Monoclonal antibody interactions with micro- and nanoparticles: adsorption, aggregation, and accelerated stress studies.

Therapeutic proteins are exposed to various wetted surfaces that could shed subvisible particles. In this work we measured the adsorption of a monoclonal antibody (mAb) to various microparticles, characterized the adsorbed mAb secondary structure, and determined the reversibility of adsorption. We also developed and used a front-face fluorescence quenching method to determine that the mAb tertiary structure was near-native when adsorbed to glass, cellulose, and silica. Initial adsorption to each of the materials tested was rapid. During incubation studies, exposure to the air-water interface was a significant cause of aggregation but acted independently of the effects of microparticles. Incubations with glass, cellulose, stainless steel, or Fe(2)O(3) microparticles gave very different results. Cellulose preferentially adsorbed aggregates from solution. Glass and Fe(2)O(3) adsorbed the mAb but did not cause aggregation. Adsorption to stainless steel microparticles was irreversible, and caused appearance of soluble aggregates upon incubation. The secondary structure of mAb adsorbed to glass and cellulose was near-native. We suggest that the protocol described in this work could be a useful preformulation stress screening tool to determine the sensitivity of a therapeutic protein to exposure to common surfaces encountered during processing and storage.

Correlation between chemical reactivity and the Hammett acidity function in amorphous solids using inversion of sucrose as a model reaction.

The goal was to evaluate the effects of acidity, expressed as the Hammett acidity function, on chemical reactivity in freeze-dried materials (lyophiles). Dextran-sucrose-citrate and polyvinyl pyrrolidone (PVP)-sucrose-citrate aqueous solutions, adjusted to pH values of 2.6, 2.8, and 3.0 were freeze dried, and characterized by X-ray powder diffractometry, DSC, isothermal microcalorimetry, and Karl Fischer titrimetry. Lyophiles were also prepared from identical solutions but containing bromophenol blue (BB). Diffuse reflectance-visible spectroscopy was used to measure the extent of BB protonation from which the Hammett acidity functions were determined. The stability studies were performed at 60 degrees C. All the freeze-dried samples were observed to be X-ray amorphous with <0.15% w/w water content. The T(g) of dextran lyophiles were approximately 20 degrees C higher than that of PVP lyophiles whereas enthalpy relaxation rates at 60 degrees C were similar. The Hammett acidity functions were significantly lower (i.e., higher acidity) for dextran systems (<2.2-2.6) when compared with PVP systems (3.3-3.9). The rate of sucrose inversion was significantly (an order of magnitude) higher in dextran lyophiles. This study showed that in amorphous matrices with comparable water content and structural relaxation times, chemical reactivity could be significantly different depending on the matrix "acidity".

Impact of freeze-drying on ionization of sulfonephthalein probe molecules in trehalose-citrate systems.

"pH memory," i.e., correlation between pH of solution before freeze-drying and chemical reactivity in the freeze-dried state, has been reported in many systems. In this study, the "pH memory" is explored by comparing the extent of protonation of sulfonephthalein probe molecules, bromophenol blue, bromocresol green, and chlorophenol red, in aqueous solution in the pH range of 3.4-6.0 and in the resulting freeze-dried amorphous matrix (lyophile) containing trehalose and sodium citrate buffer. The protonation of the probe molecules was measured in the lyophiles by diffuse reflectance visible spectroscopy, and compared with that in the solution before drying. The protonation of the indicators in the amorphous matrix correlated with solution pH, that is, an increase in solution pH resulted in a progressive decrease in the indicator protonation in the corresponding lyophile. However, the protonation was consistently higher in the lyophile than in the corresponding solution. The Hammett acidity function of lyophiles was calculated based on the extent of protonation of the probe molecules. Protonation of the probe molecules and the Hammett acidity function depended not only on prelyophilization solution pH, but also on the residual water content and the presence of amorphous sugar in the lyophile.

Raffinose crystallization during freeze-drying and its impact on recovery of protein activity.

PURPOSE: To study i) phase transitions in raffinose solution in the frozen state and during freeze-drying and ii) evaluate the impact of raffinose crystallization on the recovery of protein activity in reconstituted lyophiles. METHODS: X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions of raffinose pentahydrate. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze-drying process, in situ, in the sample chamber of the diffractometer. The activity of lactate dehydrogenase (LDH) in reconstituted lyophiles was determined spectrophotometrically. RESULTS: Raffinose formed a kinetically stable amorphous freeze-concentrated phase when aqueous solutions were frozen at different cooling rates. When these solutions were subjected to primary drying without annealing, raffinose remained amorphous. Raffinose crystallized as the pentahydrate when the solutions were annealed at a shelf temperature of -10 degrees C. Primary drying of these annealed systems resulted in the dehydration of raffinose pentahydrate to an amorphous phase. The phase separation of the protein from the amorphous raffinose in these two systems during freeze-drying resulted in a significant reduction in the recovery of LDH activity, even though the lyophile was amorphous. CONCLUSIONS: Annealing of frozen aqueous raffinose solutions can result in solute crystallization, possibly as the pentahydrate. The crystalline pentahydrate dehydrates during primary drying to yield an amorphous lyophile. Raffinose crystallization during freeze-drying is accompanied by a significant loss of protein activity.

Partially crystalline systems in lyophilization: II. Withstanding collapse at high primary drying temperatures and impact on protein activity recovery.

In an accompanying article we have described the construction of the water-rich sections of raffinose-glycine-water and trehalose-glycine-water state diagrams. In this study, we use the information obtained from the state diagrams to identify the minimum weight fraction of the crystalline component in glycine-carbohydrate systems necessary to withstand collapse at high primary drying temperatures. We also determine the impact of primary drying, substantially above T'g, on the recovery of lactate dehydrogenase (LDH) activity. Ambient and variable temperature X-ray powder diffractometry and differential scanning calorimetry were used to characterize the frozen and freeze-dried systems. Aqueous solutions with glycine to carbohydrate (raffinose pentahydrate or trehalose dihydrate) weight ratios ranging from 0.2 to 2.0 were freeze dried. The protein formulations contained 20 mM citrate buffer (pH 6.0) and LDH (20 microg/mL). A glycine to anhydrous raffinose weight ratio >or=1.18 and a glycine to anhydrous trehalose weight ratio >or=1.56 were necessary to withstand macroscopic collapse in the system, when the primary drying was carried out at a product temperature at least 10 degrees C above the T'g. The recovery of LDH activity was almost complete in the reconstituted lyophile whether the primary drying was carried out above T'g (-10 degrees C) or below T'g (-32 degrees C). Thus, by judiciously combining crystalline and amorphous components, it was possible to primary dry at temperatures substantially above the T'g.

Partially crystalline systems in lyophilization: I. Use of ternary state diagrams to determine extent of crystallization of bulking agent.

Two model ternary systems: water-glycine-raffinose and water-glycine-trehalose were investigated to determine the extent of glycine crystallization in frozen solutions. The use of such partially crystalline systems allows primary drying to be carried out substantially above the collapse temperature. Differential scanning calorimetry (DSC) and variable temperature X-ray diffractometry (XRD) were used to monitor phase transitions in frozen systems as well as to determine the T'g. Aqueous solutions containing different glycine to carbohydrate weight ratios were first cooled to -60 degrees C and then warmed to room temperature. In both raffinose and trehalose systems, when the initial glycine to sugar (raffinose pentahydrate or trehalose dihydrate) ratio was <1, glycine crystallization was not detected. When the ratio was >or=1, partial glycine crystallization was observed during warming. The presence of amorphous glycine caused the T'g to be substantially lower than that of the solution containing only the carbohydrate. To determine the extent of glycine crystallization, the solutions were annealed for 5 h just above the temperature of glycine crystallization. The T'g observed in the second warming curve was very close to that of the carbohydrate solution alone, indicating almost complete glycine crystallization. These studies enabled the construction of the water-rich sections of the raffinose-glycine-water and trehalose-glycine-water state diagrams. These diagrams consist of a kinetically stable freeze-concentrated solution and a doubly unstable glassy region, which readily crystallizes during cooling or subsequent warming. In addition, there is an intermediate region, where during the experimental timescale, there appears to be hindered glycine nucleation but unhindered crystal growth. To obtain substantially crystalline glycine in the frozen solutions, the glycine to carbohydrate ratios should be >or=1.

Acid-base characteristics of bromophenol blue-citrate buffer systems in the amorphous state.

In this study, we have examined the acid-base characteristics of various citrate buffer systems alone and in the presence of the pH indicator dye, bromophenol blue, in aqueous solution, and after lyophilization to produce amorphous material. Fourier transform Raman and solid-state nuclear magnetic resonance spectroscopy have been used to monitor the ratio of ionized to un-ionized citric acid under various conditions, as a function of initial pH in the range of 2.65-4.28. Ultraviolet-visible spectrophotometry was used to probe the extent of proton transfer of bromophenol blue in the citrate buffer systems in solution and the amorphous state. Spectroscopic studies indicated greater ionization of citric acid and bromophenol blue in solution and the solid state with increasing initial solution pH, as expected. Fourier transform Raman measurements indicated the same ratio of ionized to un-ionized citrate species in solution, frozen solution, and the amorphous state. It is shown that the ratio of species at any particular initial pH is primarily determined by the amount of sodium ion present so as to maintain electroneutrality and not necessarily to the fact that pH and pK(a) remain unchanged during freezing and freeze drying. Indeed, for bromophenol blue, the relative ultraviolet-visible intensities for ionized and un-ionized species in the amorphous sample were different from those in solution indicating that the extent of protonation of bromophenol blue was significantly lower in the solid samples. It is concluded that under certain conditions there can be significant differences in the apparent hydrogen activity of molecules in amorphous systems.

Solute crystallization in mannitol-glycine systems--implications on protein stabilization in freeze-dried formulations.

The use of mannitol in combination with glycine has resulted in stable freeze-dried protein formulations. Our objectives were to (1) study solute crystallization in ternary systems containing mannitol, glycine, and water during all the stages of freeze drying as a function of processing conditions and formulation variables; (2) investigate the effect of sodium phosphate buffer salts on the crystallization of both mannitol and glycine and vice versa; and (3) investigate the effects of these excipients in a freeze-dried lactate dehydrogenase (LDH) formulation. X-ray powder diffractometry (XRD) and differential scanning calorimetry (DSC) were used to study the frozen aqueous solutions. Phase transitions during primary and secondary drying were monitored by simulating the entire freeze-drying process in situ in the sample chamber of the diffractometer. LDH activity after freeze drying was determined spectrophotometrically. In frozen aqueous solutions containing mannitol and glycine, each solute influenced the extent of crystallization of the other. The solutes crystallized as delta-mannitol and beta-glycine during primary drying. Glycine had a stronger tendency to crystallize, while it was easier to influence mannitol crystallization. The buffer salts inhibited the crystallization of mannitol and glycine. However, in some cases, during primary drying, glycine crystallization was followed by that of disodium hydrogen phosphate dodecahydrate. The latter underwent dehydration forming an amorphous anhydrate. It was possible to correlate the extent of crystallization of mannitol and glycine in the lyophile with the retention of protein activity. An increase in buffer concentration decreased the crystallinity of mannitol and glycine. This translated to increased retention of protein activity.

Crystalline to amorphous transition of disodium hydrogen phosphate during primary drying.

PURPOSE: To monitor the phase transitions during freeze-drying of disodium hydrogen phosphate. METHODS: The variable temperature sample stage of the X-ray diffractometer (XRD) was attached to a vacuum pump, which enabled the entire freeze-drying process to be carried out in the sample chamber. The phase transitions during the freeze-drying cycle were monitored in real time by XRD. Aqueous buffer solution (containing disodium hydrogen phosphate and sodium dihydrogen phosphate) was cooled at 2 degrees C/min from room temperature to -70 degrees C. It was then heated to -25 degrees C and subjected to primary drying for 2 h at a chamber pressure of approximately 100 mTorr, followed by secondary drying at -10 degrees C. RESULTS: In the frozen solution, disodium hydrogen phosphate had crystallized as the dodecahydrate (Na2HPO4 x 12H2O) as was evident from its characteristic lines at approximately 5.37, 4.27, and 2.81 angstroms. Primary drying for 2 h resulted in ice sublimation, and the complete disappearance of the dodecahydrate peaks. CONCLUSION: The dehydration of the crystalline dodecahydrate resulted in an amorphous anhydrate. Thus the amorphous nature of the end product is a result of phase transitions during the process and do not reflect the solid-state of the ingredients during the entire process.

Characterization of physical mixtures and directly compressed tablets of sulfamerazine polymorphs: implications on in vitro release characteristics.

The present study evaluates the effects of excipients, compression pressure, and relative humidity (RH) on the stability of sulfamerazine polymorphs (referred here as SMZ I and SMZ II) and their release from directly compressed tablets using differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and dissolution analysis. SMZ I and SMZ II tablets were compressed with magnesium stearate (MGST), and microcrystalline cellulose (MCC) at 5000, 7500, and 10,000 lbs. pressures and stored at 40, 75, 95, and 100% RH conditions for 5 weeks. There were indications of possible drug-excipient interaction in the binary mixtures under different relative humidity conditions from the DSC data, but they could not be confirmed by PXRD because the crystal structures of the drug and excipients remained unaltered. The crystal structures of the polymorphs in the tablet also remained unaltered under the above conditions. There were, however, significant differences observed in the drug release properties of the two polymorphs. SMZ II was found in general to have a higher rate of drug release than SMZ I. Extensive gelation of MCC under higher moisture conditions, compression pressure during tableting, and inherent tabletability of the sulfamerazine crystals were factors that affected drug release. All these factors contributed towards prolonging the disintegration and deaggregation of the tablet particles and were therefore concluded to be the rate limiting steps for the dissolution process.

Estimating vapor pressure curves by thermogravimetry: a rapid and convenient method for characterization of pharmaceuticals.

The purpose of this study was to investigate a rapid method for the evaluation of vaporization characteristics for selected benzoic acid derivatives. The compounds studied in this context were the ortho-, meta- and para-derivatives of hydroxy and amino benzoic acids. Calculations for the order of reaction were first carried out for each of the compounds using methyl paraben as the calibration standard. Those compounds undergoing zero order, non-activated evaporation processes, were analyzed by the Antoine and Langmuir equations, conjointly. The coefficient of vaporization was obtained as 1.2 x 10(5)+/-0.8 Pakg (0.5)mol(0.5)s(-1)m(-2)K(-0.5). The vapor pressure values were used to determine the Antoine constants using the SPSS 10.0 software. This study attempts to outline a comprehensive thermogravimetric technique for vapor pressure characterization of single-component systems.

An evaporation study for phthalic acids--a rapid method for pharmaceutical characterization.

The objective of this study was to develop and analyze an analytical method in order to evaluate preformulation candidates by their thermodynamic parameters and evaporation characteristics. Ortho, meta and tere-phthalic acids were chosen as model compounds. The relative advantages and disadvantages of a rapid thermogravimetric method have been studied in detail, which would aid in the preformulation characterization for pharmaceuticals. Methyl paraben was taken as the model compound for calibration, as its evaporation characteristics are well known. Using the Antoine and the Langmuir equation for evaporation conjointly, the parameter k, known as the coefficient of evaporation was determined. The value for this constant was validated by three methods simultaneously. Previously the use of such methods for compounds having uninhibited zero order evaporation has been documented. In the present study, phthalic acid was chosen as the model compound since its evaporation is a two-step overlapping phenomenon. In this study we have shown the use of Pressure Differential Scanning Calorimetry in separating such simultaneous endothermic processes. The Clausius-Clapeyron equation seemingly has anomalous behavior for vapor pressure over high temperature ranges. In this study a modification of the equation has been suggested to take into account the changes in the heat capacities that result due to high temperature effects. This study aims at documenting a concise method for rapid pharmaceutical characterization and suggests modifications for some basic thermodynamic parameters over higher temperature ranges.