Manometric Temperature Measurement in Small-Scale Equipment.

The objective of this research was to assess the applicability of manometric temperature measurement (MTM) and SMART™ for cycle development and monitoring of critical product and process parameters in a mini-freeze dryer using a small set of seven vials. Freeze drying cycles were developed using SMART™ which automatically defines and adapts process parameters based on input data and MTM feedback information. The freeze drying behavior and product characteristics of an amorphous model system were studied at varying wall temperature control settings of the cylindrical wall surrounding the shelf in the mini-freeze dryer. Calculated product temperature profiles were similar for all different wall temperature settings during the MTM-SMART™ runs and in good agreement with the temperatures measured by thermocouples. Product resistance profiles showed uniformity in all of the runs conducted in the mini-freeze dryer, but absolute values were slightly lower compared to values determined by MTM in a LyoStar™ pilot-scale freeze dryer. The resulting cakes exhibited comparable residual moisture content and optical appearance to the products obtained in the larger freeze dryer. An increase in intra-vial heterogeneity was found for the pore morphology in the cycle with deactivated wall temperature control in the mini-freeze dryer. SMART™ cycle design and product attributes were reproducible and a minimum load of seven 10R vials was identified for more accurate MTM values. MTM-SMART™ runs suggested, that in case of the wall temperature following the product temperature of the center vial, product temperatures differ only slightly from those in the LyoStar™ freeze dryer.

Mannitol/l-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals: Effect of the l-Arginine Counter Ion and Formulation Composition on the Formulation Properties and the Physical State of Mannitol.

Previous studies have shown that protein storage stability in freeze-dried l-arginine-based systems improved in the presence of chloride ions. However, chloride ions reduced the glass transition temperature of the freeze concentrate (Tg') and made freeze drying more challenging. In this study, l-arginine was freeze dried with mannitol to obtain partially crystalline solids that can be freeze dried in a fast process and result in elegant cakes. We characterized the effect of different l-arginine counter ions on physicochemical properties of mannitol compared with mannitol/sucrose systems. Thermal properties of formulations with different compositions were correlated to thermal history during freeze drying and to physicochemical properties (cake appearance, residual moisture, reconstitution time, crystallinity). Partially crystalline solids were obtained even at the highest l-arginine level (mannitol:l-arginine of 2:1) used in this study. All l-arginine-containing formulations yielded elegant cakes. Only cakes containing l-arginine chloride and succinate showed a surface "crust" formed by phase separation. X-ray powder diffraction showed that inhibition of mannitol crystallization was stronger for l-arginine compared with sucrose and varied with the type of l-arginine counter ion. The counter ion affected mannitol polymorphism and higher levels of mannitol hemi-hydrate were obtained at high levels of l-arginine chloride.

Freeze-Drying of L-Arginine/Sucrose-Based Protein Formulations, Part 2: Optimization of Formulation Design and Freeze-Drying Process Conditions for an L-Arginine Chloride-Based Protein Formulation System.

We recently reported that the presence of chloride counter ions in freeze-dried l-arginine/sucrose formulations provided the greatest protein stability, but led to low collapse temperatures and glass transition temperatures of the freeze concentrates. The objectives of this study were to identify l-arginine chloride-based formulations and optimize freeze-drying process conditions to deliver a freeze-dried product with good physical quality attributes (including cake appearance, residual moisture, and reconstitution time). Additional properties were tested such as thermal properties, cake microstructure, and protein physical stability. Excipient concentrations were varied with and without a model protein (bovine serum albumin, BSA). Formulations were frozen with and without annealing or with and without controlled nucleation. Primary drying was conducted at high and low shelf temperature. Cakes with least defects and optimum physical attributes were achieved when protein to excipient ratios were high. Controlled nucleation led to elegant cakes for most systems at a low shelf temperature. Replacing BSA by a monoclonal antibody showed that protein (physical) stability was slightly improved under stress storage temperature (i.e., 40°C) in the presence of a low concentration of l-arginine in a sucrose-based formulation. At higher l-arginine concentrations, cake defects increased. Using optimized formulation design, addition of l-arginine chloride to a sucrose-based formulation provided elegant cakes and benefits for protein stability.

Stabilization challenges and formulation strategies associated with oral biologic drug delivery systems.

Delivery of proteins to mucosal tissues of GI tract typically utilize formulations which protect against proteolysis and target the mucosal tissues. Using case studies from literature and the authors' own work, the in-process stability and solid state storage stability of biopharmaceuticals formulated in delivery systems designed for oral delivery to the GI tract will be reviewed. Among the range of delivery systems, biodegradable polymer systems for protection and controlled release of proteins have been the most studied; hence these systems will be covered in greater depth. These delivery systems include polymeric biodegradable microspheres or nanospheres that contain proteins or vaccines, which are designed to reduce the number of administrations/inoculations and the total protein dose required to achieve the desired biological effect. Specifically, this review will include a landscape survey of the systems that have been studied, the manufacturing processes involved, stability through the manufacturing process, key pharmaceutical formulation parameters that impact stability of the encased proteins, and storage stability of the encapsulated proteins in these delivery systems.

Freeze drying of L-arginine/sucrose-based protein formulations, part I: influence of formulation and arginine counter ion on the critical formulation temperature, product performance and protein stability.

The objective of this study was to investigate product performance of freeze dried l-arginine/sucrose-based formulations under variation of excipient weight ratios, l-arginine counter ions and formulation pH as a matrix to stabilize a therapeutic monoclonal antibody (MAb) during freeze drying and shelf life. Protein and placebo formulations were lyophilized at aggressive primary drying conditions and key attributes of the freeze dried solids were correlated to their thermal properties and critical formulation temperature. Stability (physical) during processing and long-term storage of the MAb in different formulations was assessed by SE-HPLC. Thermal properties of the mixtures were greatly affected by the type of l-arginine counter ion. High glass transition temperatures were achieved by adding multivalent acids, whereas the temperature values significantly decreased in the presence of chloride ions. All mixtures were stable during freeze drying, but storage stability varied for the different preparations and counter ions. For l-arginine-based formulations, the protein was most stable in the presence of chloride ion, showing no obvious correlation to estimated global mobility of the glass. Besides drying behavior and thermal properties of the freeze dried solids, the counter ion of l-arginine must be considered relevant for protein shelf life stability.

Investigating factors leading to fogging of glass vials in lyophilized drug products.

Vial "Fogging" is a phenomenon observed after lyophilization due to drug product creeping upwards along the inner vial surface. After the freeze-drying process, a haze of dried powder is visible inside the drug product vial, making it barely acceptable for commercial distribution from a cosmetic point of view. Development studies were performed to identify the root cause for fogging during manufacturing of a lyophilized monoclonal antibody drug product. The results of the studies indicate that drug product creeping occurs during the filling process, leading to vial fogging after lyophilization. Glass quality/inner surface, glass conversion/vial processing (vial "history") and formulation excipients, e.g., surfactants (three different surfactants were tested), all affect glass fogging to a certain degree. Results showed that the main factor to control fogging is primarily the inner vial surface hydrophilicity/hydrophobicity. While Duran vials were not capable of reliably improving the level of fogging, hydrophobic containers provided reliable means to improve the cosmetic appearance due to reduction in fogging. Varying vial depyrogenation treatment conditions did not lead to satisfying results in removal of the fogging effect. Processing conditions of the vial after filling with drug product had a strong impact on reducing but not eliminating fogging.

Stability of lyophilized sucrose formulations of an IgG1: subvisible particle formation.

Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening ß-relaxations within the amorphous glass.

Structure, stability, and mobility of a lyophilized IgG1 monoclonal antibody as determined using second-derivative infrared spectroscopy.

There are many aspects of stabilization of lyophilized proteins. Of these various factors, retention of native structure, having sufficient amount of stabilizer to embed the protein within an amorphous matrix, and dampening ß-relaxations have been shown to be critical in optimizing protein stability during storage. In this study, an IgG1 was lyophilized with varying amounts of sucrose. In some formulations, a small amount of sorbitol was added as a plasticizer. The structure of the protein in dried state was monitored using infrared (IR) spectroscopy. The IR spectra indicated increasing retention of the native structure, which correlated with stability as indicated by size-exclusion chromatography as well as micro-flow imaging. Maximal stability was achieved with a 2:1 mass ratio of sucrose to protein, which is more than that would be expected based on earlier studies. Analysis of both high and low frequency bands associated with intramolecular ß-sheet structure provides additional information on the structure of antibodies in the solid state. Finally, there is a correlation between the bandwidth of the ß-sheet bands and the enthalpy of relaxation, suggesting that amide I bands can provide some indication of the degree of coupling to the sugar matrix, as well as structural heterogeneity of the protein.

The impact of drying method and formulation on the physical properties and stability of methionyl human growth hormone in the amorphous solid state.

The objective of this work was to investigate the impact of drying method and formulation on the physical stability (aggregation) and selected important physical properties of dried methionyl human growth hormone (Met-hGH) formulations. Solutions of Met-hGH with different stabilizers were dried by different methods (freeze drying, spray drying, and film drying), with and without surfactant. Properties of the dried powders included powder morphology, specific surface area (SSA), protein surface coverage, thermal analysis, and protein secondary structure. Storage stability of Met-hGH in different formulations was also studied at 50 degrees C and at 60 degrees C for 3 months. The dried powders displayed different morphologies, depending mainly on the method of drying and on the presence or absence of surfactant. Film dried powders had the lowest SSA (approximately 0.03 m(2)/g) and the lowest total protein surface accumulation (approximately 0.003%). Surfactant caused a reduction in the SSA of both spray dried and freeze dried powders. Spray dried powders showed greater protein surface coverage and SSA relative to the same formulations dried by other means. Greater in-process perturbations of protein secondary structure were observed with polymer excipients. Formulation impacted physical stability. In general, low molecular weight stabilizers provided better stability. For example, the aggregation rate at 50 degrees C of Met-hGH in a freeze dried trehalose-based formulation was approximately four times smaller than the corresponding Ficoll-70-based formulation. Drying method also influenced physical stability. In general, the film dried preparations studied showed superior stability to preparations dried by other methods, especially those formulations employing low molecular weight stabilizers.

The effect of annealing on the stability of amorphous solids: chemical stability of freeze-dried moxalactam.

The objective of this study was to investigate the effect of annealing on the chemical stability and calorimetric structural relaxation times of freeze-dried moxalactam. Moxalactam disodium was freeze dried with 12% mannitol and split into several batches after freeze drying. One batch was held as a control while others were subjected to a further heating (annealing) treatment at 60 degrees C, 70 degrees C, and 80 degrees C for different periods of time. Isothermal microcalorimetry studies using thermal activity monitor (TAM) were performed on the freeze-dried samples to measure relaxation times (tau) and stretched exponential values (beta). Modulated DSC experiments were carried out to determine T(g) and DeltaC(P) for moxalactam-12% w/w mannitol systems at various moisture contents to allow extrapolation of these quantities to zero residual moisture. Storage stability studies were performed at 25 degrees C, 40 degrees C and 50 degrees C. Decarboxylated moxalactam and parent contents after various storage times were measured by reverse phase HPLC. Annealing moxalactam-12% mannitol amorphous systems improved chemical stability of moxalactam and reduced molecular mobility, as measured by TAM. Moxalactam-12% w/w mannitol systems annealed at higher temperatures and for longer times had higher tau(beta) values than the "control" sample, with tau(beta) values increasing as annealing temperature increased. Additionally, tau(beta) value increased as annealing time at the same temperature increased. These observations indicated that higher temperature annealing decreased molecular mobility in the glass, as expected. Further, chemical stability improved as annealing temperatures and annealing times increased. For example, the rate of decarboxylation of the sample annealed at 70 degrees C for 8 h was roughly 1.7 times lower than the "control." Note that in spite of degradation during the annealing process, the level of degradation at the end of storage is actually less in the annealed sample than in the control sample; thus, annealing can result in samples having less degradation at the end of a storage period. Chemical stability and relaxation times are correlated, thus indicating that molecular mobility and structural relaxation time are coupled.

Drying-induced variations in physico-chemical properties of amorphous pharmaceuticals and their impact on Stability II: stability of a vaccine.

OBJECTIVES: To investigate the impact of drying method on the storage stability of dried vaccine formulations. MATERIALS AND METHODS: A sucrose-based formulation of a live attenuated virus vaccine of a parainfluenza strain, with and without surfactant, was dried from by different methods; freeze drying, spray drying and foam drying. Dried powders were characterized by differential scanning calorimetry, specific surface area (SSA) analysis and by electron spectroscopy for chemical analysis (ESCA) to evaluate vaccine surface coverage in the dried formulations. Dried formulations were subjected to storage stability studies at 4, 25 and 37 degrees C. The vaccine was assayed initially and at different time points to measure virus-cell infectivity, and the degradation rate constant of the vaccine in different dried preparations was determined. RESULTS: SSA was highest with the spray dried preparation without surfactant (approximately 2.8 m(2)/g) and lowest in the foam dried preparations (with or without surfactant) (approximately 0.1 m(2)/g). Vaccine surface coverage was estimated based on ESCA measurements of nitrogen content. It was predicted to be highest in the spray dried preparation without surfactant and lowest in the foam with surfactant. Stability studies conducted at 25 degrees C and 37 degrees C showed that the vaccine was most stable in the foam dried preparation with surfactant and least stable in spray dried preparations without surfactant and in all freeze dried preparations regardless of the presence of surfactant. Addition of surfactant did lower the SSA and vaccine surface coverage in freeze dried preparations but still did not improve storage stability. CONCLUSIONS: In drying methods that did not involve a freezing step, good storage stability of Medi 534 vaccine in the dried form was found with low SSA and low vaccine surface accumulation, both of which integrate into low fraction of vaccine at the surface. Ice appears to be a major destabilizing influence.

Drying-induced variations in physico-chemical properties of amorphous pharmaceuticals and their impact on stability (I): stability of a monoclonal antibody.

The present study was conducted to investigate the impact of drying method and formulation on the storage stability of IgG1. Formulations of IgG1 with varying levels of sucrose with and without surfactant were dried by different methods, namely freeze drying, spray drying, and foam drying. Dried powders were characterized by thermal analysis, scanning electron microscopy, specific surface area (SSA) analysis, electron spectroscopy for chemical analysis (ESCA), solid state FTIR, and molecular mobility measurements by both isothermal calorimetry and incoherent elastic neutron scattering. Dried formulations were subjected to storage stability studies at 40 degrees C and 50 degrees C (aggregate levels were measured by size exclusion chromatography initially and at different time points). Both drying method and formulation had a significant impact on the properties of IgG1 powders, including storage stability. Among the drying methods, SSA was highest and perturbations in secondary structure were lowest with the spray-dried preparations. Sucrose-rich foams had the lowest SSA and the lowest protein surface accumulation. Also, sucrose-rich foams had the lowest molecular mobility (both fast dynamics and global motions). Stability studies showed a log-linear dependence of physical stability on composition. Preparations manufactured by "Foam Drying" were the most stable, regardless of the stabilizer level. In protein-rich formulations, freeze-dried powders showed the poorest storage stability and the stability differences were correlated to differences in secondary structure. In stabilizer-rich formulations, stability differences were best correlated to differences in molecular mobility (fast dynamics) and total protein surface accumulation.

The challenge of drying method selection for protein pharmaceuticals: product quality implications.

Numerous drying methods are used to dry solutions of proteins in the laboratory and/or in pharmaceutical manufacturing. In this review article, we will discuss many of these drying methods. We will briefly introduce and compare the unit operations involved in the drying methods to give an insight on thermal history, and the different stresses that a drying method can present to an active ingredient, particularly for protein molecules. We will review and compare some important physico-chemical properties of the dried powder that result from using different drying methods such as specific surface area, molecular dynamics, secondary structure (for protein molecules), and composition heterogeneity. We will discuss some factors that might lead to differences in the physico-chemical properties of different powders of the same formulation prepared by different techniques. We will examine through a literature review how differences in some of these properties can affect storage stability. Also, we will review process modifications of the basic drying methods and how these modifications might impact physico-chemical properties, in-process stability and/or storage stability of the dried powders.

Properties and stability of a liquid crystal form of cyclosporine-the first reported naturally occurring peptide that exists as a thermotropic liquid crystal.

A new solid-state form of cyclosporine produced by spray-drying exhibited characteristics consistent with a liquid crystal. No sharp diffraction peaks were observed by powder X-ray diffraction; however, analysis by both small-angle X-ray diffraction (SAXR) and microscopic under polarized light (PLM) confirmed the existence of two-dimensional ordered liquid crystal. Hot stage microscopy revealed a solid-to-liquid transition, in the range of 118 to 125 degrees C. Moreover, the solid-to-liquid transition showed frequency dependence by dielectric analysis (DEA), and was coincidental with a stepwise heat capacity change measured by differential scanning Calorimetry (DSC). The two-dimensional order was maintained above the solid-to-liquid transition temperature indicated by low-angle diffraction by SAXR and birefringence by PLM. However, birefringence was lost at temperatures above 170 degrees C, indicating the conversion of the liquid crystal into an isotropic liquid. In situ annealing experiments, by DSC, revealed the presence of an endotherm, unexplained by either a phase transition or solvent loss, and it is believed to be the result of a structural rearrangement that has no impact on the macroscopic properties of the material. Spray-dried cyclosporine at room temperature is therefore a frozen thermotropic liquid crystal due to the presence of two-dimensional order and the lack of substantial residual solvent. This is, to our knowledge, the first report of the existence of a thermotropic liquid crystal of a naturally occurring peptide.

Quantitative in vitro comparison of fluorescein delivery to the eye via impregnated paper strip and volumetric techniques.

PURPOSE: To compare the quantity of fluorescein delivered to the eye via fluorescein-impregnated paper strips of various sizes and surface areas and via various microliter volumes of fluorescein sodium using an in vitro assay. METHODS: A commercially available fluorescein-impregnated strip (75 mm2) and three modified strips of reduced fluorescein-impregnated surface areas (10, 7.5, and 5.0 mm2) were used. The amount of fluorescein delivered to the eye for each of the four strips was approximated by applying each strip to a Whatman No. 1 filter paper under conditions simulating application of the strip to the eye, extracting the fluorescein from the filter paper in an aqueous solution, and performing spectrophotometric analysis at 484 nm. Similarly, this filter paper analytical system was calibrated using 1, 2, and 3 microl volumes of 2% w/v fluorescein delivered to the filter paper. RESULTS: Using calibration curves, linearity was observed between absorbance and concentration of fluorescein sodium with an R2 value > or = 0.99. Using these calibration curves, the amount of fluorescein delivered to the eye for the four strips and the three fluorescein solution samples was determined. Fluorescein-impregnated strips with surface areas of 75, 10, and 5 mm2 delivered approximately the same quantity of fluorescein to the ocular surface as 3 microl, 1 microl, and 0.5 microl of fluorescein 2% solution, respectively. CONCLUSIONS: The surface area of the fluorescein-impregnated portion of the strip can be designed to control the amount of fluorescein delivered to the eye.

A new high-performance liquid chromatography (HPLC) method for the analysis of halofantrine (HF) in pharmaceuticals.

A simple, rapid and stability-indicating reverse-phase high-performance liquid chromatography (HPLC) method was developed for the assay of halofantrine (HF) base, 1,3-dichloro-alpha-[2-(dibutylamino)ethyl]-6-(trifluoromethyl)-9-phenanthrenemethanol. The HPLC method was validated for precision, accuracy, selectivity and linearity and range. It was used to assay for HF base in solid dispersions. Excellent linearity was observed between HF base concentration and the peak area (R(2)=0.9998). The limit of detection was 1 ng (with a signal-to-noise ratio of 2:1), and the limit of quantitation was 10 ng (with a signal-to-noise ratio of 10:1). The method proved to be selective. Selectivity was validated by subjecting a stock solution of HF to acidic, basic, oxidative and thermal degradations. The peaks of the degradation products did not interfere with the peak of HF. Excipients present in the solid dispersions did not interfere with the analysis.

Preparation and in vitro evaluation of solid dispersions of halofantrine.

The low aqueous solubility of halofantrine (HF) and its low bioavailability from commercially available tablets (Halfan) suggested the formulation of solid dispersions (SDs) of HF to reduce its particle size and improve its wettability and aqueous solubility. Preformulation studies involved the development of a high performance liquid chromatography (HPLC) method for the analysis of HF. In addition, solubility studies were conducted on HF in aqueous solutions containing different concentrations of various carriers. Formulation studies included the preparation of SDs and physical mixtures (PMs) of HF with different carriers and their physicochemical characterization using differential scanning calorimetry (DSC), Fourier-Transform infra-red (FT-IR) spectroscopy and dissolution studies. A 3-month stability study at elevated temperatures was conducted on representative SDs of HF with selected carriers.