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Freezing and Annealing Phenomena in Lyophilization

Updated: Apr 27, 2021

The freezing step of lyophilization is of paramount importance. It is the principal dehydration step, and it determines the morphology and pore sizes of the ice and product phases. In general, the desired attributes of a lyophilized product are:

  • Consistent high yield of product activity through lyophilization

  • Appropriate crystallization (or not) of product and excipient(s)

  • Glass transition temperature higher than the desired storage temperature (related directly to residual moisture level)

  • Pharmaceutically elegant, mechanically strong cake

  • Rapid reconstitution

  • Short, consistent, and robust freeze-drying cycle

  • Stability of all product quality attributes through the intended shelf-life

The freezing method as well as any intentional or unintentional postfreezing annealing influence many of the above attributes.

Freezing is the major dehydration step of lyophilization. It determines the texture of the final product. Changes to the freezing protocol, thermal history during freezing, vial, volume of fill, particulate levels, ice nucleation properties, or any postfreezing temperature excursions above Tg' (intentional or not) can result in morphological changes, phase separation, product degradation, changes in the crystallization behavior of the solutes or the product, product stability, changes in drying rates (which can result in altered moisture levels or product collapse), and altered levels of residual cosolvent. Beware that many of the process factors affecting freezing can change through the scale-up/technical transfer process, leading to risk of failure at full-scale if not adequately controlled.

Two fundamental types of freezing behavior have been identified: directional solidification and global supercooling. Directional solidification results in a directional lamellar morphology with connected pores, and global supercooling creates spherulitic ice crystals. The directional morphology has a higher specific surface area, and results in higher primary drying rates due to the connected, continuous nature of the pores.

Within a given type of freezing one can say that “faster freezing” leads to higher surface areas, smaller more numerous ice crystals, and slower primary drying rates. For directional solidification, freezing rate control equates to cooling rate control, but below a critical cooling rate one would expect a loss of directional solidification at least in some portion of the volume being frozen.

However one cannot say that in general “faster freezing” means higher surface areas, smaller more numerous ice crystals, and slower primary drying rates because liquid nitrogen immersion provides much faster cooling than shelf-ramp freezing. Liquid nitrogen immersion results in higher surface areas but faster primary drying rates due to the completely different type of morphology that results.

Different freezing methods and annealing steps are powerful tools to manipulate numerous product quality and productivity attributes. By understanding how freezing and annealing affect our products and processes, we can devise better freezing and annealing protocols to increase lyophilization plant capacity utilization and improve product quality and consistency. Do not blindly accept the results of your freezing method: investigate alternative freezing methods and test the effects of annealing steps.

Pharmaceutical active ingredients generally fall into one of the two categories: characterized or uncharacterized. Can it be said that lyophilized dosage forms are fully characterized, even if the active ingredient is fully characterized? When a fully characterized active ingredient is lyophilized we must be mindful of the limited extent to which we can characterize attributes of the final lyophilized product. For example, aspects of product cake structure and surface area are not well understood or easy to measure and are part of what is accepted as the “natural” heterogeneity within a lyophilized batch. Do we understand how a shift in the freezing protocol will affect stability of the product? Without a better understanding of how freezing actually takes place, we cannot hope to fully characterize our lyophilized dosage forms. For example, how and why will cake morphology be affected by a change in vial? Why do cakes sometimes appear with a directional morphology in the bottom half of the cake and spherulitic in the top half? How do we correlate cake appearance (e.g., extent of collapse) with product quality for deviation investigations? What cooling rates during freezing will result in the “same” product? When freezing on a precooled shelf, what conditions are necessary to achieve directional solidification? How will the cake morphology change with a slight change in the freezing protocol? Which forms of crystalline mannitol will arise from a slight change in the protocol? These questions are worth answering—failing to pursue them will lead to increased regulatory scrutiny, occasional product development and manufacturing deviation failures, and inefficient use of our existing lyophilization infrastructure.

Original article: "Freeze Drying/Lyophilization of Pharmaceutical and Biologics Products"


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