September 15, 2005 (Vol. 25, No. 16)

New Products Such as Mabs and Recombinant Proteins Should Drive Advances

Lyophilization is a complex operation used to prepare stable pharmaceutical and biopharmaceutical products through freeze-drying. During lyophilization, the water fraction of the product is reduced to approximately 3% by weight through a combination of sublimation (the primary drying process) and desorption (secondary drying process).

Lyophilization equipment consists of a drying chamber, condensers (water and drying), a cooling system, and a vacuum system.

The principal steps in a typical lyophilization process include product preparation, freezing, primary drying, and secondary drying. Because lyophilized pharmaceutical products are usually injected, equipment and processing are typically carried out in a sterile environment.

Since it was first developed as a commercial process in the 1930s, and adopted over the next several decades by pharmaceutical and biotechnology companies, lyophilization has undergone several waves of refinement, resulting in a versatile, more clearly understood process. At the core of this evolution, and perhaps driving future enhancements, is a greater appreciation for chemical, physical, and thermal aspects of the process.

Although the principal change in lyophilization is water removal, the objective of freeze-drying pharmaceuticals is product stabilization. Lyophilization imparts higher stability, broader temperature tolerance, and longer shelf-life to most pharmaceutical formulations that are unstable in aqueous solution.

Lyophilization should be considered for all solid actives that are either sensitive to heat or which are chemically or biologically unstable, or when solutions are suspected to interact unfavorably with the vial or package.

Lyophilization is growing approximately as rapidly as biotechnology, although non-biotech products can also benefit. Candidates for lyophilization include injectable solutions, proteins, peptides, and vaccines.

The number of such products has been growing steadily: 370 biotechnology products are currently in development, with more than 300 sterile product approvals expected between 2005 and 2012. Approximately 25% of these products will be lyophilized.

The market for lyophilized products is expected to reach up to 151 million units annually by 2010. This growth will be driven primarily by the introduction of new products, specifically biologics such as monoclonal antibodies (Mabs) and recombinant proteins.

It is expected that 40% of the injectable NME’s approved in the next five years will require lyophilization. According to a recent survey by Pharmsource, about 75% of the overall lyophilization capacity is committed through 2008.

Integrated Lyophilization: Balance Between Cooling and Drying

Lyophilization employs a series of distinct, integrated operations. Drugs undergoing lyophilization experience significant physical and chemical transformations during freezing, primary drying, and secondary drying. As a result, every step of the process must be closely monitored, especially with respect to heat transfer, to assure proper conditions and product consistency.

Adjusting and control of heating and cooling during the process ensures products are maintained within acceptable limits (defined by the product and the process) during freezing and drying to maximize product stability and quality.

The first step in lyophilization involves freezing a solution of the product. Freezing rate is critical since it may affect the product’s structural integrity. Freezing too rapidly may induce formation of small crystals that can result in higher water vapor resistance and an extended drying time.

Slower, deliberate freezing can create larger ice crystals and a final product containing coarser pore structure, which necessitates longer drying time.

Once the product solution is frozen, the lyophilization chamber is placed under vacuum and gradually heated. The net result of heating and loss of heat through sublimation is that the product and vial will achieve a temperature that is colder than shelf temperature.

As primary drying concludes, the product temperature rises until it reaches the shelf temperature, signifying the end point of the primary drying phase. The water vapor given off by the product in the sublimation phase condenses as ice on a condenser.

Once frozen under a vacuum, the newly lyophilized drug compound, with all its water content removed, usually exists as a dry, stable powder with a very long shelf life. The product may then be reconstituted by addition of an appropriate diluent.

Chamber pressure and product and shelf temperatures during primary drying are determined by the product’s formulation’s eutectic or collapse temperature. The eutectic point is governed by the actual composition of the product and the temperature at which it can only exist as a solid.

Eutectic points vary from compound to compound. As the product solution freezes, its temperature must fall below its lowest eutectic point. Once reached, this temperature must be maintained throughout primary drying.

As the water within the solution freezes, the drug may experience supercooling, a condition related to freezing point depression. Supercooling may cause structural changes in the compound during lyophilization as well as alter the external physical characteristics of the compound, for example formation of a “skin” layer on the surface of the freezing solution, which impedes the escape of water vapor.

During primary drying, the chamber pressure is lowered and heat is introduced, promoting sublimation of frozen water. It is critical that the condenser be of high enough capacity for all sublimed water from this step, and that the frozen water remain at a temperature lower than that of the product. Otherwise, water may migrate back to the product or drying may be inhibited.

Controlled drying and heating rates during the primary drying phase are keys to lyophilization success.

Product that dries ahead of schedule has the potential to be swept out of the container as vapors escape. Another pitfall is exposing product to inappropriate heating, which can cause melting and most likely an inability to reconstitute the product to its correct form.

After primary drying, residual moisture on the product surface is reduced through secondary drying to levels that no longer support biological growth and/or chemical reaction while promoting long shelf life without melting. Moisture reduction during secondary drying is achieved by increasing shelf temperature while reducing the partial pressure of the container’s water vapor.

The product’s physical constitution determines the length of the duration of secondary drying. For example, proteins and peptides require that varying levels of water remain within the product to maintain structural integrity and pharmacologic activity. The requisite residual moisture is product-dependent and must be determined empirically. Additionally, excess heat may cause the product to char or shrink.

The accuracy and precision of pressure control are critical to successful lyophilization. Most commercial drug drying cycles employ chamber pressure control to manage the drying rate. During extremely low pressures encountered during lyophilization, heat is transferred through conduction from the product through the bottom of its container, which hinders drying.

Introducing an inert gas, such as nitrogen, improves heat transfer. Gas molecules facilitate heating of the container walls and heat conduction through the container, which increases the amount of heat applied to the product. This improves drying rate, reduces the cycle time, and also decreases energy and labor costs.

By contrast, ambient pressure that surpasses the ice vapor pressure may inhibit sublimation. Compensating for less-than-optimal pressure by heating may result in melting.

During secondary drying, temperature is increased slowly to desorb bound water until the residual water content falls to the desired range. Normally, secondary drying is performed at the highest possible vacuum level.

Although not normal, some products may require additional steps beyond primary and secondary drying. Drying beyond the secondary drying phase is usually product-specific and based on a product’s need for residual water to preserve an active ingredient’s structural integrity and/or biological activity.

In these situations, water content must be painstakingly monitored and managed. Just as with primary drying, a disproportionate amount of heat exposure may cause the product to char or shrink.

Thermal Conductivity: The Key to Successful Lyophilization

Successful lyophilization depends on efficient transfer of heat from the shelf heating element, through the glass vial, to the frozen product. Lyophilization vials must withstand extremes of pressure and temperature without breaking, and that the bottoms should be flat.

Depending upon the excipients, the angle between the bottom and the wall of the vial may be specified further to minimize breakage, for example, with mannitol solutions.

Additionally, vials must meet industry standards for protecting product from atmosphere and moisture after they are sealed. Components (vials, stoppers, vial necks, crimps) that do not match perfectly, or plastic syringes with inadequate vapor transmission barriers, can result in the loss of large batches of valuable material.

Leachables from all primary packaging components for all pharmaceuticals must of course also be tested, as they would for any standard pharmaceutical package. Additional validation may be needed to assure that vials meet industry standards for heat transfer and physical/mechanical integrity.

Lyophilization Equipment: Advancing the Process

Lyophilization has evolved in recent years, becoming more efficient, easier to use, more cost-effective, and extremely accurate. These advances have arisen from advanced freeze-drying equipment designs and have been further advanced by modern computer/instrument control automation (especially for vial loading).

This added level of automation and control has introduced an even greater need for validation of lyophilization equipment, vials, and supporting hardware and software systems.

In advanced lyophilization equipment, vials containing product are placed directly on shelves inside the drying chamber, which can be cooled to freeze the material at atmospheric pressure before applying the vacuum.

Without a controlled heat input directed at the sample, the product’s temperature falls until drying ceases. To avoid this, a heat supply is added to the product-support shelves to replace energy lost after initial freezing. Sublimation then helps maintain the product at a constant low temperature.

At atmospheric pressure, the molar volume difference between ice or water and water vapor is about 1200. Under high lyophilization pressures, the differential is closer to one million. This is a concern for drug manufacturers, given that the more energy-efficient vacuum pumps are unable to process large quantities of water vapor.

Most manufacturers will therefore augment a lyophilization process by fitting a refrigerated trap (called the ice condenser) between the lyophilization chamber and the vacuum pump.

The most exciting recent advances in lyophilization involve automation and stand-alone operation. Automatic loading and unloading systems, for example radio-controlled tray loaders and robotic systems, have resulted in simpler, more accurate process.

The Draxis (Quebec) contract lyophilization facility in Montreal employs robot-driven automatic loading that transfers filled vials from the filling line directly onto the freeze dryer shelves. Enhanced computer systems control the freeze-drying cycle, while automated CIP (clean-in-place) and SIP (sterilize-in-place) are marked improvements over the more traditional manually-controlled cleaning and cleaning validation systems.

As lyophilization is further refined, we expect to see even more efficient drying systems, more automation, routine use of in-process monitoring, and ultimately reduced cycle times and continuous processing capabilities.

Lyophilization vs. Conventional Drying

Lyophilization’s principal advantages over conventional drying methods (which may incorporate crystallization, filtration, or precipitation) is preservation of chemical and biological potency, homogeneity in the final product, and ease of dispensing/metering before final packaging.

For example, pharmaceutical solutions can be dispensed and sterile-filtered with a high degree of accuracy immediately before lyophilization, guaranteeing a precise dose.

Other less obvious advantages of lyophilization include:

Protection from solution effects and chemical degradation

Speed and completeness of rehydration compared with dry powder fills (e.g., for injectable penicillin)

Accurate, sterile dosing into final product containers

Porous, friable structure of final product, compared with crystallized or conventionally-dried materials, permit easy reconstitution

The principle disadvantage of lyophilization are:

High capital cost of equipment (about three times more than traditional sterile solutions or sterile dry powder fill methods)

High energy costs (two to three times more than those mentioned above)

Long process time (typically a minimum 24-hour drying cycle)

However, it should be pointed out that for many products, including the vast majority of high-value proteins, peptides, and vaccines, lyophilization is the only way to deliver stable, biologically active products with long shelf-life.

There is an increased demand from pharmaceutical companies for lyophilization manufacturing services as there is an increased recognition of this process’ benefits. With the growing number of drugs in development, lyophilization becomes a logical, time-effective, and cost-sensitive option.

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