U.S. patent application number 13/177834 was filed with the patent office on 2013-01-10 for methods for freezing and thawing proteins.
Invention is credited to Prerona CHAKRAVARTY, Naresh J. SUCHAK.
Application Number | 20130008191 13/177834 |
Document ID | / |
Family ID | 47437340 |
Filed Date | 2013-01-10 |
United States Patent
Application |
20130008191 |
Kind Code |
A1 |
SUCHAK; Naresh J. ; et
al. |
January 10, 2013 |
METHODS FOR FREEZING AND THAWING PROTEINS
Abstract
A method for freezing and thawing proteins is disclosed. The
proteins are rapidly frozen by introducing droplets of a protein
solution into a cryogenic freezing medium. The frozen pellets thus
formed are rapidly thawed by introducing them into a heat transfer
fluid to form a reconstitute protein solution.
Inventors: |
SUCHAK; Naresh J.; (Glen
Rock, NJ) ; CHAKRAVARTY; Prerona; (Springfield,
NJ) |
Family ID: |
47437340 |
Appl. No.: |
13/177834 |
Filed: |
July 7, 2011 |
Current U.S.
Class: |
62/64 ;
62/62 |
Current CPC
Class: |
A61K 9/1688 20130101;
A61K 9/16 20130101; A61K 38/00 20130101 |
Class at
Publication: |
62/64 ;
62/62 |
International
Class: |
F25D 31/00 20060101
F25D031/00; F25D 17/02 20060101 F25D017/02 |
Claims
1. A method comprising feeding droplets to a freezing medium
thereby freezing said droplets and forming pellets.
2. The method as claimed in claim 1 wherein said droplets comprise
a protein.
3. The method as claimed in claim 2 wherein said protein is in
solution.
4. The method as claimed in claim 1 wherein said freezing medium is
a cryogen.
5. The method as claimed in claim 1 wherein said cryogen is
selected from the group consisting of liquid nitrogen, oxygen, air,
and argon.
6. The method as claimed in claim 1 further comprising adding
stabilizers to said freezing medium.
7. The method as claimed in claim 6 wherein said stabilizers are
selected from the group consisting of sorbitol, sucrose, trelose,
and alenine.
8. The method as claimed in claim 1 further comprising adding
bulking agents and buffers to said freezing medium.
9. The method as claimed in 8 wherein said bulking agents are
selected from the group consisting of glycine and mannitol and said
buffers are selected from the group consisting of sodium citrate
and sodium phosphate.
10. The method as claimed in claim 2 wherein said protein is
pre-cooled to a temperature range of -20.degree. C. to -45.degree.
C.
11. The method as claimed in claim 1 wherein the temperature of
said freezing medium is -80.degree. C.
12. The method as claimed in claim 1 wherein said droplets are
added to said freezing medium from 0.5 to 15 seconds.
13. The method as claimed in claim 1 wherein said pellets are 0.5
to 15 mm in diameter.
14. The method as claimed in claim 1 wherein said pellets are
separated from said freezing medium.
15. The method as claimed in claim 1 wherein said pellets are
stored at temperatures of -80.degree. C. or below.
16. A method comprising reconstituting vitrified pellets by adding
said vitrified pellets to a heat transfer fluid.
17. The method as claimed in claim 16 wherein said vitrified
pellets comprise a protein solution.
18. The method as claimed in claim 16 wherein heat transfer fluid
is water.
19. The method as claimed in claim 16 wherein said heat transfer
fluid is in a stirred vessel.
20. The method as claimed in claim 16 wherein said vitrified
pellets are added to said heat transfer fluid in an amount
necessary to form a reconstituted protein solution.
21. A method comprising feeding droplets to a freezing medium
thereby freezing said droplets and forming pellets and
reconstituting said pellets by adding said pellets to a heat
transfer solution.
22. The method as claimed in claim 21 wherein said droplets
comprise a protein.
23. The method as claimed in claim 22 wherein said protein is in
solution.
24. The method as claimed in claim 21 wherein said freezing medium
is a cryogen.
25. The method as claimed in claim 21 wherein said cryogen is
selected from the group consisting of liquid nitrogen, oxygen, air,
and argon.
26. The method as claimed in claim 21 further comprising adding
stabilizers to said freezing medium.
27. The method as claimed in claim 26 wherein said stabilizers are
selected from the group consisting of sorbitol, sucrose, trelose,
and alenine.
28. The method as claimed in claim 21 further comprising adding
bulking agents and buffers to said freezing medium.
29. The method as claimed in 28 wherein said bulking agents are
selected from the group consisting of glycine and mannitol and said
buffers are selected from the group consisting of sodium citrate
and sodium phosphate.
30. The method as claimed in claim 22 wherein said protein is
pre-cooled to a temperature range of -20.degree. C. to -45.degree.
C.
31. The method as claimed in claim 21 wherein the temperature of
said freezing medium is -80.degree. C.
32. The method as claimed in claim 21 wherein said droplets are
added to said freezing medium from 0.5 to 15 seconds.
33. The method as claimed in claim 21 wherein said pellets are 0.5
to 15 mm in diameter.
34. The method as claimed in claim 21 wherein said pellets are
separated from said freezing medium.
35. The method as claimed in claim 21 wherein said pellets are
stored at temperatures of -80.degree. C. or below.
36. The method as claimed in claim 21 wherein said vitrified
pellets comprise a protein solution.
37. The method as claimed in claim 21 wherein heat transfer fluid
is water.
38. The method as claimed in claim 21 wherein said heat transfer
fluid is in a stirred vessel.
39. The method as claimed in claim 21 wherein said vitrified
pellets are added to said heat transfer fluid in an amount
necessary to form a reconstituted protein solution.
Description
BACKGROUND OF THE INVENTION
[0001] The invention provides for a method of uniformly freezing
and thawing protein solution to minimize functional damage to the
protein. More particularly, the invention provides non-equilibrium
heat transfer to the protein solutions.
[0002] Advances in biotechnology have led to increasing production
of protein-based therapeutics. This in turn has led to increased
demands for efficient methods to stabilize and store such
therapeutic proteins. In a typical biomanufacturing system, the
protein obtained from downstream processing and purification is
usually in bulk quantity, in aqueous environment, and chemically
stabilized by the addition of buffers and excipients. Sometimes
additional preformulation studies are required following downstream
processing for efficient stabilization. A protein solution that has
been chemically stabilized often requires moderate to long periods
of storage prior to final dosage formulation.
[0003] Although lyophilization is one way to improve stability of
final drug products, it is not a practical or economical method for
intermediate storage of protein solutions between bulk processing
and final dosage formulation. In addition, sometimes proteins
reconstituted from lyophilized state need to be stored for moderate
intervals. Manufacturers often use indigenous techniques where such
bulk protein solutions are frozen in smaller batches, in sacs,
pouches, jars and containers of various sizes and shapes. Freezing
is usually carried out in mechanical freezers. In many cases, the
manufacturer finds that the functional activity of a batch of
protein from the same mother solution is not the same following a
freeze and thaw cycle. Often, even batches of similar size and
freezing history differ from each other in protein activity. As
such, there is a need to understand what causes such non-uniformity
in protein activity during freeze-and-thaw and thereby devise more
efficient methods for the same.
[0004] A protein structure is quantified at three levels: primary,
secondary and tertiary. The secondary and tertiary structures are
the ones that are susceptible to changes in microenvironments and
ultimately cause the protein to change its conformation and lose
functional activity. At the molecular level, a protein will change
its conformation to acquire the lowest energy state. If the
microenvironment changes such that free energy of the protein in
its unfolded state is lower than the free energy of the protein in
its native state, then the protein would transition from its native
state and denature. See Transfusion Medicine and Hemotherapy, 2007,
34(4): 246-252. So any factor that can alter the free energy of the
protein can affect its stability, e.g. temperature, pressure, pH,
presence of co-solutes, salts, preservatives, and surfactants.
Hence, stabilization methods should aim at modifying the
thermodynamic state in the microenvironment of the protein.
Proteins experience several stresses during freezing and thawing.
Two of the most important stresses that occur are freeze
concentration and ice-induced denaturation.
[0005] Freeze concentration stresses. A protein changes its
configuration to conform to a minimum energy state. When the
microenvironment of the protein changes and water concentration
decreases surrounding the protein, the protein starts unfolding so
the inner hydrophobic groups can bind with organic solvents. This
causes deactivation of protein function. This is an equilibrium
process and occurs over long cooling times.
[0006] Ice-induced denaturation. Proteins may adsorb onto an ice
surface which leads to irreversible conformational changes.
[0007] These detrimental stresses can be minimized if cooling and
thawing is fast enough that the protein does not have enough time
to unfold and if no ice crystals are formed.
[0008] This equilibrium unfolding of the protein during freezing is
clearly a problem in terms of the storage and use of proteins. The
invention will freeze proteins by vitrification. According to the
vitrification mechanism, as a system approaches glassy state,
viscosity increases and all dynamic processes slow down. This
causes the protein in solution to become virtually immobilized, and
the protein denaturation rate is reduced (Pharm. Dev. Technol.
2007, 12(5): 505-23). As such, if the protein can be made to go
into the glassy state fast enough, it may not have time to unfold.
The retention of protein activity during thawing will also depend
upon a fast enough warming rate, and the invention is designed to
favor quick thawing with adequate mixing.
[0009] Previous methods of rapid cooling by introducing protein
droplets into liquid nitrogen resulted in fine dendritic ice
crystals that increased surface area for ice-induced denaturation.
The invention seeks to inhibit the formation of ice crystals by
reducing the time the solution spends between ice nucleation and
glass transition.
[0010] This is accomplished by:
Precooling the protein solution so that it approaches freezing
temperature. This will allow faster cooling on liquid nitrogen
contact. Adding agents that increase the glass transition
temperature. This will reduce the cooling required to achieve
glassy state. Using subcooled liquid nitrogen.
[0011] Subcooled liquid nitrogen provides very rapid cooling and
increased heat flux to precooled droplets.
[0012] Subcooled liquid nitrogen minimizes formation of nitrogen
vapor blanket around the droplet and provides more efficient
cooling than liquid nitrogen.
[0013] Subcooled liquid nitrogen minimizes formation of gaseous
nitrogen. Gaseous nitrogen, when formed, rises up as bubbles and
meets downcoming droplets to cause turbulent contact at air/liquid
interface which can damage proteins.
SUMMARY OF THE INVENTION
[0014] The invention provides for a method comprising feeding
droplets to a freezing medium thereby freezing the droplets and
forming pellets.
[0015] The droplets comprise a protein solution and the freezing
medium is a cryogen selected from the group consisting of liquid
nitrogen, oxygen, air and argon.
[0016] Stabilizers selected from the group consisting of sorbitol,
sucrose, trelose, and alenine may be added to the initial protein
solution as well as bulking agents and buffers. The bulking agents
are selected from the group consisting of glycine and mannitol and
the buffers are selected from the group consisting of sodium
citrate and sodium phosphate.
[0017] The protein solution may be pre-cooled to a temperature
range of -20.degree. C. to -45.degree. C. thereby bringing it in
temperature closer to the desired freezing medium temperature of
-80.degree. C.
[0018] The droplets are added to the freezing medium for 0.5 to 15
seconds and result in forming pellets or beads that are 0.5 to 15
millimeters in diameter. The now frozen pellets are separated from
said freezing medium and stored at temperatures of -80.degree. C.
and below.
[0019] The stored pellets may then be reconstituted by adding the
vitrified pellets to a heat transfer fluid. When the heat transfer
fluid is water, a reconstituted protein solution is formed and can
be recovered for an intended use.
[0020] Alternatively both methods for feeding and thawing can be
practiced in combination.
[0021] The invention seeks to minimize the time the protein
solution spends between ice-nucleation temperature and glass
transition temperature so that there is less time for ice crystals
to nucleate and grow. In clean environments, spontaneous ice
nucleation requires supercooling and usually occurs between
-20.degree. C. to -45.degree. C. Below -80.degree. C., ice crystal
formation is not favored so causing the system to transition fast
enough from below ice-nucleation temperature to -80.degree. C. can
minimize the ice crystal formation.
[0022] The method of free-thaw includes the following steps.
[0023] Cooling Method
Modify the microenvironment of protein to alter the glass
transition temperature by adding (a) stabilizers such as sorbitol,
sucrose, trelose, alenine, etc., (b) bulking agents such as
glycine, mannitol, etc. and (c) buffers such as sodium citrate,
sodium phosphate, etc. Pre-cool the protein solution to near but
not below ice nucleation temperature so that cold required to reach
-80.degree. C. is reduced. Droplets of pre-cooled protein solution
are introduced above or below surface of the subcooled liquid
nitrogen for 0.5 to 15 seconds, thus converting the droplets into
vitrified pellets or beads of 0.5 to 15 mm diameter. Separate the
pellets from the liquid nitrogen. Store vitrified pellets at
temperatures below -80.degree. C. or below.
Reconstituting/Thawing Vitrified Protein Pellets
[0024] Prepare the starting solution by warming a small number of
pellets in a jacketed stirred vessel. Provide a heat flux to the
solution by circulating heat transfer fluid in the jacket or via
internal coil. Continue adding the pellets slowly to the starting
solution to obtain the desired quantity of reconstituted protein
solution.
[0025] The cryogenic fluid that may be employed in the invention is
selected from the group consisting of liquid nitrogen, oxygen, air
and argon. The cryogenic fluid may not have to be subcooled should
the effects resulting from gas formation not being an issue. The
cryogenic fluid may be suitably processed such as by filtration
processes to produce sterile fluids.
[0026] The cryogenic fluid may be maintained sub cooled by
periodically subjecting to low pressure for short duration to cause
partial boil off of cryogen.
[0027] The protein solution can be introduced as drops or pellets
using any known device for generating droplets or pellets.
[0028] A variety of freeze-thaw arrangements are possible within
the scope of the present invention. Pellets can be frozen in batch
mode in a sieved vessel from which pellets can be collected at the
end of each batch, or in continuous mode using a conveyer belt or
other means. Glass transition temperatures are also possible.
The protein solution that can be frozen and thawed may be of any
type protein susceptible to freezing and thawing. The method of the
invention can be used at any stage during drug manufacture.
[0029] Alternatively, the methods of the invention could be
employed by compounds having similar properties to those of protein
solutions such as peptides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is a schematic of a freezing operation according to
the invention, particularly showing vitrifying pellets in liquid
nitrogen.
[0031] FIG. 2 is a schematic of a quick thawing process according
to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Turning to the figures, the methods of the invention are
shown in detail. FIG. 1 shows a vitrification process for
vitrifying protein pellets in liquid nitrogen. A protein solution
is fed through line 1 to a heat exchanger A which reduces the
temperature of the protein feed stream. A pre-cooled protein 2
solution having a temperature between -20.degree. C. and
-45.degree. C. is fed into line 3 so the amount of cold necessary
to reach -80.degree. C. is reduced. The combined pre-cooled protein
solution is fed through line 3 into droplet generator 4. The
droplets are introduced through line 3A into an immersion bath C
which contains sterile liquid nitrogen which is fed through line 4
into the immersion bath C.
[0033] The pre-cooled protein solution is dropped above or below
the surface of the subcooled liquid nitrogen for 0.5 to 15 seconds
through line 3A. The droplets are thus converted into vitrified
pellets or beads G having a diameter of 0.5 to 15 mm. The vitrified
pellets or beads G are carried along a conveyer belt E through
tunnel D where the vitrified pellets or beads G will collect in the
collection basin F. Gaseous nitrogen leaves the system through line
5. The recovered vitrified pellets or beads G can then be stored at
temperatures of -80.degree. C. or below.
[0034] FIG. 2 shows how the vitrified pellets or beads are
reconstituted. The vitrified pellets or beads are fed through line
6 into the jacketed stirring vessel H. The jacketed stirring vessel
H contains a stirring mechanism 7 and a heat transfer fluid such as
aqueous medium used in the formulation of the drug product. The
jacketed stirring vessel H is blanketed by a jacket which can
contain a heat transfer fluid such as water. Line 8 allows for warm
fluid to enter the jacket and line 9 allows for the warm fluid to
exit the jacket. The circulating fluid in the jacket provides a
heat flux to the heat transfer fluid and quickly thaws the
vitrified pellets or beads which can be recovered through line
10.
[0035] While this invention has been described with respect to
particular embodiments thereof, it is apparent that numerous other
forms and modifications of the invention will be obvious to those
skilled in the art. The appended claims in this invention generally
should be construed to cover all such obvious forms and
modifications which are within the true spirit and scope of the
invention.
* * * * *