INTRODUCTION
Lyophilization or freeze drying is a process in which water is
removed from a product after it is frozen and placed under a vacuum,
allowing the ice to change directly from solid to vapor without passing
through a liquid phase. The process consists of three separate, unique,
and interdependent processes; freezing, primary drying (sublimation),
and secondary drying (desorption).
The advantages of lyophilization include:
Ease of processing a liquid, which simplifies aseptic handling
Enhanced stability of a dry powder
Removal of water without excessive heating of the product
Enhanced product stability in a dry state
Rapid and easy dissolution of reconstituted product
Disadvantages of lyophilization include:
Increased handling and processing time
Need for sterile diluent upon reconstitution
Cost and complexity of equipment
The lyophilization process generally includes the following steps:
- Dissolving the drug and excipients in a suitable solvent, generally water for injection (WFI).
- Sterilizing the bulk solution by passing it through a 0.22 micron bacteria-retentive filter.
- Filling into individual sterile containers and partially stoppering the containers under aseptic conditions.
- Transporting the partially stoppered containers to the lyophilizer and loading into the chamber under aseptic conditions.
- Freezing
the solution by placing the partially stoppered containers on cooled
shelves in a freeze-drying chamber or pre-freezing in another chamber.
- Applying a vacuum to the chamber and heating the shelves in order to evaporate the water from the frozen state.
- Complete stoppering of the vials usually by hydraulic or screw rod stoppering mechanisms installed in the lyophilizers.
There are many new parenteral products, including anti-infectives,
biotechnology derived products, and in-vitro diagnostics which are
manufactured as lyophilized products. Additionally, inspections have
disclosed potency, sterility and stability problems associated with the
manufacture and control of lyophilized products. In order to provide
guidance and information to investigators, some industry procedures and
deficiencies associated with lyophilized products are identified in this
Inspection Guide.
It is recognized that there is complex technology associated with the
manufacture and control of a lyophilized pharmaceutical dosage form.
Some of the important aspects of these operations include: the
formulation of solutions; filling of vials and validation of the filling
operation; sterilization and engineering aspects of the lyophilizer;
scale-up and validation of the lyophilization cycle; and testing of the
end product. This discussion will address some of the problems
associated with the manufacture and control of a lyophilized dosage
form.
PRODUCT TYPE/FORMULATION
Products are manufactured in the lyophilized form due to their
instability when in solution. Many of the antibiotics, such as some of
the semi-synthetic penicillins, cephalosporins, and also some of the
salts of erythromycin, doxycycline and chloramphenicol are made by the
lyophilization process. Because they are antibiotics, low bioburden of
these formulations would be expected at the time of batching. However,
some of the other dosage forms that are lyophilized, such as
hydrocortisone sodium succinate, methylprednisolone sodium succinate and
many of the biotechnology derived products, have no antibacterial
effect when in solution.
For these types of products, bioburden should be minimal and the
bioburden should be determined prior to sterilization of these bulk
solutions prior to filling. Obviously, the batching or compounding of
these bulk solutions should be controlled in order to prevent any
potential increase in microbiological levels that may occur up to the
time that the bulk solutions are filtered (sterilized). The concern with
any microbiological level is the possible increase in endotoxins that
may develop. Good practice for the compounding of lyophilized products
would also include batching in a controlled environment and in sealed
tanks, particularly if the solution is to be held for any length of time
prior to sterilization.
In some cases, manufacturers have performed bioburden testing on bulk
solutions after prefiltration and prior to final filtration. While the
testing of such solutions may be meaningful in determining the bioburden
for sterilization, it does not provide any information regarding the
potential formation or presence of endotoxins. While the testing of 0.1
ml samples by LAL methods of bulk solution for endotoxins is of value,
testing of at least 100 ml size samples prior to prefiltration,
particularly for the presence of gram negative organisms, would be of
greater value in evaluating the process. For example, the presence of
Pseudomonas sp. in the bioburden of a bulk solution has been identified
as an objectionable condition.
FILLING
The filling of vials that are to be lyophilized has some problems
that are somewhat unique. The stopper is placed on top of the vial and
is ultimately seated in the lyophilizer. As a result the contents of the
vial are subject to contamination until they are actually sealed.
Validation of filling operations should include media fills and the
sampling of critical surfaces and air during active filling (dynamic
conditions).
Because of the active involvement of people in filling and aseptic
manipulations, an environmental program should also include an
evaluation of microbiological levels on people working in aseptic
processing areas. One method of evaluation of the training of operators
working in aseptic processing facilities includes the surface monitoring
of gloves and/or gowns on a daily basis. Manufacturers are actively
sampling the surfaces of personnel working in aseptic processing areas. A
reference which provides for this type of monitoring is the USP XXII
discussion of the Interpretation of Sterility Test Results. It states
under the heading of "Interpretation of Quality Control Tests" that
review consideration should be paid to environmental control data,
including...microbial monitoring, records of operators, gowns, gloves,
and garbing practices. In those situations in which manufacturers have
failed to perform some type of personnel monitoring, or monitoring has
shown unacceptable levels of contamination, regulatory situations have
resulted.
Typically, vials to be lyophilized are partially stoppered by
machine. However, some filling lines have been noted which utilize an
operator to place each stopper on top of the vial by hand. At this time,
it would seem that it would be difficult for a manufacturer to justify a
hand-stoppering operation, even if sterile forceps are employed, in any
type of operation other than filling a clinical batch or very small
number of units. Significant regulatory situations have resulted when
some manufacturers have hand-stoppered vials. Again, the concern is the
immediate avenue of contamination offered by the operator. It is well
recognized that people are the major source of contamination in an
aseptic processing filling operation. The longer a person works in an
aseptic operation, the more microorganisms will be shed and the greater
the probability of contamination.
Once filled and partially stoppered, vials are transported and loaded
into the lyophilizer. The transfer and handling, such as loading of the
lyophilizer, should take place under primary barriers, such as the
laminar flow hoods under which the vials were filled. Validation of this
handling should also include the use media fills.
Regarding the filling of sterile media, there are some manufacturers
who carry out a partial lyophilization cycle and freeze the media. While
this could seem to greater mimic the process, the freezing of media
could reduce microbial levels of some contaminants. Since the purpose of
the media fill is to evaluate and justify the aseptic capabilities of
the process, the people and the system, the possible reduction of
microbiological levels after aseptic manipulation by freezing would not
be warranted. The purpose of a media fill is not to determine the
lethality of freezing and its effect on any microbial contaminants that
might be present.
In an effort to identify the particular sections of filling and
aseptic manipulation that might introduce contamination, several
manufacturers have resorted to expanded media fills. That is, they have
filled approximately 9000 vials during a media fill and segmented the
fill into three stages. One stage has included filling of 3000 vials and
stoppering on line; another stage included filling 3000 vials,
transportation to the lyophilizer and then stoppering; a third stage
included the filling of 3000 vials, loading in the lyophilizer, and
exposure to a portion of the nitrogen flush and then stoppering. Since
lyophilizer sterilization and sterilization of the nitrogen system used
to backfill require separate validation, media fills should primarily
validate the filling, transportation and loading aseptic operations.
The question of the number of units needed for media fills when the
capacity of the process is less than 3000 units is frequently asked,
particularly for clinical products. Again, the purpose of the media fill
is to assure that product can be aseptically processed without
contamination under operating conditions. It would seem, therefore, that
the maximum number of units of media filled be equivalent to the
maximum batch size if it is less than 3000 units.
After filling, dosage units are transported to the lyophilizer by
metal trays. Usually, the bottom of the trays are removed after the
dosage units are loaded into the lyophilizer. Thus, the dosage units lie
directly on the lyophilizer shelf. There have been some situations in
which manufacturers have loaded the dosage units on metal trays which
were not removed. Unfortunately, at one manufacturer, the trays warped
which caused a moisture problem in some dosage units in a batch.
In the transport of vials to the lyophilizer, since they are not
sealed, there is concern for the potential for contamination. During
inspections and in the review of new facilities, the failure to provide
laminar flow coverage or a primary barrier for the transport and loading
areas of a lyophilizer has been regarded as an objectionable condition.
One manufacturer as a means of correction developed a laminar flow cart
to transport the vials from the filling line to the lyophilizer. Other
manufacturers building new facilities have located the filling line
close to the lyophilizer and have provided a primary barrier extending
from the filling line to the lyophilizer.
In order to correct this type of problem, another manufacturer
installed a vertical laminar flow hood between the filling line and
lyophilizer. Initially, high velocities with inadequate return caused a
contamination problem in a media fill. It was speculated that new air
currents resulted in rebound contamination off the floor. Fortunately,
media fills and smoke studies provided enough meaningful information
that the problem could be corrected prior to the manufacture of product.
Typically, the lyophilization process includes the stoppering of vials
in the chamber.
Another major concern with the filling operation is assurance of fill
volumes. Obviously, a low fill would represent a subpotency in the
vial. Unlike a powder or liquid fill, a low fill would not be readily
apparent after lyophilization particularly for a biopharmaceutical drug
product where the active ingredient may be only a milligram. Because of
the clinical significance, sub-potency in a vial potentially can be a
very serious situation.
For example, in the inspection of a lyophilization filling operation,
it was noted that the firm was having a filling problem. The gate on
the filling line was not coordinated with the filling syringes, and
splashing and partial filling was occurring. It was also observed that
some of the partially filled vials were loaded into the lyophilizer.
This resulted in rejection of the batch.
On occasion, it has been seen that production operators monitoring
fill volumes record these fill volumes only after adjustments are made.
Therefore, good practice and a good quality assurance program would
include the frequent monitoring of the volume of fill, such as every 15
minutes. Good practice would also include provisions for the isolation
of particular sections of filling operations when low or high fills are
encountered.
There are some atypical filling operations which have not been
discussed. For example, there have also been some situations in which
lyophilization is performed on trays of solution rather than in vials.
Based on the current technology available, it would seem that for a
sterile product, it would be difficult to justify this procedure.
The dual chamber vial also presents additional requirements for
aseptic manipulations. Media fills should include the filling of media
in both chambers. Also, the diluent in these vials should contain a
preservative. (Without a preservative, the filling of diluent would be
analogous to the filling of media. In such cases, a 0% level of
contamination would be expected.)
LYOPHILIZATION CYCLE AND CONTROLS
After sterilization of the lyophilizer and aseptic loading, the
initial step is freezing the solution. In some cycles, the shelves are
at the temperature needed for freezing, while for other cycles, the
product is loaded and then the shelves are taken to the freezing
temperature necessary for product freeze. In those cycles in which the
shelves are precooled prior to loading, there is concern for any ice
formation on shelves prior to loading. Ice on shelves prior to loading
can cause partial or complete stoppering of vials prior to
lyophilization of the product. A recent field complaint of a product in
solution and not lyophilized was attributed to preliminary stoppering of
a few vials prior to exposure to the lyophilization cycle.
Unfortunately, the firm's 100% vial inspection failed to identify the
defective vial.
Typically, the product is frozen at a temperature well below the eutectic point.
The scale-up and change of lyophilization cycles, including the
freezing procedures, have presented some problems. Studies have shown
the rate and manner of freezing may affect the quality of the
lyophilized product. For example, slow freezing leads to the formation
of larger ice crystals. This results in relatively large voids, which
aid in the escape of water vapor during sublimation. On the other hand,
slow freezing can increase concentration shifts of components. Also, the
rate and manner of freezing has been shown to have an affect on the
physical form (polymorph) of the drug substance.
It is desirable after freezing and during primary drying to hold the
drying temperature (in the product) at least 4-5o below the eutectic
point. Obviously, the manufacturer should know the eutectic point and
have the necessary instrumentation to assure the uniformity of product
temperatures. The lyophilizer should also have the necessary
instrumentation to control and record the key process parameters. These
include: shelf temperature, product temperature, condenser temperature,
chamber pressure and condenser pressure. The manufacturing directions
should provide for time, temperature and pressure limits necessary for a
lyophilization cycle for a product. The monitoring of product
temperature is particularly important for those cycles for which there
are atypical operating procedures, such as power failures or equipment
breakdown.
Electromechanical control of a lyophilization cycle has utilized
cam-type recorder-controllers. However, newer units provide for
microcomputer control of the freeze drying process. A very basic
requirement for a computer controlled process is a flow chart or logic.
Typically, operator involvement in a computer controlled lyophilization
cycle primarily occurs at the beginning. It consists of loading the
chamber, inserting temperature probes in product vials, and entering
cycle parameters such as shelf temperature for freezing, product freeze
temperature, freezing soak time, primary drying shelf temperature and
cabinet pressure, product temperature for establishment of fill vacuum,
secondary drying shelf temperature, and secondary drying time.
In some cases, manufacturers have had to continuously make
adjustments in cycles as they were being run. In these situations, the
lyophilization process was found to be non-validated.
Validation of the software program of a lyophilizer follows the same
criteria as that for other processes. Basic concerns include software
development, modifications and security. The
Guide to Inspection of Computerized Systems in Drug Processing contains a discussion on potential problem areas relating to computer systems. A
Guide to the Inspection of Software Development Activities is a reference that provides a more detailed review of software requirements.
Leakage into a lyophilizer may originate from various sources. As in
any vacuum chamber, leakage can occur from the atmosphere into the
vessel itself. Other sources are media employed within the system to
perform the lyophilizing task. These would be the thermal fluid
circulated through the shelves for product heating and cooling, the
refrigerant employed inside the vapor condenser cooling surface and oil
vapors that may migrate back from the vacuum pumping system.
Any one, or a combination of all, can contribute to the leakage of
gases and vapors into the system. It is necessary to monitor the leak
rate periodically to maintain the integrity of the system. It is also
necessary, should the leak rate exceed specified limits, to determine
the actual leak site for purposes of repair.
Thus, it would be beneficial to perform a leak test at some time
after sterilization, possibly at the beginning of the cycle or prior to
stoppering. The time and frequency for performing the leak test will
vary and will depend on the data developed during the cycle validation.
The pressure rise found acceptable at validation should be used to
determine the acceptable pressure rise during production. A limit and
what action is to be taken if excessive leakage is found should be
addressed in some type of operating document.
In order to minimize oil vapor migration, some lyophilizers are
designed with a tortuous path between the vacuum pump and chamber. For
example, one fabricator installed an oil trap in the line between the
vacuum pump and chamber in a lyophilizer with an internal condenser.
Leakage can also be identified by sampling surfaces in the chamber after
lyophilization for contaminants. One could conclude that if
contamination is found on a chamber surface after lyophilization, then
dosage units in the chamber could also be contaminated. It is a good
practice as part of the validation of cleaning of the lyophilization
chamber to sample the surfaces both before and after cleaning.
Because of the lengthy cycle runs and strain on machinery, it is not
unusual to see equipment malfunction or fail during a lyophilization
cycle. There should be provisions in place for the corrective action to
be taken when these atypical situations occur. In addition to
documentation of the malfunction, there should be an evaluation of the
possible effects on the product (e.g., partial or complete meltback.
Refer to subsequent discussion). Merely testing samples after the
lyophilization cycle is concluded may be insufficient to justify the
release of the remaining units. For example, the leakage of chamber
shelf fluid into the chamber or a break in sterility would be cause for
rejection of the batch.
The review of Preventive Maintenance Logs, as well as Quality
Assurance Alert Notices, Discrepancy Reports, and Investigation Reports
will help to identify problem batches when there are equipment
malfunctions or power failures. It is recommended that these records be
reviewed early in the inspection.
CYCLE VALIDATION
Many manufacturers file (in applications) their normal lyophilization
cycles and validate the lyophilization process based on these cycles.
Unfortunately, such data would be of little value to substantiate
shorter or abnormal cycles. In some cases, manufacturers are unaware of
the eutectic point. It would be difficult for a manufacturer to evaluate
partial or abnormal cycles without knowing the eutectic point and the
cycle parameters needed to facilitate primary drying.
Scale-up for the lyophilized product requires a knowledge of the many
variables that may have an effect on the product. Some of the variables
would include freezing rate and temperature ramping rate. As with the
scale-up of other drug products, there should be a development report
that discusses the process and logic for the cycle. Probably more so
than any other product, scale-up of the lyophilization cycle is very
difficult.
There are some manufacturers that market multiple strengths, vial
sizes and have different batch sizes. It is conceivable and probable
that each will have its own cycle parameters. A manufacturer that has
one cycle for multiple strengths of the same product probably has done a
poor job of developing the cycle and probably has not adequately
validated their process. Investigators should review the reports and
data that support the filed lyophilization cycle.
LYOPHILIZER STERILIZATION/DESIGN
The sterilization of the lyophilizer is one of the more frequently
encountered problems noted during inspections. Some of the older
lyophilizers cannot tolerate steam under pressure, and sterilization is
marginal at best. These lyophilizers can only have their inside surfaces
wiped with a chemical agent that may be a sterilant but usually has
been found to be a sanitizing agent. Unfortunately, piping such as that
for the administration of inert gas (usually nitrogen) and sterile air
for backfill or vacuum break is often inaccessible to such surface
"sterilization" or treatment. It would seem very difficult for a
manufacturer to be able to demonstrate satisfactory validation of
sterilization of a lyophilizer by chemical "treatment".
Another method of sterilization that has been practiced is the use of
gaseous ethylene oxide. As with any ethylene oxide treatment,
humidification is necessary. Providing a method for introducing the
sterile moisture with uniformity has been found to be difficult.
A manufacturer has been observed employing Water For Injection as a
final wash or rinse of the lyophilizer. While the chamber was wet, it
was then ethylene oxide gas sterilized. As discussed above, this may be
satisfactory for the chamber but inadequate for associated plumbing.
Another problem associated with ethylene oxide is the residue. One
manufacturer had a common ethylene oxide/nitrogen supply line to a
number of lyophilizers connected in parallel to the system. Thus, there
could be some ethylene oxide in the nitrogen supply line during the
backfilling step. Obviously, this type of system is objectionable.
A generally recognized acceptable method of sterilizing the
lyophilizer is through the use of moist steam under pressure.
Sterilization procedures should parallel that of an autoclave, and a
typical system should include two independent temperature sensing
systems. One would be used to control and record temperatures of the
cycle as with sterilizers, and the other would be in the cold spot of
the chamber. As with autoclaves, lyophilizers should have drains with
atmospheric breaks to prevent back siphonage.
As discussed, there should also be provisions for sterilizing the
inert gas or air and the supply lines. Some manufacturers have chosen to
locate the sterilizing filters in a port of the chamber. The port is
steam sterilized when the chamber is sterilized, and then the
sterilizing filter, previously sterilized, is aseptically connected to
the chamber. Some manufacturers have chosen to sterilize the filter and
downstream piping to the chamber in place. Typical
sterilization-in-place of filters may require steaming of both to obtain
sufficient temperatures. In this type of system, there should be
provisions for removing and/or draining condensate. The failure to
sterilize nitrogen and air filters and the piping downstream leading
into the chamber has been identified as a problem on a number of
inspections.
Since these filters are used to sterilize inert gas and/or air, there
should be some assurance of their integrity. Some inspections have
disclosed a lack of integrity testing of the inert gas and/or air
filter. The question is frequently asked how often should the vent
filter be tested for integrity? As with many decisions made by
manufacturers, there is a level of risk associated with the operation,
process or system, which only the manufacturer can decide. If the
sterilizing filter is found to pass the integrity test after several
uses or batches, then one could claim its integrity for the previous
batches. However, if it is only tested after several batches have been
processed and if found to fail the integrity test, then one could
question the sterility of all of the previous batches processed. In an
effort to minimize this risk, some manufacturers have resorted to
redundant filtration.
For most cycles, stoppering occurs within the lyophilizer. Typically,
the lyophilizer has some type of rod or rods (ram) which enter the
immediate chamber at the time of stoppering. Once the rod enters the
chamber, there is the potential for contamination of the chamber.
However, since the vials are stoppered, there is no avenue for
contamination of the vials in the chamber which are now stoppered.
Generally, lyophilizers should be sterilized after each cycle because of
the potential for contamination of the shelf support rods.
Additionally, the physical act of removing vials and cleaning the
chamber can increase levels of contamination.
In some of the larger units, the shelves are collapsed after
sterilization to facilitate loading. Obviously, the portions of the ram
entering the chamber to collapse the shelves enters from a non-sterile
area. Attempts to minimize contamination have included wiping the ram
with a sanitizing agent prior to loading. Control aspects have included
testing the ram for microbiological contamination, testing it for
residues of hydraulic fluid, and testing the fluid for its
bacteriostatic effectiveness. One lyophilizer fabricator has proposed
developing a flexible "skirt" to cover the ram.
In addition to microbiological concerns with hydraulic fluid, there is also the concern with product contamination.
During steam sterilization of the chamber, there should be space
between shelves that permit passage of free flowing steam. Some
manufacturers have placed "spacers" between shelves to prevent their
total collapse. Others have resorted to a two phase sterilization of the
chamber. The initial phase provides for sterilization of the shelves
when they are separated. The second phase provides for sterilization of
the chamber and piston with the shelves collapsed.
Typically, biological indicators are used in lyophilizers to validate
the steam sterilization cycle. One manufacturer of a Biopharmaceutical
product was found to have a positive biological indicator after
sterilization at 121oC for 45 minutes. During the chamber sterilization,
trays used to transport vials from the filling line to the chamber were
also sterilized. The trays were sterilized in an inverted position on
shelves in the chamber. It is believed that the positive biological
indicator is the result of poor steam penetration under these trays.
The sterilization of condensers is also a major issue that warrants
discussion. Most of the newer units provide for the capability of
sterilization of the condenser along with the chamber, even if the
condenser is external to the chamber. This provides a greater assurance
of sterility, particularly in those situations in which there is some
equipment malfunction and the vacuum in the chamber is deeper than in
the condenser.
Malfunctions that can occur, which would indicate that sterilization
of the condenser is warranted, include vacuum pump breakdown,
refrigeration system failures and the potential for contamination by the
large valve between the condenser and chamber. This is particularly
true for those units that have separate vacuum pumps for both the
condenser and chamber. When there are problems with the systems in the
lyophilizer, contamination could migrate from the condenser back to the
chamber. It is recognized that the condenser is not able to be
sterilized in many of the older units, and this represents a major
problem, particularly in those cycles in which there is some equipment
and/or operator failure.
As referenced above, leakage during a lyophilization cycle can occur,
and the door seal or gasket presents an avenue of entry for
contaminants. For example, in an inspection, it was noted that during
steam sterilization of a lyophilizer, steam was leaking from the unit.
If steam could leak from a unit during sterilization, air could possibly
enter the chamber during lyophilization.
Some of the newer lyophilizers have double doors - one for loading
and the other for unloading. The typical single door lyophilizer opens
in the clean area only, and contamination between loads would be
minimal. This clean area, previously discussed, represents a critical
processing area for a product made by aseptic processing. In most units,
only the piston raising/lowering shelves is the source of
contamination. For a double door system unloading the lyophilizer in a
non-sterile environment, other problems may occur. The non-sterile
environment presents a direct avenue of contamination of the chamber
when unloading, and door controls similar to double door sterilizers
should be in place.
Obviously, the lyophilizer chamber is to be sterilized between
batches because of the direct means of contamination. A problem which
may be significant is that of leakage through the door seal. For the
single door unit, leakage prior to stoppering around the door seal is
not a major problem from a sterility concern, because single door units
only open into sterile areas. However, leakage from a door gasket or
seal from a non-sterile area would present a significant microbiological
problem. In order to minimize the potential for contamination, it is
recommended that the lyophilizers be unloaded in a clean room area to
minimize contamination. For example, in an inspection of a new
manufacturing facility, it was noted that the unloading area for double
door units was a clean room, with the condenser located below the
chamber on a lower level.
After steam sterilization, there is often some condensate remaining
on the floor of the chamber. Some manufacturers remove this condensate
through the drain line while the chamber is still pressurized after
sterilization. Unfortunately, some manufacturers have allowed the
chamber to come to and remain at atmospheric pressure with the drain
line open. Thus, non-sterile air could contaminate the chamber through
the drain line. Some manufacturers have attempted to dry the chamber by
blowing sterile nitrogen gas through the chamber at a pressure above
atmospheric pressure.
In an inspection of a biopharmaceutical drug product, a Pseudomonas
problem probably attributed to condensate after sterilization was noted.
On a routine surface sample taken from a chamber shelf after
sterilization and processing, a high count of Pseudomonas sp. was
obtained. After sterilization and cooling when the chamber door was
opened, condensate routinely spilled onto the floor from the door. A
surface sample taken from the floor below the door also revealed
Pseudomonas sp. contamination. Since the company believed the condensate
remained in the chamber after sterilization, they repiped the chamber
drain and added a line to a water seal vacuum pump.
FINISHED PRODUCT TESTING FOR
LYOPHILIZED PRODUCTS
There are several aspects of finished product testing which are of
concern to the lyophilized dosage form. These include dose uniformity
testing, moisture and stability testing, and sterility testing.
(a) Dose Uniformity
The USP includes two types of dose uniformity testing: content
uniformity and weight variation. It states that weight variation may be
applied to solids, with or without added substances, that have been
prepared from true solutions and freeze-dried in final containers.
However, when other excipients or other additives are present, weight
variation may be applied, provided there is correlation with the sample
weight and potency results. For example, in the determination of
potency, it is sometimes common to reconstitute and assay the entire
contents of a vial without knowing the weight of the sample. Performing
the assay in this manner will provide information on the label claim of a
product, but without knowing the sample weight will provide no
information about dose uniformity. One should correlate the potency
result obtained form the assay with the weight of the sample tested.
(b) Stability Testing
An obvious concern with the lyophilized product is the amount of
moisture present in vials. The manufacturer's data for the establishment
of moisture specifications for both product release and stability
should be reviewed. As with other dosage forms, the expiration date and
moisture limit should be established based on worst case data. That is, a
manufacturer should have data that demonstrates adequate stability at
the moisture specification.
As with immediate release potency testing, stability testing should
be performed on vials with a known weight of sample. For example,
testing a vial (sample) which had a higher fill weight (volume) than the
average fill volume of the batch would provide a higher potency results
and not represent the potency of the batch. Also, the expiration date
and stability should be based on those batches with the higher moisture
content. Such data should also be considered in the establishment of a
moisture specification.
For products showing a loss of potency due to aging, there are
generally two potency specifications. There is a higher limit for the
dosage form at the time of release. This limit is generally higher than
the official USP or filed specification which is official throughout the
entire expiration date period of the dosage form. The USP points out
that compendial standards apply at any time in the life of the article.
Stability testing should also include provisions for the assay of
aged samples and subsequent reconstitution of these aged samples for the
maximum amount of time specified in the labeling. On some occasions,
manufacturers have established expiration dates without performing label
claim reconstitution potency assays at the various test intervals and
particularly the expiration date test interval. Additionally, this
stability testing of reconstituted solutions should include the most
concentrated and the least concentrated reconstituted solutions. The
most concentrated reconstituted solution will usually exhibit
degradation at a faster rate than less concentrated solutions.
(c) Sterility Testing
With respect to sterility testing of lyophilized products, there is
concern with the solution used to reconstitute the lyophilized product.
Although products may be labeled for reconstitution with Bacteriostatic
Water For Injection, Sterile Water For Injection (WFI) should be used to
reconstitute products. Because of the potential toxicities associated
with Bacteriostatic Water For Injection, many hospitals only utilize
WFI. Bacteriostatic Water For Injection may kill some of the vegetative
cells if present as contaminants, and thus mask the true level of
contamination in the dosage form.
As with other sterile products, sterility test results which show
contamination on the initial test should be identified and reviewed.
FINISHED PRODUCT INSPECTION - MELTBACK
The USP points out that it is good pharmaceutical practice to perform
100% inspection of parenteral products. This includes sterile
lyophilized powders. Critical aspects would include the presence of
correct volume of cake and the cake appearance. With regard to cake
appearance, one of the major concerns is meltback.
Meltback is a form of cake collapse and is caused by the change from
the solid to liquid state. That is, there is incomplete sublimation
(change from the solid to vapor state) in the vial. Associated with this
problem is a change in the physical form of the drug substance and/or a
pocket of moisture. These may result in greater instability and
increased product degradation.
Another problem may be poor solubility. Increased time for
reconstitution at the user stage may result in partial loss of potency
if the drug is not completely dissolved, since it is common to use
in-line filters during administration to the patient.
Manufacturers should be aware of the stability of lyophilized
products which exhibit partial or complete meltback. Literature shows
that for some products, such as the cephalosporins, that the crystalline
form is more stable than the amorphous form of lyophilized product. The
amorphous form may exist in the "meltback" portion of the cake where
there is incomplete sublimation.
GLOSSARY
ATMOSPHERE, THE EARTH
The envelope of gases surrounding the earth, exerting under gravity a
pressure at the earth's surface, which includes by volume 78% nitrogen,
21% oxygen, small quantities of hydrogen, carbon dioxide, noble gases,
water vapor, pollutants and dust.
ATMOSPHERIC PRESSURE
The pressure exerted at the earth's surface by the atmosphere. For
reference purposes a standard atmosphere is defined as 760 torr or
millimeters of mercury, or 760,000 microns.
BACKSTREAMING
A process that occurs at low chamber pressures where hydrocarbon vapors from the vacuum system can enter the product chamber.
BLANK-OFF PRESSURE
This is the ultimate pressure the pump or system can attain.
BLOWER (see Mechanical Booster Pump)
This pump is positioned between the mechanical pump and the chamber.
It operates by means of two lobes turning at a high rate of speed. It is
used to reduce the chamber pressure to less than 20 microns.
BREAKING VACUUM
Admitting air or a selected gas to an evacuated chamber, while
isolated from a vacuum pump, to raise the pressure towards, or up to,
atmospheric.
CIRCULATION PUMP
A pump for conveying the heat transfer fluid.
CONDENSER (Cold trap)
In terms of the lyophilization process, this is the vessel that
collects the moisture on plates and holds it in the frozen state.
Protects the vacuum pump from water vapor contaminating the vacuum pump
oil.
CONDENSER/RECEIVER
In terms of refrigeration, this unit condenses (changes) the hot
refrigerant gas into a liquid and stores it under pressure to be reused
by the system.
COOLING
The lowering of the temperature in any part of the temperature scale.
CONAX CONNECTION
A device to pass thermocouple wires through and maintain a vacuum tight vessel.
CONTAMINATION
ln the vacuum system, the introduction of water vapor into the oil in
the vacuum pump, which then causes the pump to lose its ability to
attain its ultimate pressure.
DEFROSTING
The removal of ice from a condenser by melting or mechanical means.
DEGREE OF CRYSTALLIZATION
The ratio of the energy released during the freezing of a solution to that of an equal volume of water.
DEGREE OF SUPERCOOLING
The number of degrees below the equilibrium freezing temperature where ice first starts to form.
DESICCANT
A drying agent.
DRY
Free from liquid, and/or moisture.
DRYING
The removal of moisture and other liquids by evaporation.
EQUILIBRIUM FREEZING TEMPERATURES
The temperature where ice will form in the absence of supercooling.
EUTECTIC TEMPERATURE
A point of a phase diagram where all phases are present and the
temperature and composition of the liquid phase cannot be altered
without one of the phases disappearing.
EXPANSION TANK
This tank is located in the circulation system and is used as a holding and expansion tank for the transfer liquid.
FILTER OR FILTER/DRIER
There are two systems that have their systems filtered or
filter/dried. They are the circulation and refrigeration systems. In the
newer dryers this filter or filter/dryer is the same, and can be
replaced with a new core.
FREE WATER
The free water in a product is that water that is absorbed on the
surfaces of the product and must be removed to limit further biological
and chemical reactions.
FREEZING
This is the absence of heat. A controlled change of the product
temperature as a function of time, during the freezing process, so as to
ensure a completely frozen form.
GAS BALLAST
Used in the vacuum system on the vacuum pump to decontaminate small amounts of moisture in the vacuum pump oil.
GAS BLEED (Vacuum control)
To control the pressure in the chamber during the cycle to help the
drying process. In freeze-drying the purpose is to improve heat-transfer
to the product.
HEAT EXCHANGER
This exchanger is located in the circulation and refrigeration
systems and transfers the heat from the circulation system to the
refrigeration system.
HEAT TRANSFER FLUID
A liquid of suitable vapor pressure and viscosity range for
transferring heat to or from a component, for example, a shelf or
condenser in a freeze-dryer. The choice of such a fluid may depend on
safety considerations. Diathermic fluid.
HOT GAS BYPASS
This is a refrigeration system. To control the suction pressure of
the BIG FOUR (20-30 Hp) compressors during the refrigeration operation.
HOT GAS DEFROST
This is a refrigeration system. To defrost the condenser plates after the lyophilization cycle is complete.
ICE
The solid, crystalline form of water.
INERT GAS
Any gas of a group including helium, radon and nitrogen, formerly considered chemically inactive.
INTERSTAGE
In a two stage compressor system, this is the cross over piping on
top of the compressor that connects the low side to the high side. One
could also think of it as low side, intermediate, and high side.
INTERSTAGE PRESSURE REGULATING VALVE
This valve controls the interstage pressure from exceeding 80 - 90
PSI. This valve opens to suction as the interstage pressure rises above
80 - 90 PSI.
LEXSOL
A heat transfer fluid (high grade kerosene).
LIQUID SUB-COOLER HEAT EXCHANGER (see Sub-cooled Liquid)
The liquid refrigerant leaving the condenser/receiver at cooling
water temperature is sub-cooled to a temperature of +15oF (-10oC) to
-15oF (-25oC).
LYOPHILIZATION
A process in which the product is first frozen and then, while still
in the frozen state, the major portion of the water and solvent system
is reduced by sublimation and desorption so as to limit biological and
chemical reactions at the designated storage temperature,
MAIN VACUUM VALVE (see Vapor Valve)
This valve is between the chamber and external condenser to isolate
the two vessels after the process is finished. This is the valve that
protects the finished product.
MATRIX
A matrix, in terms of the lyophilization process, is a system of ice
crystals and solids that is distributed throughout the product.
MECHANICAL BOOSTER PUMP (see Blower)
A roots pump with a high displacement for its size but a low
compression ratio. When backed by an oil-seal rotary pump the
combination is an economical alternative to a two-stage oil-sealed
rotary pump, with the advantage of obtaining a high vacuum.
MECHANICAL VACUUM PUMP
The mechanical pumping system that lowers the pressure in the chamber
to below atmospheric pressure so that sublimation can occur.
MELTING TEMPERATURE (Melt-back)
That temperature where mobile water first becomes evident in a frozen system.
MICRON (see Torr)
A unit of pressure used in the lyophilization process. One micron =
one Mtorr or 25,400 microns = 1" Hg., or 760,000 microns = one
atmosphere.
NONCONDENSABLES
A mixture of gases such as nitrogen, hydrogen, chlorine, and
hydrocarbons. They may be drawn into the system through leaks when part
of the system is under a vacuum. Their presence reduces the operating
efficiency of the system by increasing the condensing pressure.
NUCLEATION
The formation of ice crystals on foreign surfaces or as a result of the growth of water clusters.
OIL-MIST FILTER
In vacuum terminology a filter attached to the discharge (exhaust) of
an oil-sealed rotary pump to eliminate most of the "smoke" of suspended
fine droplets of oil which would be discharged into the environment.
OIL SEALED ROTARY PUMP
A standard type of mechanical vacuum pump used in freeze-drying with a
high compression ratio but having a relatively low displacement (speed)
for its size. A two-stage pump is effectively two such pumps in series
and can obtain an ultimate vacuum.
OIL SEPARATOR
Separates the oil from the compressor discharge gas and returns the
oil through the oil float trap and piping to the compressor crankcase.
REAL LEAK
A real leak is a source of atmospheric gases resulting from a penetration through the chamber.
RECONSTITUTE
The dissolving of the dried product into a solvent or diluent.
RELIEF VALVE
Used for safety purposes to prevent damage in case excessive pressure is encountered.
ROTARY VANE PUMP
A mechanical pumping system with sliding vanes as the mechanical seal. Can be single or two stages.
SHELF COMPRESSOR (Controlling Compressor)
Used for controlling the shelf temperature, either cooling or from overheating.
SELF LIQUID HEAT EXCHANGER
The transfer of heat from the shelf fluid to the refrigeration system
through tubes in the exchanger causing compressor suction gas to warm.
SHELVES
In terms of the lyophilization process, they are a form of heat
exchanger, within the chamber, that have a serpentine liquid flow
through them, entering one side and flowing to the other side. They are
located in the circulation system.
SINGLE STAGE COMPRESSOR
This is a normal type compressor used in refrigeration. In the
lyophilization process it is used to control the shelf temperature, both
for cooling and keeping the shelf temperature from overheating using a
temperature controller.
SILICONE OIL
A heat transfer fluid.
STERILIZATION
The use of steam and pressure to kill any bacteria that may be able to contaminate that environment or vessel.
SUBLIMATION
The conversion of a material from a solid phase directly to a vapor
phase, without passing through the liquid phase. This is referred to as
the primary drying stage.
SUB-COOLED LIQUID (See Liquid Sub-cooler Heat Exchanger)
The liquid refrigerant is cooled through an exchanger so that it
increases the refrigerating effect as well as reduces the volume of gas
flashed from the liquid refrigerant in passage through the expansion
valve.
SUCTION LINE ACCUMULATOR
To provide adequate refrigerant liquid slug protection (droplets of
liquid refrigerant) from returning to the compressor, and causing damage
to the compressor.
TCE
Trichloroethylene - A heat transfer fluid.
TEMPERATURE
The degree of hotness or coldness of a body.
THERMOCOUPLE
A metal-to-metal contact between two dissimilar metals that produces a small voltage across the free ends of the wire.
THERMOSTATIC EXPANSION VALVE
An automatic variable device controlling the flow of liquid refrigerant.
TORR (See Micron)
A unit of measure equivalent to the amount of pressure in 1000 microns.
TWO STAGE COMPRESSOR (see Interstage)
This is a specially built compressor. Its function is to be able to
attain low temperatures by being able to operate at low pressures. It is
two compressors built into one. A low stage connected internally and a
high stage connected externally with piping, called interstage.
UNLOADING VALVE
This valve connects the interstage with suction to equalize both pressures during pump-down.
VACUUM
Strictly speaking, a space in which the total pressure is less than atmospheric.
VACUUM CONTROL (Gas Bleed)
To assist in the rate of sublimation, by controlling the pressure in the lyophilizer.
VACUUM PUMP
A mechanical way of reducing the pressure in a vessel below
atmospheric pressure to where sublimation can occur. There are three
types of pumps, rotary vane, rotary piston and mechanical booster.
VAPOR BAFFLE
A target shaped object placed in the condenser to direct vapor flow and to promote an even distribution of condensate.
VACUUM VALVES
The vacuum valves used are of a ball or disk type that can seal
without leaking. The balI types are used for services to the chamber and
condenser. They are also used for drains and isolation applications.
The disk types are used in the vacuum line system and are connected to
the vacuum pump, chamber and condenser.
VAPOR VALVE (See Main Vacuum Valve)
The vacuum valve between the chamber and external condenser. When
this valve is closed the chamber is isolated from the external
condenser. Also known as the main vapor valve.
VIAL
A small glass bottle with a flat bottom, short neck and flat flange designed for stoppering.
VIRTUAL LEAK
In the vacuum system a virtual leak is the passage of gas into the
chamber from a source that is located internally in the chamber.
