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WO2013006224A1 - Methods for freezing and thawing proteins - Google Patents

Methods for freezing and thawing proteins Download PDF

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Publication number
WO2013006224A1
WO2013006224A1 PCT/US2012/033737 US2012033737W WO2013006224A1 WO 2013006224 A1 WO2013006224 A1 WO 2013006224A1 US 2012033737 W US2012033737 W US 2012033737W WO 2013006224 A1 WO2013006224 A1 WO 2013006224A1
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WIPO (PCT)
Prior art keywords
pellets
protein
freezing medium
droplets
freezing
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Ceased
Application number
PCT/US2012/033737
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French (fr)
Inventor
Naresh J. Suchak
Prerona Chakravarty
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Linde GmbH
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Linde GmbH
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1688—Processes resulting in pure drug agglomerate optionally containing up to 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Subcooled liquid nitrogen provides very rapid cooling and increased heat flux to precooled droplets.
  • Subcooled liquid nitrogen minimizes formation of nitrogen vapor blanket around the droplet and provides more efficient cooling than liquid nitrogen.
  • 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.
  • the invention provides for a method comprising feeding droplets to a freezing medium thereby freezing the droplets and forming pellets.
  • 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.
  • 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.
  • the protein solution may be pre-cooled to a temperature range of - 20°C to -45°C thereby bringing it in temperature closer to the desired freezing medium temperature of -80°C.
  • 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°C and below.
  • the stored pellets may then be reconstituted by adding the vitrified pellets to a heat transfer fluid.
  • a heat transfer fluid is water
  • reconstituted protein solution is formed and can be recovered for an intended use.
  • both methods for feeding and thawing can be practiced in combination.
  • the invention seeks to minimize the time the protein solution spends between ice-nuc!eation temperature and glass transition temperature so that thee is less time for ice crystals to nucleate and grow.
  • spontaneous ice nucleation requires supercooling and usually occurs between - 20°C to -45°C. Below -80°C, ice crystal formation is not favored so causing the system to transition fast enough from below ice-nucleation temperature to -80°C can minimize the ice crystal formation.
  • the method of free-thaw includes the following steps. [0023] Cooling Method
  • stabilizers such as sorbitol, sucrose, trelose, alenine, etc.
  • bulking agents such as glycine, mannitoi, etc.
  • buffers such as sodium citrate, sodium phosphate, etc.
  • 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.
  • 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.
  • the cryogenic fluid may be maintained sub cooled by periodically subjecting to low pressure for short duration to cause partial boil off of cryogen.
  • the protein solution can be introduced as drops or pellets using any known device for generating droplets or pellets.
  • 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.
  • the methods of the invention could be employed by compounds having similar properties to those of protein solutions such as peptides.
  • Figure 1 is a schematic of a freezing operation according
  • Figure 2 is a schematic of a quick thawing process according to the invention.
  • Figure 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-cooied protein 2 solution having a temperature between -2CTC and -45°C is fed into line 3 so the amount of cold necessary to reach -80°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.
  • 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°C or below.
  • 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 fiux to the heat transfer fluid and quickly thaws the vitrified pellets or beads which can be recovered through line 10.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

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.

Description

METHODS FOR FREEZING AND THAWING PROTEINS
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 dearly 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°C to -45°C thereby bringing it in temperature closer to the desired freezing medium temperature of -80°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°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-nuc!eation temperature and glass transition temperature so that thee is less time for ice crystals to nucleate and grow. In clean environments, spontaneous ice nucleation requires supercooling and usually occurs between - 20°C to -45°C. Below -80°C, ice crystal formation is not favored so causing the system to transition fast enough from below ice-nucleation temperature to -80°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, mannitoi, 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 °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°C or below.
[0024] Reconstituting/thawing vitrified protein pellets
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] Figure 1 is a schematic of a freezing operation according
invention, particularly showing vitrifying pellets in liquid nitrogen. [0031] Figure 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. Figure 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-cooied protein 2 solution having a temperature between -2CTC and -45°C is fed into line 3 so the amount of cold necessary to reach -80°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°C or below.
[0034] Figure 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 fiux 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.

Claims

Having thus described the invention, what we claim is:
1. A method comprising feeding droplets to a freezing medium thereby freezing said droplets and forming pellets.
2. The method as claimed in ciaim 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°C to -45°C.
11. The method as claimed in claim 1 wherein the temperature of said freezing medium is -80°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°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 fiuid 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°C to -45°C.
31 . The method as claimed in claim 21 wherein the temperature of said freezing medium is -80°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 peliets are separated from said freezing medium.
35. The method as claimed in claim 21 wherein said pellets are stored at temperatures of -80°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.
PCT/US2012/033737 2011-07-07 2012-04-16 Methods for freezing and thawing proteins Ceased WO2013006224A1 (en)

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