The Secret to Developing Lyophilized Pharmaceuticals

In the Lab: APPLICATIONS

The Secret to Developing Lyophilized Pharmaceuticals

Use more than one technique

In the development of lyophilized products, differential scanning calorimetry (DSC) can give insight into collapse temperature, glass transition temperature (Tg), storage conditions, and phase separation. But DSC actually consists of several techniques, including conventional DSC, high rate DSC, and modulated temperature DSC. Each of these techniques has unique advantages and offers a slightly different insight into the material under study. Intelligent use of these three approaches to DSC allows better characterization of your product than any one technique used alone.

Conventional DSC

Conventional DSC (c-DSC) is the heating and cooling of a sample between -180 and 500^oC at rates of 0.1 to 100^oC/minute. One common use for this technique is the measurement of glass transition temperature (Tg) of protein-excipient mixtures, which is an indicator of the collapse temperature for freeze drying. In this test, approximate 20 �l of solution is cooled to between -80 to -100^oC. The solution is then reheated to room temperature at a rate of 10 to 20^oC/minute, causing a small shift in the baseline corresponding to the glass transition of the protein sugar phase (see Figure 1, p. 47). This test indicates the temperature below which the sample must be maintained during the freeze drying process.

Once the material is in a dried cake, the DSC can be used to measure the Tg to define the storage temperature and to look for phase separation. The Tg of lyophilized products can often be hard to detect, however, because of very high protein contents and high molecular weight components. For these tests, therefore, it is often beneficial to run high rate DSC instead of c-DSC.

Conventional DSC is also used to measure the heat capacity of materials, to calculate the purity of traditional pharmaceuticals, and to determine the temperature of the unfolding of protein solutions (Tm). In the latter case, c-DSC does not have the volume and sensitivity of dedicated micro calorimeters and is therefore limited to higher concentration samples. Sample molecular weight is also important; while solutions of lysozyme can be measured to concentrations of approximately 0.5 mg/ml, bovine serum albumin is only measurable to approximately 12 mg/ml. This limits the use of c-DSC for these samples.

High Rate DSC

High rate DSC (HR DSC) involves heating or cooling the sample at rates of 100^oC or greater per minute over the same temperature range used in c-DSC. HR DSC exploits the standard heat flow equations to improve the sensitivity of the instrument by using increased heating rates. Because of the design of power compensation DSC, with its very small furnace mass, rates of up to 500^oC/min can be obtained for quantitative data. This quality makes HR DSC highly sensitive to the presence of amorphous material in a sample. In addition, high throughput can be achieved, because runs typically take less than three minutes. An auto-sampler system at 200^oC/min allows up to 268 runs a day.

Figure 1. Effects of heating and cooling a sample in conventional DSC

Heating at these rates does more than just increase the instrument'S sensitivity, however. It also allows suppression of kinetic events like decompositions, polymorphic transformations, and reactions by heating and cooling the sample faster than the event can occur. This becomes an important advantage when protein concentrations increase, because it allows for data collection before the material decomposes.

Running a lyophilized sample for its Tg using HR DSC is relatively simple: A small sample of material, typically one to three mg, is placed in a DSC pan and run from 0 to 250^oC at 300^oC/minute. Often a second run establishes a common heating and cooling history for the samples. Scanning at these rates allows the measurement of very weak glass transitions of samples like high protein concentration mixtures, pure proteins, cellular material, and even difficult samples such as hydroxyethyl starch (HES). In addition, because HR DSC allows scanning that is faster than kinetic events can occur, decomposition is retarded and glass transition results are nice and clean (see Figure 2, right).

Obviously, this ability to "outrun" kinetic changes has uses besides the more traditional pharmaceutical applications, including trapping polymorphic forms and measuring melting points in materials prone to decomposition.

Modulated Temperature DSC

Modulated temperature DSC techniques require the application of a non-linear heating or cooling cycle to a sample. There are several variations of modulated temperature DSC. One of these is the Step-Scan, in which a sample is heated in a series of micro steps consisting of a ramp followed by an isothermal. StepScan allows the separation of the heat flow signal into a thermodynamic curve, called the thermodynamic heat capacity (Cp) curve, and a kinetic curve, called the Iso-Kinetic (IsoK) curve.

This division allows the removal of kinetic noise from the data by the separation of long time scale events from short kinetic ones such as, for example, the separation of the Tg from the loss of water. In lyophilized materials, this approach allows you to get a good measure of the amount of enthalpic overshoot associated with a glass transition. This information is not only useful for estimating storage life but also helps in obtaining a cleaner glass transition. Other applications for Modulated temperature DSC include removing the affect of a moisture loss from a Tg, separating a Tg from a decomposition, and identifying polymorphic materials.

A common problem with lyophilized samples is an enthalpic change occurring at the glass transition. This can appear as a hump, but it can also appear as a dip. Running a StepScan method with its alternating heating and holding steps generates a complex curve (see Figure 3a, p. 48). When the mathematics is applied, the resultant Cp and IsoK curves are calculated, and the data becomes much simpler. The thermodynamic data, in this case showing the Tg, is cleanly resolved in the Cp curve; the energy associated with the kinetic hump is shown as a peak in the IsoK curve (see Figure 3b, above).

Figure 2. Glass transition results in HR DSC

Figure 3a. Results of a StepScan method

Figure 3b. The energy associated with the kinetic hump

This technique becomes invaluable when the kinetic noise at the Tg is exothermic and causes the Tg to have a plateau or dimple in the middle. You may ask, is this really one Tg with some kinetic noise or is it two Tgs, indicating a phase separation in the material? A StepScan approach allows you to separate the signals and see either one clean Tg with the noise in the IsoK curve or the two Tgs, indicating a phase separated material.

Differential scanning calorimetry (DSC) consists of several techniques, including conventional DSC, high rate DSC, and modulated temperature DSC.

Putting It Together

Conventional DSC allows us to look at the protein excipient solution before freeze drying and estimate if the drying temperature is low enough. It also lets us look for Tm in concentrated solutions. Once the formulation is lyophilized, high rate DSC lets us detect even the very weak Tgs associated with pure proteins or high protein formulations, as well as small amounts of phase separation. And it speeds up the quality control process by dramatically shortening testing times. Finally, Step-Scan DSC can be used to calculate the energy associated with a relaxation at the Tg or to confirm the phase separation and multiple Tgs.

While it is sometimes possible to extract more data from multiple experiments, some data only results from a specific technique. Using all three approaches to DSC makes the instrument a valuable and flexible tool for lyophilization formulation development and quality control.