Lyophilization

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Biotech Processes.
David M. Fetterolf
Lyophilization
David M. Fetterolf
“Biotech Processes” discusses fundamental information
about biotechnology manufacturing useful to practitioners in validation and compliance. Reader comments,
questions, and suggestions are needed to make this
column a useful resource for daily work applications.
The key objective for this column: Useful information.
Contact column coordinator David Fetterolf at dfetterolf@biotechlogic.com or journal coordinating editor Susan Haigney at shaigney@advanstar.com with
comments or suggestions for future discussion topics.
KEY POINTS
The following key points are discussed in this article:
•Lyophilization, or freeze-drying, is used to remove
moisture by sublimation
•Products are lyophilized to increase shelf life
•Freeze-dried products are reconstituted with water
at time of use
•Lyophilization processes are based on the physical properties of water, as described by the phase
diagram
•Sublimation is effected by control of product temperature and pressure within the lyophilization
equipment
•There are four major steps to the lyophilization
process: formulation/filling, freezing, primary
drying, and secondary drying
•The major components of a lyophilizer are the
chamber, condenser, and vacuum pump.
•Freeze-drying is ancient technology, but lyophilizers have only been around for approximately
100 years
•Lyophilizers are qualified by typical installation
qualification (IQ), operational qualification (OQ),
and performance qualification (PQ) protocols.
Lyophilization processes are qualified by the
For more Author
information,
go to
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three stages of process validation: process definition, process qualification, and continued process
verification.
INTRODUCTION
Lyophilization, more commonly known as “freeze-drying,” is a means of dehydration (desiccation) used in the
food, chemicals, pharmaceutical, and biotechnology
industries. In all cases, lyophilization is used to improve
the stability of a perishable product or make the product
easier to store or transport. In the biotechnology industry, lyophilization is used as a final processing step for
purified active pharmaceutical ingredients (APIs) or drug
products to stabilize the protein for long-term storage.
Freeze-drying is a process that removes water by first
freezing the material within a lyophilizer. The ambient
pressure is then reduced and the temperature is slowly
increased within the lyophilization chamber to allow
frozen water to sublimate (i.e., move from the solid phase
directly to gas). Many food products (e.g., coffee, fruits,
vegetables, meats, and ice cream) can be freeze-dried and
subsequently stored at room temperature. The resulting
product generally retains its original shape and is much
lighter and easier to carry. For example, hikers frequently
pack freeze-dried food to reduce weight in their packs.
The freeze-dried products are easily reconstituted with
water. Freeze-drying is also used to preserve museum
artifacts, remove moisture, and prevent degradation and
mold growth. Similarly, in the biotechnology industry,
protein products, antibodies, oligonucleotides, and vaccines are lyophilized to increase the shelf life by reducing the risk of degradation during storage. Again, these
products are much lighter and take up much less space,
which make them easier to store and ship. The end user
(i.e., doctor, patient, downstream manufacturer, etc.)
simply reconstitutes the freeze-dried powder prior to
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ABOUT THE AUTHOR
David M. Fetterolf is a consultant with BioTechLogic, Inc. He provides manufacturing and CMC
support for clients with biopharmaceutical products from development through commercial launch.
David can be reached by e-mail at dfetterolf@biotechlogic.com.
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David M. Fetterolf, Coordinator.
injection or other use. This article discusses the fundamental principles behind lyophilization and the specific
stages of the lyophilization cycle. It also briefly describes
the types of equipment used in lyophilization and the
types of validation studies that are typically performed
for this unit operation.
Figure 1: Phase diagram for water.
PHASE DIAGRAMS
The principles of lyophilization are based on the physical
properties of water that are illustrated by the phase diagram for water. A phase diagram for a substance describes
the solid, liquid, and gaseous states of a substance as a
function of temperature and pressure. In the lyophilization process, the temperature and pressure conditions
within the lyophilizer are controlled to enable the sublimation of water and its removal from the dosage form.
Water is removed from the dosage form as a gas. Figure
1 provides the phase diagram for water.
As previously stated, the phase diagram for a substance provides information on its state as a function
of temperature and pressure. In Figure 1, temperature
is on the x-axis, with values ranging from below 0°C to
above 100°C. Pressure is on the y-axis, with values from
an absolute vacuum (0 mm Hg or 0 microns) to beyond
760 mm Hg, or atmospheric pressure (760,000 microns).
The three states of water are indicated: solid (ice), liquid
(water), and gas (water vapor). The lines between each
phase represent equilibrium conditions. The following
are phases of water at specific pressures as temperature
is increased, as described in Figure 1:
•Pressure 760 mm Hg or atmospheric pressure (1
atm). We know water freezes, and ice thaws, at
0°C. Between 0°C and 100°C, water is liquid. At
100 °C, water boils and water vapor condenses.
•Pressure 380 mm Hg, or midway down the pressure scale. As temperature increases, ice melts
at slightly above 0°C. As temperature increases
further, water boils at approximately 82°C.
•Pressure 4.58 mm Hg. As temperature increases
to 0.0098°C, ice, water, and water vapor exist in
equilibrium. This is known as the triple point
of water.
•Pressure below 4.58 mm Hg. As temperature
increases, solid ice converts directly to water vapor
gas. Liquid water does not exist at these pressure
and temperature conditions.
Water Phase Diagram And The
Lyophilization Process
The various steps in lyophilization can be plotted on the
water phase diagram to understand how temperature
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and pressure enable sublimation. In sublimation, frozen
water is converted directly to water vapor gas, avoiding the
water liquid state. The following provides an example of a
lyophilization process for a product dissolved in water. The
process begins at ambient temperature and pressure and
proceeds with changes to each paramater, as follows:
•Atmospheric pressure and room temperature. Product in solution is aseptically filled into vials. Water
is in the liquid state.
•Atmospheric pressure and temperature lowered to
-10°C. Product freezes to ice.
•Pressure is reduced to approximately 4 mm Hg and
temperature remains below 0°C. Product remains
as solid ice.
•Pressure maintained at 4 mm Hg and temperature
increased to 20°C or higher. Water begins to sublime
directly into the gaseous state. Transition to the liquid
state does not occur at this pressure and temperature.
Water continues to sublime until all ice has sublimated. This is termed “primary drying.”
•Temperature is continually increased until all adsorbed
moisture is eliminated. Pressure may or may not be
increased. This is termed “secondary drying.”
These conditions enable the dosage form to maintain
its integrity without losses due to boiling. There is no
liquid state in the sublimation process. Figure 2 shows
the stepwise description of the example lyophilization
process described previously. As you can see, the steps
form a curve around the triple point, thus avoiding the
liquid state of water.
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Biotech Processes.
Figure 2: Example lyophilization process.
LYOPHILIZATION PROCESS FOR
BIOPHARMACEUTICAL PRODUCTS
There are four major stages in the lyophilization process
for biopharmaceutical products, as follows:
•Formulation/filling
•Freezing
•Primary drying
•Secondary drying.
Stage 1. Formulation And Filling
As briefly discussed in a previous article in this series
(1), formulation is exchanging the product matrix
(buffer) into the final buffer or adding water or other
raw materials (e.g., excipients) to create the final prelyophilized product in solution. For drug products,
formulation prior to lyophilization usually includes
the addition of water (i.e., water for injection) and
excipients to the drug substance to obtain the desired
product concentration. If the lyophilization is to
occur in the vials that will be used for administration
to the patient, the intent of the formulation process
is to create the actual final drug formulation, which
is a suitable matrix for stabilization of the protein.
The lyophilization process will then remove the
water, which is then re-added just prior to use (i.e.,
reconstitution).
Many times, chemicals that do not increase or
decrease product efficacy are added to the formulation
matrix to protect the product during each stage of the
lyophilization process and/or during long-term storage. These types of chemicals, also called stabilizers
(2), can prevent unwanted changes in the drug, such
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as unfolding, during lyophilization. Commonly used
stabilizers for biologics are sugars and glycols.
At the end of the lyophilization process, biotech drug
products resemble a fluffy white powder, or “cake.”
Because the aesthetics of the cake sometimes play an
important role in product marketability, bulking agents
are often added to make the cake appear fluffier. Bulking agents can also help to prevent “collapse” of the
drug product, which can occur if the product is heated
too rapidly during the drying stages. These bulking
agents are not intended to change the chemical properties of the product. Some examples of bulking agents
typically used in the biopharmaceutical industry are
mannitol, dextran, and polyethylene glycol.
Once the product is in the proper form and all excipients are added, the last step prior to placing the material
into the lyophilizer is filling the product into the proper
container. For biotechnology products, the containers
are usually glass vials, which come in a variety of shapes,
sizes, and colors. Although not always, vials are typically
used if no further processing is needed. Once the product
is filled into the vials, each vial is partially stoppered (i.e.,
not fully pushed into place) such that the vial is vented
so water vapor can escape during lyophilization. Other
types of containers such as trays can be used to lyophilize large quantities of product. Trays are typically used
when lyophilization is an intermediate processing step.
Regardless of the container choice, aseptic technique is
used during filling and lyophilization processes if the
drug product is a parenteral.
Stage 2. Freezing
Once the product is placed into the lyophilizer chamber,
the product (inside the vials or trays) is frozen. This is done
by cooling the lyophilizer shelves, which are in contact with
the product container, to freeze the contents. This freezing process separates the water from the product, and also
decreases chemical activity of the product. What results is
an amorphous (without any clear shape) solid product and
water crystals. Typical shelf temperatures for lyophilization
of protein products are around -40°C or lower.
From the simple phase diagram shown in Figure
3, lowering the temperature of a liquid at constant
pressure results in a phase change from liquid to solid
(point 1 to point 2).
It is clear that the temperature of the shelves, type of
container, amount of product in each vial or tray, height
of liquid, etc. can impact the rate of freezing, which, in
turn, impacts the cake form and structure (i.e., morphology), drying rate, and (in some cases) product stability.
In general, fast rates of freeze are harder to control, and
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David M. Fetterolf, Coordinator.
Figure 3: Phase diagram for freezing.
Figure 4: Phase diagram for drying.
therefore, are more variable. They also tend to produce a finer structure, which results in a slower rate
of water transfer during the subsequent drying step.
There is also some evidence that the higher surface area
resulting from smaller crystals can lead to increased
product degradation. These types of consequences
(i.e., increased vs. decreased cycle time, potential degradation, etc.) are kept in mind when designing and
optimizing the overall lyophilization cycle.
from the cake, the temperature will slowly increase
to the temperature of the shelf. An equivalent temperature of the product and shelves is a signal that
primary drying has ended.
As mentioned previously, the drying and heating
rate must be carefully controlled. The heating of the
product must be kept below the glass transition temperature (Tg) of the solution, which is the point in the
freezing process at which the physical state changes
from an elastic liquid to a brittle but amorphous solid
glass and the point at which ice formation ceases (3).
If heat is applied too quickly or to temperatures above
Tg, the cake can melt or collapse, which could lead to
degradation and aesthetic issues mentioned previously
(4). The cake could be difficult to reconstitute at a
later point, as well. Although rare, drying the product
too fast at this stage could result in the product being
carried off with the exiting water vapor.
Stage 3. Primary Drying
After freezing, two types of water exist within the
product; these include the following:
•Mobile water free from the amorphous solid
•Bound/trapped water within the amorphous solid.
The intent of primary drying is to remove the
mobile water from the product, which is accomplished
by lowering the lyophilizer chamber pressure (i.e.,
pulling a vacuum). By the phase diagram in Figure
4, one can see that lowering the pressure at constant
temperature results in a phase change from solid to
gas (i.e., sublimation–point 2 to point 3). Sublimation at atmospheric conditions is commonly seen
when frozen carbon dioxide (dry ice) is left at room
temperature. The solid turns to a gas without first
changing into the liquid form.
Because the product temperature decreases during
the sublimation process, heat is added via the lyophilizer shelves to keep the cake at a relatively constant
temperature—that is, the shelves are providing the
heat of sublimation. However, as water is removed
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Stage 4. Secondary Drying
At the end of primary drying, there is no mobile water
left in the product. However, the water trapped within
the amorphous solid is more difficult to remove. To
do this, temperature is increased at the low pressures
used for primary drying. Again, as in primary drying,
it is important that the temperature is not increased
too quickly, and that it stays below the Tg, which
(coincidentally) increases as water is removed (3).
This results in a porous, fluffy cake with little residual
moisture. Increasing the temperature too quickly, or
above Tg, could result in collapse and make reconstitution difficult (5).
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Secondary drying can be a lengthy process, lasting
up to several days. Typical residual moisture levels after secondary drying are less than 1%, but are
dependent on the needs of each individual product.
Karl Fisher Titration (ASTM E203-08) (6) is the most
common test used to determine residual moisture
levels. Because the dry product will act as a sponge
and pull water from ambient conditions, the product
containers are closed/capped as soon after secondary
drying as possible. Most lyophilizers have the capability of pushing stoppers into place while the product
is still under vacuum. If using trays, they are sealed
immediately upon release of the vacuum and product
removal from the lyophilizer chamber.
To fully define the lyophilization process, development studies are performed to characterize the
freeze-drying parameters. A risk-assessment is then
performed to determine potentially critical parameters, which are then carried into a DOE framework
to fully define the design and control spaces. Typical
product quality attributes that are monitored during
these types of studies include, but are not limited to,
residual moisture, potency, purity, etc. at all places
within the lyophilization chamber (i.e., product uniformity). Then, the lyophilization process is qualified
and further monitored and evaluated during continued process verification; both under prospective
protocols.
Lyophilization Cycle Optimization
CONCLUSION
The four stages of lyophilization described previously
are intended to provide a basic understanding of the
principles behind lyophilization. The discussion
of the addition of annealing steps (e.g., to modify
water crystal structure), solvents, inert gases, temperature optimization, pressure optimization, and
other parameters during lyophilization is beyond the
scope of this article; however, these are common ways
to aid in the optimization of moisture removal. See
the articles listed in the references and recommended
resources sections for more information.
All four stages of the lyophilization process (i.e.,
formulation, freezing, primary drying, and secondary drying) are equally important to the successful
performance of any lyophilization process and are
instrumental in producing a stable product for longterm storage. Any change in one step has the potential
to greatly impact the subsequent steps, overall product quality, or final moisture level. It is important
to understand the basic principles of lyophilization
and then apply them to each individual product and
lyophilization process. Proper process qualification
and continuous process monitoring can then be performed to ensure proper validation.
VALIDATION OF LYOPHILIZATION
EQUIPMENT AND PROCESSES
A freeze-dryer, or lyophilizer, is made up of a chamber,
condenser, and vacuum pump. The basics of freezedrying food were used by ancient Peruvian Incas;
however, laboratory versions of lyophilizers have only
been around for approximately 100 years. As you can
imagine, designs of laboratory and manufacturing-scale
lyophilizers have greatly evolved over the last century.
Equipment has increased in complexity, which makes
validation of the lyophilization equipment and process
a time-consuming activity. In addition to the cooling (freezing), heating, and vacuum control functions
described in the previous sections, many freeze-dryers
now incorporate computerized control and monitoring
systems, clean-in-place (CIP), and sterilize-in-place
(SIP) functionality. The reliability and reproducibility of these functions must be validated (through IQ,
OQ, and PQ protocols) to ensure consistent moisture
removal and overall product quality; therefore, typical
validation of a lyophilizer involves multiple protocols
(or multiple sections) focusing on verifying the performance of each function.
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REFERENCES
1.Houp, Rachel C., “Biotech Processes: Ultrafiltration/
Diafiltration,” Journal of Validation Technology, Autumn
2009.
2.Carpenter, J.F., Pikal, M.J., Chang, B.S., and Randoloph,
T.W., “Rational Design of Stable Lyophilized Protein
Formulations: Some Practical Advice,” Pharmaceutical
Research, Vol. 14, No. 8, 1997.
3.BioPharm International, “Guide to Formulation, Fill,
and Finish,” The BioPharm International Guide, August
2004.
4.FTS Systems, Inc., “Basic Theory of Freeze Drying,”
Dura-Dry MP Instruction Manual, February 1991.
5.Virtis, “Freeze Drying 101,” http://www.virtis.com/literature/freeze101.jsp.
6.ASTM International, “ASTM E203-08, Standard Test
Method for Water Using Volumetric Karl Fischer Titration,” http://www.astm.org/Standards/E203.htm.
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David M. Fetterolf, Coordinator.
RECOMMENDED RESOURCES
Jennings, T.A., Lyophilization—Introduction and Basic Principles, CRC Press LLC, Boca Raton, Florida, 1999.
Carpenter, J.F. and Chang, B.S., “Lyophilization of Protein
Pharmaceuticals, Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation,” Interpharm Press, Buffalo Grove, IL, pp. 199 – 264, 1996.
Carpenter, J.F. and Manning, M.C. (editors), Rational Design of
Stable Protein Formulations: Theory and Practice (Pharmaceutical Technology), Springer, 1st Edition, April 30, 2002.
Tang, X. and Pikal, M.J., “Design of Freeze Drying Processes
for Pharmaceuticals: Practical Advice,” Pharmaceutical Research, Vol. 21, No. 2, February 2004.
“Product Technologies for Lyophilization,” Genetic Engineering and Biotechnology News, November 15, 2006. JVT
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GLOSSARY
Glass transition temperature (Tg). The point
in the freezing process at which the physical state
changes from an elastic liquid to a brittle but amorphous solid glass. This is the point at which ice formation ceases.
Karl Fischer Titration. Most common method by
which residual moisture is determined in a product
sample.
Phase diagram. Information about the solid, liquid,
and gaseous states of a substance as a function of
temperature and pressure.
ARTICLE ACRONYM LISTING
CIP
DOE
IQ
OQ
PQ
SIP
Tg
Clean in Place
Design of Experiments
Installation Qualification
Operational Qualification
Performance Qualification
Sterilize in Place
Glass Transition Temperature
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