Lyophilization

PHARMACEUTICAL PRO CESSES
Lyophilization
GLOWIMAGES/GETTY IMAGES
David M. Fetterolf
52 Journal of GXP Compliance
“Pharmaceutical Processes” discusses scientific and technical principles associated with pharmaceutical unit operations useful to practitioners in compliance and validation. We intend this column to be a useful resource for daily
work applications. The primary objective for this feature: Useful information.
Reader comments, questions, and suggestions are needed to help us fulfill
our objectives. Please send your comments and suggestions to column coordinator Armin Gerhardt at [email protected] or to journal coordinating editor
Susan Haigney at [email protected].
KEY POINTS
The following key points are discussed in this article:
•Lyophilization, or freeze-drying, is used to remove moisture from
pharmaceutical and biotechnology materials by sublimation.
•Products are lyophilized to improve stability and increase their
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,
operational qualification, and performance qualification protocols.
Lyophilization processes are qualified by the three stages of process
validation, process design, process qualification, and continued
process verification.
•Compliance professionals have great involvement in the various
manufacturing stages: during processing in advance of lyophilization, during the lyophilization process, and in post-lyophilization
product and process monitoring.
Armin H. Gerhardt, Coordinator
•Compliance activities and decisions must be
technically sound and based on development
work as necessary. Product and process changes must be carefully evaluated.
•Ongoing monitoring of product attributes and
process parameters should be conducted.
•Equipment preventive maintenance and instrument calibrations must be current.
•Regulatory auditors expect cleaning validation
to be conducted for lyophilizers.
INTRODUCTION
Lyophilization, more commonly known as “freezedrying,” is a means of removing moisture from
substances in the food, chemical, pharmaceutical, and
biotechnology industries. In all cases, lyophilization
is used to improve the stability of a moisture-sensitive
product or make the product easier to store or transport. In the pharmaceutical industry, lyophilization is
used in bulk active pharmaceutical ingredient (API)
manufacturing, sterile product manufacturing, and
manufacturing of specialty solid dosage forms such as
fast-dissolving tablets. In the biotechnology industry,
lyophilization is used as a final processing step for
purified 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 or
equivalent equipment. 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 the gaseous phase). Many food products,
such as 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 (e.g., doctor, patient, downstream
manufacturer, etc.) simply reconstitutes the freezedried powder prior to injection or other use.
This discussion addresses the fundamental principles of lyophilization, discusses the specific stages
of the lyophilization cycle, and briefly describes the
types of equipment used in lyophilization and the
types of validation studies that are typically performed
for this unit operation. Important considerations for
compliance professionals are discussed.
PHASE DIAGRAMS
The principles of lyophilization are based on the
physical properties of water, which 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.
Figure 1:
Phase diagram for water.
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PHARMACEUTICAL PROCESSES
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 the 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 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, relative to pressure and
temperature, starting with ambient conditions:
1. Atmospheric pressure and room temperature.
Product in solution is aseptically filled into
vials. Water is in the liquid state.
2. Atmospheric pressure and temperature lowered
to -30 °C or lower. Product freezes to ice.
3. Pressure is reduced to approximately 4 mm Hg
54 Journal of GXP Compliance
and temperature remains below 0 °C. Product
remains as solid ice.
4. 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.
Removal of frozen water by sublimation is
termed “primary drying.”
5. Temperature is continually increased until all
adsorbed moisture is eliminated. Pressure may
or may not be increased. Pressure increase
facilitates heat transfer, but potentially lessens
removal of moisture. Removal of adsorbed
moisture is termed “secondary drying.”
These conditions enable the bulk API or dosage
form to maintain its integrity without losses due
to boiling water. There is no liquid state in the
sublimation process. Figure 2 shows the stepwise
description of the example lyophilization process.
As you can see, the steps form a curve around the
triple point, thus avoiding the liquid phase of water.
Figure 2:
Example lyophilization process.
Armin H. Gerhardt, Coordinator
LYOPHILIZATION PROCESS FOR PHARMACEUTICAL
AND BIOPHARMACEUTICAL PRODUCTS
There are four major stages in the lyophilization
process for pharmaceutical and biopharmaceutical
materials and products:
•Formulation and filling
•Freezing
•Primary drying
•Secondary drying.
Stage 1: Formulation and Filling
Stage 1 describes the preparation of the material to
be lyophilized. The material to be lyophilized may
be a pure chemical in solution; chemical with inactive ingredients to be lyophilized; active drug with
excipients such as fillers, buffers, and salts required
for a parenterial dosage form; or other combinations
of ingredients. For drug products, formulation
prior to lyophilization usually includes the addition
of the active drug and excipients to water for injection to obtain the desired product concentration. In
biotechnology products, 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 pre-lyophilized product in
solution (1). 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 that is a
suitable matrix for stabilization of the protein. The
lyophilization process will then remove the water
to yield the final commercial product. Sterile water
will then be added to the product (i.e., reconstitution) for administration to the patient.
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 or during long-term
storage. These types of chemicals, also called
stabilizers (2), can prevent unwanted changes in
the drug, such as unfolding, during lyophilization.
Commonly used stabilizers for biologics are sugars
and glycols.
At the end of the lyophilization process, 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 pharmaceutical and 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. These 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., the specially-designed stopper is 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 bulk 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 product for injection.
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 the freezing process are around -30°C
or lower.
Lowering the temperature of a liquid at constant
pressure results in a phase change from liquid to
solid (point 1 to point 2) (see Figure 3).
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PHARMACEUTICAL PROCESSES
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 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.
The intent of primary drying is to remove the
mobile water from the product, which is ac-
complished by lowering the lyophilizer chamber
pressure (i.e., pulling a vacuum). 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 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
Figure 3:
Figure 4:
Phase diagram for freezing process.
Phase diagram for primary drying.
Stage 3: Primary Drying
After freezing, the following two types of water exist within the product:
•Mobile water free from the amorphous solid
•Bound/trapped water within the amorphous solid.
56 Journal of GXP Compliance
Armin H. Gerhardt, Coordinator
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 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 (see
Figure 5, point 3 to point 4). Again, as in primary
drying, it is important that the temperature is not
increased too quickly and that it stays below the Tg
(see above), which 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).
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 or 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.
Lyophilization Cycle Optimization
The four stages of lyophilization described previously are intended to provide a basic understanding of the principles behind lyophilization. Other
processing steps such as annealing (e.g., to modify
water crystal structure), additional solvents, pres-
Figure 5:
Phase diagram for secondary drying.
sure with inert gases during freezing, temperature
optimization, pressure optimization, and other parameters during the lyophilization process may be
used to optimize moisture removal or reduce variation in the final product. These steps may also help
to reduce processing time, which reduces costs. See
the articles listed in the references and recommended resources sections for more information.
VALIDATION OF LYOPHILIZATION EQUIPMENT
AND PROCESSES
A freeze-dryer, or lyophilizer, is made up of a chamber, condenser, and vacuum pump. The basics of
freeze-drying food were used by ancient Peruvian
Incas; however, laboratory versions of lyophilizers
have only been around for about 100 years. Designs and features in laboratory and manufacturingscale 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, cleanAutumn 2010 Volume 14 Number 4
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PHARMACEUTICAL PROCESSES
in-place (CIP), and sterilize-in-place (SIP) functions.
The reliability and reproducibility of these functions must be validated (through installation qualification [IQ], operational qualification [OQ], and
performance qualification [PQ] protocols) to ensure
consistent moisture removal and overall product
quality. Typical validation of a lyophilizer involves
multiple protocols (or multiple sections) focusing on
verifying the performance of each function.
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 design of
experiments (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.
IMPLICATIONS FOR COMPLIANCE
Compliance professionals have great involvement in
the various stages of manufacturing during processing in advance of lyophilization, during the lyophilization process, and in post-lyophilization product
or process monitoring. Much of what is demonstrated during process validation and throughout
the product lifecycle is based on work performed in
the product design stage of the product lifecycle.
Risk Analysis
Compliance personnel should be knowledgeable of
high-risk products for which they are responsible.
High-risk products are products with marginal stability, critical product characteristics, low bioavailability, high toxicity, or other high-risk attributes.
Greatest attention, especially regarding evaluation
of formulation or process changes, should be given
to process monitoring and evaluation of changes to
highest risk products.
58 Journal of GXP Compliance
Product and Process Development
Activities conducted during product and process
development by research and development (R&D)
and technical support functions are fundamental to
the long-term success in lyophilized product manufacturing. Product characteristics should be well
characterized. Process steps should be carefully
defined. Rationale and justification for formulation
and processes should be documented. This work
should be utilized as needed throughout the product lifecycle. Experimental work conducted during
development may be the justification for process
adjustment and changes. Compliance personnel
should have ready access to technical reports that
support the formulation and manufacturing process
of their products. These reports should be readily
available and quickly retrieved as needed for regulatory audits.
Formulation Ingredients
All formulation ingredients (including inactive
ingredients) and manufacturing processes prior to
lyophilization may have impact on the lyophilization process. Compliance personnel should be
vigilant of even subtle changes to the formulation
and manufacturing process, and especially so for
high-risk products. For example, changes in suppliers of inactive ingredients—even though the
ingredients are technically the same—may have
significant effects on freezing in lyophilization,
which in turn, may affect the lyophilization and
final product attributes. Low levels of impurities in
excipient from a new vendor may significantly affect
product stability.
Process Changes and Change Control
Changes to formulation, processing, equipment,
facilities, and so on—anything connected with the
lyophilization process—must be carefully evaluated
to determine its effect on lyophilization. A good
change control system is fundamental to maintaining the validated state. Development scientists
should be consulted as needed to evaluate formulation and process changes.
Armin H. Gerhardt, Coordinator
Product and Process Monitoring
The following ongoing monitoring of product attributes and process parameters should be conducted:
•Annual product reviews. Annual product
reviews typically monitor product quality
attribute testing such as potency, moisture,
and other product attributes. Annual product
reviews are required by US Food and Drug
Administration current good manufacturing
practices (CGMPs). These reviews provide a
broad overview of product performance, but do
not focus on process performance. High-risk
products or newly approved products should be
monitored at more frequent intervals.
•Process reviews. Compliance professionals should be mindful of specific indicators
of lyophilization problems such as process
changes. These include significant changes in
time required to freeze product, and changes
in the times required for primary and secondary drying. Personnel reviewing these
data should be mindful of emerging trends.
Changes in yields, increased defective vials,
are “red flags” that should be investigated even
though they do not represent process failures.
Technical personnel should be contacted to
address these occurrences.
Equipment Preventive Maintenance
and Calibration
Successful lyophilization depends on properly functioning equipment. Equipment preventive maintenance (PM) and calibration of instrumentation must
be current. Often outside services must be contacted to perform equipment PM and calibration.
Equipment Cleaning
Cleaning lyophilization equipment is difficult, especially machines that are not equipped with cleanin-place capability. Even though product does not
seemingly directly contact lyophilizer shelves and
the chamber wall, these areas of the equipment may
become contaminated with active drug particles
during lyophilization, sublimation, and vacuum
release. Equipment should be carefully inspected
to assure acceptable cleaning from previous manufacturing. Regulatory auditors expect cleaning
validation to be conducted for lyophilizers.
Process Validation Guidance
Compliance professionals should be familiar with
the concepts discussed in the November 2008 FDA
draft process validation guidance (7). This guidance
identifies three stages in the lifecycle approach to
process validation: process design, performance
qualification, and continued process verification.
The guidance emphasizes that validation must not
be considered a single distinct event, but must be
continued through monitoring and maintenance of
the manufacturing process as long as the product
is manufactured. As described previously, compliance professionals have great involvement in the
various stages of manufacturing during processing
in advance of lyophilization, during the lyophilization process, and in post-lyophilization product or
process monitoring. The concepts discussed previously are consistent with the recommendations of
the FDA guidance.
CONCLUSION
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
long-term storage. Any change in one step has the
potential to greatly impact the subsequent steps or
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 continuing
maintenance of the validated state. Compliance
professionals have great involvement in the various
stages of manufacturing lyophilized products and
must be vigilant regarding process changes, equipment maintenance, and related activities. Activities
and decisions must be soundly based on scientific
and technical principles of lyophilization.
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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,” DuraDry 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
7.FDA, Process Validation: General Principles and Practices,
Draft Guidance, Center for Drug Evaluation and Research
(CDER), Center for Biologics Evaluation and Research
(CBER) and Center for Veterinary Medicine (CVM); Food
and Drug Administration, U.S. Department of Health and
Human Services, November, 2008.
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
60 Journal of GXP Compliance
for Pharmaceuticals: Practical Advice,” Pharmaceutical
Research, Vol. 21, No. 2, February 2004.
5. “Product Technologies for Lyophilization,” Genetic Engineering and Biotechnology News, November 15, 2006. GXP
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
Clean in Place
Design of Experiments
Installation Qualification
Operational Qualification
Performance Qualification
Sterilize in Place
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 at [email protected].