Issue:October 2012

LYOPHILIZATION – Making Formulations More Efficient Using the Freeze-Dry Microscopy Pre-Lyophilization Method


In the development of parenteral drugs, lyophilization, or freeze-drying, is a crucial process technology. Pharmaceutical manufacturers have traditionally found lyophilization to be a costly, complex, facility-intensive operation, even though it is a straightforward process at the lab level.

The market for lyophilized products is expected to grow significantly in the coming years. The flood of protein-based therapeutics and other injectable products being pushed through the drug development pipeline has contributed to the need for leaner, more efficient freezedrying methods. Without lyophilization, 60% of biotherapeutics, including recombinant proteins, plasma, vaccines, and antibodies, could not be commercially available. So, what kind of NMEs can be freeze-dried? See the following list:

  • Non-biologicals (small molecules)
  • Non-living biologicals such as:
    -Blood products

Pharmaceutical formulators use freeze-drying to extend the shelf -life of their drugs, as many of these products have less than 2 years of shelf-life before they expire. One key step is to determine the right conditions for the freeze-drying process. Usually, formulators use largescale machines, which can make freeze-drying the most expensive and timeconsuming step in the parenteral manufacturing process.

To minimize cost and streamline this process, many companies developing new parenterals are finding it in their best interest to optimize their lyophilization cycles by looking at pre-lyophilization methods such as freeze-dry microscopy.

Freeze-Dry Microscopy

Freeze-dry microscopy focuses on the direct examination of freezing and freezedrying via a special microscope and thermal stage. Freeze-dry microscopy also complements and supports the information gained from the differential scanning calorimetry (DSC) technique. The process’ popularity stems from its ability to save pharmaceutical companies time, money, and product compared to traditional trialand- error techniques using freeze-drying machines. Freeze-dry microscopy 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 them at less-than-optimal temperatures. The following will describe how the technique is performed, the essential equipment, and finally, show how one company has successfully used it.

Traditional Lyophilization Process

Freeze-drying typically has three stages: freezing, primary freezing, and secondary freezing. Freezing is usually performed with a freeze-drying machine in larger-scale operations, particularly in the pharmaceutical industry. During freezing, material is cooled below its eutectic point, the lowest temperature at which the solid and liquid phases of the material can coexist. The main step in the lyophilization process is primary freezing, which involves the removal of water from the frozen product and is primarily done via sublimination. Temperature is critical during this phase because if too much heat is added, the material’s structure could be altered and spoiled. Finally, any unfrozen water molecules are removed during secondary drying and the product is sealed. Basic system components consist of a vacuum pump, temperature-controlled shelves, condenser, compressible shelves, temperature-monitoring devices, vacuum monitoring devices, a bleed valve, and a recording device.

Determining Critical Temperature is Essential

Every formulation has a critical temperature – after this point, the formulation experiences processing defects during freeze-drying and may be unusable. Maintaining temperature below the formulation goes through freeze-drying (or before the frozen water is removed) 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 determine optimal lyophilization conditions using less time and product.

Critical Temperatures Deciphered

There are three kinds of critical temperatures: eutectic temperature (Te), glass-transition temperature (Tg), and collapse temperature (Tc). Te refers to crystalline systems and is measured by thermal or thermoelectric analyses, such as differential scanning calorimetry (DSC). The material will melt during processing if this temperature is exceeded. Tg is also measured by thermal or thermoelectric analyses and refers to amorphous systems. Te and Tg determine the maximum temperature that the formulated product can withstand without the loss of structure during primary drying. As the sample is warmed, the glasstransition temperature is typically followed by the collapse temperature (Tc), which is best measured by freeze-dry microscopy. Collapse temperature 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 in reconstitution, and poor product appearance. Because most formulations exist in an amorphous state, the critical temperature for freeze-drying will be their collapse temperature. Tc is best viewed via a polarized light microscope and thermal stage (Figure 1).

Setting Up a Freeze-Dry Microscopy Lab

Necessary components for the standard freeze-dry microscopy lab include a polarized-light microscope, a liquidnitrogen- cooled thermal stage, a vacuum pump, and an imaging system. Polarizedlight microscopy allows researchers to visualize collapse temperature (Tc) as well as determine if their sample is crystalline or partially crystalline based on the birefringence of anisotropic crystals within the frozen matrix (Figure 2). The optimal polarized-light microscope system includes a strong light source, preferably 12V/100W, a Bertrand lens, distance objectives, polarizer/analyzer, and compensator. Compensators correct for differences in the refractive indices of the sample and the surrounding medium. Equally important is the thermal stage (Figure 3). An ideal thermal stage possesses the following key capabilities:

“¢ Temperature range of -196°C to 125°C
“¢ Temperature stability less than 0.1°C
“¢ Temperature accuracy of 0.01°C
“¢ X-Y sample manipulation functionality
“¢ Vacuum-tight sample chamber to 10-3 mbar
“¢ Silver heating block (ensuring high thermal conductivity)

In addition, chamber pressure is monitored via a pirani gauge mounted directly on the stage. Formulators can save product and profit losses associated with trial and error freeze-dry attempts by using these components to determine the critical temperature before lyophilization begins. Formulators are able to predict how their products will react under different thermal conditions and pinpoint the critical temperature (Tc) so they can get lyophilization right the first time.

Praxair: A Case Study

Three years ago, a facility at Praxair, Inc., a global Fortune 300 company that supplies atmospheric, process, and specialty gases, started using freeze-dry microscopy. The company turned to the technique to determine primary drying temperatures on small samples of product for lyophilization cycle optimization and to predict substance stability and reaction during R&D. Praxair scientists typically work with samples of product from pharma and biopharma customers to determine lyophilization parameters, such as freeze rates, shelf and product temperatures, and sublimation rates. Determining these factors enables Praxair to optimize their processes to attain ideal moisture levels and shelf-life for lyophilized products.

Praxair began using freeze-dry microscopy to minimize the amount of product used for determining critical temperatures and lyophilization cycle optimization for formulations. Although Praxair uses lyophilization literature and industry standards to work with formulations at the correct temperatures, every sample is different, and they sometimes lose product and have to do more trial-and-error work. In ddition, some pharma clients do not give outside labs complete formulation information until confidentiality agreements are completely worked out. Without key formulation information, Praxair scientists could be delayed in working on portions of product because they do not want to damage the sample.

A Praxair facility invested in a freezedry microscopy system to better support the key stages of the lifecycle of biologic and pharmaceutical products of its clients. A Praxair scientist even attended a freezedry microscopy course at the Hooke College of Applied Sciences (Westmont, IL) to learn how to best use the new system.

Praxair finds that freeze-dry microscopy helps them better capture the process conditions and understand their clients’ products, and therefore better meet their needs. Now, they do not see why they would want to try to do cycle development without having a freeze-dry microscopy system. They claim that it is a precise, simple procedure that gives them a precise temperature to meet when running their cycles, and a process that used to take days to do now can be completed in an hour and uses less product.

The Future of Formulation

Freeze-dry microscopy enables pharmaceutical companies to save a significant amount of time and money both in process development and in commercial manufacturing. When used as part of a complete thermal-analysis study, it is an invaluable tool in the characterization of the thermal properties of any formulation.

Mr. Jeffrey D. McGinn
President & Director of Instrument Sales
McCrone Microscopes & Accessories

Mr. Jeffrey D. McGinn has more than 8 years of experience in the pharmaceutical microscopy industry with expertise in training and sales, polarized light microscopy, lyophilization techniques, and scanning electron microscopy. As a Hooke College of Applied Sciences instructor, Mr. McGinn develops and teaches the microchemical test sections. His enthusiastic and hands-on approach ensures students learn by doing.