INTRODUCTION:

Freeze drying, as a unit operation in the production of parenteral drugs, is becoming more prevalent because many of the new molecular entities coming out of discovery have less than two years of shelf life before they expire. Formulators usually use large-scale machines to determine the right conditions for freeze-drying products and extending shelf life, which can makes, this process the most expensive and time consuming (1). Each active pharmaceutical ingredient has limitations and requirements that either prevent or promote the addition of certain excipients desirable for the lyophilization process (2). A freeze dried formulation generally consists of various excipients such as sugars and polymers or proteins to stabilize biomolecule conformation against denaturation which is caused by water removal during freeze drying.

These excipients usually remain amorphous during and after freeze drying. Due to use of improper FDC protocol the excipients used in these formulations are usually expected to be in crystalline state after processing. Crystallization of excipients generally tends to eliminate any stabilizing effect of the excipients on the biomolecules(3).

An integral part of designing a formulation is the characterization of its behavior using one or more appropriate analytical methods to gain insight into the critical processing temperature for processing. The intent of low temperature thermal analysis is to identify the critical values for freezing and primary drying as well as the characteristics that contribute to the behavior of a product during processing. The freezing method has a significant effect on the structure of the ice formed, affecting both the water-vapor flow during primary drying and the final product. By increasing the primary drying shelf temperature, the rate of sublimation is increased. Understanding and controlling how a solution freezes can lead to shorter lyophilization cycles and, in some cases, more stable products (4).

Methodologies and the instrumentation for identifying the critical temperatures of a formulation to be freeze dried have technologically progressed from simple devices constructed by the user to sophisticated instrumentation now commercially available. To minimize cost and wasted efforts, many companies developing new products to optimize their freeze drying cycles. Several methods have been used to determine the thermal behavior of excipients and the final formulation (5). The most frequently used techniques are freeze-drying microscopy (FDM) and differential thermal analysis and/or electrical impendence. FDM is an efficient and cost-effective tool that allows formulators to determine how their products will react in varying thermal conditions using small samples, instead of wasting large quantities of products by freezing it at less than optimal temperatures (6). Every formulation has a definitive critical temperature, after this point the formulation experiences processing defects during freeze drying and may become unstable. Maintaining temperature below the critical limits before the formulation goes through freeze-drying is imperative or the product can be ruined during the actual process, wasting time and money. For this reason, knowing the critical temperature of a formulation before lyophilization takes place is essential. By using a microscope and thermal stage, researchers can implement these same critical conditions on a macro scale, using less time and product to determine optimal pre-lyophilization conditions.

Collapse temperature is best measured by FDM (7). Most formulations exist in an amorphous state, and the critical temperature for freeze-drying will be their collapse temperature. This is the temperature at which the formulated product weakens to the point of not being able to support its own structure, leading to incomplete drying, inadequate stability difficulty in re-constitution and poor product appearance. Electrical impedance, which is perhaps the oldest technique, measures a phase change from solid to liquid that corresponds to a change in the relative conductivity of an electrical current through a sample. EI measurements are used as a thermoanalytical method to detect the presence of a charge carrying species in a fluid phase (8). A frozen matrix containing a crystalline material exhibits a eutectic melting point at a precise temperature. Under such conditions, the use of electrical resistance is a practical thermoanalytical method for detecting a phase transition. However, not all formulations contain species of electrolytic nature, nor do most formulations consist of crystalline material to make precise measurement of a phase transition possible. With these systems, formulators can determine critical temperature before lyophilization begins, saving themselves product and profit losses associated with trial and error attempts to freeze dry (9).

This article evaluates methods used to determine these critical values and to characterize the thermal behavior of the product. Our research work of freeze drying was composed of three steps namely optimization of formulation composition, determination of critical process parameters using real time FDM and integrated DTA-EI and characterization of freeze dried formulations to investigate stability. The main focus was on use of two most sophisticated techniques FDM and DTA/EI for optimizing FDC. We have subsequently discussed how to interpret data from these characterization studies for developing an efficient and economical FDC.

EXPERIMENTAL:

Materials

Human serum albumin was a generous gift from Baxter (India) Pvt. Ltd, Gurgaon, India. Sucrose was purchased from Himedia Laboratories Pvt. Ltd. (Mumbai, India). Sodium dihydrogen phosphate and polyvinyl pyrrolidone K30 were purchased from Merck Limited (Mumbai, India). Solvents and other chemicals were of analytical grade.

Formulation variables investigated

Based on a literature reviewed various formulations for freeze drying were defined by combining three kinds of stabilizers: a glass-forming agent (sucrose); a stabilizer (sodium dihydrogen phosphate) and a polymer polyvinyl pyrrolidone K30(10). The pH of the solutions was adjusted to 6.4 by addition of 0.1M HCl or NaOH. HSA being stable at weak acidic pH its solutions for freeze drying were prepared in distilled water whose pH was adjusted to 6.4. HSA aqueous solution (pH 6.4) was prepared in a series of mixtures containing 1%w/v HSA. Aliquots of the aqueous solution of HSA with 1.0%w/v SDP and 0.3%w/v PVP were combined with 5%w/v, 10%w/v, 15%w/v, 20%w/v and 25%w/v sucrose, respectively, to produce solutions for freeze drying. Accurately measured 1 mL aliquots of the solutions were filled into clean and dried vials. The vials were partially sealed with dry vented butyl rubber closures and the solutions were dried at -40°C under vacuum (pressure 0.06 hPa) for 72 h. The dried cakes were further subjected to secondary drying by keeping vials in the vacuum chamber (pressure 0.06hPa) for 6h and sealed under vacuum. At the end a composition showing highest HSA recovery determined using RP-HPLC was used for FDC optimization.

HSA content

HSA content in the freeze dried products was determined using validated RP-HPLC method(11). The measurements were made relative to the amount of HSA added to solutions and each product was assayed in triplicate. The freeze dried products were reconstituted with distilled water to obtain HSA concentrations of 300mg/mL and analyzed by RP-HPLC for HSA content. The HPLC system (Shimadzu Corp., Tokyo, Japan) consisted of UV/VIS detector (UV 2075 plus) covering the range of 200-400nm and interfaced to a computer for data acquisition. A 20μl of sample was injected at a flow rate of 1.0mL/min and UV detection was carried out at λ_max 280 nm. At the end a composition showing highest HSA recovery determined using RP-HPLC was selected for FDC optimization.

Freeze dry microscopy

Lyostat 2 is a fully integrated FDM that enables critical events such as collapse and melting to be observed in situ, as well as characteristics such as skin or crust formation to be observed and identified. FDM system, Fig. 1, comprises a compound microscope focused onto a sample that sits in a vacuum-tight temperature-controlled stage which acts as a micro-freeze-dryer. The freezing and drying behaviour of sample was determined visually, and the system is controlled through a computer, which provides image- and data- capture and archiving functions. Self-made, precision cut spacers (brass platelets, height 0.025 mm) were used to assure a constant thickness of the sample layer. In the temperature range of interest pictures were recorded in a one second time interval using a digital firewire camera (resolution 1.3 mega pixels) and (after the measurement) analyzed using the Lyostat 2 software (Biopharma Technology, Winchester, UK).

Figure 1. Lyostat 2- a freeze-drying microscopy system (Biopharma Technology)

The process of FDM began with loading a sample of product into a stage chamber under the microscope, which acts like a miniature freeze-dryer. Amount of sample required for Lyostat2 was 2 μL which was kept on stage followed by lid closure. Liquid nitrogen used for cooling was filled in reservoir tank to attain a standard lowest freezing temperature -60°C. The sample was cooled up to -40°C at the freezing rate of 20°C/min. At 20°C a holding step for 5 min was implemented in the measurement routine to purge the system with dry nitrogen from the Dewar through the open valve. Before starting sublimation the sample was held at the lowest freezing temperature for equilibration for 5 min. The application of vacuum pump helped in drying the sample. After 2 min the vacuum pump was switched on at the rate of 0.5°C/min so that sublimation could be observed a few seconds later. Through all the cycle pictures were taken with the delay of 10s. When the collapse was initiated and when the entire sample got collapsed was concluded with the help of series of pictures. Pressure was measured using a calibrated Pirani gauge and was controlled below 0.285hpa. The saved pictures were compared by using custom-made supporting software. All software supplied as standard with Lyostat 2, allowed controlling the freeze-drying remotely via a computer. This includes displaying and recording temperature and vacuum readings, providing a live image of the sample to be viewed on the screen, as well as recording a gallery of images taken at selected time intervals.

To investigate the freeze dried behavior of a formulation, a small aliquot (1μL to 10μL) of formulation containing HSA, sucrose, sodium dihydrogen phosphate and polyvinyl pyrrolidone was frozen to –60°C between quartz coverslips in the freeze dried stage, without annealing. The behavior of the material during sublimation was observed using a polarizing microscope. Micrographs were recorded using a video camera that was mounted on the microscope. The magnification used for observation of the freeze dried front was 150-fold. All the above information is useful for screening components formulation and cycle development. The investigation of thermal properties of a formulation was done by determining collapse temperature using FDM.

Differential Thermal Analysis-Electrical Impedance

Lyotherm 2, Fig. 2, has been designed to measure glass transition, eutectic and melting temperatures relevant to freeze-drying formulations. Based on the conventional “electrical resistance” analysis that has traditionally been used for identification of softening or eutectic melting points in frozen materials, the use of impedance analysis and specifically the measurement of a derivative of impedance known as “Zsinφ” provides important information as to the behaviour of the frozen solute phase which may not be identifiable using conventional analytical techniques (12).

Figure 2. Lyotherm2- an integrated DTA and an electrical impedance system

In the Lyotherm2 studies the sample quantity required was 6 μL. Sample was filled in both the sample holders. One of them was used for recording differential thermogram and another for recording EI. One probe kept in reference holder which contained WFI and second probe kept in block. Liquid nitrogen was filled in reservoir tank which was used for cooling. After filling the sample and reference the block was kept in reservoir tank and by starting the Lyotherm2 software the data recording was started. The cooling started up to -90ºC, followed by block pulling upward in tank and the heating was started which goes to 3ºC/min. When the temperature reached 0ºC, recording was stopped and the data was taken on excel sheet to plot the graph. The Lyostat2 graph shows a sharp change in temperature/ electrical impendence indicating sample collapse.

Lyophilization Process:

The critical temperatures obtained from FDM and DTA-EI studies were applied for the freeze drying of optimized product composition. Accurately measured 2mL solutions were filled in glass vials (5mL capacity, Yantai Uech Pharmaceutical Package Co., Ltd, China). The rubber closures (Apipharma, India) were positioned as half-closed. The solutions were frozen at –35°C for 4h in deep freezer (Polar 530V, Angelantoni Industries India). The prefrozen vials were freeze dried in LyoPro3000 lyophilizer at shelf temperature of –35°Cand vacuum 0.06hPa for 48h (condenser temperature –50°C). The freeze dried products were protected from atmosphere, light and moisture.

Cake Appearance

The appearance of the freeze dried product cake was inspected visually for its structural integrity and pharmaceutically elegancy.

Residual Moisture

Moisture analysis of freeze dried products was performed using a Karl Fischer titrator (Vigo – Matic M. D.). Accurately 20 mL of anhydrous methanol was transferred to the titration vessel and titrated to the end-point. A sample of 10 μl of water, accurately measured, was used to standardize the Karl Fischer reagent. Accurately weighed samples were suspended in anhydrous methanol and titration was carried out to the electromagnetic end point.

Reconstitution Time

Reconstitution time of the freeze dried product was determined by Thiermann method (13). Each sample was reconstituted with 1 mL (0.9 %w/v NaCl) solution while putting the solution flow onto the inside of the vial. The vials were shaken horizontally at a distance between 6 inches on hard surface till solution is formed. The vials were inspected visually by measuring the solution time without visible aggregates.

XRD Analysis

The XRPD analysis of all the freeze dried products was performed using X-Ray Diffractometer (PW 3710, Philips, Netherlands). Powdered product samples were scanned between 2°–100°2θ at a scan speed of 0.1°2θ/sec using 1.524Å radiations. The XRPD patterns were recorded and analyzed.

DSC Analysis

DSC studies were performed to determine the T_g of the freeze dried products to allow proper design of the process and to investigate their stability by using a Model 821 DSC (Mettler Toledo). Samples of 1.5–7mg were analyzed in crimped, vented aluminium pans under a dry nitrogen purge with an automated liquid nitrogen-cooling accessory. Samples were heated from 25°C to 300°C with a scanning rate of 10°C/min.

RESULT AND DISCUSSION:

Lyostat2 and Lyotherm2 techniques quickly and accurately identify collapse temperature in typically less than one hour. Therefore, the efficiency of these methods was compared with conventional DSC and XRPD methods. In general, the product being freeze dried in a vial collapses at a slightly higher temperature. The effect of variation in collapse temperature is negligible in a system of low solids content due to very rapid sublimation (14). To minimize the variation between FDM experiments and production behavior in freeze dried, the collapse temperature measurements by FDM were conducted using solute concentrations comparable to the concentrations ultimately used in freeze drying. A critical stage (collapse temperature) during freeze dried of the optimized HSA composition studied by using Lyotherm2 is given in Table-1. The observed T_c was the highest allowable product temperature during primary drying. That is because the cake will collapse if the product temperature is higher than this highest allowable temperature during sublimation.

Table 1: Critical stages during freeze-drying of the composition studied by Lyostat2

Picture No.	Temp. ( °C )	Observation
1	-40.0°C	No collapse observed.
2	-34.5°C	No collapse observed.
3	-33.0°C	No collapse observed.
4	-28.7°C	Collapse symptoms initiated.
5	-28.1°C	Collapse clearly evident.
6	-27.7°C	Collapse confirmed.
7	-27.1°C	Collapse observed.
8	-26.3°C	Majority collapsed.
9	-25.9°C	Fully collapsed.

The images of an optimized HSA product by FDM (Fig-5) showing thermal behavior are given in Fig. 3. The graph of DTA/EI of optimized HSA composition run on Lyotherm2 is given in Fig. 4. In Lyotherm2 at -28.7°C there is an increase in impedance indicating a stabilisation/rearrangement of frozen structure which is a collapse temperature. Black vertical line is collapse temperature plotted from Lyostat 2. Above -16.0°C there is an increase in downward gradient of impedance curve indicating a softening of the frozen material. The composition should be essentially sublimed below -25°C.

The result of Lyostat 2 and Lyotherm 2 clearly indicate that -35°C is an appropriate primary drying temperature in freeze dried of HSA optimized composition. The HSA solutions were processed through FDC optimized by FDM and DTA/EI. The appearance of the freeze dried HSA product was colorless to yellowish freeze dried cake. Visually freeze dried cakes were pharmaceutically elegant with non-collapsed structural integrity. Generally speaking, the lower the allowable product temperature is, the less efficient the freeze dried cycle will be. DTA/EI analyses are commonly used in determining collapse temperature.

Figure 4. Graph of electrical impedance and DTA of optimized HSA composition run on Lyotherm 2.

Figure 5. Freeze dried HSA test products

Residual Moisture:

Freeze drying is a process to remove water so that the water activity or the mobility of the water, in the dried product can be reduced to an optimal level. This is achieved by using a secondary drying cycle. As a result, fully understanding the effects of the moisture content on product stability becomes the most important information necessary to develop a secondary drying. Moisture levels in all freshly prepared freeze dried products were in the range of 1.546–2.142% w/w. Moisture levels in products were well below 3%w/w as specified in the USFDA guidelines. These observations are consistent with the reported studies by Satoshi et al. (15). Generally, for any formulation there is an optimal range of moisture content that would result in the best stability of the freeze dried product. Hsu et al. (1991) reported that there is an optimum residual moisture range for a freeze dried recombinant protein (16). Overdrying may result in opalescence in the product upon reconstitution, while underdrying leads to a greater protein activity loss upon storage under temperature stress conditions. Greiff (1971) and Liu et al. (1991) revealed that aggregation of insoluble proteins can be induced by moisture content that was higher than the optimal range (17, 18).

XRPD analysis

The XRPD pattern of pure HSA confirmed the structure and absolute configuration, which had been previously determined by chemical studies (19). The diffraction patterns of pure HSA and its freeze dried products comprising stabilizers are shown in Fig. 6. The XRPD pattern of pure sucrose used in this study showed sharp peaks at 17.8º, 19.8º, 20.23º, 31º and 37.3º2θ were characteristic of crystalline state. No peaks were observed in the XRPD patterns of freeze dried samples indicating a lack of crystallinity (20). This finding indicates that freeze dried product has tendency to inhibit crystallization of components.

Figure 6. Overlain XRPD patterns of pure sucrose and freeze dried HSA product

DSC analysis

DSC measures heat flow as a function of temperature applied to a sample going through freezing, melting, crystallization, and glass transition. The thermal properties that can be measured by using DSC include eutectic T_c, T_e, T_g and T_m. Among these critical temperatures, the T_g is one of the most important thermophysical properties of the formulation (21). For a formulation that forms an amorphous cake after being freeze dried, the T_g is also the collapse temperature, which is the most critical factor in ensuring the success of the primary drying.

Figure 7. Overlain DSC thermograms of pure sucrose and freeze dried HSA product

Typical DSC thermograms of pure sucrose and freeze dried product are presented in Fig. 7. These observations confirmed that HSA with stabilizers is more likely to be in an amorphous state. DSC provides very important information not only for designing a primary drying process. For example, if -33ºC is the highest allowable temperature for the formulation, the Fig. 4 illustrates that all combinations of shelf temperature and chamber pressure resulting in a product temperature lower than -33ºC would be acceptable process parameters. In practice, we usually take at least 2-5ºC as a safety margin, and we always optimize the process by looking for the combinations that result in the highest sublimation rate and consequently, the shortest drying time.

CONCLUSION:

The real time FDM and DTA/EI techniques accurately determine the thermophysical properties of a formulation that helps to precisely design freezing and primary drying parameters. By conducting a moisture optimization study, we can optimize the secondary drying process with regard to its drying temperature, pressure and time. FDM, as part of a complete thermal-analysis study, is an invaluable tool in the characterization of the thermal properties of any formulation. The process enables pharmaceutical companies to save a significant amount of time and money both in process development and in commercial manufacturing. In summary, we have demonstrated that a FDM and DTA/EI techniques gives good understanding of formulation characteristics that assist in developing an optimal FDC with a scientific rationale. The analytical methods described in this paper have both benefits and limitations governed by the measurements and properties of the excipients and active pharmaceutical ingredients in a final product formulation. Each method measures different characteristics of a sample. Therefore, it may be beneficial to characterize a formulation by conducting multiple analyses using various methods.

ACKNOWLEDGMENT:

The authors gratefully extend their acknowledgments to Dr. S. S. Kadam, Vice-Chancellor Bharati Vidyapeeth Deemed University, Pune, for his valuable support for this research. Special thanks also to Dr. K. R. Mahadik (Principal, BVDU Poona College of Pharmacy, Pune), for providing facilities for the study.

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Received on 26.05.2012 Modified on 22.06.2012

Research J. Pharm. and Tech. 5(7): July 2012; Page 985-991