Reducing Lyophilization Time and Increasing Product Stability

Many biological materials may be frozen in the presence of protectants, and using a carefully controlled change in temperature and pressure, more than 95% of the water may be removed. This leaves the biological material intact and active. The specialized devices used to perform this process are lyophilizers or freeze dryers.

‍

Properly developed lyophilization cycles will result in an efficient (i.e., shorter) lyophilization cycle and a final product with stability that may have been extended by years.

‍

Cycle Development

‍

The first step is to develop a “lyophilization-friendly” formulation for the product, that would facilitate the process and help with long-term product stability.  Just sending a liquid formulation to lyophilization cycle development is not a good idea without scrutinizing each excipient and its role.  Liquid formulations tend to be isotonic with the addition of sodium chloride (NaCl).  NaCl is usually a no-no for lyophilized formulations as it depresses the Eutectic point (below which no liquid phase can exist) to very low levels.

‍

A lyophilized formulation usually includes as a minimum:

• A buffer system that can keep the product at a safe pH range during process and upon long-term storage.
• A bulking agent to provide a solid matrix for the dried product.
• Stability enhancing agents such as detergents and amino acids.

‍

Initial steps of freeze-drying cycle development involve mapping the thermodynamic properties of the formulated product by determining the critical parameters: freezing/melting point, recrystallization, eutectic and collapse temperatures.  The analytical methods typically used for these analyses are Digital Scanning Calorimetry (DSC) and freeze-drying microscopy.  A much lesser known, but more discerning method is Resistivity.  All of these methods look at changes in the thermodynamic properties of the formulated product as it is cooled down and heated back up.

‍

DSC detects endothermic and exothermic reactions, and is the most commonly used method, but its high equipment cost and lower sensitivity does not necessarily make it the best method to use.  Transitions with low heats of transition (∆H) such as collapse cannot be readily determined by DSC.

Freeze-Drying microscopes are custom-built and can be expensive.  This method can visually detect all of the critical temperatures listed above, but the equipment is not readily available.

‍

Resistivity measuring equipment uses relatively inexpensive off-the-shelf components, but usually still needs to be custom-assembled.  This method measures the electrical resistance between two electrodes as they are submerged in the formulated liquid, and temperature is lowered and heated back up.  Because electricity travels through the path of least resistance, and is increased with the degree of solidification of the test materials, the electrodes can easily and clearly detect all of the critical temperatures, and is much more sensitive than the other two methods for determining the eutectic and collapse temperatures.

‍

Lyophilization Process

‍

Once the critical product temperatures have been defined for the formulated product, then the freeze-drying cycle can be developed and optimized.  The freeze-drying process is typically composed of the following steps:

1.     Initial freezing:  In this step, the product is cooled to below the supercooling temperature and the freezing point, where the majority of the water in the product formulation will transform into pure ice crystals.  The size and number of ice crystals is dependent on the rate of heat removal.  Faster cooling will produce more numerous smaller crystals, which helps maintain the original shape of the frozen matrix in the final dried product, while slower cooling will promote large crystals, which can help increase sublimation rates.  The effect of the smaller crystals on stability is different for each product.  

The remaining unfrozen liquid containing all of the components of the formulation including the drug substance, becomes trapped within the interstices of the ice crystals at this stage.  It is usually desirable to not let the product stay at this stage for too long, as the drug substance is now in a super-concentrated liquid at a pH that has shifted either to a more acidic or basic environment depending on the buffer system used in the starting liquid.  The probability of damage to the drug substance is high at this time.

‍

2.     Continued freezing:  Optionally there may need to be some thermal cycling where the product is either held at a temperature for some time or reheated to a determined temperature to allow certain formulation components to also crystallize (e.g., mannitol).  This process is exothermic and may heat the product to above the melting/freezing point if temperature control is not adequate.  

‍

Upon completion of this process the product is further cooled to below the eutectic point, where all of the components are now solidified.  For all practical purposes, the product is cooled to the minimum temperature that a particular Lyophilizer can achieve.

‍

3.     Primary drying:  Once the product has fully solidified, high vacuum is pulled in the lyophilizer chamber and the shelf temperature is gradually increased until sublimation starts, where water goes directly from the solid state to the vapor phase.  This is the stage where catastrophic product failure may happen if processing conditions are not well defined and controlled.  The product temperature must be kept below two critical temperatures that must be defined in development studies, the collapse temperature, where the dried matrix softens to the point that it cannot support its own weight, and the eutectic point, where all the formulation components, except for water, becomes liquid again and “melt-back” occurs.  Of course, the melting point is also critical, but if the product has exceeded the other two temperatures, then the batch has already failed, and complete or partial product melting is now a moot point.

Primary drying is typically a heat-transfer controlled process, as sublimation is an endothermic process, and large amounts of energy must be supplied to the product to satisfy the enthalpy of sublimation.  This must be achieved while keeping the product at a safe level below the critical temperatures.  However, it is somewhat of a self-balancing process, as higher product temperatures increase sublimation rates, which will in turn cool down the product if not enough make-up energy is supplied.  Some reverse logic applies during this stage, because higher temperatures and pressures (up to a certain point) increase sublimation rates and can keep the product temperatures cooler than if lower temperatures and pressures were used.  This means that what looks like a more aggressive primary drying cycle may turn out to be more protective of the product than a cycle using low temperatures and pressures.

‍

4.     Secondary drying:  At this stage, most of the “free water” has already sublimated, and mostly “bound water”, molecules that have strong ionic interaction with the polar sites of the formulation excipients and product remain, and might need to be removed at higher temperatures.  A certain amount of residual “bound water” is critical to stability of the product at the end of the process.  The bound water helps biological molecules maintain their tertiary structure, which in turn improves stability.  The take home message here is that a lower final moisture content is not necessarily beneficial to product stability, and studies comparing the stability profiles of the products at varying levels of final moisture content should be used to set final moisture content specifications.

‍

This process stage is mass transfer-controlled, and higher temperatures and vacuum are typically used, while keeping the dried matrix below its collapse temperature.  Short-term exposure to stability impacting temperatures may be required to achieve the desired moisture content endpoint.

‍

Summary

‍

Once every step of the above-described procedures has been properly executed, the end result should be an efficient (i.e., shorter) lyophilization cycle and a final product with stability that may have been extended by years.

‍

About the Author

‍

Hoc Nguyen is a highly skilled executive with extensive experience in project management, operations, engineering, and process development in the pharmaceutical industry. With expertise spanning Technical Operations, Chemistry, Manufacturing and Controls (CMC), and regulatory compliance, Hoc has a proven track record of developing innovative solutions across gene therapy, biotechnology, drug/device combinations, and traditional pharmaceutical products. Known for strategic vision, creative problem-solving, and exceptional leadership, Hoc consistently delivers results through collaboration and communication. His areas of expertise include aseptic processing, cGMP, tech transfer, process optimization, and lyophilization.