Chapter 3
The Principles of Freeze-Drying and Application
of Analytical Technologies
Kevin R. Ward and Paul Matejtschuk
Abstract
Freeze-drying is a complex process despite the relatively small number of steps involved, since the freezing,
sublimation, desorption, and reconstitution processes all play a part in determining the success or otherwise
of the final product qualities, and each stage can impose different stresses on a product. This is particularly
the case with many fragile biological samples, which require great care in the selection of formulation
additives such as protective agents and other stabilizers. Despite this, the process is widely used, not least
because once any such processing stresses can be overcome, the result is typically a significantly more stable
product than was the case with the starting material. Indeed, lyophilization may be considered a gentler
method than conventional air-drying methods, which tend to apply heat to the product rather than starting
by removing heat as is the case here. Additionally, due to the high surface area to volume ratio, freeze-dried
materials tend to be drier than their conventionally dried counterparts and also rehydrate more rapidly. This
chapter provides an overview of freeze-drying (lyophilization) of biological specimens with particular
reference to the importance of formulation development, characterization, and cycle development factors
necessary for the commercial exploitation of freeze-dried products, and reviews the recent developments in
analytical methods which have come to underpin modern freeze-drying practice.
Key words Freeze-drying, Lyophilization, Lyoprotectants, Sublimation, Freeze-drying microscopy,
Thermal analysis, Process analytical technology
1 Introduction
1.1 General Overview
Water is essential to life, providing a universal solvent supporting
biochemical activities within cells, which enables metabolisms to
continue and sustains all living processes. Quite simply, in the
absence of water, life as we define it will cease or at best enter a
period of dormancy in live cells, while biochemical activity will be
suspended. Water determines structure and function of biomolecules, and removal of water may lead to irreversible changes in
biomolecular structure and loss of function. On the other hand,
biomolecules are prone to degradation reactions when stored in
water. Vaccines, other biological materials, and microorganisms can
be stabilized by chilling or freezing. However, maintaining and
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
99
The Principles of Freeze-Drying and Application
of Analytical Technologies
Kevin R. Ward and Paul Matejtschuk
Abstract
Freeze-drying is a complex process despite the relatively small number of steps involved, since the freezing,
sublimation, desorption, and reconstitution processes all play a part in determining the success or otherwise
of the final product qualities, and each stage can impose different stresses on a product. This is particularly
the case with many fragile biological samples, which require great care in the selection of formulation
additives such as protective agents and other stabilizers. Despite this, the process is widely used, not least
because once any such processing stresses can be overcome, the result is typically a significantly more stable
product than was the case with the starting material. Indeed, lyophilization may be considered a gentler
method than conventional air-drying methods, which tend to apply heat to the product rather than starting
by removing heat as is the case here. Additionally, due to the high surface area to volume ratio, freeze-dried
materials tend to be drier than their conventionally dried counterparts and also rehydrate more rapidly. This
chapter provides an overview of freeze-drying (lyophilization) of biological specimens with particular
reference to the importance of formulation development, characterization, and cycle development factors
necessary for the commercial exploitation of freeze-dried products, and reviews the recent developments in
analytical methods which have come to underpin modern freeze-drying practice.
Key words Freeze-drying, Lyophilization, Lyoprotectants, Sublimation, Freeze-drying microscopy,
Thermal analysis, Process analytical technology
1 Introduction
1.1 General Overview
Water is essential to life, providing a universal solvent supporting
biochemical activities within cells, which enables metabolisms to
continue and sustains all living processes. Quite simply, in the
absence of water, life as we define it will cease or at best enter a
period of dormancy in live cells, while biochemical activity will be
suspended. Water determines structure and function of biomolecules, and removal of water may lead to irreversible changes in
biomolecular structure and loss of function. On the other hand,
biomolecules are prone to degradation reactions when stored in
water. Vaccines, other biological materials, and microorganisms can
be stabilized by chilling or freezing. However, maintaining and
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
99