U.S. patent application number 13/457879 was filed with the patent office on 2013-05-09 for a method and system for cryopreservation to achieve uniform viability and biological activity.
The applicant listed for this patent is Alan T. Cheng, Nigel J. Grinter, Ying Zhou. Invention is credited to Alan T. Cheng, Nigel J. Grinter, Ying Zhou.
Application Number | 20130111931 13/457879 |
Document ID | / |
Family ID | 48222773 |
Filed Date | 2013-05-09 |
United States Patent
Application |
20130111931 |
Kind Code |
A1 |
Grinter; Nigel J. ; et
al. |
May 9, 2013 |
A METHOD AND SYSTEM FOR CRYOPRESERVATION TO ACHIEVE UNIFORM
VIABILITY AND BIOLOGICAL ACTIVITY
Abstract
A method and system for controlled rate freezing and nucleation
of biological materials is provided. The presently disclosed system
and method provides the ability to rapidly cool the materials
contained in vials or other containers within a cooling unit via
forced convective cooling and optionally simultaneous pressure drop
using uniform and unidirectional flow of cryogen in proximity to
the plurality of vials disposed within a cooling unit. The rapid
cooling of the biological materials is achieved by precisely
controlling and adjusting the temperature of the cryogen being
introduced to the system as well as the chamber pressure as a
function of time.
Inventors: |
Grinter; Nigel J.; (Buffalo
Grove, IL) ; Cheng; Alan T.; (Naperville, IL)
; Zhou; Ying; (Naperville, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Grinter; Nigel J.
Cheng; Alan T.
Zhou; Ying |
Buffalo Grove
Naperville
Naperville |
IL
IL
IL |
US
US
US |
|
|
Family ID: |
48222773 |
Appl. No.: |
13/457879 |
Filed: |
April 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12266760 |
Nov 7, 2008 |
|
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13457879 |
|
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61480647 |
Apr 29, 2011 |
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Current U.S.
Class: |
62/62 ;
62/440 |
Current CPC
Class: |
F25D 13/00 20130101;
F25D 3/10 20130101; F25D 29/001 20130101; A01N 1/0252 20130101;
B01L 2300/1838 20130101; F25D 2600/06 20130101; F25D 3/102
20130101; B01L 2300/0829 20130101; A01N 1/0257 20130101; B01L 7/50
20130101; B01L 2300/14 20130101; B01L 1/025 20130101 |
Class at
Publication: |
62/62 ;
62/440 |
International
Class: |
F25D 13/00 20060101
F25D013/00; F25D 3/10 20060101 F25D003/10 |
Claims
1. A method of controlling a chilling or freezing process of
biological material disposed in a plurality of containers,
comprising the steps of: (i) placing said plurality of containers
of said biological materials in a cooling area defined as an area
between a gas distribution surface and a parallel gas collection
surface within a cooling chamber; (ii) mixing a liquid cryogen with
a warmer gas to produce a cold cryogenic gas at a selected
temperature profile, said temperature profile corresponding to a
desired cooling rate of said biological materials within said
containers; (iii) delivering a unidirectional flow of said cold
cryogenic gas through the gas distribution surface to said cooling
area between said parallel gas distribution and said gas collection
surfaces and generally parallel to each of said plurality of
containers to uniformly cool said biological materials within said
containers; and (iv) promptly exhausting said gas from said cooling
chamber via said gas collection surface so as to prevent
recirculation of said gas within said cooling area; wherein greater
uniformity of said biological materials in each of said plurality
of containers is achieved.
2. The method of claim 1, wherein said collection surface and/or
said gas distribution surface are porous surfaces.
3. The method of claim 1, wherein said step of mixing liquid
cryogen with said warmer gas further comprises mixing liquid
nitrogen with either room temperature nitrogen gas from a nitrogen
supply source or with recycled gas exiting from the cooling chamber
or a combination thereof and said mixing occurs in cryogen intake
circuits to produce a cold nitrogen gas at said selected
temperature profile, said temperature profile corresponding to a
desired cooling rate of said biological materials within said
containers.
4. The method of claim 1 wherein the step of delivering a
unidirectional flow of said cold cryogenic gas further comprises
performing a temperature quench of said biological material in each
of said plurality of containers by delivering a unidirectional flow
of cold cryogenic gas having a temperature of 40.degree. C. or more
below the temperature of said biological material in said plurality
of containers to induce nucleation of freezing in said biological
materials.
5. The method of claim 1 further comprising the step of rapidly
reducing the pressure in the cooling chamber during the step of
delivering a unidirectional flow of said cold cryogenic gas to
induce nucleation of freezing in said biological materials.
6. The method of claim 1 wherein greater uniformity of said
biological materials further comprises greater uniformity of the
cell viability of said biological materials in said plurality of
containers.
7. The method of claim 1 wherein greater uniformity of said
biological materials further comprises greater uniformity of the
biological activity of said biological materials in said plurality
of containers.
8. The method of claim 1, wherein said desired cooling rate of said
biological materials within said containers is between about
-2.5.degree. C./min to about -5.0.degree. C./min.
9. The method of claim 1, wherein said plurality of containers
comprise at least 10,000 vials.
10. The method of claim 1, wherein said plurality of containers
comprise at least 50,000 vials.
11. The method of claim 1, wherein said plurality of containers
comprise a plurality of bags.
12. The method of claim 1, wherein said biological material in each
of a plurality of containers comprises; microorganisms, tissues,
organs, stem cells, primary cells, cell lines, small multicellular
organisms, complex cellular structures, live or attenuated viruses,
nucleic acids, monoclonal antibodies, polyclonal antibodies,
biomolecules, non-peptide analogues, peptides, proteins, RNA, DNA,
oligonucleotides, and/or viral particles.
13. A cooling unit, comprising a uniform flow cryogenic chiller
including a cryogen intake circuit coupled to a source of cryogen
wherein said cryogenic chiller further includes a base gas
injection box, a porous metal plate disposed or set in or near the
top surface of said gas injection box, and a corresponding gas
removal box positioned immediately above said base gas injection
box with said porous metal plate disposed therein.
14. A cooling unit comprising a chilling or freezing control system
for controlling a cryogen source, an intake circuit coupled to said
cryogen source and adapted for providing a uniform flow and
temperature of a cryogenic cold gas to said cooling chamber, and
wherein said cooling unit also comprises an intake plenum, an
exhaust manifold, and two or more parallel porous surfaces that
define a cooling area between adjacent parallel surfaces with one
of said parallel porous surfaces disposed adjacent to said intake
plenum and in fluid communication with said intake plenum and
another of said parallel porous surfaces disposed adjacent to said
exhaust manifold, said parallel porous surfaces and associated
cooling area adapted to retain, or hold, a plurality of containers
of biological materials.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present invention is a continuation-in-part application
of U.S. patent application Ser. No. 12/266,760 filed Nov. 7, 2008
and also claims priority from U.S. provisional patent application
Ser. No. 61/480,647 filed Apr. 29, 2011 the disclosure of both
applications are also fully incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention broadly relates to a cryopreservation
process, and more particularly, to a method and system for
providing controlled rate freezing and nucleation control of
biological materials to minimize cell damage resulting from
intercellular ice formation and solute effects that arise during
the cryopreservation process.
BACKGROUND
[0003] Cryopreservation is a process used to stabilize biological
materials at very low temperatures. Previous attempts to freeze
biological materials, such as living cells often results in a
significant loss of cell viability and in some cases as much as 80%
or more loss of cell activity and viability.
[0004] Cell damage during cryopreservation usually occurs as a
result of intracellular ice formation within the living cell during
the freezing step or during subsequent recrystallization. Rapid
cooling often leads to formation of more intracellular ice since
water molecules are not fully migrated out of the cell during the
short timeframe associated with the rapid cool-down rates.
Intercellular ice formation also can arise during recrystallization
that occurs during the warming or thawing cycles. If too much water
remains inside the living cell, damage due to initial ice crystal
formation during the rapid cooling phase and subsequent
recrystallization during warming phases can occur and such damage
is usually lethal.
[0005] On the other hand, slow cooling profiles during
cryopreservation often results in an increase in the solute effects
where excess water is migrated out of the cells. Excess water
migrating out of the cells adversely affects the cells due to an
increase in osmotic imbalance. Thus, cell damage occurs as a result
of osmotic imbalances which can be detrimental to cell survival and
ultimately lead to cell damage and a loss of cell viability.
[0006] Current cryopreservation techniques involve using either
conductive based cryogenic cooling equipment such as a cold shelf
or lyophilizer type freezer unit or convective based cryogenic
cooling equipment such as controlled rate freezers and cryo-cooler
units. Such equipment, however, is only suitable for relatively
small volume capacities and is not suitable for commercial scale
production and preservation of biological materials such as
therapeutic cell lines. For example, the largest commercially
available controlled rate freezer suitable for use with biological
materials holds only about 8000 or so closely packed vials. One
such system is the Kryo 1060-380 capable of storing 8000.times.2 ml
ampoules. Such existing controlled rate freezers, including the
Kryo 1060 series, also suffer from the non-uniformity in cooling
vial to vial due, in part, to the non-uniform flow of cryogen
within the freezers and the requirement for close packing of the
vials within the freezer. The size of individual conventional
freezers is limited due to these non-uniform effects. As
conventional controlled rate freezers are scaled up in size, the
non-uniformities in cooling increase. Consequently, the size of
conventional controlled rate freezers must be limited to prevent
non-uniform sample-to-sample properties due to non-uniform cooling
of each sample. The only effective way to further increase the
quantity of samples processed at once using conventional controlled
rate freezers is to use multiple controlled rate freezers.
[0007] Many conventional freezing systems utilize internal fans to
disperse cryogen around the unit and deliver the refrigeration to
the vials via convection. Such convection based cooling or freezing
systems cannot achieve temperature uniformity as the vials are
often located at various distances from the internal fan or packed
in the shadow of other vials or trays. Vials of biological material
exposed to high velocity turbulent flow of cryogen are typically
cooled at a different rate and often much faster than vials
situated further away from the fan.
[0008] There are also existing lyophilizer type of control rate
freezers that can handle large volumes of vials but typically rely
on thermal conduction between cold shelves in the lyophilizer unit
and the vials. However, it is impossible to provide a uniform
conductive surface area on the bottom of each glass vial since most
glass vial bottoms are concave. Therefore, temperature variations
during the freezing process from vial to vial are the biggest
drawback for these types of equipment. Furthermore, the cooling
rate can be painfully slow due to the very small conductive surface
of the vial that remains in contact with the cold shelves.
[0009] Prior attempts to mitigate the loss of cell activity and
viability involved the use of cryoprotective additives such as DSMO
and glycerol. Use of such cryoprotectives during the
cryopreservation process has demonstrated a reduction in cell
losses attributable to more suitable freezing and subsequent
thawing cycles. However, many cryoprotectants such as DSMO are
toxic to human cells and are otherwise not suitable for use in
whole cell therapies. Disadvantageously, cryoprotectants also add a
degree of complexity and associated cost to the cell production and
preservation process. Also, cryoprotectants alone, have not
eradicated the problem of loss of cell activity and viability.
[0010] Another problem associated with the above mentioned systems
is a lack of control with respect to the uniformity of the
nucleation temperature between the multiple vials. This variability
in the nucleation temperature of the multiple vials can lead to
non-uniform vial-to-vial properties. Such properties can include
cell activity and viability as well as the crystal structure of the
frozen material and the time needed to complete a freeze drying
process. Consequently, controlling the generally random process of
nucleation in the freezing stage of a cryopreservation,
lyophilization, or freeze-drying process to increase the product
uniformity from vial-to-vial in the finished product would be
highly desirable in the art.
[0011] In a typical pharmaceutical freeze-drying process, multiple
vials containing a common aqueous solution are placed on shelves
that are cooled, generally at a controlled rate, to low
temperatures. The aqueous solution in each vial is cooled below the
thermodynamic freezing temperature of the solution and remains in a
sub-cooled metastable liquid state until nucleation occurs.
[0012] The range of nucleation temperatures across the vials is
distributed randomly between a temperature near the thermodynamic
freezing temperature and some value significantly (e.g., up to
about 30.degree. C.) lower than the thermodynamic freezing
temperature. This distribution of nucleation temperatures causes
vial-to-vial variation in ice crystal structure and ultimately the
physical properties of the lyophilized product. Furthermore, the
drying stage of the freeze-drying process must be excessively long
to accommodate the range of ice crystal sizes and structures
produced by the natural stochastic nucleation phenomenon.
[0013] Additives have been used to increase the nucleation
temperature of sub-cooled solutions. These additives can take many
forms. It is well known that certain bacteria (e.g., Pseudomonas
syringae) synthesize proteins that help nucleate ice formation in
sub-cooled aqueous solutions. Either the bacteria or their isolated
proteins can be added to solutions to increase the nucleation
temperature. Several inorganic additives also demonstrate a
nucleating effect; the most common such additive is silver iodide,
AgI. In general, any additive or contaminant has the potential to
serve as a nucleating agent. For instance, lyophilization vials
prepared in environments containing high particulate levels will
generally nucleate and freeze at a lower degree of sub-cooling than
vials prepared in low particulate environments.
[0014] All the nucleating agents described above are known as or
labeled "additives," because they change the composition of the
medium in which they nucleate a phase transition. These additives
are not typically acceptable or desirable for FDA regulated and
approved freeze-dried pharmaceutical products. These additives also
do not provide control over the time and temperature during which
the vials nucleate and freeze. Rather, the additives operate
primarily to increase the average nucleation temperature of the
vials (e.g. as freezing temperature depressants).
[0015] Ice crystals can themselves act as nucleating agents for ice
formation in sub-cooled aqueous solutions. In the "ice fog" method,
a humid freeze-dryer is filled with a cold gas to produce a vapor
suspension of small ice particles. The ice particles are
transported into the vials and initiate nucleation when they
contact the fluid interface.
[0016] The "ice fog" method does not control the nucleation of
multiple vials simultaneously at a controlled time and temperature.
In other words, the nucleation event does not occur concurrently or
instantaneously within all vials upon introduction of the cold
vapor into the freeze-dryer. The ice crystals will take some time
to work their way into each of the vials to initiate nucleation,
and transport times are likely to be different for vials in
different locations within the freeze-dryer. For large scale
industrial freeze-dryers, implementation of the "ice fog" method
would require system design changes as internal convection devices
may be required to assist in a more uniform distribution of the
"ice fog" throughout the freeze-dryer. When the freeze-dryer
shelves are continually cooled, the time difference between when
the first vial freezes and the last vial freezes creates a
difference in the temperature between vials, which will also
increase the vial-to-vial non-uniformity in the final freeze-dried
products.
[0017] Vial pre-treatment by scoring, scratching, or roughening has
also been used to lower the degree of sub-cooling required for
nucleation. As with the other prior art methods, vial pre-treatment
also does not impart any degree of control over the time and
temperature when the individual vials nucleate and freeze, but
instead only increases the average nucleation temperature of all
vials.
[0018] Vibration has also been used to nucleate a phase transition
in a metastable material. Vibration sufficient to induce nucleation
occurs at frequencies above 10 kHz and can be produced using a
variety of equipment. Often vibrations in this frequency range are
termed "ultrasonic," although frequencies in the range 10 kHz to 20
kHz are typically within the audible range of humans. Ultrasonic
vibration often produces cavitation, or the formation of small gas
bubbles, in a sub-cooled solution. In the transient or inertial
cavitation regime, the gas bubbles rapidly grow and collapse,
causing very high localized pressure and temperature fluctuations.
The ability of ultrasonic vibration to induce nucleation in a
metastable material is often attributed to the disturbances caused
by transient cavitation. The other cavitation regime, termed stable
or non-inertial, is characterized by bubbles that exhibit stable
volume or shape oscillations without collapse. U.S. Patent
Application 20020031577 A1 discloses that ultrasonic vibration can
induce nucleation even in the stable cavitation regime, but no
explanation of the phenomenon is offered. GB Patent Application
2400901A also discloses that the likelihood of causing cavitation,
and hence nucleation, in a solution using vibrations with
frequencies above 10 kHz may be increased by reducing the ambient
pressure around the solution or dissolving a volatile fluid in the
solution.
[0019] An electrofreezing method has also been used in the past to
induce nucleation in sub-cooled liquids. Electrofreezing is
generally accomplished by delivering relatively high electric
fields (1 V/nm) in a continuous or pulsed manner between narrowly
spaced electrodes immersed in a sub-cooled liquid or solution.
Drawbacks associated with an electrofreezing process in typical
lyophilization applications include the relative complexity and
cost to implement and maintain, particularly for lyophilization
applications using multiple vials or containers. Also,
electrofreezing cannot be directly applied to solutions containing
ionic species (e.g., NaCl).
[0020] Recently, there have been studies that examined the concept
of `vacuum-induced surface freezing` (See e.g., U.S. Pat. No.
6,684,524). In such `vacuum induced surface freezing`, vials
containing an aqueous solution are loaded on a temperature
controlled shelf in a freeze-dryer and held initially at about 10
degrees Celsius. The freeze-drying chamber is then evacuated to
near vacuum pressure (e.g., 1 mbar) which causes surface freezing
of the aqueous solutions to depths of a few millimeters. Subsequent
release of vacuum and decrease of shelf temperature below the
solution freezing point allows growth of ice crystals from the
pre-frozen surface layer through the remainder of the solution. A
major drawback for implementing this `vacuum induced surface
freezing` process in a typical lyophilization application is the
high risk of violently boiling or out-gassing the solution under
stated conditions.
[0021] Improved control of the nucleation process could enable the
freezing of all unfrozen solution containers in a cryogenic chiller
or freeze-dryer to occur within a narrower temperature and time
range, thereby yielding a product with greater uniformity from
sample-to-sample. With regard to freeze-drying systems, controlling
the minimum nucleation temperature affects the ice crystal
structure formed within the vial and allows for a greatly
accelerated freeze-drying process.
[0022] In view of the above, what is needed is a method and system
to control the uniformity of the temperature profiles and
nucleation temperatures of the multiple containers so as to provide
a more uniform finished product sample-to-sample. Moreover, the
system and method should be both efficient and readily scalable to
handle commercial scale production.
SUMMARY OF THE INVENTION
[0023] The invention may be characterized as a method of
controlling a chilling or freezing process of biological material
disposed in a plurality of containers, comprising the steps of: (i)
placing said plurality of containers of said biological materials
in a cooling area defined as an area between parallel porous
surfaces within a cooling chamber; (ii) mixing a liquid cryogen
with a warmer gas to produce a cold cryogenic gas at a selected
temperature profile, said temperature profile corresponding to a
desired cooling rate of said biological materials within said
containers; (iii) delivering a unidirectional flow of said cold
cryogenic gas through one of the porous surfaces to said cooling
area between said parallel porous surfaces and generally parallel
to each of said plurality of containers to uniformly cool said
biological materials within said containers; and (iv) promptly
exhausting said gas from said cooling chamber via another parallel
porous surface so as to prevent recirculation of said gas within
said cooling area; wherein greater uniformity of said biological
materials in each of said plurality of containers is achieved.
[0024] The inventors have recognized and appreciated a need for
delivering uniform viability and/or desired biological activity to
a plurality of containers each containing a biological material
during a cryopreservation process on a commercial scale.
Furthermore, the inventors have recognized and appreciated that it
is possible to provide a uniform enhanced viability and/or
biological activity to the biological material in each of the
containers by uniformly controlling the temperature profile and
nucleation of freezing for each container. More generally, the
inventors have recognized the advantages of a method capable of
providing large scale commercial volumes of frozen biological
material exhibiting uniform enhanced viabilities and/or biological
activities. For purposes of this application, biological activity
is defined as the therapeutic, biologic, or biochemical effect of a
material or constituents of the material on living matter. Such a
method is capable of being used for any number of different
applications in addition to cryopreservation.
[0025] In one exemplary embodiment, the uniformity of the viability
and/or biological activity in each container is maintained within
.+-.5% during the freezing process regardless of the location in
the cooling chamber where the material is frozen. The above example
of uniformity of viability and/or biological activity should not be
construed as limiting with regard to the current disclosure.
[0026] In another exemplary embodiment, a method of controlling a
freezing process of biological material in a plurality of
containers includes providing a plurality of containers each
holding a biological material in a cooling chamber within a single
system. The plurality of containers may be at least 10,000, and
often as many as 20,000, 50,000, or 100,000. The biological
material in each of the plurality of containers is frozen while the
containers are in the cooling chamber. The freezing process is such
that there is a uniform viability and/or biological activity of the
frozen biological material in each of the plurality of
containers.
[0027] In a further exemplary embodiment, the uniform viability
and/or biological activity of the frozen biological material in
each of the plurality of containers provides a uniformly enhanced
viability and/or biological activity.
[0028] It should be appreciated that all combinations of the
foregoing concepts and additional concepts discussed in greater
detail below (provided such concepts are not mutually inconsistent)
are contemplated as being part of the inventive subject matter
disclosed herein. In particular, all combinations of claimed
subject matter appearing at the end of this disclosure are
contemplated as being part of the inventive subject matter
disclosed herein.
[0029] The foregoing and other aspects, embodiments, and features
of the present teachings can be more fully understood from the
following description in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The accompanying drawings are not intended to be drawn to
scale. For purposes of clarity, not every component may be labeled
in every drawing. In the drawings:
[0031] FIG. 1 is a schematic illustration of an embodiment of a
uniform flow cryogenic chiller unit;
[0032] FIG. 2 is a detailed view of a cut-away portion of the
uniform flow cryogenic chiller unit of FIG. 1 depicting the uniform
flow characteristics of the cryogen gas proximate the vials of
biological materials;
[0033] FIG. 3 is a diagram of an embodiment of a single batch
uniform flow cryogenic chiller unit;
[0034] FIG. 4 is a schematic view of an embodiment of a multi-batch
or large commercial scale uniform flow cooling chamber;
[0035] FIG. 5 is a schematic view of another embodiment of a
continuous type uniform flow cooling unit;
[0036] FIGS. 6 through 8 depict selected temperature profiles of
the cryogenic cold gas and corresponding relationship to the
cooling rates of biological materials contained in multiple
vials;
[0037] FIG. 9 depicts an embodiment of a multi-batch or commercial
scale uniform flow cooling system with more detailed views of the
process and instrumentation;
[0038] FIG. 10 depicts another embodiment of a multi-batch or
commercial scale uniform flow cooling system with more detailed
views of the process and instrumentation;
[0039] FIG. 11 is an illustrative temperature profile of the
cryogenic cold gas during a temperature quench induced nucleation
freezing process;
[0040] FIG. 12 is a graph depicting the temperature profiles of
different samples during a temperature quench induced nucleation
freezing process;
[0041] FIG. 13 is a graph depicting the temperature profiles of
different samples during a freezing process with no nucleation
control;
[0042] FIG. 14 is a graph depicting the temperature versus time
plot of a solution undergoing a stochastic nucleation process and
further showing the range of nucleation temperatures of the
solution;
[0043] FIG. 15 is a graph depicting the temperature versus time
plot of a solution undergoing an equilibrated cooling process with
depressurized nucleation;
[0044] FIG. 16 is a graph depicting the temperature versus time
plot of a solution undergoing a dynamic cooling process with
depressurized nucleation;
[0045] FIG. 17 is a schematic representation of a lyophilization
system in accordance with the present invention;
[0046] FIG. 18a depicts illustrative temperature and pressure
profiles versus time of the cryogenic cold gas during a
depressurization induced nucleation freezing process;
[0047] FIG. 18b is a graph depicting the pressure profile and the
temperature profiles of different samples during a depressurization
induced nucleation freezing process;
[0048] FIG. 19 is a graph depicting cell viability versus
pre-nucleation temperature;
[0049] FIG. 20 is a graph depicting cell viability versus cold
spike temperature;
[0050] FIG. 21 is a graph depicting temperature profiles of samples
subjected to the different cold spike temperatures of FIG. 20;
[0051] FIG. 22 is a graph depicting cell viability versus cold
spike temperature and hold time;
[0052] FIG. 23 is a graph depicting temperature profiles of samples
subjected to the different cold spike conditions of FIG. 22;
[0053] FIG. 24 is a graph depicting cell viability versus
post-nucleation holding temperature;
[0054] FIG. 25 is a graph depicting temperature profiles of samples
subjected to the different post-nucleation holding temperatures of
FIG. 24;
[0055] FIG. 26 is a graph depicting cell viability versus
post-nucleation holding time;
[0056] FIG. 27 is a graph depicting temperature profiles of samples
subjected to the different post-nucleation holding times of FIG.
26;
[0057] FIG. 28 is a graph depicting cell viability versus
post-nucleation cooling rate following a 10 minute hold at
-35.degree. C.;
[0058] FIG. 29 is a graph depicting temperature profiles of samples
subjected to the different post-nucleation cooling rates of FIG. 28
after holding at -35.degree. C.;
[0059] FIG. 30 is a graph depicting cell viability versus
post-nucleation cooling rate following a 10 minute hold at
-10.degree. C.;
[0060] FIG. 31 is a graph depicting temperature profiles of samples
subjected to the different post-nucleation cooling rates of FIG. 30
after holding at -10.degree. C.; and
[0061] FIG. 32 is a chart comparing cell recovery following
freezing in the controlled rate freezing system in accordance of
the present invention.
DETAILED DESCRIPTION
[0062] It should be understood that aspects of the invention are
described herein with reference to the figures, which show
illustrative embodiments in accordance with aspects of the
invention. The illustrative embodiments described herein are not
necessarily intended to show all aspects of the invention, but
rather are used to describe a few illustrative embodiments. Thus,
aspects of the invention are not intended to be construed narrowly
in view of the illustrative embodiments. It should be appreciated,
then, that the various concepts and embodiments introduced above
and those discussed in greater detail below may be implemented in
any of numerous ways, as the disclosed concepts and embodiments are
not limited to any particular manner of implementation. In
addition, it should be understood that aspects of the invention may
be used alone or in any suitable combination with other aspects of
the invention.
Controlled Rate Freezing
[0063] Cryopreservation of biological materials typically involves
rapid cooling of biological specimens from temperatures of
40.degree. C. or more to temperatures of about -100.degree. C. or
lower. The specified temperatures, cool-down rates, and cooling
profiles, expressed as temperature of the materials as a function
of time, are highly dependent on the specific biological materials
to be frozen. In most cryopreservation of biological materials, the
freezing process must be precisely controlled. Uniformity in
temperatures, cool-down rates, and cooling profiles from container
to container and batch to batch is of utmost importance in the
production process.
[0064] The presently disclosed method and system represents an
improvement to current cryopreservation processes for biological
materials. The presently disclosed system and method provides the
ability to rapidly cool the biological materials contained in vials
or other containers within a cooling unit primarily via forced
convective cooling simultaneously using a uniform flow of cryogen
in proximity to each of the plurality of vials disposed within the
cooling unit. In addition, the present system and methods are
capable of providing the rapid cooling of the biological materials
over a wide range of cooling rates while simultaneously holding the
temperature of the biological materials at the prescribed and
specified temperature.
[0065] More specifically, the rapid cooling of the biological
materials is achieved by precisely controlling and adjusting the
temperature of the cryogen being introduced to the system as a
function of time. In one mode, the disclosed embodiments of the
system are adapted to provide a stepwise (quick) drop in cryogen
temperature 102 (See FIG. 6) to generate a higher degree of
sub-cooling within the sample materials 100 thereby minimizing the
exothermic effects of the phase transition (e.g. water-to-ice
transformation) in the vials. In another mode, the disclosed
embodiments of the present controlled rate freezing or cryogenic
chilling system and method are adapted to provide a ramp down of
cryogen cold gas temperature at a rate of about -4.5.degree. C. per
minute 112 (See FIG. 7) and of about -5.0.degree. C. per minute
(See FIG. 8), respectfully in order to provide rapid cooling of the
sample biological materials 110, 120 yet minimize any vial to vial
variations in temperature.
[0066] Temperatures of the cold cryogen gas introduced to the
cooling chamber or unit are adjusted or otherwise controlled by
mixing a source of liquid nitrogen with a source of warmer nitrogen
gas just prior to introduction of the cold cryogen gas to the
cooling unit. The mixed flow is then introduced and dispersed
throughout the cooling unit by means of suitable cryogen intake
circuits, as described herein. The warmer nitrogen gas is
preferably either room temperature nitrogen gas from a supply
source or nitrogen gas exiting from the cooling unit and recycled
to the cryogen intake circuit. The warmer nitrogen gas mixed with
the cold nitrogen liquid or gas also acts as a motive gas and
preferably has a volumetric flow rate many times that of the liquid
or cold nitrogen. Through the appropriate mixing of the warmer
nitrogen gas with the cooler nitrogen flow, the present system
creates a uniform flow of the cryogen across the entire cooling
area targeted by the cold cryogen gas. By recycling the nitrogen
gas exiting the cooling unit(s), the presently disclosed system and
method also offers a higher utilization efficiency of the cryogen
(e.g. nitrogen) than existing controlled rate freezers.
[0067] Given the uniform flow of the cold cryogen gas across all
samples or vials of the biological material, it has been found that
precise control of the cold cryogen gas temperature and cryogen
temperature gradient has a direct correlation to the observed
cooling rates of the biological material within the cooling unit,
for a given biological material. For example, when the cold cryogen
gas temperature provided to the present cooling unit is varied or
ramped at about -4.5.degree. C./min to about -5.0.degree. C./min,
an average cooling rate of the biological material of approximately
-2.5.degree. C./min is achieved with minimum vial-to-vial
temperature variations. (See FIGS. 7 and 8).
[0068] Turning now to FIGS. 1 and 2, there are depicted selected
views of a cooling unit, referred to as a uniform flow cryogenic
chiller 10. As seen therein, the uniform flow cryogenic chiller 10
includes a cryogen intake circuit 12 or conduit coupled to a source
of cryogen (not shown). The uniform flow cryogenic chiller 10
further includes a base gas injection box 14, a porous metal plate
16 disposed or set in or near the top surface 17 of the gas
injection box 14, and a corresponding gas removal box 18 positioned
immediately above the base gas injection box 14 and porous metal
plate 19 disposed therein. Alternatively, various arrangements of
supported polymeric membranes suitable to withstand the cryogenic
temperatures or other perforated plates with mechanically punctured
or chemically etched holes can be used in lieu of the porous metal
plates. Alternatively, the gas removal box 18 may include other
collection surfaces in lieu of the porous metal plate, such as a
mesh, screen or open surface or area leading to the exhaust
manifold 34.
[0069] The porous metal plate 16 associated with the gas injection
box 14 is adapted to receive and hold a plurality of vials 20
containing biological materials. Also disposed in or near the vials
20 is a plurality of temperature sensors 25 to be used as inputs to
the system controller (not shown). The cryogen intake circuit 12 or
conduit is further coupled to the gas injection box 14 that is
adapted to receive the cryogen intake flow and evenly distribute
the cryogen across the porous metal plate 16. The cold cryogen gas
flows in a uniform manner into an intake plenum 32 in the gas
injection box 14 through the lower porous metal plate 16 holding
the vials 20 into the cooling space 30 and then to the gas removal
box 18 which also includes an upper collection surface or area
(e.g. shown as the optional porous metal plate 19) and an exhaust
manifold 34. From the exhaust manifold 34, the spent nitrogen gas
exits via the gas exhaust circuit 28 or conduit.
[0070] As discussed above, the cooling of the vials 20 is provided
by the heat transfer between the vials 20 and the cryogenic cold
gas 27 flowing through the cooling area 30. The cryogenic cold gas
27 is produced in the cryogen intake circuit 12 by mixing liquid
nitrogen with a warmer nitrogen gas or recirculating spent nitrogen
gas from the gas exhaust circuit 28 with appropriate mixing
apparatus or valves 36. The vials 20 are cooled generally at a
slightly slower rate than the cryogenic cold gas. The temperature
difference between the vials 20 and the cryogenic cold gas 27 is
the thermal driving force to cool down the vials 20. Therefore, it
is possible to freeze the vials 20 with any temperature profile by
precisely controlling the temperature of the cryogenic cold gas 27
at a particular temperature profile.
[0071] Preferably, the cryogenic cold gas temperature, and more
particularly, the temperature profile is actively controlled in
response to the average temperatures indicated by the temperature
or thermal sensors 25 disposed at or near the vials 20. In the
present embodiment, the average temperatures in a plurality of
vials 20 are being used as the inputs for the active control of the
system. Preferably, a cascade based control methodology where the
system temperatures including vial temperatures are monitored and
controlled by a primary system controller, which transfers set
point signals and other commands to a slave controller responsible
for modulating the cryogenic cold gas temperatures in the intake
circuit. As discussed in more detail below, the cryogenic cold gas
temperature profile is created through the operative control of a
mixing valve that blends a specified volume of cold liquid nitrogen
with a specified volume of warmer nitrogen gas. The blending or
mixing is preferably a continuous operation that changes as a
function of time to yield a cryogenic cold gas having a temperature
that follows a prescribed temperature profile (i.e. temperature
that changes as a function of time). In short, operative
temperature control of the uniform flow cryogenic chiller is
achieved by controlling the temperature profile of the cryogenic
cold gas in the intake circuits. As discussed above, it has been
found that precise control of the cryogenic cold gas temperatures
and temperature gradients has a direct correlation to the observed
cooling rates of the given biological material.
[0072] In the illustrated embodiment, as the cryogenic cold gas
enters the lower gas injection box 14, the cryogenic cold gas 27 is
dispersed into an intake plenum 32 through a series of downward
oriented sparger pipes or channels within the gas injection box
(not shown). This dispersion in the intake plenum 32 promotes an
even distribution of the cryogenic cold gas 27 across the entire
surface of the porous metal plate 16. The downward oriented
distribution of cryogenic cold gas 27 in the intake plenum 32
avoids the direct impingement of the cryogenic cold gas 27 on the
porous metal plate 16, resulting in cold spots and non-uniform
cooling. The porous metal plate 16 in the gas injection box 14
forces the cryogenic cold gas 27 to distribute uniformly across the
entire cooling area 30 of the uniform flow cryogenic chiller 10,
where the vials or other containers of biological material are
held. Alternative means or devices for cryogenic cold gas
dispersion or distribution across the entire surface of the porous
metal plate are contemplated and use of the above described
downward oriented sparger pipes or channels within the gas
injection box is an additional example of such gas dispersion or
distribution for the present disclosure.
[0073] In one illustrated embodiment, the spent nitrogen is
collected in an exhaust manifold 34 disposed above the gas
collection area or surface (e.g. porous plate 19) in the gas
removal box 18. As illustrated, the cryogenic cold gas 27 has only
a short path to traverse from the intake plenum 32 through the
porous plate 16 upward into the cooling area 30, through the upper
porous plate 19 and into the exhaust manifold 34. The uniform
direction and short distance of the cryogenic cold gas flow results
in a high level of uniformity in vial 20 cooling within the
cryogenic chiller 10. Pore sizes for the porous metallic plates 16,
19 are preferably on the order of about 2 to 50 microns in
diameter, as small pores enhance the dispersion and resulting
uniformity in cooling. By cooling or freezing the biological
material at the optimized rate, the survival rate of the cells is
enhanced yielding potentially higher drug potency.
[0074] At the freezing point of the solutions, the heat of
crystallization keeps the solution temperature from dropping, and
sometimes the temperature within the vial can also rise. Using one
or more thermal or temperature sensors 25 embedded in or near
selected control vials, the temperature of cryogenic cold gas can
be adjusted to minimize temperature deviation from the optimized
cooling rate, as needed. In other words, control of the system may
be either pre-programmed or may be a real-time feedback based
operation.
[0075] Pharmaceutical, biopharmaceutical or biologic solutions
contained in vials or containers for cryopreservation would benefit
from the present system and methods. Such biological or
biopharmaceutical materials may include microorganisms, tissues,
organs, stem cells, primary cells, established cell lines, small
multicellular organisms, complex cellular structures such as
embryos, or a solution or mixture that includes: live or attenuated
viruses; nucleic acids; monoclonal antibodies; polyclonal
antibodies; biomolecules; nonpeptide analogues; peptides, proteins,
including fusion and modified proteins; RNA, DNA and subclasses
thereof, oligonucleotides; viral particles; and similar such
materials or components thereof. Also, the containers used for
holding the biological materials may include vials, straws,
polymeric bags, or other form of suitable container.
[0076] FIGS. 3, 4, and 5 depict various embodiments of the present
uniform flow controlled rate freezer or cryogenic chiller
incorporating the uniform flow approach or concept. More
specifically, FIG. 3 is a diagram of a single modular unit 40 of
the controlled rate freezer adapted to hold one of the uniform flow
cryogenic chillers. The external housing for the unit 40 shown in
FIG. 3 is comprised of a solid stainless steel housing with a gas
injection box 44 having an intake conduit 42, a plenum, and porous
plate 46 as well as a gas removal box 48 having a porous plate, an
exhaust manifold, and an exhaust conduit. The unit shown is
dimensioned to hold a single uniform flow cryogenic chiller as
described above with reference to FIGS. 1 and 2.
[0077] FIG. 4 depicts a multi-batch or commercial scale unit 50
that includes a cooling chamber 52 that includes a plurality of
shelves or rails 54 adapted to hold multiple uniform flow cryogenic
chiller assemblies. Such a multi-batch or commercial scale unit 50
is preferably capable of cryopreserving 50,000 or more vials or
other such containers per production run. As seen in FIG. 4, the
cryogen intake circuit 56 and spent gas exhaust circuit 58 are
designed and sized to circulate sufficient cryogen to the multiple
individual cryogenic chillers 60. Control system 70 is used to
operatively control the temperature profile of the cold cryogen gas
provided to each shelf 54, or to each cryogenic chiller assembly 60
depending on the inputs from the thermal sensors disposed within
the system.
[0078] FIG. 5 depicts yet another possible commercial scale
embodiment of the controlled rate freezer or chiller system 80 that
operates in a continuous or conveyorized manner. Again, the unit 80
and cryogenic cold gas intake circuit 90 and gas exhaust circuit 92
are designed and sized to circulate sufficient cryogenic cold gas
to individualized containers or tray assemblies 88 disposed along a
conveyor 86 within the tunnel-type freezer chamber 82 having an
entrance and exit means 84. In this continuous operation, the
cooling profiles of different containers, vials or trays could be
either time based, as described above, or spatially based.
[0079] The ability to precisely control the cooling rate of
biological material provides many benefits. For example, biological
material frozen in an aqueous solution may experience various
stresses during the freezing and subsequent thawing process that
may impair the function or activity of the material. Ice formation
may physically disrupt the material or create severe changes in the
interfacial bonding, osmotic forces, solute concentrations, etc.
experienced by the material. Proper design of the freezing process
can mitigate such stresses and the present system and method allows
for the precise control of the freezing process to achieve
uniformity in the frozen material in all vials in accordance with
the designed freezing profile.
[0080] One exemplary system includes a cryogen source, an intake
circuit coupled to the cryogen source and adapted for providing a
uniform flow and temperature of a cryogenic cold gas to a cooling
chamber, an exhaust circuit and a control system. The cooling
chamber comprises an intake plenum, an exhaust manifold, and two or
more parallel surfaces that define a cooling area between adjacent
parallel surfaces with one of the parallel surfaces (i.e
distribution surface) disposed adjacent to the intake plenum and in
fluid communication with the intake plenum and another of the
parallel surface (i.e. collection surface) disposed adjacent to the
exhaust manifold, the parallel collection surfaces and cooling area
adapted to retain, or hold, a plurality of containers of biological
materials. The exhaust circuit of the freezing or chilling system
is adapted to remove the cryogen gas from the exhaust manifold of
the cooling chamber whereas the control system is adapted to adjust
the flow rates of the cryogen source in the intake circuit and any
cryogen gas in the exhaust circuit to adjust the temperature of the
cold cryogen gas delivered to the cooling chamber in response to a
desired cooling rate of the biological materials and measured
temperatures within the cooling chamber. In this manner, a uniform,
unidirectional flow of temperature adjusted cryogenic cold gas is
delivered to the cooling area between the parallel porous surfaces
and parallel to each of the plurality of containers to uniformly
cool the biological materials.
[0081] The operation of the above disclosed exemplary system
includes the steps of: (i) placing a plurality of containers of the
biological materials in a cooling area defined as the area between
parallel porous surfaces within a cooling chamber; (ii) mixing a
liquid cryogen with a warmer gas to produce a cold cryogenic gas at
a selected temperature profile, the temperature profile
corresponding to a desired cooling rate of the biological materials
within the containers; (iii) delivering a unidirectional flow of
the temperature adjusted cryogenic cold gas through one of the
porous surfaces to the cooling area between the parallel porous
surfaces and parallel to each of the plurality of containers to
uniformly cool the biological materials; and (iv) promptly
exhausting the gas from cooling chamber via another parallel porous
surface so as to prevent recirculation of the gas within the
cooling area.
[0082] Turning now to FIG. 9, a detailed embodiment is presented.
The illustrated cryogenic chiller system 210 includes a cooling
chamber 220 adapted to receive a cryogenic cold gas 260 from a
cryogen cold gas circuit 262, a source of liquid nitrogen 230, a
liquid supply circuit 232 including a phase separator 234, a supply
of gaseous nitrogen 240, a gas supply circuit 242, a recirculating
cryogenic gas 250 and a gas recirculation circuit 252. The
cryogenic chiller system 210 further includes a programmable logic
controller (PLC) based control system 270 that operatively controls
the fluid circuits in response to measured temperatures and
pressures as well as certain user defined parameters including the
desired cooling profiles.
[0083] The illustrated cooling chamber 220 has a plurality of
cooling shelves 222 used to cool a large number of vials containing
pharmaceutical active ingredients or active biological molecules. A
cryogenic cold gas 260 is supplied to the cooling chamber 220 from
a static in-line mixer 263 that mixes liquid nitrogen from the
source of liquid nitrogen 230 via the liquid supply circuit 232
with a precisely metered gaseous nitrogen gas stream from the gas
supply circuit 242 and recirculating cryogenic gas 250 from the gas
recirculation circuit 252.
[0084] The temperature of the cryogenic cold gas 260 is preferably
measured with a temperature sensor 264 disposed downstream of the
static in-line mixer 263. By precisely adjusting the flow of
nitrogen from the liquid supply circuit 232 with nitrogen gas from
the gas supply circuit 242 and the gas recirculation circuit 252 it
is possible to rapidly shift the temperature of the cryogenic cold
gas 260 which allows cooling of the vials in the cooling chamber
220 with a wide range of cooling profiles to optimize operating
conditions and maximize cell viability, biological activity, drug
uniformity, as well as drug potency.
[0085] Once a cryogenic cold gas 260 is formed by mixing this
nitrogen gas with liquid nitrogen, it is split into multiple levels
of cooling shelves 222 in a single cooling chamber 220. To provide
the exact split of the cryogenic cold gas 260 to the multiple
cooling shelves 222, a plurality of critical flow orifices 265 are
used to split cryogenic cold gas 260 into multiple gas streams.
Under critical choke flow conditions, the cryogenic cold gas flow
to the cooling shelves 222 is maintained independent of the
downstream pressure. A large cryogenic cold gas manifold 266 is
used to eliminate or minimize pressure differences upstream of the
critical flow orifices 265 while the downstream gas flow resistance
has no impact on the gas flow through the critical flow orifices
265. In this manner, the cryogenic cold gas flow to each of cooling
shelves 222 in the cooling chamber 220 is nearly identical.
[0086] The cryogenic chiller system 210 is a direct contact cooling
system with a cryogenic cold gas 260 flowing in the same direction
with respect to each vial and preferably along the longitudinal
axis of the vials, thus creating a uniform cooling profile for all
the vials. A porous metallic membrane (See FIGS. 1 and 2) provides
uniform resistance across all the cooling surfaces, thus allowing
the individual vials to receive uniform amounts of
refrigeration.
[0087] The nitrogen gas supply 240 is preferably received from a
bulk storage tank and is directed through a filter 244 to remove
particulate materials. The nitrogen gas supply 240 is then
regulated down to the desired pressure through a discharge pressure
regulator 245. Line pressures before and after the pressure
regulator 245 are preferably monitored using one or more pressure
indicators 246. A mass flow controller 247 including a mass flow
sensor 248 with electro-pneumatic control valve 249 is preferably
used to control and maintain a precisely metered nitrogen gas flow
rate through the gas supply circuit 242 to the static in-line mixer
263. An electrical solenoid valve 243 is also included in the gas
supply circuit 242 to provide positive shut off capability when the
cryogenic chiller system 210 is not operating. Alarms can be set in
the control system 270 to deactivate this solenoid valve 243 if
emergency shutdown of the cryogenic chiller system 210 is
required.
[0088] The illustrated system depicts an additional source of gas,
namely air, to be used to operate various control valves. The
illustrated air supply circuit 215 includes a filter 216 adapted to
remove any particulates from the line, a pressure regulator 218
that is adapted to reduce the air pressure to about 25 psig for
safe operation, and one or more pressure indicators 219 used to
monitor the pressure in the air supply circuit 215.
[0089] The liquid nitrogen supply circuit 232 includes a source of
liquid nitrogen 230, a phase separator 234, one or more temperature
and pressure sensors 233, a liquid nitrogen manifold 235, one or
more safety/relief valves 236, a strainer 237, and a primary
cryogenic flow control valve 238. All liquid nitrogen piping is
preferably insulated so as to minimize any phase change of the
liquid nitrogen to nitrogen gas and the resulting two-phase flow in
any of the pipes within the liquid nitrogen supply circuit 232.
[0090] The liquid nitrogen phase separator 234 is designed to
remove any nitrogen gas that forms in the liquid nitrogen supply
circuit 232 due to heat leakage or changes in pipeline pressures.
The illustrated phase separator 234 is a double-walled, vertically
mounted, cylindrical tank. The inner liquid vessel has a maximum
allowable working pressure (MAWP) rating of 250 psig, with the
outer vessel providing a vacuum insulation. The gas phase vent
valve 239 operatively controls the filling of the phase separator
234 with liquid nitrogen from the source of liquid nitrogen 230. At
a low liquid level, the gas phase vent valve 239 opens to vent 280
vapor pressure from the phase separator 234, allowing liquid
nitrogen to transfer from the source of liquid nitrogen 230. As the
liquid nitrogen level increases in the phase separator 234, gas
phase vent valve 239 begins to close and the fill rate decreases
until the valve 239 is completely closed and filling of the phase
separator 234 with liquid nitrogen stops.
[0091] The strainer 237 is coupled to a blow-down relief valve 236A
that is operated as required to clean the strainer 237 or purge any
vaporized nitrogen gases from the liquid nitrogen supply circuit
232. The strainer 237 also serves to filter out any particulates in
the liquid nitrogen so as to avoid adverse performance or damage to
the primary cryogenic control valve or relief valves.
[0092] One of the illustrated safety valves is a cryogenic
electrical solenoid valve 236B that provides positive shutoff of
the liquid nitrogen supply. Deactivating the electrical solenoid
valve 236B shuts off all liquid nitrogen flow through the liquid
nitrogen supply circuit and to the static in-line mixer 263. This
electrical solenoid valve 236B is configured such that cutting
electrical power immediately stops the liquid nitrogen flow through
the liquid nitrogen supply circuit 232 circuit and vent 280 any
trapped liquid nitrogen from the circuit. In addition, other
process shutdown and the emergency shutoff procedures within the
control system 270 generate command signals to the one or more
safety valves 236 as, for example, when the cryogenic chiller
system 210 has stopped operating at the end of the freezing cycle
or for other reasons including preset alarm conditions. The control
system 270 stops the liquid nitrogen flow in the liquid nitrogen
supply circuit 232 by shutting off one or more of the safety valves
236.
[0093] The primary cryogenic flow control valve 238 receive signals
from the control system 270 to control the amount of liquid
nitrogen supplied to the cryogenic cold gas circuit 262 in response
to measured temperatures and pressures within the cryogenic
chilling system 210 as well as certain user defined parameters
including the desired cooling profiles.
[0094] Liquid nitrogen from the liquid nitrogen supply circuit 232
is directed to the static in-line mixer 263. The liquid nitrogen
evaporates into a cryogenic cold gas 260 by mixing with the
nitrogen gas directed from the gas supply circuit 242 and the gas
recirculation circuit 252. The static in-line mixer 263 is used to
ensure that no slug of unevaporated liquid nitrogen enters the
cooling chamber 220. The temperature in the cryogenic cold gas
circuit 262 is monitored with a temperature sensor 264 disposed at
or near the exit of the static in-line mixer 263. The control
system 270 receives this measured temperature and regulates the
liquid nitrogen flow rate and gas flow rates to the static in-line
mixer 263 in response thereto based on programmed temperature
profiles and preset parameters to adjust the temperature of the
cryogenic cold gas.
[0095] Downstream of the static in-line mixer 263, the cryogenic
cold gas 260 is directed to a large cryogenic cold gas manifold 266
and then to the multiple cooling shelves 222 in the cooling chamber
220 via a plurality of critical flow orifices 265. The large
cryogenic cold gas manifold 266 is used to ensure that all the gas
distribution points realize the same or similar pressures. The
actual cryogenic cold gas flow rate delivered to each of the
cooling shelves 222 of the cooling chamber 220 is determined by the
size of the critical flow orifice 265 associated with each cooling
shelf 222.
[0096] Inside the cooling chamber 220 at each level, there are a
series of gas distribution pipes with downward oriented nozzles.
The purpose of the additional gas distribution pipes inside the
cooling chamber is to avoid or minimize velocity generated local
pressure gradients that may impact the cryogenic cold gas
distribution across any large porous metallic membrane. With the
critical flow orifices 265 and gas distribution networks, a large
cooling chamber can be used holding thousands of vials or packages
with very high degree of cooling uniformity.
[0097] The cooling surfaces within the multiple levels of the
cooling chamber 220 are made of porous metallic membranes 227
adapted to generate uniform gas flow across the plurality of vials.
Due to the small pore size and high flux in the metallic membranes
227, a generally laminar flow rising from the entire cooling
surface is generated. While a generally laminar flow from the
cooling surface is preferred, other flows, including a turbulent
gas flow are tolerable so long as the flow remains generally
parallel to the vials and that macro recirculation of the gas does
not occur inside the cooling chamber 220.
[0098] Above the porous metallic membranes at each level in the
cooling chamber 220 is an exhaust manifold 225 with a perforated
plate disposed in a parallel orientation with the porous metallic
membranes 227 to maintain the uniform flow of the cryogenic cold
gas 260 during the cooling of the vials. The gas received in the
exhaust manifold 225 is immediately removed from the cooling
chamber 220 in order to avoid or minimize any internally
recirculating flow of the spent nitrogen gas. It is important to
avoid the internal recirculation of the nitrogen gas as such
recirculated gas is generally at a warmer temperature than the
cryogenic cold gas 260 supplied to the cooling chamber 220. Such
internally recirculating flow is the main cause of temperature
non-uniformity with edge effects in prior art or conventional
cooling devices.
[0099] The exhausted gas removed from the cooling chamber 220 is
preferably diverted to a gas recirculation circuit 252. The
illustrated gas recirculation circuit 252 includes a recirculating
gas manifold 253 disposed between the exhaust manifolds 225 in the
cooling chamber 220 and a recirculating blower 254 that starts
automatically during the later part of the freezing cycle. The gas
recirculation circuit 252 also includes a mass flow meter 255
coupled to the control system 270 that measures the flow through
the gas recirculation circuit 252 so as to adjust the make-up gas
flow rate from the gas supply circuit 242 to maintain a desired
level of cryogenic cold gas 260 flow in the cryogenic cold gas
circuit 262. Back pressure regulator 256 maintains the pressure
from the recirculating blower 254 while check valve 258 keeps the
make-up nitrogen gas from the gas supply circuit 242 from entering
the gas recirculation circuit 252 when the recirculation blower 254
is not operating. Safety relief valve 259 provides
over-pressurization protection for cooling chamber 220 in case
there are blockages in gas recirculation circuit 252.
[0100] The pressure and temperature inside the cooling chamber 220
are monitored with pressure gauge 228 and temperature sensors 229
or thermocouples disposed within the cooling chamber 220 proximate
some of the vials. The pressure gauges 228, temperature sensors 229
as well as the thermocouples are coupled to and provide inputs to
the control system 270.
[0101] The above disclosed cryogenic chiller system is able to
provide uniform temperatures and flows of cryogenic cold gas to
each vial due to the disclosed unidirectional and uniform flow of
cryogenic cold gas generally parallel to the longitudinal axis of
the vials or containers. In some embodiments, this flow may be
approximated as a plug flow through cooling chamber 220 delivering
a uniform temperature and flow rate of cryogenic cold gas to each
of the vials of material.
[0102] FIG. 10 presents another embodiment of a cryogenic chiller
system 300. In this particular embodiment, a source of liquid
cryogen 302 acts as the supply source for both the liquid cryogen
and gas flows in the cryogenic chiller system. The liquid cryogen
source may include, but is not limited to liquid: argon, nitrogen,
air, permissible mixtures thereof, and any other appropriate
cryogen that is non-reactive with the system and material to be
cryopreserved. The source of liquid cryogen 302 is connected to a
safety valve 304. In one embodiment safety valve 304 is an
electronically controlled solenoid valve. Safety valve 304 shuts
off the flow of cryogen to the system when the system is
de-energized, the pressure in the exhaust system is greater than or
equal to the pressure in the cooling chamber, a manual safety
switch has been thrown, a preset alarm activates, or other
appropriate situations. When safety valve 304 is open liquid
cryogen is supplied to phase separator 306. Phase separator 306 has
two output flows to conduits 308a and 308b.
[0103] The first flow provided from phase separator 306 flows
through conduit 308a. This first flow provides liquid cryogen or a
mix of liquid cryogen and gas to vaporizer 314. Vaporizer 314
vaporizes any liquid cryogen present in the flow to create a flow
of gas. The vaporizer is connected to a pressure relief valve 316
and heater 318. In a preferred embodiment, pressure relief valve
316 is set to 100 psi (gauge). Heater 318 heats the gas flow to
adjust the temperature to that indicated by the control systems.
Temperature sensor 324 monitors the temperature of the gas flow
exiting heater 318 and outputs a signal to temperature controller
326. Temperature controller 326 actuates a relay 320 to control
heater 318. In a preferred embodiment relay 320 is a solid state
relay. However, relay 320 may be replaced with a circuit or other
suitable component capable of receiving instructions from
temperature controller 326 and controlling heater 318. The flow of
temperature adjusted gas is provided from heater 318 through safety
valve 322 to flow control valve 332. A flow sensor 330 monitors the
flow of temperature adjusted gas and outputs a signal to flow
controller 328. Flow controller 328 controls flow control valve 332
which in turn controls the flow rate of temperature adjusted gas
provided to mixer 334.
[0104] The second flow provided from phase separator 306 is liquid
cryogen flowing through conduit 308b. The flow of liquid cryogen is
directed through flow control valve 310. Flow control valve 310
controls the flow rate of liquid cryogen. The flow of liquid
cryogen is combined with the flow of temperature adjusted gas
provided to mixer 334. All conduits, valves, and control systems
associated with transporting the liquid cryogen are preferably
insulated to minimize any unwanted phase change of the liquid
cryogen to a gas.
[0105] The separate flows of temperature adjusted gas from flow
control valve 332 and liquid cryogen from flow control valve 310
are mixed together in mixer 334 to provide cryogenic cold gas. In
some embodiments, mixer 334 may be a static mixer, an arrangement
of valves, an impeller, or any other appropriate structure adapted
for mixing the flows. The temperature of the cryogenic cold gas is
monitored by temperature sensor 336 after exiting mixer 334.
Temperature sensor 336 provides a signal to temperature controller
312. Temperature controller 312 adjusts the flow of cryogen through
flow control valve 310 so as to adjust the temperature of the
cryogenic cold gas exiting mixer 334.
[0106] The flow of cryogenic cold gas is provided to cooling
chamber 338 holding a plurality of vials or containers. The
cryogenic cold gas flows through the chamber so as to transfer heat
with the vials or containers. To provide uniform cooling of each
vial or container, the cryogenic cold gas is uniformly distributed
throughout cooling chamber 338 by at least one gas injection box
340. The cryogenic cold gas is immediately exhausted from the
cooling chamber by at least one gas exhaust box 342 preferably
arranged above the at least one gas injection box 340. The
cryogenic cold gas is immediately exhausted to avoid any
recirculation of the cryogenic cold gas since this will lead to
non-uniformities in the cooling chamber's 338 temperature. Cooling
chamber 338 is further provided with pressure relief valve 346
preferably set at 50 psig. Pressure sensor 348 monitors the
pressure in cooling chamber 338. A signal is output from pressure
sensor 348 to liquid nitrogen controller 312, gas flow controller
328, and pressure controller 350. Pressure controller 350 controls
flow control valves 354 and 358. In some embodiments, the system
may reduce or shut down the liquid and gas flows during
pressurization and depressurization steps in response to a signal
from pressure sensor 348.
[0107] In a preferred embodiment, porous membranes 344 suitable for
cryogenic use are attached to the gas injection box 340 and gas
exhaust box 342. The porous membranes 344 are adapted to generate a
uniform flow of gas across the plurality of vials. Due to the small
pore size and high flux across the porous membranes 344, a uniform
flow rising from the entire cooling surface is generated. In a
preferred embodiment, porous membranes 344 are porous metallic
plates. While a laminar flow from the cooling surface is preferred,
a turbulent gas flow or other gas flow is tolerable so long as the
flow remains parallel to the vials and that macro recirculation of
the gas does not occur inside the cooling chamber. While the flow
is disclosed as flowing upwards it should be understood that the
flow of gas may be oriented in any direction so long as each of the
vials receives a uniform flow and temperature of cryogenic cold
gas.
[0108] The cryogenic cold gas exhausted through the at least one
exhaust hood 342 flows through conduit 352 to adjustable pressure
control valve 354 prior to being exhausted to system exhaust 362.
Adjustable pressure control valve 354 enables pressurization of
cooling chamber 338. The pressure of the exhausted cryogenic cold
gas is monitored by pressure switch 360. Pressure switch 360
actuates safety valves 304 and 322 when the pressure of the
exhausted cryogenic cold gas is lower than the pressure in cooling
chamber 338. Closing safety valves 304 and 322 stops the flows of
liquid and gas into the system thus preventing nitrogen leakage due
to abnormal system operation.
[0109] Cooling chamber 338 further includes an adjustable flow
control valve 356 connected to flow control valve 358. Adjustable
flow control valve 356 may be adapted for either manual or
electronic adjustment. In one possible embodiment, flow control
valve 358 is an on/off control valve actuated by a pneumatic
solenoid. In other alternative embodiments, flow control valve 358
may be actuated by an electrical solenoid, an electrical switch, a
pneumatic control system, a hydraulic control system, or any other
appropriate mechanism. When open, flow control valve 358 enables
the depressurization of cooling chamber 338 to system exhaust 362.
The rate of depressurization is controlled by the setting of
adjustable flow control valve 356.
[0110] In a preferred embodiment, at least a portion of the
cryogenic cold gas exhausted through system exhaust 362 is recycled
into the system. The recycled gas is preferably input into the gas
flow between vaporizer 314 and heater 318 depicted as recycled
exhaust gas input 364. However, it is possible to place the
recycled exhaust gas input 364 in other alternative positions
within the system.
[0111] It should be understood that the separate valves and systems
depicted herein may be powered and controlled in a variety of ways.
Possible methods of providing power and control include, but are
not limited to, manual, electrical, pneumatic, and hydraulic
control. In addition, cryogenic chiller system 300 may implement
any combination of the above methods to provide power and control
to the separate components. The selection and application of these
different control and power methods merely represents a design
choice and can be easily implemented by one of skill in the
art.
[0112] While the above described methods and systems have been
shown with regards to providing uniform temperature profiles during
a freezing process, the same methods and systems may be applied to
provide a uniform temperature profile for a plurality of vials or
containers for uniformly thawing the material in each of the
plurality of vials or containers. In both a uniform freezing and
thawing process, each vial or container sees substantially the same
temperature profile regardless of its location within the cooling
chamber. Similar to providing a uniform temperature profile during
freezing, uniform thawing of a plurality of vials or containers
will result in more repeatable uniform properties. Such properties
include, but are not limited to, cell viability, functionality,
and/or biological activity. Depending upon the type of biological
material present in each container or vial, the biological material
is revived during the thawing process. Reviving the biological
material entails returning the biological material to its original
state prior to the freezing process. Furthermore, while such
systems are typically used for one type of material at a time it is
possible to freeze multiple biological materials at once inside the
system. Therefore, it is possible that at least two of the
containers inside of the system would contain different biological
materials during a freezing process. Such a use is considered
within the scope of this disclosure.
[0113] The above disclosed systems and methods are particularly
well-suited for commercial type or large scale biological
production operations since the process is conducted using the same
equipment and process parameters that are easily scaled or adapted
to manufacture a wide range of biological products. The presently
disclosed process and system provides for the controlled rate
freezing of biological materials using a process that achieves a
high degree of uniformity in cooling or freezing of the biological
material from sample to sample, vial to vial, container to
container, and batch to batch.
Nucleation Control
[0114] In addition to the temperature profile seen by each vial or
container, the final uniformity of properties and structure from
sample to sample, vial to vial, container to container, and batch
to batch may also depend on the nucleation temperature. As stated
above, this variability in the nucleation temperature can impact
properties such as cell activity and viability as well as the
crystal structure of the frozen material and the time needed to
complete a freeze drying process. Advantageously, the currently
disclosed systems and processes may be applied to provide control
over the nucleation temperature of the material in the plurality of
vials or containers using two possible methods. The methods
include, but are not limited to, pressure control induced
nucleation and temperature quench induced nucleation, although
nucleation control need not be used in all embodiments with respect
to improving or otherwise enhancing cell viability and/or other
biological activity. For example, substantially uniform cooling
features of aspects of the invention may be used to freeze
materials in a plurality of vessels without the use of nucleation
control, yet still provide uniform and enhanced viability and/or
biological activity features for the materials. Also, it should be
understood that enhanced viability and/or biologic activity
provided by aspects of the invention may vary according to the
materials being frozen or otherwise processed, and may be measured
in relation to prior techniques.
[0115] For example, when working with a first set of materials,
prior techniques may be capable of achieving cell viability and/or
biological activity in an 80% range, whereas viability and/or
biological activity of only 50% may be achievable with a second set
of materials. Thus, aspects of the invention may provide
enhancements to viability and/or biological activity of 85% or more
for the first set of materials, which is an improvement over prior
techniques, and enhancements to viability and/or biological
activity of 55% or more for the second set of materials, which
likewise provides an improvement over prior techniques even though
a 55% viability and/or biological activity for the second set of
materials is less than the 80% viability and/or biological activity
provided by prior techniques for the first set of materials. The
above disclosed processes and systems may also be modified to apply
any other appropriate nucleation method including, use of
additives, ice fog, vial pretreatment, vibration, and vacuum
freezing.
[0116] Nucleation is the onset of a phase transition in a small
region of a material. For example, the phase transition can be the
formation of a crystal from a liquid. The crystallization process
(i.e., formation of solid crystals from a solution) often
associated with freezing of a solution starts with a nucleation
event followed by crystal growth.
[0117] In the crystallization process, nucleation is the step where
selected atoms and/or molecules dispersed in the solution or other
material start to gather to create clusters in the nanometer scale
so as to become stable under the current operating conditions.
These stable clusters constitute the nuclei. The clusters need to
reach a critical size in order to become stable nuclei. Such
critical size is usually dictated by the operating conditions such
as temperature, contaminants, degree of supersaturation, etc. and
can vary from one sample of the solution to another. It is during
the nucleation event that the atoms in the solution arrange in a
defined and periodic manner that defines the crystal structure.
[0118] Crystal growth is the subsequent growth of the nuclei that
succeed in achieving the critical cluster size. Depending upon the
conditions either nucleation or crystal growth may predominate over
the other, and as a result, crystals with different sizes and
shapes are obtained. Control of crystal size and shape constitutes
one of the main challenges in industrial manufacturing, such as for
pharmaceuticals.
[0119] In addition to temperature control, the present methods and
systems may be used for controlling the time and/or temperature at
which a nucleated phase transition occurs in a material. In
freezing applications, the probability that a material will
spontaneously nucleate and begin changing phase is related to the
degree of sub-cooling of the material and the absence or presence
of contaminants, additives, structures, or disturbances that
provide a site or surface for nucleation.
[0120] The freezing or solidification step is particularly
important in cryopreservation and freeze-drying processes where
existing techniques result in nucleation temperature differences
across a multitude of vials or containers. The nucleation
temperature differences tend to produce a non-uniform product and
an excessively long drying time for freeze-drying processes. The
present methods, on the other hand, provide a higher degree of
process control in batch solidification processes (e.g.,
freeze-drying) and produce a product with more uniform structure
and properties Unlike some of the prior art techniques to induce
nucleation, the present methods require minimal equipment and
operational changes for implementation.
[0121] In principle, the present methods can be applied to any
material processing step that involves a nucleated phase
transition. Examples of such processes include the freezing of a
liquid, crystallization of ice from an aqueous solution,
crystallization of polymers and metals from melts, crystallization
of inorganic materials from supersaturated solutions,
crystallization of proteins, artificial snow production, deposition
of ice from vapor, food freezing, freeze concentration, fractional
crystallization, cryopreservation, or condensation of vapors to
liquids. From a conceptual standpoint, the present methods may also
be applied to phase transitions such as melting and boiling.
[0122] The presently disclosed methods represent an improvement to
current pharmaceutical cryopreservation and lyophilization
processes. For example, within a large industrial system there can
be over 100,000 vials containing a pharmaceutical product that
needs to be frozen and/or dried. Current practice in the industry
is to cool the solution to a very high degree so that the solution
in all vials or containers in the freeze-dryer are guaranteed to
freeze. However, as discussed above, the non-uniform cooling and
lack of a uniform and consistent nucleation control method, the
contents of each vial or container freezes randomly over a range of
temperatures below the freezing point.
[0123] Turning now to a closer examination of FIGS. 6-8, the
plotted temperature profiles illustrate that the presently
disclosed freezing or chilling process and system can be used to
initiate and control the nucleation of freezing in materials using
a temperature quench. As illustrated in FIGS. 6-8, the nucleation
of freezing of the materials in all vials monitored occurred at
roughly the same time and same temperature. Nucleation of freezing
is exhibited by the concurrent short spike in sample temperature
(see 100, 110, 120) as a result of the exothermic process occurring
during the phase change occurring in the samples. Thus, nucleation
control is possible by precisely controlling the timing and
magnitude of a sharp or rapid temperature quench using the above
described controlled freezing systems and methods. Alternatively, a
temperature quench may be referred to as a temperature spike or
cold spike in reference to the same physical process described
above. In certain embodiments the change in temperature during the
temperature quench may be a step-wise change in temperature or it
may change at a predetermined rate.
[0124] In a broad sense, the presently disclosed methods for
inducing nucleation of a phase transition within a material via
temperature quench nucleation control comprise the steps of: (i)
uniformly cooling the material to a temperature near or below a
phase transition temperature of the material; and (ii) uniformly
and rapidly decreasing the temperature of the cryogenic cold gas to
induce nucleation of the material. Each of these important steps
will be discussed in more detail below.
[0125] FIG. 11 presents a more detailed exemplary temperature
profile of the cryogenic gas during a freezing process using a
temperature quench to control nucleation. The temperature profile
versus time comprises an equilibrium step 402, a cooling step 404,
a pre-nucleation temperature step 406, a temperature quench step
408, a temperature quench hold step 410, a post-nucleation
temperature hold step 412, and a final cooling step 414.
[0126] During equilibrium step 402 each of the vials or containers
in the system are brought to a uniform equilibrium temperature near
or below the freezing point (i.e. the phase transition temperature)
of the material prior to beginning the remaining steps in the
freezing process. After each vial or container reaches a uniform
temperature, the vials or containers are further cooled by
decreasing the cryogenic cold gas to a pre-nucleation temperature
during steps 404 and 406.
[0127] The change in temperature of the cryogenic cold gas may
decrease linearly as indicated in step 404 and then hold steady as
shown in step 406, or alternatively the temperature of the
cryogenic cold gas may stepwise change to the pre-nucleation
temperature indicated in step 406 without the need for the linear,
or optionally non-linear, temperature change shown in step 404.
Furthermore, the cryogenic cold gas may be held at the
pre-nucleation temperature for a predetermined time as indicated in
step 406 to ensure all vials or containers reach a uniform
temperature prior to nucleating the phase change. Optionally, steps
404 and 406 may be performed dynamically without the hold time
shown in step 406.
[0128] The material is nucleated in temperature quench step 408.
During step 408, the temperature of the cryogenic cold gas is
adjusted to a temperature sufficiently low to ensure nucleation of
the material in each vial or container. In a preferred embodiment,
the temperature adjustment of the cryogenic cold gas is
sufficiently fast such that the temperature of the material in each
of the vials or containers does not substantially change during the
temperature adjustment. After nucleating the material in each vial
or container an optional temperature quench hold may be applied at
step 410.
[0129] After performing temperature quench hold step 410 the
temperature of the cryogenic cold gas is subsequently raised to a
temperature below the freezing point of the material and held for a
set amount of time during the post-nucleation temperature hold step
412 to ensure uniform temperature. After step 412, the cryogenic
cold gas is cooled at a predetermined rate to a final desired
temperature during final cooling step 414.
[0130] FIG. 12 depicts the temperature profiles 416 of six separate
vials and the cryogenic cold gas temperature profile 418 during a
freezing process implementing temperature quench induced nucleation
control. The cryogenic cold gas profile 418 included a
pre-nucleation temperature of -5.degree. C., a quench temperature
of -80.degree. C., a post nucleation temperature of -35.degree. C.,
a post-nucleation hold time of 10 minutes, and a post-nucleation
cooling rate of 2.5.degree. C./min. In contrast FIG. 13 depicts the
temperature profiles 420 of six separate vials and the cryogenic
cold gas temperature profile 422 during a freezing process without
nucleation control. The cryogenic cold gas profile 422 had a
cooling rate of 5.degree. C./min. As indicated by the sharp
increase in temperature along each temperature profile, the samples
that underwent the freezing process with nucleation control exhibit
nucleation temperatures and times in a narrower range than those in
the freezing process without nucleation control. Consequently, the
freezing process with nucleation control enables more uniform and
repeatable sample temperature profiles.
[0131] Further examples of specific temperatures and cooling rates
are discussed in detail below with regards to cell viability
testing of normal dermal human fibroblast cells during cryogenic
preservation.
[0132] When compared to the wide spectrum of times and temperatures
in the nucleation of freezing that results from use of conventional
controlled rate freezers, the present system and method applying
nucleation control via a temperature quench provides a greater
degree of control which likely impacts other performance aspects
and characteristics of the preserved biological material. Also, as
the contemplated nucleation initiation and control is temperature
driven, it works equally well in open or closed containers or
vials.
[0133] As shown above in FIG. 10, pressure control systems may be
included to permit pressurization and depressurization control of
the system and freezing process in addition to uniform temperature
control. Therefore, in addition to controlling the nucleation
temperature via a temperature quench method, the nucleation
temperature may be controlled using pressure induced nucleation.
FIG. 14 depicts a temperature versus time plot of six vials of an
aqueous solution undergoing a conventional stochastic nucleation
process while in a conventional freeze dryer using a cold shelf
arrangement. The plot shows the typical range of nucleation
temperatures of the solution within the vials (511,512,513,514,515,
and 516). As seen therein, the vial contents have a thermodynamic
freezing temperature of about 0.degree. C., yet the solution within
each vial naturally nucleates over the broad temperature range of
about -7.degree. C. to -20.degree. C. or more, as highlighted by
area 518. Plot 519 represents the shelf temperature inside the
freeze-drying chamber.
[0134] Conversely, FIG. 15 and FIG. 16 depict temperature versus
time plots of a solution undergoing a freezing process with
depressurized nucleation in accordance with the present methods. In
particular, FIG. 15 shows the temperature versus time plot of six
vials of an aqueous solution undergoing an equilibrated cooling
process (See Example 2) with nucleation induced via
depressurization of the chamber (521,522,523,524,525, and 526). The
vial contents have a thermodynamic freezing temperature of about
0.degree. C. yet the solution within each vial nucleates at the
same time upon depressurization and within a very narrow
temperature range (i.e., -4.degree. C. to -5.degree. C.) as seen in
area 528. Plot 529 represents the shelf temperature inside the
freeze-drying chamber and depicts an equilibrated freezing process,
one where the temperature of the shelves is held more or less
steady prior to depressurization.
[0135] Similarly, FIG. 16 shows the temperature versus time plot of
three vials of an aqueous solution undergoing a dynamic cooling
process (See Example 7) with nucleation induced via
depressurization of the chamber (531,532, and 533). Again, the vial
contents have a thermodynamic freezing temperature of about
0.degree. C. yet the solution within each vial nucleates at the
same time upon depressurization at a temperature range of about
-7.degree. C. to -10.degree. C., as seen in area 538. Plot 539
represents the shelf temperature inside the freeze-drying chamber
and generally depicts a dynamic cooling process, one where the
temperature of the shelves is actively lowered during or prior to
depressurization.
[0136] As illustrated in FIGS. 14-16, the present pressure induced
nucleation method provides improved control of the nucleation
process by enabling the freezing of solutions to occur within a
more narrow temperature range (e.g., about 0.degree. C. to
-10.degree. C.) and/or concurrently, thereby yielding a product
with greater uniformity from vial-to-vial. While not demonstrated,
it is foreseeable that the induced nucleation temperature range may
even extend slightly above the phase transition temperature and may
also extend to about 40.degree. C. of sub-cooling.
[0137] In a broad sense, the presently disclosed methods for
inducing nucleation of a phase transition within a material via
pressure control comprise the steps of: (i) uniformly cooling the
material to a temperature near or below a phase transition
temperature of the material; and (ii) rapidly decreasing the
pressure to induce nucleation of the material. Each of these
important steps will be discussed in more detail below.
Step 1--Cooling the Material
[0138] Illustrative materials useful in the present method include
pure substances, gases, suspensions, gels, liquids, solutions,
mixtures, or components within a solution or mixture. Suitable
materials for use in the present method may include, for example,
pharmaceutical materials, biopharmaceutical materials, foodstuffs,
chemical materials, and may include products such as wound-care
products, cosmetics, veterinary products and in vivo/in vitro
diagnostics related products and the like. When the material is a
liquid, it may be desirable to dissolve gases into the liquid.
Liquids in a controlled gas environment will generally have gases
dissolved in them.
[0139] Other illustrative materials useful in the present method
include biological or biopharmaceutical material such as tissues,
organs and multi-cellular structures. For certain biological and
pharmaceutical applications, the material may be a solution or
mixture that includes: a live or attenuated viruses; nucleic acids;
monoclonal antibodies; polyclonal antibodies; biomolecules;
nonpeptide analogues; peptides, including polypeptides, peptide
mimetics and modified peptides; proteins, including fusion and
modified proteins; RNA, DNA and subclasses thereof
oligonucleotides; viral particles; and similar such materials or
components thereof.
[0140] Pharmaceutical or biopharmaceutical solutions contained in
vials or containers for freeze-drying would be a good example of a
material that would benefit from the present method. The solutions
are mostly water and are substantially incompressible. Such
pharmaceutical or biopharmaceutical solutions are also highly pure
and generally free of particulates that may form sites for
nucleation. Uniform nucleation temperature is important to creating
a consistent and uniform ice crystal structure from vial to vial or
container to container. The ice crystal structure developed also
greatly affects the time required for drying during a freeze drying
process.
[0141] As applied to a freeze-drying process, the material is
preferably placed in a chamber, such as a freeze-drying chamber.
Preferably, the chamber is configured so as to allow control of the
temperature, pressure, and gas atmosphere within the chamber. The
gas atmosphere may include, but is not limited to: argon, nitrogen,
helium, air, water vapor, oxygen, carbon dioxide, carbon monoxide,
nitrous oxide, nitric oxide, neon, xenon, krypton, methane,
hydrogen, propane, butane, and the like, including permissible
mixtures thereof. The preferred gas atmosphere comprises an inert
gas, such as argon, at a pressure between about 7 to about 50 psig
or more. Temperatures within the freeze-dryer chamber are often
dictated by the freeze-drying process and are easily controlled via
the use of a heat transfer fluid that cools or warms the shelves
within the chamber to drive the temperature of the vials or
containers and the material within each vial or container.
[0142] In accordance with the present methods, the material is
cooled to a temperature near or below its phase transition
temperature. In the case of an aqueous based solution undergoing a
freeze-drying process, the phase transition temperature is the
thermodynamic freezing point of the solution. Where the solution
reaches temperatures below the thermodynamic freezing point of the
solution, it is said to be sub-cooled. When applied to a freezing
process of an aqueous-based solution, the present method is
effective when the degree of sub-cooling ranges from near or below
the phase transition temperature up to about 40.degree. C. of
sub-cooling, and more preferably between about 3.degree. C. of
sub-cooling and 10.degree. C. of sub-cooling. In some of the
examples described below, the present method of inducing nucleation
works desirably even where the solution has only about 1.degree. C.
of sub-cooling below its thermodynamic freezing point.
[0143] Where the material is at a temperature below its phase
transition temperature, it is often referred to as being in a
metastable state. A metastable state is an unstable and transient,
but relatively long-lived, state of a chemical or biological
system. A metastable material temporarily exists in a phase or
state that is not its equilibrium phase or state. In the absence of
any changes in the material or its environment, a metastable
material will eventually transition from its non-equilibrium state
to its equilibrium state. Illustrative metastable materials include
super-saturated solutions and sub-cooled liquids.
[0144] A typical example of a metastable material would be liquid
water at atmospheric pressure and a temperature of -10.degree. C.
With a normal freezing point of 0.degree. C., liquid water should
not thermodynamically exist at this temperature and pressure, but
it can exist in the absence of a nucleating event or structure to
begin the ice crystallization process. Extremely pure water can be
cooled to very low temperatures (-30.degree. C. to -40.degree. C.)
at atmospheric pressure and still remain in the liquid state. Such
sub-cooled water is in a non-equilibrated thermodynamically
metastable state. It only lacks a nucleation event to cause it to
begin the phase transition whereby it will return to
equilibrium.
[0145] As discussed above, the present methods of inducing
nucleation of a phase transition within a material or freezing a
material can be utilized with various cooling profiles, including,
for example, an equilibrated or a dynamic cooling environment (See
FIGS. 15 and 16).
Step 2--Rapidly Decreasing the Pressure
[0146] When the material has reached the desired temperature near
or below the phase transition temperature, the chamber is quickly
or rapidly depressurized. This depressurization triggers the
nucleation and phase transition of the solution within the vials or
containers. In the preferred embodiment, chamber depressurization
is accomplished by opening or partially opening a large control
valve that separates the high pressure chamber from either the
ambient environment or a lower pressure chamber or environment. The
elevated pressure is quickly lowered by mass flow of gas atmosphere
out of the chamber. The depressurization needs to be fairly fast to
induce the nucleation. The depressurization should be finished in
several seconds or less, preferably 40 seconds or less, more
preferably 20 seconds or less, and most preferably 10 seconds or
less.
[0147] In typical freeze-drying applications, the pressure
difference between the initial chamber pressure and the final
chamber pressure, after depressurization, should be greater than
about 7 psi, although smaller pressure drops may induce nucleation
in some situations. Most commercial freeze-dryers can readily
accommodate the range of pressure drops needed to control
nucleation. Many freeze-dryers are designed with pressure ratings
in excess of 25 psig to withstand conventional sterilization
procedures employing saturated steam at 121.degree. C. Such
equipment ratings provide an ample window to induce nucleation
following protocols that depressurize from starting pressures above
ambient pressure or the pressure in the surrounding environment.
The elevated pressure and subsequent depressurization can be
achieved through any known means (e.g., pneumatic, hydraulic, or
mechanical). In the preferred embodiments, operating pressures for
the present methods should remain below the supercritical pressure
of any applied gas, and subjecting the material to extreme low
pressures (i.e., about 10 mTorr or less) should be avoided during
nucleation of the material.
[0148] While not wishing to be bound to any particular mechanism,
one possible mechanism to explain the controlled nucleation
observed in the practice of the presently disclosed depressurized
nucleation method is that gases in solution in the material come
out of solution upon depressurization and form bubbles that
nucleate the material. An initial elevated pressure increases the
concentration of dissolved gas in the solution. The rapid decrease
in pressure after cooling reduces the gas solubility, and the
subsequent release of gas from the sub-cooled solution triggers
nucleation of the phase transition.
[0149] Another possible mechanism is that the temperature decrease
of the gas proximate the material during depressurization causes a
cold spot on the surface of the material that initiates nucleation.
Another possible mechanism is that the depressurization causes
evaporation of some liquid in the material and the resultant
cooling from the endothermic evaporation process may initiate the
nucleation. Another possible mechanism is that the depressurized
cold gas proximate the material freezes some vapor either in
equilibrium with the material prior to depressurization or
liberated from the material by evaporation during depressurization;
the resultant solid particles re-enter the material and act as
seeds or surfaces to initiate nucleation. One or more of these
mechanisms may contribute to initiation of nucleation of freezing
or solidification to differing extents depending on the nature of
the material, its environment and the phase transition being
nucleated.
[0150] The process may be carried out entirely at a pressure
greater than ambient pressure or over a range of pressures spanning
ambient pressure. For example, initial chamber pressure can be
above ambient pressure and the final chamber pressure, after
depressurization, can be above ambient pressure but less than the
initial chamber pressure; the initial chamber pressure can be above
ambient pressure and the final chamber pressure, after
depressurization, can be about ambient pressure or slightly below
ambient pressure. In addition, the chamber pressure may be
increased, prior to depressurization, while uniformly cooling the
samples or the step of increasing the pressure may be carried out
in a separate distinct step.
[0151] The rate and magnitude of the pressure drop are also
believed to be an important aspect of the present methods.
Experiments have shown that nucleation will be induced where the
pressure drop (.DELTA.P) is greater than about 7 psi.
Alternatively, the magnitude of the pressure drop may be expressed
as an absolute pressure ratio, R.dbd.P.sub.i/P.sub.f, where P.sub.i
is initial absolute pressure and P.sub.f is final absolute
pressure. It is believed that nucleation may be induced upon
depressurization where the absolute pressure ratio, R, is greater
than about 1.2 in many practical applications of the present
methods. The rate of pressure drop also plays an important role in
the present methods. One method of characterizing the rate of
pressure drop is through use of a parameter, A, where
A=.DELTA.P/.DELTA.t. Again, it is surmised that nucleation will be
induced for values of A greater than a prescribed value, such as
about 0.2 psi/sec. Empirical data through experimentation should
aid one to ascertain the preferred pressure drop and rate of
pressure drop.
[0152] The above disclosed method of inducing nucleation via
pressure control may be implemented in a conventional freeze-dryer
or the currently disclosed uniform flow cryogenic chiller. Turning
now to FIG. 17, a freeze-dryer is illustrated as freeze-dryer unit
(600) which has various main components plus additional auxiliary
systems to carry out a lyophilization cycle. In particular, the
freeze-dryer unit (600) includes a lyophilization chamber 602 that
contains the shelves 604 adapted to hold vials or containers of the
solution to be lyophilized (not shown). The solution to be
lyophilized is specially formulated and typically contains the
active ingredient, a solvent system and several stabilization
agents or other pharmaceutically acceptable carriers or additives.
Lyophilization of this formulation takes place from specialized
containers located on hollow shelves. These containers may include
vials with stoppers, ampoules, syringes, or, in the case of bulk
lyophilization, pans.
[0153] The illustrated freeze-dryer unit 600 also includes a
condenser 606 that is adapted to remove the sublimated and desorbed
solvent from the vapor phase by condensing or freezing it out as
ice to maintain adequate vacuum inside the freeze-dryer. The
condenser 606 can be internally located in the lyophilization
chamber 602 or as a separate external unit in communication with
the lyophilization chamber 602 through a so-called isolation valve.
The freeze-dryer unit 600 also preferably includes a vacuum pump
608 operatively coupled to the condenser 606 and adapted to pull a
vacuum on lyophilization chamber 602 and condenser 606.
[0154] The cryogenic refrigeration system 610 provides the
temperature control means for the freeze-dryer unit 600 by cooling
a prescribed heat transfer fluid which is circulated to the shelves
604 within the lyophilization chamber 602 and the condenser 606. As
illustrated, the cryogenic refrigeration system 610 comprises a
source of cryogen 618, such as liquid nitrogen, a cryogenic heat
exchanger 620, and a heat transfer fluid circuit 622, a vent 624, a
heater 626 and pumps 627,628.
The cryogenic heat exchanger 620 is preferably an NCOOL.TM.
Non-Freezing Cryogenic Heat Exchange System available from Praxair,
Inc. An important aspect of the cryogenic heat exchanger 620 is the
vaporization of the liquid nitrogen within or internal to the heat
exchanger yet in a manner that avoids direct contact of the liquid
nitrogen on cooling surfaces exposed to the heat transfer fluid.
Details of the structure and operation of such a heat exchanger can
be found in U.S. Pat. No. 5,937,656; the disclosure of which is
incorporated by reference herein.
[0155] The prescribed heat transfer fluid circuit 622 is adapted to
circulate a heat transfer fluid and is operatively coupled to both
the lyophilization chamber 602 as well as the condenser 606. More
specifically, the heat transfer fluid circulates inside the hollow
shelves 604 within the lyophilization chamber 602 to precisely
communicate the cooling or heating through the shelves 604 to the
solution as needed. In addition the prescribed heat transfer fluid
also flows through the condenser 606 to provide the cooling means
necessary to sublimate the ice and further desorb the solvent.
[0156] Pump 627 and heater 626 are disposed along the heat transfer
fluid circuit 622 upstream of the lyophilization chamber 602 and
downstream of the cryogenic heat exchanger 620. The pump 627 is
sized to move the heat transfer fluid through the heat transfer
circuit 622 at the required flow rates. The heater 626 is
preferably an electric heater adapted to provide supplemental heat
to the heat transfer fluid and the lyophilization chamber 602 as
may be required during the drying processes.
[0157] As seen in the embodiment of FIG. 17, the condenser 606 is
also cooled by a recirculation low temperature heat transfer fluid.
Refrigeration of the heat transfer fluid flowing through the
condenser 606 is also provided by a cryogenic heat exchanger 620.
The cryogenic heat exchanger 620 is capable of cooling heat
transfer fluid continuously without freezing. During the drying
phases, the cryogenic heat exchanger 620 is set or adapted to
achieve the lowest temperature required for the condenser 606. As
described above, the cryogenic heat exchanger 620 pre-evaporates
liquid nitrogen into a cryogenic cold gas for heat transfer to the
heat transfer fluid. Through pre-evaporation of the liquid
nitrogen, this assures the liquid nitrogen avoids boiling off
directly over a heat exchange surface where the heat transfer fluid
is disposed on the other side. Such an arrangement avoids freezing
of the cryogenic heat exchanger 620 since liquid nitrogen boils at
about -195 degrees Centigrade at atmospheric pressure.
[0158] The illustrated embodiment of FIG. 17 also includes a means
for controlling the gas atmosphere of the lyophilization chamber
650, and in particular the gas composition and pressure within the
chamber 602. Controlling the pressure of the chamber 602 allows for
the pressurization and rapid depressurization of the chamber to
induce nucleation of the solution. The disclosed embodiment
preferably uses one or more flow control valves 652 controllably
adapted to facilitate the introduction of a pressurized gas
atmosphere to the chamber 602 from a source of gas (not shown) and
to depressurize the chamber by venting the pressurized gas
atmosphere away from the chamber 602 in a controlled and preferably
rapid manner thereby inducing the nucleation of the solution in the
various containers or vials.
[0159] Although not shown, the freeze-dryer unit 600 also includes
various control hardware and software systems adapted to command
and coordinate the various parts of the freeze-drying equipment,
and carry out the pre-programmed lyophilization cycle. The various
control hardware and software systems may also provide
documentation, data logging, alarms, and system security
capabilities as well. In addition, auxiliary systems to the
freeze-dryer unit 600 may include various subsystems to clean and
sterilize the lyophilization chamber 602, auto-load and unload the
product in the lyophilization chamber 602; and associated cryogenic
system accessories such as refrigeration skids, liquid nitrogen
tanks, piping, valves, sensors, etc. Furthermore, the freeze-dryer
unit 600 may be used optionally with or include the currently
disclosed uniform flow controlled rate freezer system to cool and
freeze the vials or containers in the system prior to initiating
the freeze-drying process.
[0160] FIG. 18a presents a plot of an illustrative temperature and
pressure profile versus time of the cryogenic cold gas during a
depressurized nucleation freezing process as might be applied in
the above disclosed cryogenic chiller, freeze-dryer unit, or other
appropriate system. During equilibrium step 702 all vials in the
chamber are brought to the same temperature prior to further
cooling. During equilibrium step 702 the cooling chamber is
preferably not actively pressurized. However the invention is not
limited in this regards, in certain embodiments the cooling chamber
may be pressurized above or below atmospheric pressure. After
equilibrating all of the vials or containers to the same
temperature the cryogenic cold gas is cooled at a predetermined
rate to the pre-nucleation temperature (PNT) and held for a
selected amount of time during steps 704 and 706. The vials cool in
response to the decreased temperature of the cryogenic cold gas and
approach the PNT. While the cryogenic cold gas is held at the PNT a
pressurization and depressurization step 708 is applied. During
pressurization and depressurization step 708, the chamber pressure
is increased and held for a predetermined amount of time prior to
depressurizing the system. Nucleation is induced by the
depressurization. Alternatively, the prior steps could be conducted
at a first pressure and nucleation can be induced by depressurizing
the system to a lower second pressure without the need to
pressurize the system. After nucleation is induced, the cryogenic
cold gas is further cooled during step 710 to a desired final
temperature (i.e. -80.degree. C.) at various rates. Without wishing
to be bound by theory it is believed that the nucleation control is
relatively insensitive to the rate of pressurization. However, it
is necessary to ensure that a sufficient pressure drop occurs over
a short enough time period to induce nucleation. Furthermore, it
will be necessary to determine optimal conditions for each new
material to be frozen. Examples of testing regarding the above
discussed nucleation process is provided below.
[0161] FIG. 18b presents temperature and pressure profiles for
samples undergoing the process described above in connection with
FIG. 18a. Four separate 2 ml vials were filled with 1 ml of
phosphate buffered saline and 10 (v/v) % Dimethyl sulfoxide (DMSO)
solution. The four vials were then placed in randomly selected
spaced apart locations on a tray. The tray was then placed into a
uniform flow cryogenic chiller with pressure control. The
temperatures of the four vials were monitored using the
thermocouples placed inside the liquid contained in each vial. The
cryogenic cold gas temperature profile 712 and chamber pressure
profile 714 were then applied to freeze the four vials. The chamber
pressure was raised to 30 psig and held at 30 psig for 5 min prior
to depressurization. The sharp temperature rise in vial
temperatures 716 after depressurization indicates that all four
vials nucleated and began freezing immediately after
depressurization. The vial temperatures 716 were substantially
uniform for the four samples throughout the freezing process.
[0162] Turning again to FIGS. 12 and 18b, these figures provide an
example of sample container temperatures prior to and after
nucleation of the material during a freezing process. The
differences in temperature between the samples are greater after
nucleation. This change in uniformity of the temperature is due to
the latent heat of freezing being slightly different for each
sample since each sample nucleates at a slightly different
temperature. Thus since each sample has a different latent heat of
freezing and each sample starts at a slightly different
temperature, the resulting differences in temperature
sample-to-sample are larger after nucleation.
[0163] In one embodiment, the uniformity of the container
temperatures are maintained within .+-.2.5.degree. C. of each other
during a freezing process, prior to nucleation, regardless of a
location in the cooling chamber where the material is frozen. After
nucleation, the uniformity of the container temperatures may be
maintained within .+-.5.degree. C. of each other during the
remainder of the freezing process regardless of a location in the
cooling chamber where the material is frozen. In another
embodiment, the uniformity of the container temperatures are
maintained within the measurement error of thermocouple during a
freezing process, prior to nucleation, regardless of a location in
the cooling chamber where the material is frozen. Typical
measurement errors are on the order of .+-.1.degree. C. After
nucleation, the uniformity of the container temperatures may be
maintained within .+-.2.5.degree. C. of each other during the
remainder of the freezing process regardless of a location in the
cooling chamber where the material is frozen. The above examples of
temperature uniformity prior to and after nucleation are
non-limiting with respect to the current disclosure.
[0164] As seen in FIGS. 12, 15, 16, and 18b nucleation may be
induced using either the above described temperature quench or
pressure induced nucleation control methods. Nucleation occurs over
a smaller time period as compared to the stochastic nucleation
processes presented in FIGS. 13 and 14. Furthermore, the above
described uniform controlled rate freezer may be adapted to
implement either, or both, the temperature quench and pressure
induced nucleation methods to selectively induce nucleation
substantially at the same time in each sample. Therefore, the
current systems may provide a substantially uniform nucleation time
and temperature for each sample regardless of location within the
cooling chamber.
[0165] In addition to the above, FIGS. 13 and 14 specifically show
the differences between a stochastic nucleation process using the
currently disclosed uniform flow cryogenic chiller and a stochastic
nucleation process using a prior art system. As illustrated in the
figures the samples nucleated over a smaller temperature range in
the uniform flow cryogenic chiller as compared to the prior art
system. Without wishing to be bound by theory, this difference in
the uniformity of the stochastic nucleation temperatures may be due
to the uniform temperature of the cryogenic cold gas applied to the
samples in the uniform flow cryogenic chiller. Therefore, in one
embodiment, the currently disclosed uniform flow cryogenic chiller
may provide a uniform stochastic nucleation temperature to a
plurality of containers. In a further embodiment, a stochastic
nucleation temperature may be provided to 10,000, 20,000, 50,000,
or 100,000 containers in the same system.
[0166] It should be noted that the current invention is not limited
to the specific temperature and pressure profiles described herein.
Any number of variations of the described freezing and nucleation
processes will be apparent to one of skill in the art and can be
implemented without departing from the spirit of the current
disclosure.
EXAMPLES
[0167] The following examples highlight various aspects and
features of the presently disclosed method of inducing nucleation
via pressure control in a material and are not to be taken in a
limiting sense. Rather, these examples are illustrative only and
the scope of the invention should be determined only with respect
to the claims, appended hereto.
[0168] All examples described herein were performed in a
pilot-scale VirTis 51-SRC freeze-dryer having four shelves with
approximately 1.0 square meter total shelf space and an internal
condenser. This unit was retrofitted to hold positive pressures of
up to about 15 psig. A 1.5'' diameter circular opening also was
added to the rear wall of the freeze-drying chamber with 1.5''
diameter stainless steel tubing extending from the hole through the
rear wall insulation to emerge from the back of the freeze-dryer.
Two 1.5'' full-port, air-actuated ball valves were attached to this
tubing via sanitary fittings. One ball valve allowed gas to flow
into the freeze-drying chamber and thereby provide positive
pressures up to 15 psig. The second ball valve allowed gas to flow
out of the freeze-drying chamber and thereby reduce chamber
pressure to atmospheric conditions (0 psig). All refrigeration of
the freeze-dryer shelves and condenser was accomplished via
circulation of Dynalene MV heat transfer fluid cooled by liquid
nitrogen using the Praxair NCool.TM.-HX system.
[0169] All solutions were prepared in a class 100 clean room. The
freeze-dryer was positioned with the door, shelves, and controls
all accessible from the clean room while the other components
(pumps, heaters, etc.) were located in a non-clean room
environment. All solutions were prepared with HPLC grade water
(Fisher Scientific, filtered through 0.10 .mu.m membrane). The
final solutions were filtered through a 0.22 .mu.m membrane prior
to filling the vials or lyophilization containers. All gases were
supplied via cylinders and were filtered through 0.22 .mu.m filters
to remove particulates. The glass containers (5 mL vials and 60 mL
bottles) were obtained pre-cleaned for particulates from Wheaton
Science Products. Pharmaceutically acceptable carriers were used
where appropriate. The above steps were taken to ensure the
materials and methods met conventional pharmaceutical manufacturing
standards for particulates, which act as nucleating agents.
[0170] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, antioxidants,
salts, coatings, surfactants, preservatives (e.g., methyl or propyl
p-hydroxybenzoate, sorbic acid, antibacterial agents, antifungal
agents), isotonic agents, solution retarding agents (e.g.,
paraffin), absorbents (e.g., kaolin clay, bentonite clay), drug
stabilizers (e.g., sodium lauryl sulphate), gels, binders (e.g.,
syrup, acacia, gelatin, sorbitol, tragacanth, polyvinyl
pyrrolidone, carboxy-methyl-cellulose, alginates), excipients
(e.g., lactose, milk sugar, polyethylene glycol), disintegration
agent (e.g., agar-agar, starch, lactose, calcium phosphate, calcium
carbonate, alginic acid, sorbitol, glycine), wetting agents (e.g.,
cetyl alcohol, glycerol monostearate), lubricants, absorption
accelerators (e.g., quaternary ammonium salts), edible oils (e.g.,
almond oil, coconut oil, oily esters or propylene glycol),
sweetening agents, flavoring agents, coloring agents, fillers,
(e.g., starch, lactose, sucrose, glucose, mannitol), tabletting
lubricants (e.g., magnesium stearate, starch, glucose, lactose,
rice flower, chalk), carriers for inhalation (e.g., hydrocarbon
propellants), buffering agents, or such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art.
[0171] For the experimental conditions described herein and all
lyophilization formulations studied, stochastic nucleation was
typically observed to occur at container temperatures between about
-8.degree. C. and -20.degree. C. and occasionally as warm as
-5.degree. C. The containers could generally be held at
temperatures warmer than -8.degree. C. for long periods of time
without nucleating. The onset of nucleation and subsequent crystal
growth (i.e., freezing) was determined by temperature measurement
as the point at which the container temperature quickly increased
in response to the exothermic latent heat of fusion. The initiation
of freezing also could be visually determined through a sight-glass
on the freeze-dryer chamber door.
Example 1
Controlling the Nucleation Temperature
[0172] Four separate vials were filled with 2.5 mL of 5 wt %
mannitol solution. The predicted thermodynamic freezing point of
the 5 wt % mannitol solution is approximately -0.5.degree. C. The
four vials were placed on a freeze-dryer shelf in close proximity
to one another. The temperatures of the four vials were monitored
using surface mounted thermocouples. The freeze-dryer was
pressurized with argon to 14 psig.
[0173] The freeze-dryer shelf was cooled to obtain vial
temperatures of between approximately -1.3.degree. C. and about
-2.3.degree. C. (+1.degree. C. measurement accuracy of the
thermocouples). The freeze-dryer was then depressurized from about
14 psig to about atmospheric pressure in less than five seconds to
induce nucleation of the solution within the vials. All four vials
nucleated and began freezing immediately after depressurization.
Results are summarized in Table 1 below.
[0174] As seen in Table 1, the controlled nucleation temperatures
in this example (i.e., Initial Vial Temperatures) are quite close
to the predicted thermodynamic freezing point of the solution. Thus
the present method allows control of the nucleation to occur in
solutions that have a very low degree of sub-cooling or at
nucleation temperatures near or only slightly colder than their
freezing points.
TABLE-US-00001 TABLE 1 Controlling the Nucleation Temperature
Initial Vial Temperature Pressure Depressurization Vial # Solution
Atmos [.degree. C.] Drop [psi] Outcome 1 2.5 mL of 5 wt % mannitol
Argon -2.3 14 Nucleation 2 2.5 mL of 5 wt % mannitol Argon -1.3 14
Nucleation 3 2.5 mL of 5 wt % mannitol Argon -2.1 14 Nucleation 4
2.5 mL of 5 wt % mannitol Argon -1.7 14 Nucleation
Example 2
Controlling the Nucleation Temperature
[0175] In this example, ninety-five vials were filled with 2.5 mL
of 5 wt % mannitol solution. The thermodynamic freezing point of
the 5 wt % mannitol solution is approximately -0.5.degree. C. The
ninety-five vials were placed on a freeze-dryer shelf in close
proximity to one another. The temperatures of six vials positioned
at different locations in the freeze-dryer shelf were continuously
monitored using surface mounted thermocouples. The freeze-dryer was
pressurized in an argon atmosphere to about 14 psig. The
freeze-dryer shelf was then cooled to obtain vial temperatures of
near -5.degree. C. The freeze-dryer was then depressurized from
about 14 psig to about atmospheric pressure in less than five
seconds to induce nucleation of the solution within the vials. All
ninety-five vials were visually observed to nucleate and begin
freezing immediately after depressurization. Thermocouple data for
the six monitored vials confirmed the visual observation. The
results are summarized in Table 2.
[0176] As seen therein, controlled nucleation temperatures in this
example (i.e., Initial Vial Temperatures) are somewhat below the
predicted thermodynamic freezing point of the solution. Thus the
present method allows control of the nucleation to occur in
solutions that have a moderate degree of sub-cooling. This example
also demonstrates scalability of the present method to a multiple
vial application.
TABLE-US-00002 TABLE 2 Controlling the Nucleation Temperature
Initial Vial Pressure Depressurization Vial # Solution Atmos Temp
[.degree. C.] Drop [psi] Outcome 1 2.5 mL of 5 wt % mannitol Argon
-4.2 14 Nucleation 2 2.5 mL of 5 wt % mannitol Argon -4.4 14
Nucleation 3 2.5 mL of 5 wt % mannitol Argon -4.6 14 Nucleation 4
2.5 mL of 5 wt % mannitol Argon -4.4 14 Nucleation 5 2.5 mL of 5 wt
% mannitol Argon -4.6 14 Nucleation 6 2.5 mL of 5 wt % mannitol
Argon -5.1 14 Nucleation
Example 3
Controlling the Depressurization Magnitude
[0177] In this example, multiple vials were filled with 2.5 mL of 5
wt % mannitol solution. Again, the predicted thermodynamic freezing
point of the 5 wt % mannitol solution is approximately -0.5.degree.
C. For each test run, the vials were placed on a freeze-dryer shelf
in close proximity to one another. As with the earlier described
examples, the temperatures of vials were monitored using surface
mounted thermocouples. The argon atmosphere in the freeze-dryer was
pressurized to differing pressures and the freeze-dryer shelf was
cooled to obtain vial temperatures of about -5.degree. C. In each
test run, the freeze-dryer was then rapidly depressurized (i.e., in
less than five seconds) from the selected pressure to atmospheric
pressure in an effort to induce nucleation of the solution within
the vials. Results are summarized in Table 3.
[0178] As seen in Table 3, the controlled nucleation occurred where
the pressure drop was about 7 psi or greater and the nucleation
temperature was between about -4.7.degree. C. and -5.8.degree.
C.
TABLE-US-00003 TABLE 3 Effect of Depressurization Magnitude Initial
Vial Pressure Depressurization Vial # Solution Atmos Temp [.degree.
C.] Drop [psi] Outcome 1 2.5 mL of 5 wt % mannitol Argon -4.7 7
Nucleation 2 2.5 mL of 5 wt % mannitol Argon -5.1 7 Nucleation 3
2.5 mL of 5 wt % mannitol Argon -5.3 7 Nucleation 4 2.5 mL of 5 wt
% mannitol Argon -5.6 7 No Nucleation 5 2.5 mL of 5 wt % mannitol
Argon -5.6 7 Nucleation 6 2.5 mL of 5 wt % mannitol Argon -5.8 7
Nucleation 7 2.5 mL of 5 wt % mannitol Argon -5.4 6 No Nucleation 8
2.5 mL of 5 wt % mannitol Argon -5.7 6 No Nucleation 9 2.5 mL of 5
wt % mannitol Argon -5.8 6 No Nucleation 10 2.5 mL of 5 wt %
mannitol Argon -5.1 5 No Nucleation 11 2.5 mL of 5 wt % mannitol
Argon -5.4 5 No Nucleation 12 2.5 mL of 5 wt % mannitol Argon -5.5
5 No Nucleation 13 2.5 mL of 5 wt % mannitol Argon -4.7 4 No
Nucleation 14 2.5 mL of 5 wt % mannitol Argon -5.1 4 No Nucleation
15 2.5 mL of 5 wt % mannitol Argon -5.3 4 No Nucleation
Example 4
Controlling the Depressurization Rates
[0179] For this example, multiple vials were filled with about 2.5
mL of 5 wt % mannitol solution having a predicted thermodynamic
freezing point of approximately -0.5.degree. C. For each test run
of varying depressurization time, the vials were placed on a
freeze-dryer shelf in close proximity to one another. As with the
earlier described examples, the temperatures of vials were
monitored using surface mounted thermocouples. Like the
above-described examples, the argon atmosphere in the freeze-dryer
was pressurized to about 14 psig and the shelf was cooled to obtain
vial temperatures of approximately -5.degree. C. In each test run,
the freeze-dryer was then depressurized at different
depressurization rates from 14 psig to atmospheric pressure in an
effort to induce nucleation of the solution within the vials.
[0180] To study the effect of depressurization rate or
depressurization time, a restricting ball valve was placed on the
outlet of the depressurization control valve at the rear of the
freeze-dryer. When the restricting valve is completely open,
depressurization from about 14 psig to about 0 psig is accomplished
in approximately 2.5 seconds. By only partially closing the
restricting valve, it is possible to variably increase the chamber
depressurization time. Using the restricting ball valve, several
test runs were performed with the freeze-dryer chamber
depressurized at differing rates to ascertain or determine the
effect of depressurization rate on nucleation. The results are
summarized in Table 4.
TABLE-US-00004 TABLE 4 Effect of Depressurization Time Vial Temp
Pressure Time Depressurization Vial # Solution Atmos [.degree. C.]
Drop [psi] [sec] Outcome 1 2.5 mL of 5 wt % mannitol Argon -4.6 14
300 No Nucleation 2 2.5 mL of 5 wt % mannitol Argon -5.4 14 300 No
Nucleation 3 2.5 mL of 5 wt % mannitol Argon -5.8 14 300 No
Nucleation 4 2.5 mL of 5 wt % mannitol Argon -4.6 14 200 No
Nucleation 5 2.5 mL of 5 wt % mannitol Argon -5.4 14 200 No
Nucleation 6 2.5 mL of 5 wt % mannitol Argon -5.4 14 200 No
Nucleation 7 2.5 mL of 5 wt % mannitol Argon -4.6 14 100 No
Nucleation 8 2.5 mL of 5 wt % mannitol Argon -5.2 14 100 No
Nucleation 9 2.5 mL of 5 wt % mannitol Argon -5.2 14 100 No
Nucleation 10 2.5 mL of 5 wt % mannitol Argon -4.7 14 60 No
Nucleation 11 2.5 mL of 5 wt % mannitol Argon -5.1 14 60 No
Nucleation 12 2.5 mL of 5 wt % mannitol Argon -5.1 14 60 No
Nucleation 13 2.5 mL of 5 wt % mannitol Argon -5.1 14 50 No
Nucleation 14 2.5 mL of 5 wt % mannitol Argon -5.3 14 50 No
Nucleation 15 2.5 mL of 5 wt % mannitol Argon -4.9 14 50 No
Nucleation 16 2.5 mL of 5 wt % mannitol Argon -5.4 14 42 No
Nucleation 17 2.5 mL of 5 wt % mannitol Argon -5.5 14 42 No
Nucleation 18 2.5 mL of 5 wt % mannitol Argon -5.0 14 42 No
Nucleation 19 2.5 mL of 5 wt % mannitol Argon -5.1 14 32 Nucleation
20 2.5 mL of 5 wt % mannitol Argon -5.7 14 32 Nucleation 21 2.5 mL
of 5 wt % mannitol Argon -5.6 14 32 Nucleation 22 2.5 mL of 5 wt %
mannitol Argon -4.7 14 13 Nucleation 23 2.5 mL of 5 wt % mannitol
Argon -5.3 14 13 Nucleation 24 2.5 mL of 5 wt % mannitol Argon -5.5
14 13 Nucleation
[0181] As seen in Table 4, nucleation only occurred where the
depressurization time was less than 42 seconds, the pressure drop
was about 14 psi or greater and the nucleation temperature (i.e.,
initial vial temperature) was between about -4.6.degree. C. and
about -5.8.degree. C. These results indicate that the
depressurization needs to be accomplished relatively quickly for
the method to be effective.
Example 5
Controlling the Gas Atmosphere
[0182] Again, multiple vials were each filled with about 2.5 mL of
5 wt % mannitol solution and placed on a freeze-dryer shelf in
close proximity to one another. As with earlier described examples,
temperature of the test vials were monitored using surface mounted
thermocouples. For the different test runs, the gas atmosphere in
the freeze-dryer was varied always maintaining a positive pressure
of about 14 psig. In this example, the freeze-dryer shelf was
cooled to obtain vial temperatures of approximately -5.degree. C.
to -7.degree. C. In each test run, the freeze-dryer was then
rapidly depressurized from about 14 psig to atmospheric pressure in
an effort to induce nucleation of the solution within the vials.
The results are summarized in Table 5.
[0183] As seen therein, controlled nucleation occurred in all gas
atmospheres except for helium gas atmosphere where the pressure
drop was about 14 psi and the nucleation temperature (i.e., initial
vial temperature) was between about -4.7.degree. C. and about
-7.4.degree. C. Although not shown in the examples, it is believed
that alternate conditions will likely enable controlled nucleation
in a helium atmosphere.
TABLE-US-00005 TABLE 5 Effect of Gas Atmosphere Composition Initial
Vial Pressure Depressurization Vial # Solution Atmos Temp [.degree.
C.] Drop [psi] Outcome 1 2.5 mL of 5 wt % mannitol Argon -4.9 14
Nucleation 2 2.5 mL of 5 wt % mannitol Argon -5.2 14 Nucleation 3
2.5 mL of 5 wt % mannitol Nitrogen -4.7 14 Nucleation 4 2.5 mL of 5
wt % mannitol Nitrogen -5.1 14 Nucleation 5 2.5 mL of 5 wt %
mannitol Xenon -4.8 14 Nucleation 6 2.5 mL of 5 wt % mannitol Xenon
-5.0 14 Nucleation 7 2.5 mL of 5 wt % mannitol Air -7.4 14
Nucleation 8 2.5 mL of 5 wt % mannitol Air -7.2 14 Nucleation 9 2.5
mL of 5 wt % mannitol Helium -5.8 14 No Nucleation 10 2.5 mL of 5
wt % mannitol Helium -5.5 14 No Nucleation
Example 6
Large Volume Solutions
[0184] In this example, six lyophilization bottles (60 mL capacity)
were filled with about 30 mL of 5 wt % mannitol solution having a
predicted thermodynamic freezing point of approximately
-0.5.degree. C. The six lyophilization bottles were placed on a
freeze-dryer shelf in close proximity to one another. The
temperature of six bottles positioned at different locations in the
freeze-dryer shelf was monitored using surface mounted
thermocouples. The freeze-dryer was pressurized in an argon
atmosphere to about 14 psig. The freeze-dryer shelf was then cooled
to obtain bottle temperatures of near -5.degree. C. The
freeze-dryer was then depressurized from 14 psig to about
atmospheric pressure in less than five seconds to induce nucleation
of the solution within the bottles. The results are summarized in
Table 6.
[0185] In a separate experiment, a plastic bulk freeze-drying tray
(Gore LYOGUARD, 1800 mL capacity) was filled with about 1000 mL of
5 wt % mannitol solution. The tray was obtained pre-cleaned to meet
USP low particulate requirements. The tray was placed on a
freeze-dryer shelf, and the temperature of the tray was monitored
by a thermocouple mounted on the exterior surface of the tray near
the center of one side. The freeze-dryer shelf was then cooled to
obtain a tray temperature of near -7.degree. C. The freeze-dryer
was then depressurized from 14 psig to about atmospheric pressure
in less than five seconds to induce nucleation of the solution
within the tray. The results are also summarized in Table 6.
[0186] Like the above-described examples, all containers nucleated
and began freezing immediately after depressurization. Also like
the above-described examples, the nucleation temperatures (i.e.,
Container Temperatures) in this example were very much controllable
to be somewhat near the thermodynamic freezing temperature of the
solution. More importantly, this example illustrates that the
present method allows control of the nucleation to occur in larger
volume solutions and various container formats. It should be noted
that one would expect the efficacy of the depressurization method
to improve as formulation volume increases, because the nucleation
event is more likely to occur when more molecules are present to
aggregate and form critical nuclei.
TABLE-US-00006 TABLE 6 Effect of Solution Volume and Container Type
Container Temperature Pressure Depressurization Container Solution
Atmos [.degree. C.] Drop [psi] Outcome Bottle # 1 30 mL of 5 wt %
mannitol Argon -5.3 14 Nucleation Bottle # 2 30 mL of 5 wt %
mannitol Argon -5.1 14 Nucleation Bottle # 3 30 mL of 5 wt %
mannitol Argon -5.9 14 Nucleation Bottle # 4 30 mL of 5 wt %
mannitol Argon -5.2 14 Nucleation Bottle # 5 30 mL of 5 wt %
mannitol Argon -5.9 14 Nucleation Bottle # 6 30 mL of 5 wt %
mannitol Argon -6.1 14 Nucleation Tray 1000 mL of 5 wt % mannitol
Argon -6.9 14 Nucleation
Example 7
Dynamic Cooling vs. Equilibrated Cooling
[0187] The present methods of controlling nucleation can be used in
various modes. Examples 1-6, described above, each demonstrate the
aspect of controlling the nucleation temperature of a
lyophilization solution that is essentially equilibrated at a
temperature below its thermodynamic freezing point (i.e., very
slowly changing temperature). This example demonstrates that
nucleation can also occur at a temperature below the thermodynamic
freezing point in a dynamic cooling environment (i.e., the solution
is undergoing rapid changes in temperature).
[0188] In this example, vials 1 through 6 represent the samples
described above with reference to Example 2. In addition, three
separate vials (Vials 7-9) were also filled with 2.5 mL of 5 wt %
mannitol solution. In a separate test run, the three additional
vials were placed on a freeze-dryer shelf in close proximity to one
another. The freeze-dryer shelf was cooled rapidly towards a final
shelf temperature of -45.degree. C. When one of the vials reached a
temperature of about -5.degree. C., as measured by the surface
mounted thermocouples, the freeze-dryer was depressurized rapidly
from about 14 psig to 0 psig in an effort to induce nucleation. All
three vials nucleated and began freezing immediately after
depressurization. The vial temperatures decreased significantly to
between -6.8.degree. C. and -9.9.degree. C. prior to nucleation as
a result of the dynamic cooling environment. Comparative results
are summarized in Table 7 below.
TABLE-US-00007 TABLE 7 Test Results - Effect of Dynamic Cooling on
Nucleation Nucleation Pressure Depressurization Vial # Solution
Mode Temp. [.degree. C.] Drop [psi] Outcome 1 2.5 mL of 5 wt %
mannitol Equilibrated -4.2 14 Nucleation 2 2.5 mL of 5 wt %
mannitol Equilibrated -4.4 14 Nucleation 3 2.5 mL of 5 wt %
mannitol Equilibrated -4.6 14 Nucleation 4 2.5 mL of 5 wt %
mannitol Equilibrated -4.4 14 Nucleation 5 2.5 mL of 5 wt %
mannitol Equilibrated -4.6 14 Nucleation 6 2.5 mL of 5 wt %
mannitol Equilibrated -5.1 14 Nucleation 7 2.5 mL of 5 wt %
mannitol Dynamic -6.8 14 Nucleation 8 2.5 mL of 5 wt % mannitol
Dynamic -7.2 14 Nucleation 9 2.5 mL of 5 wt % mannitol Dynamic -9.9
14 Nucleation
[0189] The efficacy of the present methods for controlling
nucleation in lyophilization solutions equilibrated in a given
temperature range or lyophilization solutions being dynamically
cooled, provides the end-user with two potential modes of
application with different benefits and trade-offs. By allowing the
lyophilization solutions to equilibrate, the range of nucleation
temperatures will be narrow or minimized to the performance limits
of the freeze-dryer itself. The equilibration step may require
extra time to achieve relative to conventional or dynamic freezing
protocols where the chamber and vial temperatures are dropped to
less than about -40.degree. C. in one step. However, employing the
equilibration step should yield much improved nucleation uniformity
across all vials or containers as well as realization of the other
benefits associated with precisely controlling the nucleation
temperature of the material.
[0190] Alternatively, if equilibrating the material or
lyophilization solution temperatures is undesirable, one may simply
implement the depressurization step at an appropriate time during
the normal freezing or dynamic cooling protocol. Depressurization
during a dynamic cool down will produce a wider spread in
nucleation temperatures for the material within the lyophilization
containers, but will add minimal time to the freezing protocol and
still allow one to mitigate the problems of extreme
sub-cooling.
Example 8
Effect of Different Excipients
[0191] The present method of controlling or inducing nucleation in
a material can be used to control the nucleation temperature of
sub-cooled solutions containing different lyophilization
excipients. This example demonstrates the use of the present
methods with the following excipients: mannitol; hydroxyethyl
starch (HES); polyethylene glycol (PEG); polyvinyl pyrrolidone
(PVP); dextran; glycine; sorbitol; sucrose; and trehalose. For each
excipient, two vials were filled with 2.5 mL of a solution
containing 5 wt % of the excipient. The vials were placed on a
freeze-dryer shelf in close proximity to one another. The
freeze-dryer was pressurized in an argon atmosphere to about 14
psig. The freeze-dryer shelf was cooled to obtain vial temperatures
near -3.degree. C. and then depressurized rapidly to induce
nucleation. Results are summarized in Table 8.
TABLE-US-00008 TABLE 8 Effect of Different Lyophilization
Excipients Initial Vial Temperature Pressure Depressurization Vial
# Solution/Excipient Atmos [.degree. C.] Drop [psi] Outcome 1 2.5
mL of 5 wt % mannitol Argon -3.3 14 Nucleation 2 2.5 mL of 5 wt %
mannitol Argon -3.0 14 Nucleation 3 2.5 mL of 5 wt % HES Argon -3.1
14 Nucleation 4 2.5 mL of 5 wt % HES Argon -3.7 14 Nucleation 5 2.5
mL of 5 wt % PEG Argon -3.8 14 Nucleation 6 2.5 mL of 5 wt % PEG
Argon -3.4 14 Nucleation 7 2.5 mL of 5 wt % PVP Argon -3.5 14
Nucleation 8 2.5 mL of 5 wt % PVP Argon -3.3 14 Nucleation 9 2.5 mL
of 5 wt % dextran Argon -4.0 14 Nucleation 10 2.5 mL of 5 wt %
dextran Argon -3.1 14 Nucleation 11 2.5 mL of 5 wt % glycine Argon
-3.8 14 Nucleation 12 2.5 mL of 5 wt % glycine Argon -3.9 14
Nucleation 13 2.5 mL of 5 wt % sorbitol Argon -3.6 14 Nucleation 14
2.5 mL of 5 wt % sorbitol Argon -3.4 14 Nucleation 15 2.5 mL of 5
wt % sucrose Argon -3.3 14 Nucleation 16 2.5 mL of 5 wt % sucrose
Argon -3.4 14 Nucleation 17 2.5 mL of 5 wt % trehalose Argon -3.7
14 Nucleation 18 2.5 mL of 5 wt % trehalose Argon -3.1 14
Nucleation
Example 9
Controlling Nucleation of Protein Solutions
[0192] The methods disclosed herein can be used to control the
nucleation temperature of sub-cooled protein solutions without
negative or adverse effects on protein solubility or enzymatic
activity. Two proteins, bovine serum albumin (BSA) and lactate
dehydrogenase (LDH) were used in this example.
[0193] BSA was dissolved in 5 wt % mannitol at a concentration of
10 mg/mL. Three lyophilization vials were filled with 2.5 mL of the
BSA-mannitol solution and placed on a freeze-dryer shelf in close
proximity to one another. The freeze-dryer was pressurized in an
argon atmosphere to about 14 psig. The freeze-dryer shelf was
cooled to obtain vial temperatures near -5.degree. C. The
freeze-dryer was depressurized rapidly to induce nucleation. All
vials of BSA solution nucleated and began freezing immediately
after depressurization. No precipitation of the protein was
observed upon thawing.
[0194] The LDH proteins were obtained from two different suppliers
and for purposes of clarity are designated as LDH-1 or LDH-2 to
distinguish the two distinct batches. LDH-1 was dissolved in 5 wt %
mannitol at a concentration of 1 mg/mL. Six lyophilization vials
were filled with 2.5 mL of the LDH-1/mannitol solution and placed
on a freeze-dryer shelf in close proximity to one another. The
freeze-dryer was pressurized in an argon atmosphere to about 14
psig. The freeze-dryer shelf was cooled starting from room
temperature to obtain vial temperatures near -4.degree. C. The
freeze-dryer was then depressurized rapidly to induce nucleation.
All vials nucleated and began freezing immediately after
depressurization. The vials were held at this state for about 15
minutes. The freeze-dryer shelf was then cooled at a rate of
approximately 1.degree. C./min to obtain vial temperatures near
-45.degree. C. and held for an additional 15 minutes to ensure
completion of the freezing process. After the freezing step, the
freeze-dryer shelf was then warmed at a rate of about 1.degree.
C./min to raise the vial temperatures to near 5.degree. C. No
precipitation of the protein was observed upon thawing. The vial
contents were assayed for enzymatic activity, and the results were
compared to a control sample of unfrozen LDH-1/mannitol
solution.
[0195] As part of Example 9, the depressurized nucleated samples of
the LDH-1/mannitol solution were compared to stochastically
nucleated samples. In the stochastically nucleated samples of
LDH-1, the procedure was repeated without pressurization and
depressurization and without the argon atmosphere. Specifically,
LDH-1 was dissolved in 5 wt % mannitol at a concentration of 1
mg/mL. Six lyophilization vials were filled with 2.5 mL of the
LDH-1/mannitol solution and placed on a freeze-dryer shelf in close
proximity to one another. The freeze-dryer shelf was cooled
starting from room temperature at a rate of about 1.degree. C./min
to obtain vial temperatures near -45.degree. C. and held for 15
minutes to ensure completion of the freezing process. After the
freezing step, the freeze-dryer shelf was warmed at a rate of about
1.degree. C./min to raise the vial temperatures to near 5.degree.
C. No precipitation of the protein was observed upon thawing. The
vial contents were assayed for enzymatic activity, and the results
were compared to the same control sample of unfrozen LDH-1/mannitol
solution. Also as part of Example 9, the experiments described
above for LDH-1 were repeated using LDH-2. The only difference was
a nucleation temperature near -3.degree. C. for LDH-2 rather than
-4.degree. C. for LDH-1.
[0196] As seen in Table 9, the controlled nucleation and freezing
process achieved via depressurization clearly does not decrease
enzymatic activity relative to a comparable stochastic nucleation
and freezing protocol. In fact, the controlled nucleation process
achieved via depressurization appears to better preserve enzyme
activity with a mean activity loss of only 17.8% for LDH-1 and
26.5% for LDH-2 compared to the mean activity loss of 35.9% for
LDH-1 and 41.3% for LDH-2 after stochastic nucleation.
TABLE-US-00009 TABLE 9 Controlling the Nucleation Temperature of
Sub-Cooled Protein Solutions Vial Temp Pressure Enzyme Activity
Depressurization Vial # Solution Atmos [.degree. C.] Drop[psi] Loss
[%] Outcome 1 2.5 mL of BSA solution Argon -4.9 14 -- Nucleation 2
2.5 mL of BSA solution Argon -4.3 14 -- Nucleation 3 2.5 mL of BSA
solution Argon -5.3 14 -- Nucleation 4 2.5 mL of LDH-1 solution
Argon -3.8 14 9.0 Nucleation 5 2.5 mL of LDH-1 solution Argon -4.0
14 16.2 Nucleation 6 2.5 mL of LDH-1 solution Argon -3.7 14 18.4
Nucleation 7 2.5 mL of LDH-1 solution Argon -4.0 14 23.4 Nucleation
8 2.5 mL of LDH-1 solution Argon -3.9 14 18.5 Nucleation 9 2.5 mL
of LDH-1 solution Argon -4.0 14 21.2 Nucleation 10 2.5 mL of LDH-1
solution Air -10.4 0 35.7 Nucleation 11 2.5 mL of LDH-1 solution
Air -16.5 0 35.4 Nucleation 12 2.5 mL of LDH-1 solution Air -15.5 0
36.1 Nucleation 13 2.5 mL of LDH-1 solution Air -10.5 0 43.9
Nucleation 14 2.5 mL of LDH-1 solution Air -9.8 0 24.9 Nucleation
15 2.5 mL of LDH-1 solution Air -11.0 0 39.2 Nucleation 16 2.5 mL
of LDH-2 solution Argon -3.1 14 29.9 Nucleation 17 2.5 mL of LDH-2
solution Argon -2.9 14 18.9 Nucleation 18 2.5 mL of LDH-2 solution
Argon -3.1 14 23.3 Nucleation 19 2.5 mL of LDH-2 solution Argon
-2.7 14 19.6 Nucleation 20 2.5 mL of LDH-2 solution Argon -3.1 14
32.1 Nucleation 21 2.5 mL of LDH-2 solution Argon -2.6 14 35.2
Nucleation 22 2.5 mL of LDH-2 solution Air -5.0 0 38.3 Nucleation
23 2.5 mL of LDH-2 solution Air -5.5 0 40.0 Nucleation 24 2.5 mL of
LDH-2 solution Air -2.3 0 36.5 Nucleation 25 2.5 mL of LDH-2
solution Air -3.8 0 42.0 Nucleation 26 2.5 mL of LDH-2 solution Air
-5.1 0 50.2 Nucleation 27 2.5 mL of LDH-2 solution Air -5.9 0 40.6
Nucleation
[0197] It should be noted that the stochastic nucleation
temperatures observed for LDH-2 were substantially warmer than the
stochastic nucleation temperatures for LDH-1. This difference may
be due to some contaminant acting as a nucleating agent in the
LDH-2. The stochastic nucleation temperatures are much closer to
the controlled nucleation temperatures for LDH-2 compared to LDH-1,
yet the improvements in retention of enzyme activity obtained via
controlled nucleation for LDH-1 and LDH-2 are similar at 18.1% and
14.8%, respectively. This result suggests that the improvements in
retention of enzyme activity can be partially attributed to the
characteristics of the controlled nucleation process itself, not
just to the prescribed warmer nucleation temperatures obtained via
depressurization.
Example 10
Reducing Primary Drying Time
[0198] A 5 wt % mannitol solution was prepared by mixing about
10.01 grams of mannitol with about 190.07 grams of water. Vials
were filled with 2.5 mL of the 5 wt % mannitol solution. The vials
were weighed empty and with the solution to determine the mass of
water added to the vials. The twenty vials were placed in a rack on
a freeze-dryer shelf in close proximity to one another. The
temperatures of six vials were monitored using surface mounted
thermocouples; all monitored vials were surrounded by other vials
to improve uniformity of vial behavior. The freeze-dryer was
pressurized to about 14 psig in a controlled gas atmosphere of
argon gas. The freeze-dryer shelf was cooled from room temperature
to about -6.degree. C. to obtain vial temperatures of between
approximately -1.degree. C. and -2.degree. C. The freeze-dryer was
then depressurized from about 14 psig to about atmospheric pressure
in less than five seconds to induce nucleation of the solution
within the vials. All vials observed visually or monitored via
thermocouples nucleated and began freezing immediately after
depressurization. The shelf temperature was then lowered rapidly to
about -45.degree. C. to complete the freezing process. Once all
vial temperatures were about -40.degree. C. or less, the
freeze-drying chamber was evacuated and the process of primary
drying (i.e., sublimation) was initiated. During this drying
process, the freeze-dryer shelf was warmed to about -14.degree. C.
via a one hour ramp and held at that temperature for 16 hours. The
condenser was maintained at about -60.degree. C. throughout the
drying process. Primary drying was stopped by turning off the
vacuum pump and backfilling the chamber with argon to atmospheric
pressure. The vials were promptly removed from the freeze-dryer and
weighed to determine how much water was lost during the primary
drying process.
[0199] In a separate experiment as part of Example 10, other vials
were filled with 2.5 mL of the same 5 wt % mannitol solution. The
vials were weighed empty and with the solution to determine the
mass of water added to the vials. The vials were loaded into the
freeze-dryer in the same manner described above, and the
temperatures of six vials were once again monitored using
surface-mounted thermocouples. The freeze-dryer shelf was cooled
rapidly from room temperature to about -45.degree. C. to freeze the
vials. Nucleation occurred stochastically between about -15.degree.
C. and about -18.degree. C. during the cooling step. Once all vials
temperatures were about -40.degree. C. or less, the vials were
dried in a manner identical to the method described above. Upon
conclusion of primary drying, the samples were promptly removed
from the freeze-dryer and weighed to determine how much water was
lost during the primary drying process.
TABLE-US-00010 TABLE 10 Increasing the Nucleation Temperature
Improves Primary Drying Initial Vial Water Temp. Pressure Loss
Depressurization Vial # Solution Atmos [.degree. C.] Drop [psi] [%]
Outcome 1 2.5 mL of 5 wt % mannitol Argon -1.3 14 89.9 Nucleation 2
2.5 mL of 5 wt % mannitol Argon -1.9 14 85.2 Nucleation 3 2.5 mL of
5 wt % mannitol Argon -1.3 14 87.1 Nucleation 4 2.5 mL of 5 wt %
mannitol Argon -2.3 14 88.8 Nucleation 5 2.5 mL of 5 wt % mannitol
Argon -2.1 14 85.0 Nucleation 6 2.5 mL of 5 wt % mannitol Argon
-1.1 14 80.7 Nucleation 7 2.5 mL of 5 wt % mannitol Air -15.7 0
65.7 -- 8 2.5 mL of 5 wt % mannitol Air -16.7 0 66.9 -- 9 2.5 mL of
5 wt % mannitol Air -14.5 0 64.6 -- 10 2.5 mL of 5 wt % mannitol
Air -15.6 0 64.7 -- 11 2.5 mL of 5 wt % mannitol Air -16.5 0 64.1
-- 12 2.5 mL of 5 wt % mannitol Air -17.9 0 65.7 --
[0200] Results of the freeze-drying process with controlled
nucleation and stochastic nucleation are summarized in Table 10. It
should be noted that these two experiments only differ in the
addition of the controlled nucleation via depressurization step to
one experiment. As seen in Table 10, the controlled nucleation
process achieved via depressurization allows nucleation at very low
degrees of sub-cooling, between about -1.1.degree. C. and
-2.3.degree. C. in this example. The much warmer nucleation
temperatures for the controlled nucleation case compared to the
stochastic nucleation case yields an ice structure and resultant
lyophilized cake with dramatically improved drying properties. For
the same amount of drying time, the vials nucleated using the
disclosed depressurization methods between about -1.1.degree. C.
and -2.3.degree. C. lost an average of 86.1% of their water while
the vials nucleated stochastically between about -14.5.degree. C.
and -17.9.degree. C. only lost an average of 65.3%. Hence, the
vials nucleated stochastically would require much more primary
drying time to achieve the same degree of water loss as the vials
nucleated in a controlled manner in accordance with the presently
disclosed methods. The improvement in drying time is likely
attributed to the formation of larger ice crystals at warmer
nucleation temperatures. These larger ice crystals leave behind
larger pores upon sublimation, and the larger pores offer less
resistance to the flow of water vapor during further
sublimation.
[0201] Another benefit associated with the above presented
temperature quench and pressure induced nucleation control methods
is that by controlling the lowest nucleation temperature and/or the
precise time of nucleation one can affect the ice crystal structure
formed within the frozen vials or containers. The ice crystal
structure is a variable that affects various properties of the
preserved material, including but not limited to, activity,
functionality, and viability as well as the time it takes for ice
to sublimate during a freeze-drying process. Thus, controlling the
ice crystal structure is important for both cryopreservation and
freeze-drying processes.
Enhanced Viability and Biological Activity
[0202] As detailed above, FIG. 11 is an illustrative temperature
profile of the cryogenic cold gas used to cool samples in the
presently disclosed uniform flow controlled rate freezer. The
temperature profile includes an equilibrium step 402, a cooling
step 404, a pre-nucleation temperature step 406, a temperature
quench step 408, a temperature quench hold step 410, a
post-nucleation temperature hold step 412, and a final cooling step
414. The testing presented below was done to optimize the presented
temperature profile to maximize the cell viability during a
cryopreservation process utilizing a temperature quench method to
induce nucleation. While results are presented for a temperature
quench controlled nucleation, a pressure controlled nucleation
process could be used to obtain similar results with regards to
cell viability.
[0203] Furthermore, while viability results are presented for
living cells, it would be apparent to one of skill in the art that
the described systems and methods could be used to provide a
uniform biological activity for non-living materials.
[0204] Cell cultures of Normal Human Dermal Fibroblasts (NHDF) were
obtained from Lonza (Walkersville, Md.). Stock cultures were
maintained at 37.degree. C. in 95% air/5% CO.sub.2 in Falcon T-75
cm.sup.2-flasks. NHDF cultures were grown in Fibroblast Basal
Medium (FBM) supplemented with Fibroblast Growth Medium (FGM
SingleQuots supplied by Lonza). Stock cultures were subcultured
every 5-6 days at approximately 95% confluence, and media was
replenished every 3 days. Experiments were performed using cell
cultures between passages 2 and 10. One day prior to
experimentation, cultures were supplemented with fresh culture
media.
[0205] In-house media/Dimethyl sulfoxide (DMSO) solutions were
prepared by the addition of DMSO (5% v/v) to the complete FGM
(having 10% fetal bovine serum). For testing purposes the solution
containing in-house media with 5% v/v DMSO is labeled as "Media+5%
DMSO". The intracellular-like CryoStor solution (BioLife Solutions,
WA) is serum-free and protein-free and is premixed with 5% v/v
DMSO. For testing purposes the solution containing CryoStor
solution premixed with 5% v/v DMSO is labeled as "CS5".
[0206] To test solutions for cryopreservation efficacy, standard
cryopreservation methods were performed. Briefly, cells
(1.times.10.sup.6 cells/ml) were re-suspended in 0.5 ml of the
respective solutions to be tested and placed into 1.2 ml cryovials.
Cryopreservation studies were performed using the uniform
controlled rate freezer currently disclosed. Samples were stored
for 10 minutes at 2-8.degree. C. in the freezing chamber to allow
each of the samples to equilibrate in temperature prior to being
subjected to various freezing protocols. Following the cryogenic
freezing process, samples were immediately transferred into liquid
nitrogen for 18-24 hours. Samples were thawed in a 37.degree. C.
water bath, immediately re-suspended in culture media (1:10
dilution), plated in a well sample plate, and allowed to recover
for one day prior to assessment.
[0207] Cell viability was assessed for each freezing process both
qualitatively and quantitatively. Qualitative assessment was
achieved by visualization using light microscopy. Quantitative
assessment was conducted using AlamarBlue.TM. (AbD Serotec) for
florescence spectroscopy evaluation. AlamarBlue.TM. was diluted in
a ratio of 1:20 in Hank's Balanced Salt Solution without phenol red
(HBSS) available from Life Technologies, Gaithersburg, Md. 100
.mu.l of the culture medium was removed from each well in the well
sample plates and 100 .mu.l of the working AlamarBlue.TM. solution
was added to each well for analysis. Samples were subsequently
incubated in the dark at 37.degree. C. for 60 min (+1 min) The
fluorescence of each sample was then evaluated using a Tecan
SPECTRAFluor Plus plate reader (TECAN Austria GmbH) with a 530-nm
excitation/590-nm emission filter set. This assessment was
performed 24 hours post-preservation for each experiment.
[0208] The above disclosed testing methods were used to optimize
the cooling rates, hold times, and temperatures of the cryogenic
cold gas temperature profile with respect to the cell viability of
NHDF cells during a temperature quench controlled nucleation
freezing process. The results of the testing are presented in FIGS.
11, 12-13, and 19-31. The testing results in FIGS. 19-31 correspond
to the cryogenic cold gas temperature profile steps defined in FIG.
11.
[0209] The samples presented in FIGS. 12 and 13 were prepared using
CS5 solution and the above disclosed method. Testing was conducted
using the currently disclosed uniform flow cryogenic chiller unit.
As detailed above, FIG. 12 depicts temperature profiles of samples
that have undergone a freezing process with temperature quench
nucleation control. FIG. 13 depicts temperature profiles of samples
that have undergone a freezing process with no nucleation control
and a constant cooling rate of 5.degree. C./min. Tables 11 and 12
present measured nucleation temperature and percent viability of
the samples depicted in FIGS. 12 and 13 for samples with
temperature quench nucleation control and without nucleation
control, respectively.
TABLE-US-00011 TABLE 11 Nucleation Temperature and Viability of
Samples with Temperature Quench Nucleation Control Nucleation Vial
# Temperature (.degree. C.) % Viability Vial 1 7.2 80 Vial 2 8.1 84
Vial 3 8.0 75 Vial 4 7.0 78 Vial 5 7.5 79 Vial 6 7.5 78
TABLE-US-00012 TABLE 12 Nucleation Temperature and Viability of
Samples without Nucleation Control Nucleation Vial # Temperature
(.degree. C.) % Viability Vial 1 11.8 62 Vial 2 15.2 64 Vial 3 10.2
68 Vial 4 9.3 65 Vial 5 10.3 68 Vial 6 11.9 65
[0210] Table 13 presents additional data from testing of NHDF cells
prepared using CS5 solution and the above detailed method and the
currently disclosed uniform flow cryogenic chiller unit. Table 13
presents data for samples frozen without nucleation control at
different constant cooling rates. Optimal NHDF cell recovery, for a
process without nucleation control, was observed for cooling rates
of 1.degree. C./min, 5.degree. C./min, and 10.degree. C./min
Decreased cell recovery was observed with a slower cooling rate of
0.5.degree. C./min. Decreased cell recovery was also observed for
faster cooling rates of 15 and 25.degree. C./min.
TABLE-US-00013 TABLE 13 Viability of Samples without Nucleation
Control and Different Cooling Rates Cooling Rate (.degree. C./min)
% Viability 0.5 58 1 69 5 67 10 68 15 60 25 41
[0211] As expected the samples with nucleation control, presented
in Table 11, exhibited a smaller variance in the observed
nucleation temperature as compared to the samples without
nucleation, presented in Table 12. Furthermore, the samples that
underwent the nucleation control exhibit an enhanced viability as
compared to the data presented in Tables 12 and 13. Specifically,
the average viability from the temperature quench induced
nucleation samples is 79% as compared to the range of viabilities
shown in Tables 12 and 13 ranging from 41% to 69% without
nucleation control. As stated above testing was conducted with the
above disclosed uniform flow cryogenic chiller unit. Therefore, if
the above freezing process with nucleation control were to be
scaled up using the uniform flow cryogenic chiller unit there will
be minimal sample to sample variation in temperature profile
between the plurality of samples for any number of samples. Such a
process would result in the plurality of samples having a uniform
enhanced viability with minimal differences from vial to vial and
batch to batch for any number of samples. In a certain embodiments
such a system could include at least 10,000, 20,000, 50,000, or
100,000 samples.
[0212] FIG. 19 illustrates the effect of Pre-nucleation temperature
on the cell viability of NHDF cells following cryopreservation.
Curve 800 corresponds to the tests conducted with CS5 solution and
curve 802 corresponds to the tests conducted with Media+5% DMSO
solution. For this test, the pre-nucleation temperature was set at
-2.5.degree. C., -5.degree. C., -7.5.degree. C. and 10.degree. C.
respectively while the remaining cooling parameters were kept the
same. Optimal NHDF cell viability in both solutions was observed
for a pre-nucleation temperature of -5.degree. C. Decreased cell
viability was observed for warmer and colder pre-nucleation
temperatures.
[0213] FIG. 20 shows the effect of the quench temperature on the
cell viability of NHDF cells following cryopreservation. Curve 804
corresponds to the tests conducted with CS5 solution and curve 806
corresponds to the tests conducted with Media+5% DMSO solution.
After being held at the pre-nucleation temperature, the cryogenic
cold gas was rapidly decreased to -40.degree. C., -60.degree. C.,
-70.degree. C. and -80.degree. C. to initiate nucleation. After
performing the temperature quench, the cryogenic cold gas
temperature was raised to -20.degree. C. at a rate of 30.degree.
C./min and held at -20.degree. C. for 10 minutes. The cryogenic
cold gas was then cooled to -80.degree. C. at a rate of
-2.5.degree. C./min Optimal NHDF cell viability in both solutions
was observed for a quench temperature of -80.degree. C. NHDF cell
viability slightly decreased for quench temperatures of -60.degree.
C. and -70.degree. C., while a significant decrease was observed
for a quench temperature of -40.degree. C.
[0214] FIG. 21 compares the sample temperature profiles for the
tests depicted in FIG. 20 with different quench temperatures for
nucleation control for those samples tested in CS5 solution. The
tested quench temperatures were -40.degree. C. (curve 808),
-60.degree. C. (curve 810), -70.degree. C. (curve 812), and
-80.degree. C. (curve 814). Combined with FIG. 20, it was observed
that nucleation temperature and the sample post-nucleation cooling
rate were related to the quench temperature. Furthermore, the
combination of the nucleation temperature and post-nucleation
cooling rate affect cell recovery. The moderate sample cooling rate
observed for a quench temperature of about -80.degree. C. gave the
optimal recovery.
[0215] FIG. 22 illustrates the effect of the quench temperature and
the hold time of the quench on the viability of NHDF cells
following cryopreservation. Curve 816 corresponds to the tests
conducted with CS5 solution and curve 818 corresponds to the tests
conducted with Media+5% DMSO solution. After being held at the
pre-nucleation temperature, the cryogenic cold gas temperature was
rapidly decreased to -80.degree. C. with no hold time, -80.degree.
C. with a two minute hold, and -100.degree. C. with a one-minute
hold. After performing the respective temperature quenches and
hold, the cryogenic cold gas was warmed up to -35.degree. C. at
30.degree. C./min and held for 10 minutes. The cryogenic cold gas
was then cooled down to -80.degree. C. at 2.5.degree. C./min. The
different temperatures and hold times were evaluated to determine
the effect of latent heat on cell viability. Looking at FIG. 22,
applying colder quench temperatures (e.g. -100.degree. C.) or
increasing the holding time at the quench temperature to minimize
latent heat resulted in decreased cell viability. Optimal NHDF cell
viability in both solutions was observed for a quench temperature
of -80.degree. C. with no quench holding time.
[0216] FIG. 23 shows the temperature profiles associated with the
tests depicted in FIG. 22 with different quench temperatures and
hold times for nucleation control for those samples tested in CS5
solution. The profiles parameters were -80.degree. C. with no hold
(curve 820), -80.degree. C. with a 2 minute hold (curve 822), and
-100.degree. C. with a one minute hold (curve 824).
[0217] FIG. 24 presents the effect of the post-nucleation hold
temperature on the viability of NHDF cells following
cryopreservation. Curve 826 corresponds to the tests conducted with
CS5 solution and curve 828 corresponds to the tests conducted with
Media+5% DMSO solution. After being held at the pre-nucleation
temperature, the cryogenic cold gas was rapidly decreased to
-80.degree. C. with no hold time, then the cryogenic cold gas was
warmed up to a post-nucleation holding temperature of -10.degree.
C., -20.degree. C., -35.degree. C. or -50.degree. C. at a rate of
30.degree. C./min and held for 10 minutes prior to further cooling
to -80.degree. C. at a rate of 2.5.degree. C./min No significant
differences were observed in the post thaw viability of NHDF cells
for post-nucleation holding temperatures of -10.degree. C.,
-20.degree. C., -35.degree. C. or -50.degree. C. for the CS5 media
solution. A decreased viability of NHDF cells was observed at a
post-nucleation holding temperature of -10.degree. C. for the
media+5% DMSO solution.
[0218] FIG. 25 shows the temperature profiles associated with the
tests depicted in FIG. 24 with different post-nucleation holding
temperatures for those samples tested in CS5 solution. The
different post-nucleation hold temperatures were -10.degree. C.
(curve 830), -20.degree. C. (curve 832), -35.degree. C. (curve
834), and -50.degree. C. (curve 836).
[0219] FIG. 26 shows the effect of post-nucleation holding time on
the viability of NHDF cells following cryopreservation. Curve 838
corresponds to the tests conducted with CS5 solution and curve 840
corresponds to the tests conducted with Media+5% DMSO solution.
After reaching the pre-nucleation temperature, the cryogenic cold
gas was plunged to -80.degree. C. with no hold time. The cryogenic
cold gas was then warmed up to a post nucleation temperature of
-35.degree. C. at a rate of 30.degree. C./min and held at
-35.degree. C. for different times ranging from no hold time to
twenty minutes prior to further cooling to -80.degree. C. at a rate
of 2.5.degree. C./min. The test run without a post-nucleation hold
time resulted in the lowest cell viability. There was a roughly
10%-20% difference in viability observed between the tests with
various hold times at a post-nucleation hold temperature of
-35.degree. C. for CS5 media solution and media+5% DMSO. Optimal
cell viability was observed in both solutions at a post-nucleation
hold temperature of -35.degree. C. with a 10 minute post-nucleation
holding time.
[0220] FIG. 27 shows the temperature profiles associated with the
tests depicted in FIG. 26 with different post-nucleation hold times
at -35.degree. C. for those samples tested in CS5 solution. The
different post-nucleation hold times were no hold (curve 842), 2
minutes (curve 844), 5 minutes (curve 846), 10 minutes (curve 848),
and 20 minutes (curve 850).
[0221] FIGS. 28 and 30 illustrate the effect of the post-nucleation
cooling rate following the post-nucleation temperature hold on the
viability of NHDF cells following cryopreservation. FIG. 28 was
conducted with a -35.degree. C. post-nucleation hold temperature.
FIG. 30 was conducted with a -10.degree. C. post-nucleation hold
temperature. Curves 852 and 862 correspond to the tests conducted
with CS5 solution and curves 854 and 864 correspond to the tests
conducted with Media+5% DMSO solution. After being held at the
pre-nucleation temperature, the cryogenic cold gas was rapidly
decreased to -80.degree. C. with no hold time, The cryogenic cold
gas was then warmed up to a post nucleation temperature of
-35.degree. C. or -10.degree. C. at a rate of 30.degree. C./min and
held at the post-nucleation temperature for 10 minutes. The samples
were then further cooled to -80.degree. C. at different rates
ranging from 2.5 to 150.degree. C./min. It was observed that the
warmer post-nucleation temperature of -10.degree. C. resulted in
increased sensitivity of the cell viability to the applied cooling
rate. The difference in cell viability between a cooling rate of
10.degree. C./min and 150.degree. C./min was approximately 5% for
the post-nucleation temperature of -35.degree. C. and approximately
10% for the post-nucleation temperature of -10.degree. C.
[0222] FIG. 29 shows the temperature profiles associated with the
post nucleation cooling rates shown in FIG. 28 and tested with a
-35.degree. C. post-nucleation hold temperature in CS5 solution.
The different post-nucleation cooling rates were 2.5.degree. C./min
(curve 856), 10.degree. C./min (curve 858), and 150.degree. C./min
(curve 860).
[0223] FIG. 31 shows the temperature profiles associated with the
post nucleation cooling rates shown in FIG. 30 and tested with a
-10.degree. C. post-nucleation hold temperature in CS5
solution.
[0224] The different post-nucleation cooling rates were 2.5.degree.
C./min (curve 866), 10.degree. C./min (curve 868), 20.degree.
C./min (curve 870), and 150.degree. C./min (curve 872).
[0225] In combination, the above disclosed cooling and nucleation
methods and the uniform flow controlled rate freezer are capable of
providing uniformly enhanced viability of biological material
frozen in any number of vials or containers located within a single
system. One such system might be a uniform flow controlled rate
freezer incorporating multiple uniform flow cryogenic chiller unit
modules. Such a system would be capable of providing uniformly
enhanced viability to at least 10,000, 20,000, 50,000, or 100,000
vials or containers containing biological material.
[0226] Alternatively, the uniform flow controlled rate freezer
could be used to implement simpler cooling profiles without
nucleation control such as the cooling rate presented in FIG. 13.
Such a method would result in similar results for the viability of
the frozen material as provided by prior art methods and systems.
However, as detailed above, in contrast to prior art systems the
uniform flow controlled rate freezer is capable of providing a
uniform temperature and flow of cryogenic cold gas to each sample
which will result in a uniform viability for each sample regardless
of location within the system or cooling chamber in which the
plurality of containers were frozen. Therefore, even when only
applying a simple cooling profile without nucleation control, the
currently disclosed devices and methods are capable of providing a
uniform viability of biological material frozen in any number of
vials or containers located within a single system. In a first
embodiment the number of samples exhibiting a uniform viability is
at least 50,000. In a second embodiment the number of samples
exhibiting a uniform viability is at least 100,000.
[0227] The present method provides an improved method for
controlling the temperature and/or time at which sub-cooled
materials, namely liquids or solutions, nucleate and then freeze.
Although this application focuses in part on freeze-drying, a
similar problem occurs for any material processing step that
involves a nucleated phase transition. Examples of such processes
include the crystallization of polymers and metals from melts,
crystallization of materials from supersaturated solutions,
crystallization of proteins, artificial snow production, food
freezing, freeze concentration, fractional crystallization,
cryo-preservation, or condensation of vapors to liquids.
[0228] The most immediate benefit of controlling the nucleation
temperature of a liquid or solution is the ability to control the
number and size of the solid domains produced by the phase
transition. In freezing water, for example, the nucleation
temperature directly controls the size and number of ice crystals
formed. Generally speaking, the ice crystals are fewer in number
and larger in size when the nucleation temperature is warmer.
[0229] The ability to control the number and size of the solid
domains produced by a phase transition may provide additional
benefits. In a freeze-drying process, for example, the number and
size of the ice crystals strongly influences the drying properties
of the lyophilized cake. Larger ice crystals produced by warmer
nucleation temperatures leave behind larger pores upon sublimation,
and the larger pores offer less resistance to the flow of water
vapor during subsequent sublimation. Hence, the present methods
provide a means of increasing primary drying (i.e., sublimation)
rates in freeze-drying processes by increasing the nucleation
temperature.
[0230] Another possible benefit may be realized in applications
where sensitive materials are preserved via freezing processes
(i.e., cryopreserved). For example, a biological material including
but not limited to, mammalian tissue samples (e.g., cord blood,
tissue biopsy, egg and sperm cells, etc.), cell lines (e.g.,
mammalian, yeast, prokaryotic, fungal, etc.) and biological
molecules (e.g., proteins, DNA, RNA and subclasses thereof) frozen
in an aqueous solution may experience various stresses during the
freezing process that may impair the function or activity of the
material. Ice formation may physically disrupt the material or
create severe changes in the interfacial bonding, osmotic forces,
solute concentrations, etc. experienced by the material. Since
nucleation controls the structure and kinetics of ice formation, it
can significantly influence these stresses. The presently disclosed
methods therefore provides a unique means of mitigating stresses
associated with cryopreservation processes and enhancing the
recovery of function or activity from cryopreserved materials. The
present methods also represent improvement over conventional
nucleation control methods (e.g., seeding or contact with cold
surfaces) used to initiate extracellular ice formation in two-step
cryopreservation algorithms designed for living cells for small to
large commercial scale.
[0231] As an example of this improved biological activity resulting
from the present process, Jurkat A3 lymphoblasts (ATCC No.
CRL-2570) obtained from ATCC (Manassas, Va.) were subjected to the
present process. Stock cultures of the Jurkat A3 lymphoblasts were
maintained at 37.degree. C. in 95% air and 5% CO.sub.2 in Falcon
T-75 cm.sup.2 flasks using a complete RPMI-1640 media containing
2.5 mM glutamine (HyClone) and supplemented with 10% v/v FetalClone
III serum (HyClone). For cell expansion, stock cultures were
sub-cultured every 2 days when cell density reached approximately
5.times.10.sup.6 cells/ml and then re-suspended in fresh T-75
flasks at a seeding density of about 0.5.times.10.sup.6 cells/ml.
For the Jurkat cell line, an in-house media/DMSO solution was
prepared by the addition of 5% v/v DMSO to the complete RPMI-1640
media containing 20% v/v FetalClone III.
[0232] Briefly, the Jurkat A3 lymphoblast cells were processed
after cell expansion for preservation in bags by pooling cultures
and re-suspending them to a cell density of 2.times.10.sup.6
cells/ml. Cryopreservation studies employing present process were
performed using the advanced controlled rate freezing system
designed for bags and used to process the Jurkat A3 lymphoblast
cell line. Samples were held for 10 min at 2-8.degree. C. in the
advanced controlled rate freezing chamber prior to freezing to
allow cell samples to equilibrate. Following the freezing program,
samples were immediately transferred to liquid nitrogen for up to
48 hrs. Samples were thawed in a 37.degree. C. water bath,
immediately re-suspended in appropriate culture media (1:10
dilution), plated, and allowed to recover prior to cell viability
assessment.
[0233] Cell viability of the cryopreserved Jurkat A3 lymphoblast
cells was assessed both qualitatively and quantitatively prior to
freezing and post-thaw. For both cell lines, qualitative assessment
was achieved by visualization using light microscopy. For the
Jurkat A3 lymphoblast cell line, quantitative assessment was
accomplished using Trypan Blue staining through an automated CEDEX
cell analyzer (Roche Applied Science). After thawing, a sample from
the 25 ml bag was re-suspended in fresh media and allowed to
recover in well plates prior to viability assessments at 3 hrs and
24 hrs post-preservation. From each well, a sample was taken and
directly measured using the CEDEX cell counter.
[0234] As discussed above, the Jurkat A3 lymphoblast cell recovery
was assessed at 3 hr and at 24 hr post-thaw viability following
freezing in the advanced controlled rate freezing system with and
without nucleation control, see FIG. 32. For the evaluated trials,
the bags containing said materials containing the live cells were
equilibrated at 4.degree. C. in the advanced controlled rate
freezing process within the controlled rate freezing chamber prior
to freezing. For the case without nucleation control, after
equilibration the bags were cooled to -45.degree. C. at a cooling
rate of 1.degree. C./min and then further cooled to -100.degree. C.
at a cooling rate of 10.degree. C./min For the case of cold spike
nucleation control, after equilibration the bags went through the
following steps: (i) cooled to -5.degree. C. at a cooling rate of
1.degree. C./min, (ii) nucleated by a cold spike, (iii) warmed to
-20.degree. C. at a warming rate of 10.degree. C./min, (iv) cooled
to -45.degree. C. at a cooling rate of 1.degree. C./min, and (v)
cooled to -100.degree. C. at a cooling rate of 10.degree. C./min.
In FIG. 32, the results show that the cryopreservation algorithm
with controlled nucleation yielded approximately 15% more cell
recovery at about 3 hrs post-thaw and approximately 10% more cell
recovery at 24 hrs post-thaw when compared to the cryopreservation
algorithm with uncontrolled nucleation. This cryopreservation
example shows the significant impact of nucleation control behavior
and thus the freezing process uniformity on cell recovery
post-thaw.
[0235] The presently disclosed controlled rate freezing and
nucleation control methods may be also applied to complex solutions
or mixtures containing several constituents both in cryopresevation
and lyophilization applications. These formulations are often
solutions with an aqueous, organic, or mixed aqueous-organic
solvent containing a pharmaceutically active ingredient (e.g., a
synthetic chemical, protein, peptide, or vaccine) and optionally,
one or more mitigating constituents, including bulking agents that
help prevent physical loss of the active ingredient during drying
(e.g., dextrose, glucose, glycine, lactose, maltose, mannitol,
polyvinyl pyrrolidone, sodium chloride, and sorbitol); buffering
agents or toxicity modifiers that help maintain the appropriate
environmental pH or toxicity for the active constituent (e.g.,
acetic acid, benzoic acid, citric acid, hydrochloric acid, lactic
acid, maleic acid, phosphoric acid, tartaric acid, and the sodium
salts of the aforementioned acids); stabilizing agents that help
preserve the structure and function of the active constituent
during processing or in its final liquid or dried form (e.g.,
alanine, dimethylsulfoxide, glycerol, glycine, human serum albumin,
polyethylene glycol, lysine, polysorbate, sorbitol, sucrose, and
trehalose); agents that modify the glass transition behavior of the
formulation (e.g., polyethylene glycol and sugars), and
anti-oxidants that protect the active constituent from degradation
(e.g., ascorbate, sodium bisulfite, sodium formaldehyde, sodium
metabisulfite, sodium sulfite, sulfoxylate, and thioglycerol).
[0236] Since nucleation is typically a random process, a plurality
of the same material subjected to identical processing conditions
might nucleate at different temperatures. As a result, the
properties of those materials that depend on nucleation behavior
will likely differ despite the identical processing conditions. The
disclosed methods provide a means for controlling the nucleation
temperatures of a plurality of materials simultaneously and thereby
offers a way to increase the uniformity of those product properties
that depend on nucleation behavior. In a typical freeze-drying
process, for example, the same solution in separate vials may
nucleate stochastically over a wide range of temperatures, and as a
result, the final freeze-dried products may possess significant
variability in critical properties like residual moisture, activity
and reconstitution time. By controlling the nucleation temperature
via the presently disclosed process, the vial-to-vial uniformity of
product properties from a freeze-drying can process can be
dramatically improved.
[0237] The ability to control the nucleation behavior of a material
may also provide substantial benefit in reducing the time necessary
to develop an industrial process that hinges upon a normally
uncontrolled nucleation event. For example, it often takes many
months to develop a successful freeze-drying cycle that can be
accomplished in a reasonable amount of time, yields desired product
properties within the specified uniformity, and preserves
sufficient activity of the active pharmaceutical ingredient (API).
By providing a means of controlling nucleation and thereby
potentially improving primary drying time, product uniformity, and
API activity, the present methods should dramatically reduce the
time necessary to develop successful freeze-drying protocols.
[0238] In particular, the potential benefits of the present
nucleation process provide increased flexibility in specifying the
composition of the formulation to be freeze-dried. Since controlled
nucleation can better preserve the API during the freezing step,
users should be able to minimize the addition of mitigating
constituents (e.g., stabilizing agents) to the formulation or chose
simpler combinations of formulation constituents to achieve
combined stability and processing goals. Synergistic benefits may
arise in cases where controlled nucleation minimizes the use of
stabilizing agents or other mitigating constituents that inherently
lengthen primary drying times (e.g., by decreasing glass transition
temperatures of aqueous solutions).
[0239] The disclosed methods are particularly well-suited for large
scale production or manufacturing operations since it can be
conducted using the same equipment and process parameters that can
easily be scaled or adapted to manufacture a wide range of
products. The process provides for the nucleation of materials
using a process where all manipulations can be carried out in a
single chamber (e.g., a freeze-dryer) and where the process does
not require use of a vacuum, use of additives, vibration,
electrofreezing or the like to induce nucleation.
[0240] In contrast to the prior art, the present method does not
add anything to the lyophilized product. It only requires that the
materials, (e.g., liquids in the vials), be held initially at a
specified pressure under a gas environment and that the pressure is
rapidly reduced to a lower pressure or the temperature is uniformly
controlled to induce nucleation. Any applied gas will be removed
from the vials during the lyophilization cycle. The vials or their
contents are not contacted or touched with anything except the gas.
Simple manipulation of the ambient pressure and gas environment or
the uniform cryogenic cold gas temperature is sufficient on its own
to achieve that goal. By relying only on ambient pressure change to
induce nucleation or a temperature change of the uniform cryogenic
cold gas, the present method disclosed herein uniformly and
simultaneously affects all vials within a freeze-dryer or uniform
flow controlled rate freezer.
[0241] The present embodiment is also less expensive and easier to
implement and maintain than prior art methods of influencing
nucleation in materials in lyophilization applications. The present
method enables significantly faster primary drying in
lyophilization processes, thereby reducing processing costs for
freeze-dried pharmaceuticals. The present method produces much more
uniform lyophilized products than prior art methods, thereby
reducing product losses and creating barriers to entry for
processors unable to meet tighter uniformity specifications. This
method achieves these benefits without contaminating the
lyophilized product. Greater process control should lead to an
improved product and shortened process times.
[0242] While the present teachings have been described in
conjunction with various embodiments and examples, it is not
intended that the present teachings be limited to such embodiments
or examples. On the contrary, the present teachings encompass
various alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art. Accordingly, the
foregoing description and drawings are by way of example only.
* * * * *