Michael J. Pikal

This article evaluates the procedures for determining the vial heat transfer coefficient and the extent of primary drying through manometric temperature measurement (MTM). The vial heat transfer coefficients (K_v) were calculated from the MTM-determined temperature and resistance and compared with K_v values determined by a gravimetric method. The differences between the MTM vial heat transfer coefficients and the gravimetric values are large at low shelf temperature but smaller when higher shelf temperatures were used. The differences also became smaller at higher chamber pressure and smaller when higher resistance materials were being freeze-dried. In all cases, using thermal shields greatly improved the accuracy of the MTM K_v measurement. With use of thermal shields, the thickness of the frozen layer calculated from MTM is in good agreement with values obtained gravimetrically. The heat transfer coefficient “error” is largely a direct result of the error in the dry layer resistance (ie, MTM-determined resistance is too low). This problem can be minimized if thermal shields are used for freeze-drying. With suitable use of thermal shields, accurate K_v values are obtained by MTM; thus allowing accurate calculations of heat and mass flow rates. The extent of primary drying can be monitored by real-time calculation of the amount of remaining ice using MTM data, thus providing a process analytical tool that greatly improves the freeze-drying process design and control.

Predicting onsets of crystallization at temperatures below T _g, from data above T _g, would require that the correlation between crystallization onset and mobility is same above and below T _g, and the techniques being used to measure mobility above and below T _g are measuring essentially the same kind of mobility. The aim of this work is to determine if the relaxation times obtained using different techniques (DSC, TAM) below T _g correlate with relaxation time obtained above T _g using dielectric spectroscopy. Model compounds for this work were chosen based on their varied ΔH _f, ΔC _p(T _g) and H-bonding in crystalline state vs. amorphous state. Relaxation times above T _g were determined by the simultaneous fit of real and imaginary permittivity to the Cole-Davidson model. Tau and beta below T _g were determined using isothermal microcalorimetry (TAM) or MDSC. MDSC was used to calculate Kauzmann temperature and strength of the glass using established relationships. Indomethacin, nifedipine and flopropione showed Arrhenius temperature dependence throughout the entire temperature range and extrapolation of τ ^β measured above T _g by dielectric relaxation agreed with τ ^β measured below T _g by TAM/MDSC. Ketoconazole, however, showed the expected VTF behavior. For at least two compounds compared (indomethacin and ketoconazole), relaxation times measured by TAM and MDSC did not agree, with TAM giving significantly lower values of τ ^β , but TAM and MDSC relaxation times appeared to extrapolate to a common value at T _g. It was found that, for all cases studied, relaxation time constants determined above and below T _g did appear to extrapolate to the same value around T _g indicating that molecular mobility measured above and below T _g using different techniques is highly correlated.

The objective of this research was to estimate differences in heat and mass transfer between freeze dryers due to inherent design characteristics using dat

This study examines the factors that may cause systematic errors in the manometric temperature measurement (MTM) procedure used to evaluate product tempera

The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying. The cold denaturation of both phosphoglycerate kinase (PGK) and β-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (T _zero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution. Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at −10°C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with β-lactoglobulin. The half-lives of PGK and β-lactoglobulin unfolding are estimated to be 5.5 × 10¹¹ and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20°C above T _g′. Thus, freeze-drying below T _g′ should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60°C above the system T _g′ in a thermodynamically unstable system during freeze-drying. Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above T _g′. Thus, it is not always necessary to freeze-dry protein formulations at temperature below T _g′ to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the T _g′, thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.

The aim of the study is to investigate the effects of stabilizers and denaturants on the thermal and cold denaturation temperatures of selected proteins in systems of interest to freeze-drying. β-Lactoglobulin and phosphoglycerate kinase (PGK) were chosen as model proteins. Protein thermal and cold denaturation temperatures were determined by both conventional and modulated differential scanning calorimetry and verified by tryptophan emission spectroscopy in selected systems. The cold denaturation of β-lactoglobulin was reversible, whereas the thermal denaturation was only reversible at high scanning rate (10°C/min). The cold denaturation temperatures of β-lactoglobulin decreased with an increase in protein concentration (self-stabilization). The cold denaturation temperature increased with increases in pH (from pH 2 to 7) with about 4.6°C increase per unit pH change. All stabilizers studied (i.e., sucrose, trehalose and glycerol) increased the thermal denaturation temperature of the proteins studied and decreased the cold denaturation temperature. The effect of sucrose in decreasing the PGK cold denaturation temperature [40°C per molar concentration increase (40°C/M)] was of the same magnitude as for β-lactoglobulin (36°C/M). The effect of stabilizers on cold denaturation temperatures is much greater than the effect on thermal denaturation temperatures. With sucrose, the β-lactoglobulin thermal denaturation temperature increases only about 5°C from 0 to 2.7 M, whereas the decrease in cold denaturation temperature was more than 35°C even at sucrose concentrationsas low as 0.9 M. Denaturants (urea and guanidine hydrochloride) increased the cold denaturationtemperatures of proteins and thereby destabilized protein; the magnitudes were 9°C/M (urea on T _cd of β-lactoglobulin) and 65°C/M (guanidine hydrochloride on PGK) compared with literature data of 16°C/M (guanidine hydrochloride on β-lactoglobulin). The cold denaturation temperatures of β-lactoglobulinand PGK extrapolated to zero concentration of denaturants were −14 and −26°C, respectively. The protein cold denaturation temperature was pH-, protein concentration-, and additive-dependent. Stabilizers, such as sugars and/or polyols, can stabilize both protein thermal and cold denaturation, whereas the denaturants destabilize protein cold denaturation. The stabilization effect on protein cold denaturation is much larger than on thermal denaturation, a result of great importance in protein freeze-drying.

To develop a procedure based on manometric temperature measurement (MTM) and an “expert system” for good practices in freeze drying that will allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment.

Introduction. Theoretical models of the freeze-drying process are potentially useful to guide the design of a freeze-drying process as well as to obtain information not readily accessible by direct experimentation, such as moisture distribution and glass transition temperature, Tg, within a vial during processing. Previous models were either restricted to the steady state and/or to one-dimensional problems. While such models are useful, the restrictions seriously limit applications of the theory. An earlier work from these laboratories presented a nonsteady state, two-dimensional model (which becomes a three-dimensional model with an axis of symmetry) of sublimation and desorption that is quite versatile and allows the user to investigate a wide variety of heat and mass transfer problems in both primary and secondary drying. The earlier treatment focused on the mathematical details of the finite element formulation of the problem and on validation of the calculations. The objective of the current study is...