Basic Principles of Freeze Drying

Freeze-drying—technically known as lyophilisation,lyophilization, or cryodesiccation—is

a dehydration process typically used to preserve a perishable material or make the material more
convenient for transport. Freeze-drying works by freezing the material and then reducing the
surrounding pressure to allow the frozen water in the material to sublime directly from the solid
phase to the gas phase.

Stages

In a typical phase diagram, the boundary between gas and liquid runs from the triple point to the critical point.
Freeze-drying (blue arrow) brings the system around the triple point, avoiding the direct liquid-gas transition
seen in ordinary drying time (green arrow).

There are four stages in the complete drying process: pretreatment, freezing, primary drying, and
secondary drying.

Pretreatment[edit]
Pretreatment includes any method of treating the product prior to freezing. This may include
concentrating the product, formulation revision (i.e., addition of components to increase stability,
preserve appearance, and/or improve processing), decreasing a high-vapor-pressure solvent, or
increasing the surface area. Food pieces are often IQF treated to make it free flowing prior to freeze
drying. In many instances the decision to pretreat a product is based on theoretical knowledge of
freeze-drying and its requirements, or is demanded by cycle time or product quality considerations.[7]

Freezing[edit]
In a lab, this is often done by placing the material in a freeze-drying flask and rotating the flask in a
bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice in
aqueous methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a freeze-
drying machine. In this step, it is important to cool the material below its triple point, the lowest
temperature at which the solid, liquid and gas phases of the material can coexist. This ensures that
sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-
dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in
temperature. This cycling process is called annealing. However, in the case of food, or objects with
formerly-living cells, large ice crystals will break the cell walls (a problem discovered, and solved,
by Clarence Birdseye), resulting in the destruction of more cells, which can result in increasingly
poor texture and nutritive content. In this case, the freezing is done rapidly, in order to lower the
material to below its eutectic point quickly, thus avoiding the formation of ice crystals. Usually, the
freezing temperatures are between −50 °C and −80 °C (-58 °F and -112 °F) . The freezing phase is
the most critical in the whole freeze-drying process, because the product can be spoiled if improperly
done.
Amorphous materials do not have a eutectic point, but they do have a critical point, below which the
product must be maintained to prevent melt-back or collapse during primary and secondary drying.

Primary drying
During the primary drying phase, the pressure is lowered (to the range of a few millibars), and
enough heat is supplied to the material for the ice to sublime. The amount of heat necessary can be
calculated using the sublimating molecules' latent heat of sublimation. In this initial drying phase,
about 95% of the water in the material is sublimated. This phase may be slow (can be several days
in the industry), because, if too much heat is added, the material's structure could be altered.
In this phase, pressure is controlled through the application of partial vacuum. The vacuum speeds
up the sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser
chamber and/or condenser plates provide a surface(s) for the water vapour to re-solidify on. This
condenser plays no role in keeping the material frozen; rather, it prevents water vapor from reaching
the vacuum pump, which could degrade the pump's performance. Condenser temperatures are
typically below −50 °C (−58 °F).
It is important to note that, in this range of pressure, the heat is brought mainly by conduction or
radiation; the convection effect is negligible, due to the low air density.

Secondary drying[edit]

Miniature quadrupole mass spectrometer used for accurate secondary endpoint detection in aseptic
lyophilization application

The secondary drying phase aims to remove unfrozen water molecules, since the ice was removed
in the primary drying phase. This part of the freeze-drying process is governed by the
material's adsorption isotherms. In this phase, the temperature is raised higher than in the primary
drying phase, and can even be above 0 °C, to break any physico-chemical interactions that have
formed between the water molecules and the frozen material. Usually the pressure is also lowered in
this stage to encourage desorption (typically in the range of microbars, or fractions of a pascal).
However, there are products that benefit from increased pressure as well.
After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as
nitrogen, before the material is sealed.
At the end of the operation, the final residual water content in the product is extremely low, around
1% to 4%.
OVERVIEW
Freeze drying is the removal of ice or other frozen solvents from a material through the process of
sublimation and the removal of bound water molecules through the process of desorption.

Lyophilization and freeze drying are terms that are used interchangeably depending on the industry and
location where the drying is taking place. Controlled freeze drying keeps the product temperature low
enough during the process to avoid changes in the dried product appearance and characteristics. It is an
excellent method for preserving a wide variety of heat-sensitive materials such as proteins, microbes,
pharmaceuticals, tissues & plasma.

SUBLIMATION
Sublimation is when a solid (ice) changes directly to a vapor without first going through a liquid (water)
phase. Thoroughly understanding the concept of sublimation is a key building block to gaining knowledge
of freeze drying.

As shown below on the phase diagram for water, low pressures are required for sublimation to take place.

Sublimation is a phase change and heat energy must be added to the frozen product for it to occur.

Sublimation in the freeze drying process can be described simply as:

1. FREEZE - The product is completely frozen, usually in a vial, flask or tray.

2. VACUUM - The product is then placed under a deep vacuum, well below the triple point of water.
3. DRY – Heat energy is then added to the product causing the ice to sublime.
The steps required to lyophilize a product in a batch process can be summarized as follows:

 Pretreatment / Formulation
 Loading / Container (Bulk, Flask, Vials)
 Freezing (Thermal Treatment) at atmospheric pressure
 Primary Drying (Sublimation) under vacuum
 Secondary Drying (Desorption) under vacuum…….Desorption is a phenomenon whereby a
substance is released from or through a surface.
 Backfill & Stoppering (for product in vials) under partial vacuum
 Removal of Dried Product from Freeze Dryer
In addition to providing an extended shelf-life, successful freeze-drying should yield a product that has a
short reconstitution time with acceptable potency levels. The process should be repeatable with well
defined temperature, pressure and time parameters for each step. Visual and functional characteristics of
the dried product are also important for many applications.
FREEZE DRYING EQUIPMENT
The main components of freeze drying equipment are:

 Refrigeration System
 Vacuum System
 Control System
 Product Chamber or Manifold
 Condenser
The refrigeration system cools the (ice) condenser located inside the freeze dryer. The refrigeration
system can also be employed to cool shelves in the product chamber for the freezing of the product.

The vacuum system consists of a separate vacuum pump connected to an airtight condenser and
attached product chamber.

Control systems vary in complexity and usually include temperature and pressure sensing ability.
Advanced controllers will allow the programming of a complete “recipe” for freeze drying and will include
options to monitor how the freeze drying process is progressing. Choosing a control system for the freeze
dryer depends on the application and use (i.e. lab vs. production).

Product chambers are typically either a manifold with attached flasks, or, a larger chamber with a system
of shelves on which to place the product.

The purpose of the condenser is to attract the vapors being sublimed off of the product. Because the
condenser is maintained at a lower energy level relative to the product ice, the vapors condense and turn
back into solid form (ice) in the condenser. The sublimated ice accumulates in the condenser and is
manually removed at the end of the freeze drying cycle (defrost step). The condenser temperature
required is dictated by the freezing point and collapse temperature of the product. The refrigeration
system must be able to maintain the temperature of the condenser substantially below the temperature of
the product.
In shelf freeze dryers, the condenser can be located inside the product chamber (internal condenser) or in
a separate chamber (external condenser) connected to the product chamber by a vapor port.

Manifold freeze dryers rely on ambient conditions to provide the heat of sublimation to the product. This
heat input does not melt the product because an equivalent amount of heat is removed by vaporization of
the solvent. Advanced shelf freeze dryers can provide a heat source to control/expedite the drying
process and they can also employ the refrigeration system to allow freezing of product inside the unit.

Freeze dryers can be informally classified by the type of product chamber: (1) Manifold dryers where the
product is typically pre-frozen & in flasks (2) Shelf dryers where the product is placed in a tray or directly
on a shelf (3) Combination units with both drying options.

Freeze-dryers can also be grouped by size & use: (1) laboratory bench-top units for R&D (2) pilot units for
process development and scale-up, and (3) larger production-sized units. It should be noted that in
addition to process scale-up work, pilot-sized freeze dryers are often used for product R&D as well as
small volume production applications.
Choosing a freeze dryer depends on the product characteristics as well as many other application-based
variables including the container that the product will be dried in, the shelf area or number of ports
required to accommodate the quantity to be dried in each batch, the total volume of ice to be condensed
and whether there are any organic solvents. The type and shape of product being dried and its end-use
also need to be considered.

PRODUCT CONTAINERS AND CONTAINMENT SYSTEMS

A suitable container system must be chosen for the product. The most common product containers are
flasks, vials and trays. If possible, it is advisable to pick a container that keeps the maximum thickness of
the product to less than ¾” (2 cm). Special containers made of Gore-Tex® & Tyvek® are also available
for specific applications where product contamination is a concern.

Product trays with removable-bottoms are available when working with vials. The tray is loaded with vials,
placed on a shelf in the freeze dryer and then the bottom part of the tray is slid out. This allows the vials
to rest directly on the shelf and increases the heat transfer to the product.

Special containment systems such as glove boxes are required for freeze drying certain products,
especially when toxic materials are present.

PHYSICAL PROPERTIES OF MATERIALS AND FORMULATION

Understanding the physical properties of materials that are freeze-dried is a key part in developing a
successful lyophilization process. Although a few products are simple crystalline materials, the vast
majority of products that are lyophilized are amorphous and form glassy states when frozen.

Processing and formulation development are important steps often taken to make a product ready for
freeze drying and usable for its specific application. The choice of excipients added to a formulation can
severely affect the thermal characteristics of the product and its ability to be freeze dried in a reasonable
amount of time.
RECIPE FOR FREEZE DRYING
Lyophilization in a shelf freeze dryer requires the design of a working process or cycle which is
sometimes referred to as a “recipe”. Typically, there are multiple steps involved for both freezing and
drying of the product. Individual temperature, pressure and time settings need to be determined for each
step.

Each specific product or formulation that is lyophilized requires the development of a freeze drying
process that is based on the unique characteristics of the product, the amount of product and the
container used. There is no universal “safe” recipe that will work with every product.

FREEZING
It is extremely important that the sample be fully and completely frozen prior to pulling a vacuum and
starting the drying process. Unfrozen product may expand outside of the container when placed under a
vacuum.

With simple manifold freeze dryers, the product is placed in a vial or flask depending on quantity, and
then frozen in a separate piece of equipment. Options include standard laboratory freezers, shell baths,
and direct immersion in liquid nitrogen.

Shell (bath) freezing involves rotating a flask containing the sample in a freezing bath so the sample
freezes on the walls of the flask. This freezing method maximizes the product surface area and minimizes
its thickness. It is best not to freeze a large block of sample in the bottom of a flask because the sample
will be too thick for efficient water removal. Also, the flask might break due to uneven stress.
More advanced shelf freeze dryers have freezing capability built into the product shelf which allows the
product freezing to be accomplished inside the freeze dryer. Product is either pre-loaded into vials which
are then transferred to the shelf or it is loaded in bulk form directly onto a product tray.

Shelf freeze dryers allow the precise control of cooling rates which affects product freezing rates and
crystal size. Larger ice crystals improve the speed of the freeze drying process because of the larger
vapor pathways left behind in the dried portion of the product as the ice crystals are sublimated.

Slower shelf cooling rates do not necessarily yield larger ice crystals because of the effects of super-
cooling. When the super-cooled liquid finally freezes, it happens extremely quickly resulting in smaller ice
crystals. In a clean room environment with very few particulates for ice nucleation, there is a significantly
greater amount of super-cooling.

Some biological products can not tolerate large ice crystals and they must be freeze dried with smaller ice
crystal sizes.

EUTECTIC / COLLAPSE TEMPERATURE

Determination of the critical collapse temperature of a product is an important step in establishing and
optimizing a freeze drying process. This critical temperature determines the maximum temperature that
the product can withstand during primary drying without it melting or collapsing. Thermal analysis
(Differential Scanning Calorimetry & Freeze Dry Microscopy) and Dielectric Resistance analysis and are
common methods used to determine this critical temperature of the product.

Frozen products can be categorized as either crystalline or amorphous glass in structure. Crystalline
products have a well defined “eutectic” freezing/melting point that is its collapse temperature. Amorphous
products have a corresponding “glass transition” temperature and they are much more difficult to freeze
dry. The collapse temperature of amorphous products is typically a few degrees warmer than its glass
transition temperature. Although most materials that are freeze dried are actually amorphous, the term
“eutectic” is often used (erroneously) to describe the freezing/melting point any product.
The US FDA Guide To Inspections Of Lyophilization Of Parenterals (
http://www.fda.gov/ora/inspect_ref/igs/lyophi.html ) states that the manufacturer should know the eutectic
point (critical collapse temperature) of the product. It is good practice to characterize the collapse
temperature for all new injectable or ingestible drug formulations to be freeze dried.

Without knowing the critical temperature of the product, a trial and error approach is required to determine
appropriate primary drying temperatures. A slow conservative cycle with low temperatures & pressures
can be used initially. The temperature & pressure can then be raised on subsequent cycles until evidence
of collapse or melt-back is seen – indicating that the product was too warm.

ANNEALING
Some amorphous products (such as mannitol or glycine) form a metastable glass with incomplete
crystallization when first frozen. These products can benefit from a thermal treatment process, which is
also called annealing. During annealing, the product temperature is cycled (for example: from -40C to -
20C for a few hours and then back to -40C) to obtain more complete crystallization. Annealing has the
added advantage of larger crystal growth and corresponding shorter drying times.

ORGANIC SOLVENTS
The use of organic solvents requires more attention in the freeze drying process. Lower temperatures are
required to freeze and condense solvents and they can easily bypass the condenser and end up causing
damage to the vacuum pump. Freeze dryer refrigeration designs are available to provide the lower shelf
and condenser temperatures needed to freeze and then condense some organic solvents.

Special filter cartridges or liquid nitrogen (LN2) traps may be required to catch/condense certain solvents
with very low freezing temperatures. Safety considerations must be made when handling volatile and/or
potentially harmful materials.
PRIMARY DRYING
The drying portion of freeze drying is actually a two part process consisting of Primary Drying and
Secondary Drying. The bulk of water removed from the product during freeze drying is via sublimation of
all of the free ice crystals during the primary drying step. Organic solvents are also removed during
primary drying.

Primary drying (sublimation) is a slow process conducted at cooler temperatures, safely below the
product’s critical collapse temperature. Sublimation requires heat energy to drive the phase change
process from solid to gas. All three methods of heat transfer - conduction, convection and radiation, must
be considered when freeze drying a product.

In a simple manifold dryer, heat is transferred to the flask/product primarily through convection and
radiation from the surrounding environment. With little control over heat flow into the product, it is more
difficult to control the process. When working with products with low collapse temperatures, it may be
necessary to wrap or insulate the flask to slow down the rate of heat transfer and avoid collapse.

In a shelf freeze dryer, most of the heat is transferred into the product through conduction and it is
important to maximize the surface contact of the product/container/tray with the shelf. However, the
effects of radiation and convection also need to be considered for product uniformity and process control
purposes.

Radiant heat from the inside walls of the product chamber will cause product/vials on the perimeter of the
shelf to dry more quickly than product in the center of the shelf (known in freeze-drying as the “edge
effect”). Radiation coming through the acrylic doors commonly used on pilot and R&D freeze dryers has
an even greater effect and product located in the front of these dryers will typically dry the fastest of all.
For this reason, production freeze dryers are designed with metal doors and small view ports. A piece of
aluminum foil can be hung in front of the product on the inside of a pilot freeze dryer as a shield – of
course this will block the view of the product and not allow observation during the process.

Because shelf contact is often inconsistent, convective heat transfer can help promote uniform product
drying. System pressures in the 100 mTorr to 300 mTorr range will usually promote an adequate amount
of convection. At ultra low system pressures less than 50 mTorr, there are fewer gas molecules present to
provide convection and uneven / slower drying is likely.

Primary drying is a top-down process with a well-defined sublimation front moving through the product as
it dries. Above the ice surface interface is dried product, or “cake”; below the interface is product with ice
crystals still remaining to be sublimed. At the end of primary drying when all of the free ice crystals have
been sublimed, the product will appear to be dried. However, the moisture content can still be in the 5-
10% range due to the presence of “sorbed” water molecules attached to the product.

PRESSURE & TEMPERATURE DURING PRIMARY DRYING

As mentioned earlier, each frozen product has a unique critical temperature. It is necessary to keep the
product temperature safely below this critical temperature during primary drying to avoid collapse. The
product temperature is dependant on the vapor pressure at the ice interface and in turn, this vapor
pressure is dependant on both the rate of heat transfer into the product (which is controlled by adjusting
the shelf temperature) and the system vacuum level set point.

Once a target product temperature is identified (typically several degrees colder than the critical
temperature), the only two variables left to determine/control are the shelf temperature and system
vacuum level. During primary drying, the system pressure and the shelf temperature are set and
controlled in combination to yield the appropriate product temperature.

A recommended approach is to first set the system pressure using the vapor pressure of ice table. The
product temperature is monitored using thermocouples and then the shelf temperature set point is slowly
increased until the product reaches its target temperature. When the target product temperature is
obtained, the shelf temperature is held constant for the balance of primary drying. Certain products with
high resistance to vapor flow in the dried portion of the cake may require that the shelf temperature be
reduced towards the end of primary drying to keep the product temperature at its target and to avoid
collapse.

It is not recommended to arbitrarily and repetitively increase the shelf temperature during primary drying,
as is seen on some older legacy cycles.

Using the vapor pressure of ice table is a scientific way to determine an appropriate pressure for freeze
drying. A general guideline is to choose a system pressure that is 20% to 30% of the vapor pressure of
ice at the target product temperature. When the vacuum level set point is deeper than the vapor pressure
of ice at the current product temperature, sublimation can take place. Typically, vacuum levels for freeze
drying are between 50mTorr and 300mTorr with 100mTorr to 200mTorr being the most common range.

With the temperature and pressure parameters set, primary drying is then continued for a length of time
sufficient for all of the ice crystals to be sublimed.
Because most commercial freeze dryers can not consistently control vacuum much below 30mTorr, at
very cold product temperatures (less than -40ºC), it becomes impossible to have a system pressure set
point that is 20% to 30% of the vapor pressure of ice. Freeze drying occurs extremely slowly at these cold
product temperatures.

With manifold freeze drying, the process is driven by the system pressure set point and the ambient
temperature in the room. Because of the lack of control over the rate of heat transfer into the product,
most manifold dryers are operated conservatively at lower pressures to help keep the product
temperature lower.

DETERMINATION OF THE END OF PRIMARY DRYING

Several analytical methods are available for determining that primary drying is complete. The most basic
method is to monitor the product temperature with a thermocouple probe. The measured product
temperature will be colder than the shelf temperature set point during active primary drying because the
heat from the shelf is being used for the sublimation phase change. When sublimation of ice crystals is
complete, the product temperature will increase and approach the shelf temperature. When the product
temperature equals the shelf temperature, it can be inferred that primary drying is complete.

Note: the specific vial that contains the thermocouple wire will typically dry faster than the other vials on
the shelf because the wire will conduct more heat into that specific vial. Similarly, if bulk drying, the area
around the thermocouple wire will dry more quickly than other areas in the product tray. It is important to
allow a modest amount of additional drying time (30 min to 2 hrs, depending on the product
characteristics) after the product thermocouple temperature increases to ensure that all of the ice in the
entire batch of product has been completely sublimated.

Because product will dry from the top down, the tip of the thermocouple should always be placed at the
very bottom and center of the container. It is OK if the thermocouple touches the bottom of the container.
If drying in vials, it is good practice to insert the thermocouple in a vial located in the middle of the shelf.
Radiant heating effects will cause vials/product on the perimeter of the shelf to dry more quickly.
Additional primary drying endpoint determination tools are available on larger freeze dryers equipped with
advanced process control systems. One such method entails comparison of parallel pressure readings
between a Pirani gauge and a capacitance manometer. A capacitance manometer always gives a true
pressure reading in the product chamber. The Pirani gauge, however, will give a false high reading in the
presence of water vapor. When the Pirani pressure reading decreases and approaches the true pressure
reading of the capacitance manometer, little or no water vapor is present and it can be concluded that
primary drying is complete.

Another tool is available with freeze dryer designs that have external condensers. An isolation valve can
be added to the vapor port that connects the product chamber to the condenser. This valve can be closed
for a short period of time and the subsequent rise in pressure in the product chamber can be measured.
When this pressure rise approaches zero, no more water vapor is being generated via sublimation.

SECONDARY DRYING
In addition to the free ice that is sublimed during primary drying, there remains a substantial amount of
water molecules that are bound to the product. This is the water that is removed (desorbed) during
secondary drying. Since all of the free ice has been removed in primary drying, the product temperature
can now be increased considerably without fear of melting or collapse.

Secondary drying actually starts during the primary phase, but at elevated temperatures (typically in the
30ºC to 50ºC range), desorption proceeds much more quickly. Secondary drying rates are dependant on
the product temperature. System vacuum may be continued at the same level used during primary drying;
lower vacuum levels will not improve secondary drying times.

Amorphous products may require that the temperature increase from primary to secondary drying be
controlled at a slow ramp rate to avoid collapse.
Secondary drying is continued until the product has acceptable moisture content for long term storage.
Depending on the application, moisture content in fully dried products is typically between 0.5% and 3%.
In most cases, the more dry the product, the longer its shelf life will be. However, certain complex
biological products may actually become too dry for optimum storage results and the secondary drying
process should be controlled accordingly.

During secondary drying, a “sample thief” mechanism may be used to periodically remove vials from the
freeze dryer for residual moisture content determination.

CYCLE OPTIMIZATION
In addition to designing a recipe that successfully dries a product, it is also extremely valuable to optimize
(shorten) the length of the cycle, especially if there is potential for process repetition or scale-up for
production. Freeze drying can be a multi-day process. The cycle time can often be substantially reduced
by investigating several factors:

 Freezing and annealing – maximize crystal size and crystallization to increase drying rates.
 Thickness of product - water vapor molecules experience resistance as they exit from the dried
portion of the product. Thinner samples yield less resistance to vapor flow and lead to faster drying.
Shell freezing can help when drying bulk product in flasks.
 Critical Collapse Temperature – this is the most important piece of information for cycle optimization.
The ability to run primary drying at higher product temperatures greatly reduces drying time by
creating a larger pressure differential between the vapor pressure over ice in the product and the
pressure at the condenser. Each 1oC increase in product temperature can decrease primary drying
time by 13%.
Cycle optimization using eutectic/collapse temperature information requires an iterative approach of
taking real-time measurements of the product temperature during primary drying and then making
corresponding adjustments to the shelf temperature settings. This can be accomplished manually using
product thermocouples or, if drying in vials, an automated SMART system can be used.
PROCESS SCALE-UP CONSIDERATIONS
Laboratory pilot-sized shelf freeze dryers are often used to develop a cycle to be used for process scale-
up to a larger production sized unit. Similarity in heat transfer characteristics and shelf temperature
uniformity is important to ensure that a lyophilization process developed in the lab can be successfully
transferred to a production freeze dryer.

One of the most important factors to consider is the difference between the clean room environment
typical of a production freeze dryer and the lab environment that most pilot units are operated in. The
difference in particulates can greatly affect product freezing and ice crystal size.

Production freeze dryers are usually configured for operation in a clean room environment and can have
the ability for clean-in-place (CIP) and steam sterilization (SIP). Another production consideration is
process compliance to US FDA regulation 21 CFR 11, if required. This regulation requires certain
standards of process control and security.

STORAGE OF DRIED PRODUCT

Lyophilized products are extremely hydroscopic and they must be sealed in air tight containers following
freeze drying to prevent rehydration from atmospheric exposure. Freeze dryers can be configured with a
“stoppering” capability to seal the product while it is still under partial vacuum inside the unit. Typically,
stoppering is done on vials with partially inserted stoppers. The shelves are collapsed so that each shelf
pushes down the vials/stoppers located on the adjacent shelf. It is also common to backfill with an inert
gas such as dry nitrogen before sealing/stoppering the product.

FREEZE DRYER CARE AND MAINTENANCE

In addition to defrosting the condenser and cleaning the system after each cycle, routine freeze dryer
maintenance typically includes periodic changing of vacuum pump oil and visually checking all seals and
gaskets. Advanced controllers offer the capability of running a periodic system test and/or leak test to
ensure that the unit is performing to original factory specifications.