WO1993000807A1 - Method for stabilization of biomaterials - Google Patents

Method for stabilization of biomaterials Download PDF

Info

Publication number
WO1993000807A1
WO1993000807A1 PCT/US1992/005643 US9205643W WO9300807A1 WO 1993000807 A1 WO1993000807 A1 WO 1993000807A1 US 9205643 W US9205643 W US 9205643W WO 9300807 A1 WO9300807 A1 WO 9300807A1
Authority
WO
WIPO (PCT)
Prior art keywords
drying
protein
freezing
group
biomaterial
Prior art date
Application number
PCT/US1992/005643
Other languages
French (fr)
Inventor
John F. Carpenter
Original Assignee
Cryolife, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cryolife, Inc. filed Critical Cryolife, Inc.
Publication of WO1993000807A1 publication Critical patent/WO1993000807A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the invention described herein relates to a method of stabilizing biomaterials, especially labile proteins, during drying or freeze-drying using stabilizing components to achieve enhanced protection.
  • Freeze-drying processes have been used with limited degrees of success in protein preservation due to protein denaturation and/or inactivation caused by the freezing or sublimation steps. Even under conditions where the freezing phase of the process is not damaging (i.e., the protein can withstand freezing stress or a cryoprotectant is used) , the subsequent removal of water from the sample by sublimation of the frozen sample (drying phase) can lead to irreversible damage.
  • additives are also used to improve the stability of the protein during long- term storage of the dried product.
  • proteins are inherently stable against acute freeze-drying stress, and only need to be protected during the subsequent storage of the dried product.
  • Human growth hormone and ribonuclease A are examples of this type of protein.
  • the current invention relates to a method of employing solutes which stabilize labile proteins and other biomaterials during the acute stresses encountered during freeze- drying. Additionally, the compositions of the present invention enhance overall protein recovery after freeze- drying and storage of the dried product. The solute mixture that confers stability against acute stresses during freeze-drying also aids in long-term storage of the protein or other biomaterial.
  • Damage to freeze-dried proteins is manifested after rehydration, for example, as a loss of protein solubility, aggregation upon rehydration, loss of activity in appropriate bioassays (e.g., stimulated or depressed mitosis or activity of cells induced by growth factors, or antigen binding by an antibody) or in the case of enzymes, a loss of catalytic activity.
  • bioassays e.g., stimulated or depressed mitosis or activity of cells induced by growth factors, or antigen binding by an antibody
  • enzymes e.g., a loss of catalytic activity.
  • the latter parameter is an extremely sensitive measure of protein damage.
  • An enzyme that is fully soluble and has not aggregated may still display a loss of catalytic activity if it has undergone damaging conformational changes.
  • assays for characterizing catalytic activity are straightforward and are not subjected to the variability and vagaries of bioassays, such as those employed for hormones and growth factors.
  • enzymes are ideal model systems for studies on the stabilization of proteins during freeze drying.
  • One object of the present invention is to provide a method of stabilizing proteins against both the freezing and drying stresses encountered during lyophilization, preferably by employing a two-component system. Whereas component one or two when used alone may not provide adequate protection of the protein both during freezing and drying, when these components are used in combination, protection during freezing and drying is obtained. Thus with embodiments of this method, synergistic protection of the protein can be realized during freeze-drying, with component one providing primary stabilization against freezing and component two primarily protecting during the drying phase.
  • One advantage of the present invention is to obtain freeze- drying stabilization by employing solutes that would not normally be expected to protect labile proteins during freeze-drying.
  • An object of the present invention is to speed and simplify development of freeze-drying protocols for the stabilization of labile proteins.
  • An improved method for stabilizing biomaterials, especially labile proteins, during freeze- drying is disclosed, wherein stabilization is realized employing an additive system, preferably a two-component system.
  • one component may serve as a cryoprotectant to protect the protein against damage due to freezing, while conferring minimal protection during the subsequent drying phase.
  • Component two may be selected and employed to confer minimal protection during freezing, and when used alone affords no protection or is damaging to the protein during drying.
  • DETAILED DESCRIPTION Freeze-drying involves lyophilization and refers to the process by which an aqueous solution of a protein and additives is frozen, after which the water is removed by sublimation at low pressure. Subsequent rehydration of the dried protein preparation is typically necessary to use the protein and to assay the effect of the processing steps on protein activity.
  • the drying phase typically involves all stages of the sublimation process following freezing.
  • Excipients and stabilizing solutes or additives are compounds that are added to the protein solution or suspension (broadly referred to herein as a "protein solution") prior to freeze-drying to improve the stability of the protein to the freeze-drying cycle, and to improve the storage stability of the dried product.
  • the invention described herein relates to a method of employing preferably a two-component mixture of solutes to stabilize labile proteins against the stresses encountered by freeze-drying.
  • the method also aids in overall protein recovery after freeze-drying and storage of the dried product. That is, the solutes that confer stability against acute stresses also aid in the long-term storage of the protein.
  • Component one, used to stabilize the protein comprises a polymer which provides at least some cryoprotection for the protein.
  • the cryoprotectant is preferably a compound that has the capacity to protect the protein during freezing and subsequent thawing or during the freezing phase of a freeze-drying process. Some cryoprotectants also protect during the subsequent drying step of freeze-drying.
  • Preferred examples of polymers that can serve as cryoprotectants for proteins include: polyethylene glycol, polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll.
  • the polymers used herein can include compounds having a molecular weight of greater than about 500 daltons.
  • Polyethylene glycol is a preferred polymer useful as component one because it is commonly used during protein purification and thus it often present in solution with a protein to be freeze-dried. Polyethylene glycol is useful in that t prevents the protein from sticking to the surface of the container in which it is present. A preferred level of polyethylene glycol is from about 0.5 to about 20%(w/v) of the suspension.
  • Component two is a compound that when used alone may provide minimal if any protection for the protein during either freezing or drying, and may actually potentiate the protein damage when used alone. Examples of compounds that can be used as component two include:
  • sugars e.g., trehalose, lactose, sucrose, glucose, galactose, maltose, mannose and fructose
  • polyhydroxy alcohols e.g., mannitol, sorbitol and inositol
  • amino acids e.g., glycine, alanine, proline and lysine
  • methylamines e.g., trimethylamine-N- oxide, betaine and sarcosine.
  • Some compounds suitable for use as the second component protect freeze-dried proteins under certain conditions, such as at specific initial concentrations (e.g., the influence of trehalose on freeze-dried phosphofructokinase; Carpenter and Crowe, Biochemistry 28:3916-3922, 1989), whereas others appear to be virtually ineffective at protecting labile proteins during freeze-drying (e.g., the influence of glycine on freeze-dried phosphofructokinase; Carpenter et al., Biochem, Biophys. Acta, 923: 109-115, 1987) .
  • component two protects the protein during the drying phase of the freeze-drying process.
  • a preferred level is from about 1 to about 300 mM.
  • freeze-drying stabilization is not limited to sugars.
  • Compounds such as polyhydroxy alcohols, amino acids and methylamines are surprisingly effective at stabiLizing proteins during the drying phase.
  • component one can be comprised of one or more of the cryoprotectant solutes defined above.
  • component two can be comprised of one or mo:-e of the above defined solutes.
  • the invention described herein can also be used to stabilize other biological materials such as viruses or human, animal, or bacterial cells during freeze drying.
  • proteins such as a growth factors, hormones, antibodies, antigens, enzymes, clotting factors, structural proteins and complement factors can be combined with components one and two as described herein and subjected to freeze- drying. Specifically, such proteins would include:
  • CAMPATH-1, anti-TAC, etc. Polyclonal antibodies; Human IgM; and Human IgG.
  • a labile biomaterial such as a liposome, cell organelle, vaccine, virus, bacteria, eukaryotic cell, platelet, living-cell viral vaccine, other subcellular element, genetic material or other biomaterial can be freeze-dried according to the embodiments described herein.
  • a further embodiment of the present invention involves the use of the two component system described herein to stabilize proteins and other biomaterials against the detrimental effects of alternative means of drying, including spray drying or vacuum centrifugation.
  • EXAMPLE 1 Rabbit muscle lactate dehydrogenase (M isozyme, 99+% homogeneous) was purchased as a crystalline suspension in ammonium sulfate (Sigma; St. Louis, Mo) . Prior to each experiment the lactate dehydrogenase (“LDH”) was dialyzed (4°C) for several hours against 10 rriM potassium phosphate buffer (pH 7.5 at 23°C) . An aliquot of the stock enzyme was added to the appropriate preparation of polyethylene glycol (MW 8000) (prepared in the 10 mM potassium phosphate buffer) to a final LDH concentration of 25 g/ml and 10% (wt/vol) of polyethylene glycol (PEG) .
  • LDH lactate dehydrogenase
  • Samples of various concentrations of PEG were obtained by diluting the above mixture with a solution containing 25 ⁇ g/ml of LDH in the buffer alone.
  • 25 / -g/ml of LDH was prepared in the buffer containing 1% (wt/vol) PEG and 100 mM trehalose.
  • Various concentrations of trehalose were obtained by diluting this mixture with a buffer solution containing 25 /-g/ml LDH and 1% PEG without trehalose.
  • FREEZE-THA EXPERIMENTS A 75 ⁇ l aliquot of a given mixture was transferred to a 1.5 ml polypropylene Eppendorf test tube. A 10 ⁇ l sample was removed and assayed for LDH catalytic activity. This served as the pre-treatment control value. LDH catalytic activity was measured at 25°C.
  • the 2.0 ml reaction mixture contained 80 mM Tris/Hcl buffer (pH 7.5), 100 mM KC1, 2 mM pyruvate, and 0.15 mM NADH. The remaining 65 ⁇ l aliquot in the test tube was frozen by immersion into liquid nitrogen for 30 seconds. Samples were then thawed at room temperature, immediately mixed and assayed for residual activity. The results are expressed as the percentage of the pre- treatment activity recovered after thawing.
  • Fig. 1A demonstrate that PEG completely protects LDH from inactivation during freeze- thawing. Essentially full activity is recovered at all PEG concentrations tested. By contrast, PEG provides essentially no stabilization to the enzyme during freeze- drying. These results indicate that PEG is an ideal cryoprotectant for proteins, but that when used alone it does not have the capacity to stabilize freeze-dried proteins.
  • trehalose either alone or in combination with 1% PEG
  • Fig. IB The influence of trehalose (either alone or in combination with 1% PEG) on LDH stability during freeze- thawing is shown in Fig. IB.
  • the combination of trehalose and 1% PEG provides almost full protection for the enzyme during freeze-thawing.
  • trehalose alone provides almost no protection during freeze-thawing.
  • Fig. 1C shows the effect of trehalose alone and trehalose in combination with 1% PEG on the stability of LDH during freeze-drying.
  • Trehalose alone provides no stabilization and actually appears to cause additional damage to the enzyme during freeze-drying.
  • trehalose is used in combination with 1% PEG (which alone provides only minimal protection) almost 100% of the LDH activity is retained after freeze-drying.
  • PEG is the solute comprising component one, which fully protects against damage induced by freezing, while conferring minimal if any protection during the subsequent drying phase.
  • Trehalose is the solute comprising component two, which confers minimal protection during freezing, and when used alone affords no protection and/or is damaging during drying.
  • trehalose provides almost complete protection during the drying phase.
  • Fig. 2A The influence of lactose (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is shown in Fig. 2A.
  • the combination of lactose and 1% PEG provides protection for the enzyme during freeze-thawing.
  • lactose alone provides almost no protection during freeze-thawing.
  • Fig. 2B shows the effect of lactose alone and lactose in combination with 1% PEG on the stability of LDH during freeze-drying. Lactose alone provides no stabilization and appears to cause additional damage to the enzyme during freeze-drying.
  • lactose is used in combination with 1% PEG (which alone provides only minimal protection) the LDH is stabilized during freeze-drying.
  • Synergistic protection by the two components is realized during freeze-drying, with PEG providing stabilization against damage during freezing and lactose protecting during the drying phase.
  • EXAMPLE 3 Samples were prepared and treated as described in Example 1, except that glucose was substituted for trehalose and served as component two.
  • the influence of glucose (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is documented in Fig. 3A.
  • the combination of glucose and 1% PEG provides protection for the enzyme during freeze-thawing.
  • Fig. 3B shows the effects of glucose alone and glucose in combination with 1% PEG on the stability of LDH during freeze-drying.
  • Glucose alone provides no stabilization and actually appears to cause additional damage to the enzyme during freeze-drying.
  • glucose is used in combination with 1% PEG (which alone provide only minimal protection) the LDH is stabilized during freeze-drying.
  • Fig. 4A The influence of glycine (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is shown in Fig. 4A.
  • the combination of glycine and 1% PEG provides protection for the enzyme during freeze-thawing.
  • Fig. 4B shows the effects of glycine alone and in combination with 1% PEG on the stability of LDH during freeze-drying.
  • Glycine alone provides no stabilization and actually fosters additional damage to the enzyme during freeze-drying.
  • glycine is used in combination with 1% PEG (which alone provide only minimal protection) the LDH is greatly stabilized during freeze-drying.
  • Synergistic protection is realized during freeze-drying, with PEG providing stabilization against damage during freezing and glycine protection during the drying phase.
  • EXAMPLE 5 Samples were prepared and treated as described in Example 1. Mannitol was substituted for trehalose and served as component two. The influence of mannitol (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is shown in Fig. 5A. The combination of mannitol and 1% PEG provides protection for the enzyme during freeze-thawing. By contrast, mannitol alone provides almost no protection during freeze-thawing. Fig. 5B shows the effects of mannitol alone and in combination with 1% PEG on stability of LDH during freeze-drying. Mannitol alone provides no stabilization and actually fosters additional damage to the enzyme during freeze-drying. By contrast, when mannitol is used in combination with 1% PEG (which alone provide only minimal protection) the LDH is greatly stabilized during freeze-drying.

Abstract

A method for stabilizing biomaterials during lyophilization using a two component additive. One component serves as a cryoprotectant such as polyethylene glycol, polyvinyl pyrrolidone, hydroxyethyl starch, dextran or ficoll which protects the protein during freezing. The second component such as sugars, polyhydroxy alcohols, amino acids or methylamine protects the biomaterial during drying.

Description

METHOD FOR STABILIZATION OF BIO ATERIALS
BACKGROUND
The invention described herein relates to a method of stabilizing biomaterials, especially labile proteins, during drying or freeze-drying using stabilizing components to achieve enhanced protection. Freeze-drying processes have been used with limited degrees of success in protein preservation due to protein denaturation and/or inactivation caused by the freezing or sublimation steps. Even under conditions where the freezing phase of the process is not damaging (i.e., the protein can withstand freezing stress or a cryoprotectant is used) , the subsequent removal of water from the sample by sublimation of the frozen sample (drying phase) can lead to irreversible damage.
Carpenter et al, Biochem. Biophys. Acta 923:109-115, 1987 relates to cryoprotectants for proteins which do not stabilize the same proteins during freeze- drying. Additives which have the capacity to protect labile proteins during both freezing and drying can fail to provide freeze-drying stabilization if used under inappropriate conditions (e.g., too high of an initial concentration of additive used; Carpenter and Crowe, Biochemistry, 28:3916-3922, 1989).
Freeze-drying preservation and the uncertainties involved with protein stabilization during freeze-drying make the combined effects of ciryopreservation and protein stabilization during freeze- drying largely empirical. Clearly there is a need for a reliable, effective and predictable method of stabilizing proteins during freeze-drying. This is because any protein product that is insufficiently stable in aqueous solution during distribution, storage, and use will desirably be freeze-dried (lyophilized) . Freeze-dried protein products are seldom composed of pure protein. This is due to the instability of proteins under the stresses encountered during freezing and drying. Stabilizing solutes (excipients) are added to proteins to improve their resistance to damage induced by the acute stresses encountered during freeze- drying and rehydration. In addition, additives are also used to improve the stability of the protein during long- term storage of the dried product. In some cases proteins are inherently stable against acute freeze-drying stress, and only need to be protected during the subsequent storage of the dried product. Human growth hormone and ribonuclease A are examples of this type of protein.
Proteins must withstand the acute effects of freeze-drying and rehydration for storage stability to become significant. It is to this aspect of the art that the current invention is directed. Namely, the invention described herein relates to a method of employing solutes which stabilize labile proteins and other biomaterials during the acute stresses encountered during freeze- drying. Additionally, the compositions of the present invention enhance overall protein recovery after freeze- drying and storage of the dried product. The solute mixture that confers stability against acute stresses during freeze-drying also aids in long-term storage of the protein or other biomaterial.
Damage to freeze-dried proteins is manifested after rehydration, for example, as a loss of protein solubility, aggregation upon rehydration, loss of activity in appropriate bioassays (e.g., stimulated or depressed mitosis or activity of cells induced by growth factors, or antigen binding by an antibody) or in the case of enzymes, a loss of catalytic activity. The latter parameter is an extremely sensitive measure of protein damage. An enzyme that is fully soluble and has not aggregated may still display a loss of catalytic activity if it has undergone damaging conformational changes. In addition, assays for characterizing catalytic activity are straightforward and are not subjected to the variability and vagaries of bioassays, such as those employed for hormones and growth factors. Hence enzymes are ideal model systems for studies on the stabilization of proteins during freeze drying.
One object of the present invention is to provide a method of stabilizing proteins against both the freezing and drying stresses encountered during lyophilization, preferably by employing a two-component system. Whereas component one or two when used alone may not provide adequate protection of the protein both during freezing and drying, when these components are used in combination, protection during freezing and drying is obtained. Thus with embodiments of this method, synergistic protection of the protein can be realized during freeze-drying, with component one providing primary stabilization against freezing and component two primarily protecting during the drying phase. One advantage of the present invention is to obtain freeze- drying stabilization by employing solutes that would not normally be expected to protect labile proteins during freeze-drying.
An object of the present invention is to speed and simplify development of freeze-drying protocols for the stabilization of labile proteins. These and other objectives will be apparent to those of ordinary skill in the art from the teachings herein.
SUMMARY OF THE INVENTION An improved method for stabilizing biomaterials, especially labile proteins, during freeze- drying is disclosed, wherein stabilization is realized employing an additive system, preferably a two-component system. In embodiments of the invention one component may serve as a cryoprotectant to protect the protein against damage due to freezing, while conferring minimal protection during the subsequent drying phase. Component two may be selected and employed to confer minimal protection during freezing, and when used alone affords no protection or is damaging to the protein during drying. Thus, with this method, protection is realized during freeze-drying, with component one providing stabilization against damage due to freezing and component two protecting during the drying phase when it is employed in the solute mixture of the present inventio .
BRIEF DESCRIPTION OF THE FIGURES
The invention is described in detail in conjunction with the accompanying drawings. Figure 1.
A. Comparison of the influence of polyethylene glycol on the stability of lactate dehydrogenase ("LDH") during freeze-thawing versus during freeze-drying and rehydration.
B. Comparison of the influence of trehalose alone versus trehalose in combination with 1% (wt/vol.) polyethylene glycol on the stability of lactate dehydrogenase during freeze-thawing.
C. Comparison of the influence of trehalose alone versus trehalose in combination with 1% (wt/vol.) polyethylene glycol on the stability of lactate dehydrogenase during freeze-drying and rehydration. Figure 2.
A. Comparison of the influence of lactose alone versus lactose in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-thawing.
B. Comparison of the influence of lactose alone versus lactose in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-drying and rehydration. Figure 3.
A. Comparison of the influence of glucose alone versus glucose in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-thawing.
B. Comparison of the influence of glucose alone versus glucose in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-drying and rehydration. Figure 4.
A. Comparison of the influence of glycine alone versus glycine in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-thawing.
B. Comparison of the influence of glycine alone versus glycine in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-drying and rehydration. Figure 5.
A. Comparison of the influence of mannitol alone versus mannitol in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-thawing.
B. Comparison of the influence of mannitol alone versus mannitol in combination with 1% (wt/vol) polyethylene glycol on the stability of lactate dehydrogenase during freeze-drying and rehydration.
DETAILED DESCRIPTION Freeze-drying involves lyophilization and refers to the process by which an aqueous solution of a protein and additives is frozen, after which the water is removed by sublimation at low pressure. Subsequent rehydration of the dried protein preparation is typically necessary to use the protein and to assay the effect of the processing steps on protein activity. The drying phase typically involves all stages of the sublimation process following freezing.
Excipients and stabilizing solutes or additives are compounds that are added to the protein solution or suspension (broadly referred to herein as a "protein solution") prior to freeze-drying to improve the stability of the protein to the freeze-drying cycle, and to improve the storage stability of the dried product. The invention described herein relates to a method of employing preferably a two-component mixture of solutes to stabilize labile proteins against the stresses encountered by freeze-drying. However, the method also aids in overall protein recovery after freeze-drying and storage of the dried product. That is, the solutes that confer stability against acute stresses also aid in the long-term storage of the protein.
Component one, used to stabilize the protein, comprises a polymer which provides at least some cryoprotection for the protein. The cryoprotectant is preferably a compound that has the capacity to protect the protein during freezing and subsequent thawing or during the freezing phase of a freeze-drying process. Some cryoprotectants also protect during the subsequent drying step of freeze-drying. Preferred examples of polymers that can serve as cryoprotectants for proteins include: polyethylene glycol, polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll. The polymers used herein can include compounds having a molecular weight of greater than about 500 daltons.
Polyethylene glycol is a preferred polymer useful as component one because it is commonly used during protein purification and thus it often present in solution with a protein to be freeze-dried. Polyethylene glycol is useful in that t prevents the protein from sticking to the surface of the container in which it is present. A preferred level of polyethylene glycol is from about 0.5 to about 20%(w/v) of the suspension.
Although thawing is not typically a step in the freeze-drying process, by thawing a frozen sample one can assess the effects of the freezing step, independent of a subsequent drying step, on the stability of the protein. Component two is a compound that when used alone may provide minimal if any protection for the protein during either freezing or drying, and may actually potentiate the protein damage when used alone. Examples of compounds that can be used as component two include:
(1) sugars, (e.g., trehalose, lactose, sucrose, glucose, galactose, maltose, mannose and fructose) ; (2) polyhydroxy alcohols (e.g., mannitol, sorbitol and inositol) ;
(3) amino acids (e.g., glycine, alanine, proline and lysine) ; and
(4) methylamines (e.g., trimethylamine-N- oxide, betaine and sarcosine) .
Some compounds suitable for use as the second component protect freeze-dried proteins under certain conditions, such as at specific initial concentrations (e.g., the influence of trehalose on freeze-dried phosphofructokinase; Carpenter and Crowe, Biochemistry 28:3916-3922, 1989), whereas others appear to be virtually ineffective at protecting labile proteins during freeze-drying (e.g., the influence of glycine on freeze-dried phosphofructokinase; Carpenter et al., Biochem, Biophys. Acta, 923: 109-115, 1987) . However, when used in combination with component one, component two protects the protein during the drying phase of the freeze-drying process.
It has been found that compounds having substituents, e.g. hydroxyl, amino or substituted amines, capable of hydrogen bonding to the protein are useful as component two in the present invention. While not wishing to be bound by this theory, it may be that such compounds may serve to replace water which is removed from the protein during drying, and thereby prevent the protein from aggregating, refolding, or undergoing some other transformation that reduces its activity.
When a sugar is used as component two, a preferred level is from about 1 to about 300 mM. One advantage of the present invention, is that when a polymeric cryoprotectant is used as component one, however, freeze-drying stabilization is not limited to sugars. Compounds such as polyhydroxy alcohols, amino acids and methylamines are surprisingly effective at stabiLizing proteins during the drying phase.
It should be apparent to one skilled in the art that during the practice of the present invention, component one can be comprised of one or more of the cryoprotectant solutes defined above. Similarly, component two can be comprised of one or mo:-e of the above defined solutes. In addition, one skilled in the art will recognize that the invention described herein can also be used to stabilize other biological materials such as viruses or human, animal, or bacterial cells during freeze drying. In embodiments of the present invention, proteins, such as a growth factors, hormones, antibodies, antigens, enzymes, clotting factors, structural proteins and complement factors can be combined with components one and two as described herein and subjected to freeze- drying. Specifically, such proteins would include:
Erythropoietin; Factor VIII; G-Colony Stimulating Factor; GM-Colony Stimulating Factor, Antithrombin III; insulin; Epidermal Growth Factor; Acidic Fibroblast Growth Factor; Basic Fibroblast Growth Factor; Interleukins (IL 1 alpha, IL 1 beta, IL 2, IL 3, IL 4, IL 6, IL 7, IL 8, IL 9, IL 10) ; Tumor Necrosis Factor-alpha; Tumor Necrosis Factor- beta; Interferon-alpha; Interferon-gamma; Transforming Growth Factor-beta; Tissue Plasminogen Activator; Platelet-derived Growth Factor; Urokinase; Streptokinase; Peroxidase; RNA polymerase; T7 DNA polymerase; Taq DNA polymerase; Fibrinogen; Thrombin; Alcohol dehydrogenase; Alkaline phosphatase; Arginase; Ascorbate oxidase;
Cholesterol esterase; Cholinesterase; Collagenase; DNase I; DNase II; Enterokinase; Glucose-6-phosphate dehydrogenase; Glucose oxidase; Glucose Isomerase; Glutamate dehydrogenase; Glyceraldehyde-3-phosphate dehydrogenase; Hexokinase; Lactate Dehydrogenase; Malate dehydrogenase; PEP carboxylase; RNase A; Soybean trypsin inhibitor; Urease; Xanthine oxidase; Superoxide dismutase; Fibronectin; Restriction endonucleases; Reverse transcriptase, AMV; Reverse transcriptase, M- MuLV; Monoclonal antibodies: OKT3, HA-1A, BMA 031,
CAMPATH-1, anti-TAC, etc.; Polyclonal antibodies; Human IgM; and Human IgG.
Similarly, a labile biomaterial such as a liposome, cell organelle, vaccine, virus, bacteria, eukaryotic cell, platelet, living-cell viral vaccine, other subcellular element, genetic material or other biomaterial can be freeze-dried according to the embodiments described herein.
An assay of structural or functional integrity after rehydration would indicate stabilization of the protein or other biomaterial over that which would occur had the two component system not been used. Furthermore, the presence of the additives would increase the stability of a protein or other biomaterial once it has been rehydrated.
A further embodiment of the present invention involves the use of the two component system described herein to stabilize proteins and other biomaterials against the detrimental effects of alternative means of drying, including spray drying or vacuum centrifugation.
EXAMPLE 1 Rabbit muscle lactate dehydrogenase (M isozyme, 99+% homogeneous) was purchased as a crystalline suspension in ammonium sulfate (Sigma; St. Louis, Mo) . Prior to each experiment the lactate dehydrogenase ("LDH") was dialyzed (4°C) for several hours against 10 rriM potassium phosphate buffer (pH 7.5 at 23°C) . An aliquot of the stock enzyme was added to the appropriate preparation of polyethylene glycol (MW 8000) (prepared in the 10 mM potassium phosphate buffer) to a final LDH concentration of 25 g/ml and 10% (wt/vol) of polyethylene glycol (PEG) . Samples of various concentrations of PEG were obtained by diluting the above mixture with a solution containing 25 μg/ml of LDH in the buffer alone. In order to test a two-component system in which PEG served as component one and trehalose served as component two, 25 /-g/ml of LDH was prepared in the buffer containing 1% (wt/vol) PEG and 100 mM trehalose. Various concentrations of trehalose were obtained by diluting this mixture with a buffer solution containing 25 /-g/ml LDH and 1% PEG without trehalose.
FREEZE-THA EXPERIMENTS A 75 μl aliquot of a given mixture was transferred to a 1.5 ml polypropylene Eppendorf test tube. A 10 μl sample was removed and assayed for LDH catalytic activity. This served as the pre-treatment control value. LDH catalytic activity was measured at 25°C. The 2.0 ml reaction mixture contained 80 mM Tris/Hcl buffer (pH 7.5), 100 mM KC1, 2 mM pyruvate, and 0.15 mM NADH. The remaining 65 μl aliquot in the test tube was frozen by immersion into liquid nitrogen for 30 seconds. Samples were then thawed at room temperature, immediately mixed and assayed for residual activity. The results are expressed as the percentage of the pre- treatment activity recovered after thawing.
FREEZE-DRYING EXPERIMENTS
Triplicate 75 μl aliquots (which were identical to the sample used for freeze-thawing) were placed into Eppendorf test tubes. The samples were frozen in liquid nitrogen and then placed on a VirTis lyophilizer for a minimum of 16 hours at 15-40 mTorr. The freeze-dried samples were rehydrated in distilled water, and residual LDH activity was assayed. The results are expressed as the percentage of the pre-treatment activity recovered after rehydration.
The results in Fig. 1A demonstrate that PEG completely protects LDH from inactivation during freeze- thawing. Essentially full activity is recovered at all PEG concentrations tested. By contrast, PEG provides essentially no stabilization to the enzyme during freeze- drying. These results indicate that PEG is an ideal cryoprotectant for proteins, but that when used alone it does not have the capacity to stabilize freeze-dried proteins.
The influence of trehalose (either alone or in combination with 1% PEG) on LDH stability during freeze- thawing is shown in Fig. IB. The combination of trehalose and 1% PEG provides almost full protection for the enzyme during freeze-thawing. By contrast, trehalose alone provides almost no protection during freeze-thawing.
Fig. 1C shows the effect of trehalose alone and trehalose in combination with 1% PEG on the stability of LDH during freeze-drying. Trehalose alone provides no stabilization and actually appears to cause additional damage to the enzyme during freeze-drying. In contrast, when trehalose is used in combination with 1% PEG (which alone provides only minimal protection) almost 100% of the LDH activity is retained after freeze-drying.
These results support synergistic stabilization of proteins during freeze-drying. In this example, PEG is the solute comprising component one, which fully protects against damage induced by freezing, while conferring minimal if any protection during the subsequent drying phase. Trehalose is the solute comprising component two, which confers minimal protection during freezing, and when used alone affords no protection and/or is damaging during drying. Thus, when either component is used alone there is at best minimal stabilization of labile proteins during freeze-drying. Surprisingly, however, when used in combination with PEG, trehalose provides almost complete protection during the drying phase. Thus, with this method synergistic protection is realized during freeze- drying. EXAMPLE 2
Samples were prepared and treated as described in Exa pLe 1, except that lactose was substituted for trehalose and used as component two. The influence of lactose (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is shown in Fig. 2A. The combination of lactose and 1% PEG provides protection for the enzyme during freeze-thawing. By contrast, lactose alone provides almost no protection during freeze-thawing. Fig. 2B shows the effect of lactose alone and lactose in combination with 1% PEG on the stability of LDH during freeze-drying. Lactose alone provides no stabilization and appears to cause additional damage to the enzyme during freeze-drying. By contrast, when lactose is used in combination with 1% PEG (which alone provides only minimal protection) the LDH is stabilized during freeze-drying.
Synergistic protection by the two components is realized during freeze-drying, with PEG providing stabilization against damage during freezing and lactose protecting during the drying phase.
EXAMPLE 3 Samples were prepared and treated as described in Example 1, except that glucose was substituted for trehalose and served as component two. The influence of glucose (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is documented in Fig. 3A. The combination of glucose and 1% PEG provides protection for the enzyme during freeze-thawing.
Fig. 3B shows the effects of glucose alone and glucose in combination with 1% PEG on the stability of LDH during freeze-drying. Glucose alone provides no stabilization and actually appears to cause additional damage to the enzyme during freeze-drying. By contrast, when glucose is used in combination with 1% PEG (which alone provide only minimal protection) the LDH is stabilized during freeze-drying.
Synergistic protection by the two components is therefore realized during freeze-drying, with PEG providing stabilization against damage during freezing and glucose protecting during the drying phase. EXAMPLE 4
Samples were prepared and treated as described in Example 1, except that glycine was substituted for trehalose as component 2. The influence of glycine (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is shown in Fig. 4A. The combination of glycine and 1% PEG provides protection for the enzyme during freeze-thawing. By contrast, glycine alone provides almost no protection during freeze- thawing. Fig. 4B shows the effects of glycine alone and in combination with 1% PEG on the stability of LDH during freeze-drying. Glycine alone provides no stabilization and actually fosters additional damage to the enzyme during freeze-drying. By contrast, when glycine is used in combination with 1% PEG (which alone provide only minimal protection) the LDH is greatly stabilized during freeze-drying.
Synergistic protection is realized during freeze-drying, with PEG providing stabilization against damage during freezing and glycine protection during the drying phase.
EXAMPLE 5 Samples were prepared and treated as described in Example 1. Mannitol was substituted for trehalose and served as component two. The influence of mannitol (either alone or in combination with 1% PEG) on LDH stability during freeze-thawing is shown in Fig. 5A. The combination of mannitol and 1% PEG provides protection for the enzyme during freeze-thawing. By contrast, mannitol alone provides almost no protection during freeze-thawing. Fig. 5B shows the effects of mannitol alone and in combination with 1% PEG on stability of LDH during freeze-drying. Mannitol alone provides no stabilization and actually fosters additional damage to the enzyme during freeze-drying. By contrast, when mannitol is used in combination with 1% PEG (which alone provide only minimal protection) the LDH is greatly stabilized during freeze-drying.
Synergistic protection by the two components is realized during freeze-drying, with PEG (component 1) providing stabilization against damage during freezing and -mannitol (component 2) protecting during the drying phase.

Claims

1. A method for stabilizing a protein during freezing and drying of a solution of the protein comprising freezing and drying the protein solution in the presence of polyethylene glycol and a sugar.
2. A method according to claim 1 wherein the sugar is selected from the group consisting of trehalose, lactose, sucrose, glucose, galactose, maltose, mannose and fructose.
3. A method according to claim 2 wherein said polyethylene glycol is present in the protein solution at a level of about 0.5 to about 20% weight/volume of the solution and said sugar is present in the protein solution at a level of from about 1 to about 300 mM.
4. A method for stabilizing a protein during freezing and drying of a solution of the protein comprising freezing and drying the protein solution in the presence of polyethylene glycol and a polyhydroxy alcohol.
5. A method according to claim 4 wherein the polyhydroxy alcohol is selected from the group consisting of mannitol, sorbitol and inositol.
6. A method for stabilizing a protein during freezing and drying of a solution of the protein comprising freezing and drying the protein solution in the presence of polyethylene glycol and at least one amino acid.
7. A method according to claim 6 wherein the amino acid is selected from the group consisting of glycine, alanine, proline and lysine.
8. A method for stabilizing a protein during freezing and drying of a solution of the protein comprising freezing and drying the protein solution in the presence of polyethylene glycol and a methylamine.
9. A method according to claim 8 wherein the methylamine is selected from the group consisting of trimethylamine-N-oxide, betaine and sarcosine.
10. A method for stabilizing a protein during freezing and drying of a suspension of the protein comprising freezing and drying the protein solution in the presence of a cryoprotectant and at least one further compound selected from the group consisting of sugars, polyhydroxy alcohols, amino acids and methylamines, wherein neither said cryoprotectant nor said further compound when used alone is capable of stabilizing said protein during freeze drying.
11. A method according to claim 10 wherein said cryoprotectant is selected from the group consisting of polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll.
12. A method for stabilizing a protein during freezing and drying of a solution of the protein comprising freezing and drying the protein solution in the presence of a cryoprotectant and at least one further compound capable of hydrogen bonding to the protein.
13. A method according to claim 12 wherein said cryoprotectant is selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll.
14. A method according to claim 1 wherein said freezing and drying comprises freezing the protein solution and thereafter drying the solution by sublimation.
15. A method for stabilizing a biomaterial during drying of a solution of the biomaterial comprising drying the biomaterial solution in the presence of a cryoprotectant selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll and at least one second component selected from the group consisting of sugars, amino acids, polyhydroxy alcohols and methylamines.
16. A method according to claim 15 wherein said biomaterial is selected from the group consisting of liposomes, cell organelles, vaccines, viruses, bacteria, eukaryotic cells, platelets, living-cell viral vaccines, other subcellular elements, and genetic materials.
17. A composition suitable for freezing and thereafter drying a biomaterial comprising the biomaterial, polyethylene glycol and a sugar.
18. A composition according to claim 17 wherein said biomaterial is selected from the group consisting of liposomes, cell organelles, vaccines, viruses, bacteria, eukaryotic cells, platelets, living-cell viral vaccines, other subcellular elements, and genetic materials.
19. A composition according to claim 17 wherein said biomaterial is a protein.
20. A composition according to claim 19 wherein said protein is a growth factor, hormone, antibody, antigen, enzyme, clotting factor, structural protein or complement factor.
21. A composition according to claim 19 wherein said protein is selected from the group consisting of Erythropoietin; Factor VIII; G-Colony Stimulating Factor; GM-Colony Stimulating Factor, Antithro bin III; Insulin; Epidermal Growth Factor; Acidic Fibroblast Growth Factor; Basic Fibroblast Growth Factor; Interleukin (IL 1 alpha, IL 1 beta, IL 2, IL 3, IL 4, IL 6, IL 7, IL 8, IL 9, IL 10) ; Tumor Necrosis Factor-alpha; Tumor Necrosis Factor- beta; Interferon-alpha; Interferon-gamma; Transforming Growth Factor-beta; Tissue Plasminogen Activator; Platelet-derived Growth Factor; Urokinase; Streptokinase; Peroxidase; RNA polymerase; T7 DNA polymerase; Taq DNA polymerase; Fibrinogen; Thrombin; Alcohol dehydrogenase; Alkaline phosphatase; Arginase; Ascorbate oxidase; Cholesterol esterase; Cholinesterase; Collagenase; DNase I; DNase II; Enterokinase; Glucose-6-phosphate dehydrogenase; Glucose oxidase; Glucose Isomerase; Glutamate dehydrogenase; Glyceraldehyde-3-phosphate dehydrogenase; Hexokinase; Lactate dehydrogenase; Malate dehydrogenase; PEP carboxylase; RNase A; Soybean trypsin inhibitor; Urease; Xanthine oxidase; Superoxide dismutase; Fibronectin; Restriction endonucleases; Reverse transcriptase, AMV; Reverse transcriptase, M- MuLV; Monoclonal antibodies: 0KT3, HA-1A, BMA 031, CAMPATΗ-1, anti-TAC, etc.; Polyclonal antibodies; Human IgM; and Human IgG.
22. A composition according to claim 17 wherein the sugar is selected from the group consisting of trehalose, lactose, sucrose, glucose, galactose, maltose, mannose and fructose.
23. A composition according to claim 22 comprising about 0.5 to about 20% (w/v) polyethylene glycol and about 1 to about 300 mM sugar.
24. A composition suitable for freezing and thereafter drying a biomaterial comprising the biomaterial, polyethylene glycol and a polyhydroxy alcohol.
25. A composition according to claim 24 wherein said polyhydroxy alcohol is selected from the group consisting of mannitol, sorbitol and inositol.
26. A composition suitable for freezing and thereafter drying a biomaterial comprising the biomaterial, polyethylene glycol and at least one amino acid.
27. A composition according to claim 26 wherein said amino acid is selected from the group consisting of glycine, alanine, proline and lysine.
28. A composition suitable for freezing and thereafter drying a biomaterial comprising the biomaterial, polyethylene glycol and a methylamine.
29. A composition according to claim 28 wherein said methylamine is selected from the group consisting of trimethylamine-N-oxide, betaine and sarcosine.
30. A composition suitable for freezing and thereafter drying a biomaterial comprising the biomaterial, a cryoprotectant and a further compound selected from the group consisting of sugars, polyhdroxy alcohols, amino acids and methylamines.
31. A composition according to claim 30 wherein said cryoprotectant is selected from the group consisting of polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll.
32. A composition suitable for freezing and thereafter drying a protein comprising the protein, a cryoprotectant and at least one further compound capable of hydrogen bonding to the protein.
33. A composition according to claim 32 wherein said cryoprotectant is selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, hydroxyethyl starch, dextran and ficoll.
PCT/US1992/005643 1991-07-03 1992-07-02 Method for stabilization of biomaterials WO1993000807A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US72559391A 1991-07-03 1991-07-03
US725,593 1991-07-03

Publications (1)

Publication Number Publication Date
WO1993000807A1 true WO1993000807A1 (en) 1993-01-21

Family

ID=24915184

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/005643 WO1993000807A1 (en) 1991-07-03 1992-07-02 Method for stabilization of biomaterials

Country Status (2)

Country Link
AU (1) AU2309692A (en)
WO (1) WO1993000807A1 (en)

Cited By (99)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994013135A1 (en) * 1992-12-04 1994-06-23 Development Biotechnological Processes S.N.C. Di Pelliccia Maria Teresa Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial sheets
BE1006379A3 (en) * 1992-11-26 1994-08-09 Raymond Gilles Method for preserving living cells, groups of cells and/or derivatives ofsaid cells by means of freeze-drying
US5424204A (en) * 1993-08-26 1995-06-13 Kyowa Medex Co., Ltd. Method for stabilizing glucose 6-phosphate dehydrogenase with hydroxylamines, aldehyde scavengers, dimethylthiocarbamoyl chloride or 2-(2-aminoethylamino) ethanol
DE19503685A1 (en) * 1995-01-30 1996-08-01 Invitek Gmbh Storage-stable reaction mixt. contg. many enzymes, reactants and stabilisers
EP0726310A1 (en) * 1995-02-10 1996-08-14 Gen-Probe Incorporated Stabilized enzyme compositons for nucleic acid amplification
US5580856A (en) * 1994-07-15 1996-12-03 Prestrelski; Steven J. Formulation of a reconstituted protein, and method and kit for the production thereof
WO1996041870A1 (en) * 1995-06-08 1996-12-27 Societa' Cooperativa Centro Ricerche Poly-Tech A Responsabilita' Limitata Hydrosoluble compositions of stabilized collagenase and the process for their preparation
US5602023A (en) * 1992-03-24 1997-02-11 Csatary; Laszlo K. Pharmaceutical product containing live, stabilized virus for the therapy of viral and malignant diseases and process for preparing the same
WO1998000530A1 (en) * 1996-07-03 1998-01-08 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
WO1998006421A1 (en) * 1996-08-16 1998-02-19 Cancer Treatments International Therapeutic compositions for treatment of cancer
EP0833667A1 (en) * 1995-06-07 1998-04-08 The Regents Of The University Of California Stabilization of polynucleotide complexes
EP0836645A1 (en) * 1995-06-09 1998-04-22 The Regents Of The University Of California Dry powder formulations of polynucleotide complexes
WO1998022136A2 (en) 1996-11-19 1998-05-28 Roche Diagnostics Gmbh Stable lyophilized pharmaceutical substances from monoclonal or polyclonal antibodies
EP0868916A2 (en) * 1997-03-04 1998-10-07 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Reduction inhibitory agent for active-oxygen eliminating activity
EP0875253A2 (en) * 1997-05-02 1998-11-04 Seikagaku Corporation Chondroitinase compositions
US5879875A (en) * 1996-06-14 1999-03-09 Biostore New Zealand Compositions and methods for the preservation of living tissues
WO1999045966A1 (en) * 1998-03-13 1999-09-16 American Home Products Corporation Polynucleotide composition, method of preparation, and use thereof
US5962213A (en) * 1996-06-14 1999-10-05 Biostore New Zealand Limited Compositions and methods for the preservation of living tissues
WO2000009086A2 (en) * 1998-08-14 2000-02-24 Valentis, Inc. Protected one-vial formulation for nucleic acid molecules, methods of making the same by in-line mixing, and related products and methods
US6037116A (en) * 1996-06-14 2000-03-14 Biostore New Zealand, Ltd. Compositions comprising betaine, sodium citrate and sodium chloride and methods for the preservation of biological materials
US6040132A (en) * 1996-06-14 2000-03-21 Biostore New Zealand, Ltd. Methods for the lyophilization of living biological materials
EP0997734A1 (en) * 1998-10-29 2000-05-03 Council of Scientific and Industrial Research A composition useful for the early diagnosis of viseral leishmaniasis and a process for preparing the same
US6060233A (en) * 1996-06-14 2000-05-09 Biostore New Zealand, Ltd Methods for the lyophilization of platelets, platelet membranes or erythrocytes
US6114107A (en) * 1996-06-14 2000-09-05 Biostore New Zealand Limited Composition comprising raffinose, TMAO, sodium citrate and methods for the preservation of living tissues
US6221385B1 (en) 1996-05-10 2001-04-24 Vrije Universiteit Brussel Freeze dried liposome encapsulated amphiphilic drug compositions and a process for the preparation thereof
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
US6361933B1 (en) 1996-06-14 2002-03-26 Biostore New Zealand Limited Solutions for the preservation of tissues
US6423529B1 (en) 1997-12-08 2002-07-23 Council Of Scientific & Industrial Research Composition useful for the early diagnosis of visceral leishmaniasis and a process for preparing the same
US6440747B2 (en) * 1998-10-30 2002-08-27 Dade Behring Marburg Gmbh Stabilization of biological fluids by addition of sterol esters
WO2002072002A2 (en) 2001-03-12 2002-09-19 Biotools Biotechnological & Medical Laboratories, S.A. Method for preparing stabilised reaction mixtures, which are totally or partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A
WO2004000347A1 (en) 2002-06-21 2003-12-31 Novo Nordisk A/S Stabilised solid compositions of factor vii polypeptides
ES2214144A1 (en) * 2003-02-26 2004-09-01 BIOTOOLS BIOTECHNOLOGICAL & MEDICAL LABORATORIES, S.A. Stabilised composition for fluorimetric, colorimetric or chemoluminescent assays, kits containing same and production method thereof
EP1455822A2 (en) * 2001-11-09 2004-09-15 Centocor, Inc. Lyophilized monoclonal antibody compositions
JP2004256545A (en) * 2000-02-08 2004-09-16 Allergan Inc Pharmaceutical composition of botulinus toxin
EP1555033A2 (en) * 1998-03-13 2005-07-20 Wyeth Polynucleotide composition, method of preparation, and use thereof
US6982080B2 (en) * 2002-03-15 2006-01-03 Wyeth Hydroxyethyl starch—containing polypeptide compositions
EP1663292A1 (en) * 2003-08-06 2006-06-07 CJ Corp. Formulation of albumin-free erythropoietin
WO2006071373A1 (en) * 2004-12-23 2006-07-06 Aurx, Inc. A Maryland Corporation Stabilization of viral compositions
WO2006023665A3 (en) * 2004-08-17 2006-08-03 Regeneron Pharma Il-1 antagonist formulations
JP2007507422A (en) * 2003-07-14 2007-03-29 ナンニン メイプル リーフ ファーマシューティカル カンパニー リミテッド A stable lyophilized pharmaceutical formulation of tetrodotoxin
US7276359B1 (en) 1998-03-13 2007-10-02 Wyeth Polynucleotide composition, method of preparation, and use thereof
US7323297B1 (en) * 1992-04-03 2008-01-29 The Regents Of The University Of California Stabilized polynucleotide complexes and methods
US7338810B2 (en) * 2002-06-18 2008-03-04 Canon Kabushiki Kaisha Substrate processing method and method for manufacturing probe carrier
WO2008090340A2 (en) * 2007-01-23 2008-07-31 Cambridge Enterprise Limited Nucleic acid amplification and testing
WO2008101179A2 (en) * 2007-02-16 2008-08-21 Wyeth Use of sucrose to suppress mannitol-induced protein aggregation
EP1974723A2 (en) 2003-11-29 2008-10-01 Merck Patent GmbH Secure forms of anti-EGFR antibodies
WO2008155529A1 (en) * 2007-06-16 2008-12-24 Enigma Diagnostics Limited Compositions
US7572893B2 (en) 2004-08-17 2009-08-11 Regeneron Pharmaceuticals, Inc. IL-1 antagonist formulations
US7655758B2 (en) 2004-08-17 2010-02-02 Regeneron Pharmaceuticals, Inc. Stable liquid IL-1 antagonist formulations
US7790852B2 (en) 2003-06-25 2010-09-07 Novo Nordisk Health Care A/G Liquid composition of factor VII polypeptides
JP2010213699A (en) * 1998-11-06 2010-09-30 Sterrenbeld Biotechnologie North America Inc Host cell expressing recombinant human erythropoietin
US7897734B2 (en) 2003-03-26 2011-03-01 Novo Nordisk Healthcare Ag Method for the production of proteins
WO2011048227A1 (en) 2009-10-22 2011-04-28 Biotools Biotechnological & Medical Laboratories, S.A. Composition, method and kit for detecting bacteria by means of sequencing
US8022031B2 (en) 2001-12-21 2011-09-20 Novo Nordisk Health Care A/G Liquid composition of factor VII polypeptides
US8026214B2 (en) 2003-08-14 2011-09-27 Novo Nordisk Health Care Ag Liquid, aqueous pharmaceutical compositions of factor VII polypeptides
US8372800B2 (en) 1999-02-22 2013-02-12 Baxter International Inc. Albumin-free factor VIII formulations
US20130059380A1 (en) * 2010-02-17 2013-03-07 Hememics Biotechnologies, Inc. Preservation solutions for biologics and methods related thereto
US8404638B2 (en) 2005-03-25 2013-03-26 Regeneron Pharmaceuticals, Inc. Dimer VEGF antagonist formulations
US20130129685A1 (en) * 2010-03-31 2013-05-23 Stabilitech Ltd. Stabilisation of Viral Particles
JP2013527832A (en) * 2010-03-22 2013-07-04 ジェネンテック， インコーポレイテッド Compositions and methods useful for stabilizing protein-containing formulations
WO2013112897A1 (en) * 2012-01-27 2013-08-01 The Regents Of The University Of California Stabilization of biomolecules using sugar polymers
US8536127B2 (en) 2003-05-23 2013-09-17 Novo Nordisk Healthcare Ag Protein stabilization in solution
US8795669B2 (en) 2011-07-28 2014-08-05 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-PCSK9 antibodies
US8815232B2 (en) 2008-08-26 2014-08-26 Kyon Biotech Ag Compositions and methods for treating cancer
JP2015500029A (en) * 2011-12-09 2015-01-05 テゴ サイエンス インコーポレイテッド A method for stable storage of useful substances in cells at room temperature
US9283273B2 (en) 1995-07-27 2016-03-15 Genentech, Inc. Protein formulation
US9402898B2 (en) 2012-01-23 2016-08-02 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-Ang2 antibodies
US9550837B2 (en) 2008-12-15 2017-01-24 Regeneron Pharmaceuticals, Inc. Therapeutic uses of anti-PCSK9 antibodies
US9561155B2 (en) 2011-01-28 2017-02-07 Sanofi Biotechnology Method of reducing cholesterol levels using a human anti-PCSK9 antibody
US9642353B2 (en) 2007-09-24 2017-05-09 Hememics Biotechnologies, Inc. Desiccated biologics and methods of preparing the same
US9675692B2 (en) 2012-05-31 2017-06-13 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-DLL4 antibodies
CZ306818B6 (en) * 2015-01-29 2017-07-26 Masarykova Univerzita A method of suppressing freeze-induced protein denaturation when freezing and/or lyophilizing biologically active substances
US9724411B2 (en) 2008-12-15 2017-08-08 Regeneron Pharmaceuticals, Inc. Methods for treating hypercholesterolemia and reducing LDL-C using antibodies to PCSK9
US9738923B2 (en) 2014-06-18 2017-08-22 Luminex Corporation Methods for generating stabilized lyophilized materials
US10029007B2 (en) 2011-10-05 2018-07-24 Stabilitech Biopharma Ltd Stabilisation of polypeptides
US10076571B2 (en) 2011-09-16 2018-09-18 Regeneron Pharmaceuticals, Inc. Methods for reducing lipoprotein(a) levels by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9)
US10111953B2 (en) 2013-05-30 2018-10-30 Regeneron Pharmaceuticals, Inc. Methods for reducing remnant cholesterol and other lipoprotein fractions by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9)
US10428157B2 (en) 2013-11-12 2019-10-01 Sanofi Biotechnology Dosing regimens for use with PCSK9 inhibitors
US10494442B2 (en) 2013-06-07 2019-12-03 Sanofi Biotechnology Methods for inhibiting atherosclerosis by administering an inhibitor of PCSK9
US10501523B2 (en) 2014-07-18 2019-12-10 Sanofi IL-8 level based method of predicting the outcome of colon cancer treatment
US10512674B2 (en) 2008-11-07 2019-12-24 Baxalta Incorporated Factor VIII formulations
CN110699346A (en) * 2019-11-13 2020-01-17 河北省微生物研究所 Preparation method of glucose oxidase freeze-dried powder
US10544232B2 (en) 2014-07-16 2020-01-28 Sanofi Biotechnology Methods for treating patients with heterozygous familial hypercholesterolemia (heFH) with an anti-PCSK9 antibody
US10716859B2 (en) 2010-03-31 2020-07-21 Stabilitech Biopharma Ltd Excipients for stabilising viral particles, polypeptides or biological material
US10765552B2 (en) 2016-02-18 2020-09-08 Zeltiq Aesthetics, Inc. Cooling cup applicators with contoured heads and liner assemblies
US10772956B2 (en) 2015-08-18 2020-09-15 Regeneron Pharmaceuticals, Inc. Methods for reducing or eliminating the need for lipoprotein apheresis in patients with hyperlipidemia by administering alirocumab
US10806783B2 (en) 2014-04-11 2020-10-20 Stabilitech Biopharma Ltd Vaccine compositions
US10806500B2 (en) 2014-01-31 2020-10-20 Zeltiq Aesthetics, Inc. Treatment systems, methods, and apparatuses for improving the appearance of skin and providing other treatments
US10980871B2 (en) 2017-05-08 2021-04-20 Iosbio Ltd Vaccine compositions
US11033606B2 (en) 2011-04-26 2021-06-15 Sanofi Composition comprising aflibercept, folinic acid, 5-fluorouracil (5-FU) and irinotecan (FOLFIRI)
US11076879B2 (en) 2017-04-26 2021-08-03 Zeltiq Aesthetics, Inc. Shallow surface cryotherapy applicators and related technology
US11154418B2 (en) 2015-10-19 2021-10-26 Zeltiq Aesthetics, Inc. Vascular treatment systems, cooling devices, and methods for cooling vascular structures
US11179269B2 (en) 2006-09-26 2021-11-23 Zeltiq Aesthetics, Inc. Cooling device having a plurality of controllable cooling elements to provide a predetermined cooling profile
US11395760B2 (en) 2006-09-26 2022-07-26 Zeltiq Aesthetics, Inc. Tissue treatment methods
US11446175B2 (en) 2018-07-31 2022-09-20 Zeltiq Aesthetics, Inc. Methods, devices, and systems for improving skin characteristics
US11452634B2 (en) 2009-04-30 2022-09-27 Zeltiq Aesthetics, Inc. Device, system and method of removing heat from subcutaneous lipid-rich cells
US11583438B1 (en) 2007-08-21 2023-02-21 Zeltiq Aesthetics, Inc. Monitoring the cooling of subcutaneous lipid-rich cells, such as the cooling of adipose tissue
US11732024B2 (en) 2006-06-16 2023-08-22 Regeneron Pharmaceuticals, Inc. VEGF antagonist formulations suitable for intravitreal administration

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3177117A (en) * 1961-12-26 1965-04-06 Joseph F Saunders Process for freezing blood
US4439421A (en) * 1982-08-30 1984-03-27 Baxter Travenol Laboratories, Inc. Stabilized gamma globulin concentrate
US4574081A (en) * 1984-09-25 1986-03-04 Colgate-Palmolive Co. Antiplaque dentifrice having improved flavor
US4874690A (en) * 1988-08-26 1989-10-17 Cryopharm Corporation Lyophilization of red blood cells
WO1989009610A1 (en) * 1988-04-13 1989-10-19 Cetus Corporation Tumor necrosis factor formulations
US4931385A (en) * 1985-06-24 1990-06-05 Hygeia Sciences, Incorporated Enzyme immunoassays and immunologic reagents

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3177117A (en) * 1961-12-26 1965-04-06 Joseph F Saunders Process for freezing blood
US4439421A (en) * 1982-08-30 1984-03-27 Baxter Travenol Laboratories, Inc. Stabilized gamma globulin concentrate
US4574081A (en) * 1984-09-25 1986-03-04 Colgate-Palmolive Co. Antiplaque dentifrice having improved flavor
US4931385A (en) * 1985-06-24 1990-06-05 Hygeia Sciences, Incorporated Enzyme immunoassays and immunologic reagents
WO1989009610A1 (en) * 1988-04-13 1989-10-19 Cetus Corporation Tumor necrosis factor formulations
US4874690A (en) * 1988-08-26 1989-10-17 Cryopharm Corporation Lyophilization of red blood cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL DIARY SCIENCE, Volume 73, Number 12, 1-33, issued 1990, CARPENTER et al., "Comparison of Solute-Induced Protein Stabilization in Aqueous Solution and in the Frozen and Dried States", pages 3627-36. *
TRANSPLANTATION, Volume 49, Number 2, issued February 1990, W. WICOMB et al., "Optimal Cardioplegia and 24-Hour Heart Storage with Simplified UW Solution Containing Polyethylene Glycol", pages 261-264. *

Cited By (183)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5602023A (en) * 1992-03-24 1997-02-11 Csatary; Laszlo K. Pharmaceutical product containing live, stabilized virus for the therapy of viral and malignant diseases and process for preparing the same
US7323297B1 (en) * 1992-04-03 2008-01-29 The Regents Of The University Of California Stabilized polynucleotide complexes and methods
BE1006379A3 (en) * 1992-11-26 1994-08-09 Raymond Gilles Method for preserving living cells, groups of cells and/or derivatives ofsaid cells by means of freeze-drying
WO1994013136A1 (en) * 1992-12-04 1994-06-23 Development Biotechnological Processes S.N.C. Di Pelliccia Maria Teresa Cryoprotective solutions
WO1994013135A1 (en) * 1992-12-04 1994-06-23 Development Biotechnological Processes S.N.C. Di Pelliccia Maria Teresa Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial sheets
US5670308A (en) * 1992-12-04 1997-09-23 Development Biotechnological Processes Snc Di Pelliccia Maria Teresa Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial sheets
US5424204A (en) * 1993-08-26 1995-06-13 Kyowa Medex Co., Ltd. Method for stabilizing glucose 6-phosphate dehydrogenase with hydroxylamines, aldehyde scavengers, dimethylthiocarbamoyl chloride or 2-(2-aminoethylamino) ethanol
US5580856A (en) * 1994-07-15 1996-12-03 Prestrelski; Steven J. Formulation of a reconstituted protein, and method and kit for the production thereof
DE19503685C2 (en) * 1995-01-30 2000-05-31 Invitek Gmbh Process for the preparation of complex multi-enzymatic storage-stable reaction mixtures and their use
DE19503685A1 (en) * 1995-01-30 1996-08-01 Invitek Gmbh Storage-stable reaction mixt. contg. many enzymes, reactants and stabilisers
US5614387A (en) * 1995-02-10 1997-03-25 Gen-Probe Incorporated Method of making stabilized enzyme compositions for nucleic acid amplification
WO1996024664A1 (en) * 1995-02-10 1996-08-15 Gen-Probe Incorporated Stabilized enzyme compositions for nucleic acid amplification
EP0726310A1 (en) * 1995-02-10 1996-08-14 Gen-Probe Incorporated Stabilized enzyme compositons for nucleic acid amplification
US5834254A (en) * 1995-02-10 1998-11-10 Gen-Probe Incorporated Stabilized enzyme compositions for nucleic acid amplification
AU699590B2 (en) * 1995-02-10 1998-12-10 Gen-Probe Incorporated Stabilized enzyme compositions for nucleic acid amplification
EP1491217A1 (en) * 1995-06-07 2004-12-29 The Regents Of The University Of California Stabilization of polynucleotide complexes
EP0833667A1 (en) * 1995-06-07 1998-04-08 The Regents Of The University Of California Stabilization of polynucleotide complexes
EP0833667A4 (en) * 1995-06-07 2001-11-21 Univ California Stabilization of polynucleotide complexes
WO1996041870A1 (en) * 1995-06-08 1996-12-27 Societa' Cooperativa Centro Ricerche Poly-Tech A Responsabilita' Limitata Hydrosoluble compositions of stabilized collagenase and the process for their preparation
EP0836645A1 (en) * 1995-06-09 1998-04-22 The Regents Of The University Of California Dry powder formulations of polynucleotide complexes
EP0836645A4 (en) * 1995-06-09 2001-11-21 Univ California Dry powder formulations of polynucleotide complexes
US9283273B2 (en) 1995-07-27 2016-03-15 Genentech, Inc. Protein formulation
US6221385B1 (en) 1996-05-10 2001-04-24 Vrije Universiteit Brussel Freeze dried liposome encapsulated amphiphilic drug compositions and a process for the preparation thereof
US6060233A (en) * 1996-06-14 2000-05-09 Biostore New Zealand, Ltd Methods for the lyophilization of platelets, platelet membranes or erythrocytes
US5879875A (en) * 1996-06-14 1999-03-09 Biostore New Zealand Compositions and methods for the preservation of living tissues
US5962213A (en) * 1996-06-14 1999-10-05 Biostore New Zealand Limited Compositions and methods for the preservation of living tissues
US6361933B1 (en) 1996-06-14 2002-03-26 Biostore New Zealand Limited Solutions for the preservation of tissues
US6114107A (en) * 1996-06-14 2000-09-05 Biostore New Zealand Limited Composition comprising raffinose, TMAO, sodium citrate and methods for the preservation of living tissues
US6037116A (en) * 1996-06-14 2000-03-14 Biostore New Zealand, Ltd. Compositions comprising betaine, sodium citrate and sodium chloride and methods for the preservation of biological materials
US6040132A (en) * 1996-06-14 2000-03-21 Biostore New Zealand, Ltd. Methods for the lyophilization of living biological materials
WO1998000530A1 (en) * 1996-07-03 1998-01-08 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US5876992A (en) * 1996-07-03 1999-03-02 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
US6294365B1 (en) 1996-07-03 2001-09-25 Molecular Biology Resources, Inc. Method and formulation for stabilization of enzymes
WO1998006421A1 (en) * 1996-08-16 1998-02-19 Cancer Treatments International Therapeutic compositions for treatment of cancer
EP0852951A1 (en) 1996-11-19 1998-07-15 Roche Diagnostics GmbH Stable lyophilized monoclonal or polyclonal antibodies containing pharmaceuticals
WO1998022136A2 (en) 1996-11-19 1998-05-28 Roche Diagnostics Gmbh Stable lyophilized pharmaceutical substances from monoclonal or polyclonal antibodies
EP0868916A3 (en) * 1997-03-04 2004-09-15 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Reduction inhibitory agent for active-oxygen eliminating activity
US7186824B2 (en) 1997-03-04 2007-03-06 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Reduction inhibitory agent for active-oxygen eliminating activity
EP0868916A2 (en) * 1997-03-04 1998-10-07 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Reduction inhibitory agent for active-oxygen eliminating activity
EP0875253A3 (en) * 1997-05-02 2000-11-22 Seikagaku Corporation Chondroitinase compositions
EP0875253A2 (en) * 1997-05-02 1998-11-04 Seikagaku Corporation Chondroitinase compositions
US6007810A (en) * 1997-05-02 1999-12-28 Seikagaku Corporation Chondroitinase compositions
US6423529B1 (en) 1997-12-08 2002-07-23 Council Of Scientific & Industrial Research Composition useful for the early diagnosis of visceral leishmaniasis and a process for preparing the same
WO1999045966A1 (en) * 1998-03-13 1999-09-16 American Home Products Corporation Polynucleotide composition, method of preparation, and use thereof
EP1555033A3 (en) * 1998-03-13 2005-08-17 Wyeth Polynucleotide composition, method of preparation, and use thereof
EP1555033A2 (en) * 1998-03-13 2005-07-20 Wyeth Polynucleotide composition, method of preparation, and use thereof
US7276359B1 (en) 1998-03-13 2007-10-02 Wyeth Polynucleotide composition, method of preparation, and use thereof
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
WO2000009086A3 (en) * 1998-08-14 2000-06-08 Valentis Inc Protected one-vial formulation for nucleic acid molecules, methods of making the same by in-line mixing, and related products and methods
US6534483B1 (en) 1998-08-14 2003-03-18 Valentis, Inc. Protected one-vial formulation for nucleic acid molecules, methods of making the same by in-line mixing, and related products and methods
WO2000009086A2 (en) * 1998-08-14 2000-02-24 Valentis, Inc. Protected one-vial formulation for nucleic acid molecules, methods of making the same by in-line mixing, and related products and methods
EP0997734A1 (en) * 1998-10-29 2000-05-03 Council of Scientific and Industrial Research A composition useful for the early diagnosis of viseral leishmaniasis and a process for preparing the same
US6440747B2 (en) * 1998-10-30 2002-08-27 Dade Behring Marburg Gmbh Stabilization of biological fluids by addition of sterol esters
JP2010213699A (en) * 1998-11-06 2010-09-30 Sterrenbeld Biotechnologie North America Inc Host cell expressing recombinant human erythropoietin
US8765665B2 (en) 1999-02-22 2014-07-01 Baxter International Inc. Albumin-free factor VIII formulations
US9669076B2 (en) 1999-02-22 2017-06-06 Baxalta Incorporated Albumin-free factor VIII formulations
US9352027B2 (en) 1999-02-22 2016-05-31 Baxalta Incorporated Albumin-free factor VIII formulations
US8372800B2 (en) 1999-02-22 2013-02-12 Baxter International Inc. Albumin-free factor VIII formulations
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A
JP2004256545A (en) * 2000-02-08 2004-09-16 Allergan Inc Pharmaceutical composition of botulinus toxin
WO2002072002A3 (en) * 2001-03-12 2002-11-28 Biotools Biotechnological & Me Method for preparing stabilised reaction mixtures, which are totally or partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
WO2002072002A2 (en) 2001-03-12 2002-09-19 Biotools Biotechnological & Medical Laboratories, S.A. Method for preparing stabilised reaction mixtures, which are totally or partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
US7919294B2 (en) 2001-03-12 2011-04-05 Biotools Biotechnological & Medical Laboratories, S.A. Process for preparing stabilized reaction mixtures which are partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
ES2180416A1 (en) * 2001-03-12 2003-02-01 Biotools Biotechnological & Me Method for preparing stabilised reaction mixtures, which are totally or partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
AU2002246125B2 (en) * 2001-03-12 2008-02-07 Biotools Biotechnological & Medical Laboratories, S.A. Process for preparing stabilized reaction mixtures which are partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
JP2004519242A (en) * 2001-03-12 2004-07-02 ビオトールズ ビオテクノロジカル アンド メディカル ラボラトリーズ，エス．エー． Method for producing a stabilized reaction mixture containing one or more enzymes which are completely or partially dried, and the reaction mixture and a kit containing the same
EP1455822A4 (en) * 2001-11-09 2004-12-29 Centocor Inc Lyophilized monoclonal antibody compositions
EP1455822A2 (en) * 2001-11-09 2004-09-15 Centocor, Inc. Lyophilized monoclonal antibody compositions
US8022031B2 (en) 2001-12-21 2011-09-20 Novo Nordisk Health Care A/G Liquid composition of factor VII polypeptides
US8461116B2 (en) 2001-12-21 2013-06-11 Novo Nordisk Healthcare Ag Liquid composition of factor VII polypeptides
US7449444B2 (en) 2002-03-15 2008-11-11 Wyeth Hydroxyethyl starch-containing polypeptide compositions
US6982080B2 (en) * 2002-03-15 2006-01-03 Wyeth Hydroxyethyl starch—containing polypeptide compositions
US7338810B2 (en) * 2002-06-18 2008-03-04 Canon Kabushiki Kaisha Substrate processing method and method for manufacturing probe carrier
EP2283856A1 (en) * 2002-06-21 2011-02-16 Novo Nordisk Health Care AG Stabilised solid compositions of factor VII polypeptides
JP2006513983A (en) * 2002-06-21 2006-04-27 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Stabilized solid composition of factor VII polypeptide
WO2004000347A1 (en) 2002-06-21 2003-12-31 Novo Nordisk A/S Stabilised solid compositions of factor vii polypeptides
JP4648002B2 (en) * 2002-06-21 2011-03-09 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Stabilized solid composition of factor VII polypeptide
US8299029B2 (en) 2002-06-21 2012-10-30 Novo Nordisk Health Care Ag Stabilised solid compositions of factor VII polypeptides
US8729022B2 (en) 2002-06-21 2014-05-20 Novo Nordisk Healthcare Ag Stabilised solid compositions of factor VII polypeptides
WO2004076656A1 (en) * 2003-02-26 2004-09-10 Biotools Biotechnological & Medical Laboratories, S.A. Stabilised composition for fluorimetric, colorimetric or chemoluminescent assays, kits containing same and production method thereof
ES2214144A1 (en) * 2003-02-26 2004-09-01 BIOTOOLS BIOTECHNOLOGICAL & MEDICAL LABORATORIES, S.A. Stabilised composition for fluorimetric, colorimetric or chemoluminescent assays, kits containing same and production method thereof
US8084587B2 (en) 2003-03-26 2011-12-27 Novo Nordisk Health Care Ag Method for the production of proteins
US7897734B2 (en) 2003-03-26 2011-03-01 Novo Nordisk Healthcare Ag Method for the production of proteins
US8536127B2 (en) 2003-05-23 2013-09-17 Novo Nordisk Healthcare Ag Protein stabilization in solution
US7790852B2 (en) 2003-06-25 2010-09-07 Novo Nordisk Health Care A/G Liquid composition of factor VII polypeptides
US8124608B2 (en) * 2003-07-14 2012-02-28 Wex Medical Limited Stable pharmaceutical composition of freeze-dried tetrodotoxin powder
US8530481B2 (en) 2003-07-14 2013-09-10 Wex Medical Limited Stable pharmaceutical composition of freeze-dried tetrodotoxin powder
US8222258B2 (en) 2003-07-14 2012-07-17 Wex Medical Limited Stable pharmaceutical composition of freeze-dried tetrodoxin powder
JP2007507422A (en) * 2003-07-14 2007-03-29 ナンニン メイプル リーフ ファーマシューティカル カンパニー リミテッド A stable lyophilized pharmaceutical formulation of tetrodotoxin
EP1663292A4 (en) * 2003-08-06 2007-11-21 Cj Corp Formulation of albumin-free erythropoietin
EP1663292A1 (en) * 2003-08-06 2006-06-07 CJ Corp. Formulation of albumin-free erythropoietin
US8318904B2 (en) 2003-08-14 2012-11-27 Novo Nordisk Health Care Ag Liquid, aqueous pharmaceutical compositions of factor VII polypeptides
US8026214B2 (en) 2003-08-14 2011-09-27 Novo Nordisk Health Care Ag Liquid, aqueous pharmaceutical compositions of factor VII polypeptides
EP1974723A2 (en) 2003-11-29 2008-10-01 Merck Patent GmbH Secure forms of anti-EGFR antibodies
AU2005277357B2 (en) * 2004-08-17 2011-08-04 Regeneron Pharmaceuticals, Inc. IL-1 antagonist formulations
WO2006023665A3 (en) * 2004-08-17 2006-08-03 Regeneron Pharma Il-1 antagonist formulations
US7655758B2 (en) 2004-08-17 2010-02-02 Regeneron Pharmaceuticals, Inc. Stable liquid IL-1 antagonist formulations
US7572893B2 (en) 2004-08-17 2009-08-11 Regeneron Pharmaceuticals, Inc. IL-1 antagonist formulations
US7365165B2 (en) 2004-08-17 2008-04-29 Regeneron Pharmaceuticals, Inc. IL-1 antagonist formulations
JP2008510716A (en) * 2004-08-17 2008-04-10 リジェネロン・ファーマシューティカルズ・インコーポレイテッド IL-1 antagonist preparation
WO2006071373A1 (en) * 2004-12-23 2006-07-06 Aurx, Inc. A Maryland Corporation Stabilization of viral compositions
US10406226B2 (en) 2005-03-25 2019-09-10 Regeneron Pharmaceuticals, Inc. Method of manufacturing VEGF antagonist fusion proteins
US10857231B2 (en) 2005-03-25 2020-12-08 Regeneron Pharmaceuticals, Inc. Formulations of VEG antagonist fusion proteins and method of manufacturing them
US8921316B2 (en) 2005-03-25 2014-12-30 Regeneron Pharmaceuticals, Inc. Stable liquid VEGF antagonist formulations
US9636400B2 (en) 2005-03-25 2017-05-02 Regeneron Pharmaceuticals, Inc. VEGF specific fusion protein antagonist formulations
US9511140B2 (en) 2005-03-25 2016-12-06 Regeneron Pharmaceuticals, Inc. Stable VEGF antagonist formulations
US9416167B2 (en) 2005-03-25 2016-08-16 Regeneron Pharmaceuticals, Inc. Stable liquid formulations of dimer VEGF antagonists
US8404638B2 (en) 2005-03-25 2013-03-26 Regeneron Pharmaceuticals, Inc. Dimer VEGF antagonist formulations
US8710004B2 (en) 2005-03-25 2014-04-29 Regeneron Pharmaceuticals, Inc. Stable liquid VEGF antagonist formulations
US11806398B2 (en) 2005-03-25 2023-11-07 Regeneron Pharmaceuticals, Inc. Citrate buffered VEGF antagonist formulations
US11732024B2 (en) 2006-06-16 2023-08-22 Regeneron Pharmaceuticals, Inc. VEGF antagonist formulations suitable for intravitreal administration
US11179269B2 (en) 2006-09-26 2021-11-23 Zeltiq Aesthetics, Inc. Cooling device having a plurality of controllable cooling elements to provide a predetermined cooling profile
US11395760B2 (en) 2006-09-26 2022-07-26 Zeltiq Aesthetics, Inc. Tissue treatment methods
US11219549B2 (en) 2006-09-26 2022-01-11 Zeltiq Aesthetics, Inc. Cooling device having a plurality of controllable cooling elements to provide a predetermined cooling profile
US11447821B2 (en) 2007-01-23 2022-09-20 Cambridge Enterprise Limited Nucleic acid amplification and testing
WO2008090340A2 (en) * 2007-01-23 2008-07-31 Cambridge Enterprise Limited Nucleic acid amplification and testing
US10563254B2 (en) 2007-01-23 2020-02-18 Cambridge Enterprise Limited Nucleic acid amplification and testing
EP2527470A3 (en) * 2007-01-23 2012-12-05 Cambridge Enterprise Limited Nucleic acid amplification and testing
WO2008090340A3 (en) * 2007-01-23 2009-01-08 Diagnostics For The Real World Nucleic acid amplification and testing
WO2008101179A2 (en) * 2007-02-16 2008-08-21 Wyeth Use of sucrose to suppress mannitol-induced protein aggregation
WO2008101179A3 (en) * 2007-02-16 2008-12-24 Wyeth Corp Use of sucrose to suppress mannitol-induced protein aggregation
WO2008155529A1 (en) * 2007-06-16 2008-12-24 Enigma Diagnostics Limited Compositions
US11583438B1 (en) 2007-08-21 2023-02-21 Zeltiq Aesthetics, Inc. Monitoring the cooling of subcutaneous lipid-rich cells, such as the cooling of adipose tissue
US9642353B2 (en) 2007-09-24 2017-05-09 Hememics Biotechnologies, Inc. Desiccated biologics and methods of preparing the same
US8815232B2 (en) 2008-08-26 2014-08-26 Kyon Biotech Ag Compositions and methods for treating cancer
US10512674B2 (en) 2008-11-07 2019-12-24 Baxalta Incorporated Factor VIII formulations
US11020459B2 (en) 2008-11-07 2021-06-01 Baxalta Incorporated Factor VIII formulations
US10941210B2 (en) 2008-12-15 2021-03-09 Regeneron Pharmaceuticals, Inc. Anti-PCSK9 antibodies
US10023654B2 (en) 2008-12-15 2018-07-17 Regeneron Pharmaceuticals, Inc. Anti-PCSK9 antibodies
US9724411B2 (en) 2008-12-15 2017-08-08 Regeneron Pharmaceuticals, Inc. Methods for treating hypercholesterolemia and reducing LDL-C using antibodies to PCSK9
US9550837B2 (en) 2008-12-15 2017-01-24 Regeneron Pharmaceuticals, Inc. Therapeutic uses of anti-PCSK9 antibodies
US11452634B2 (en) 2009-04-30 2022-09-27 Zeltiq Aesthetics, Inc. Device, system and method of removing heat from subcutaneous lipid-rich cells
WO2011048227A1 (en) 2009-10-22 2011-04-28 Biotools Biotechnological & Medical Laboratories, S.A. Composition, method and kit for detecting bacteria by means of sequencing
US20130059380A1 (en) * 2010-02-17 2013-03-07 Hememics Biotechnologies, Inc. Preservation solutions for biologics and methods related thereto
US9943075B2 (en) * 2010-02-17 2018-04-17 Hememics Biotechnologies, Inc. Preservation solutions for biologics and methods related thereto
JP2016193909A (en) * 2010-03-22 2016-11-17 ジェネンテック， インコーポレイテッド Compositions and methods useful for stabilizing protein-containing formulations
US9662395B2 (en) 2010-03-22 2017-05-30 Genentech, Inc. Compositions and methods useful for stabilizing protein-containing formulations
JP2013527832A (en) * 2010-03-22 2013-07-04 ジェネンテック， インコーポレイテッド Compositions and methods useful for stabilizing protein-containing formulations
US10716859B2 (en) 2010-03-31 2020-07-21 Stabilitech Biopharma Ltd Excipients for stabilising viral particles, polypeptides or biological material
US20130129685A1 (en) * 2010-03-31 2013-05-23 Stabilitech Ltd. Stabilisation of Viral Particles
US10206960B2 (en) * 2010-03-31 2019-02-19 Stabilitech Biopharma Ltd Stabilisation of viral particles
US11246925B2 (en) 2011-01-28 2022-02-15 Sanofi Biotechnology Human antibodies to PCSK9 for use in methods of treating particular groups of subjects
US9561155B2 (en) 2011-01-28 2017-02-07 Sanofi Biotechnology Method of reducing cholesterol levels using a human anti-PCSK9 antibody
US9682013B2 (en) 2011-01-28 2017-06-20 Sanofi Biotechnology Pharmaceutical compositions comprising human antibodies to PCSK9
US11033606B2 (en) 2011-04-26 2021-06-15 Sanofi Composition comprising aflibercept, folinic acid, 5-fluorouracil (5-FU) and irinotecan (FOLFIRI)
US8795669B2 (en) 2011-07-28 2014-08-05 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-PCSK9 antibodies
US10752701B2 (en) 2011-07-28 2020-08-25 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-PCSK9 antibodies
US10472425B2 (en) 2011-07-28 2019-11-12 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-PCSK9 antibodies
US9193801B2 (en) 2011-07-28 2015-11-24 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-PCSK9 antibodies
US11673967B2 (en) 2011-07-28 2023-06-13 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-PCSK9 antibodies
US10076571B2 (en) 2011-09-16 2018-09-18 Regeneron Pharmaceuticals, Inc. Methods for reducing lipoprotein(a) levels by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9)
US11116839B2 (en) 2011-09-16 2021-09-14 Regeneron Pharmaceuticals, Inc. Methods for reducing lipoprotein(a) levels by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9)
US10029007B2 (en) 2011-10-05 2018-07-24 Stabilitech Biopharma Ltd Stabilisation of polypeptides
JP2015500029A (en) * 2011-12-09 2015-01-05 テゴ サイエンス インコーポレイテッド A method for stable storage of useful substances in cells at room temperature
US9402898B2 (en) 2012-01-23 2016-08-02 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-Ang2 antibodies
US10543280B2 (en) 2012-01-27 2020-01-28 The Regents Of The University Of California Stabilization of biomolecules using sugar polymers
WO2013112897A1 (en) * 2012-01-27 2013-08-01 The Regents Of The University Of California Stabilization of biomolecules using sugar polymers
US9901648B2 (en) 2012-01-27 2018-02-27 The Regents Of The University Of California Stabilization of biomolecules using sugar polymers
US9675692B2 (en) 2012-05-31 2017-06-13 Regeneron Pharmaceuticals, Inc. Stabilized formulations containing anti-DLL4 antibodies
US10111953B2 (en) 2013-05-30 2018-10-30 Regeneron Pharmaceuticals, Inc. Methods for reducing remnant cholesterol and other lipoprotein fractions by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9)
US10995150B2 (en) 2013-06-07 2021-05-04 Regeneron Pharmaceuticals, Inc. Methods for inhibiting atherosclerosis by administering an anti-PCSK9 antibody
US10494442B2 (en) 2013-06-07 2019-12-03 Sanofi Biotechnology Methods for inhibiting atherosclerosis by administering an inhibitor of PCSK9
US10428157B2 (en) 2013-11-12 2019-10-01 Sanofi Biotechnology Dosing regimens for use with PCSK9 inhibitors
US10912599B2 (en) 2014-01-31 2021-02-09 Zeltiq Aesthetics, Inc. Compositions, treatment systems and methods for improved cooling of lipid-rich tissue
US10806500B2 (en) 2014-01-31 2020-10-20 Zeltiq Aesthetics, Inc. Treatment systems, methods, and apparatuses for improving the appearance of skin and providing other treatments
US11819257B2 (en) 2014-01-31 2023-11-21 Zeltiq Aesthetics, Inc. Compositions, treatment systems and methods for improved cooling of lipid-rich tissue
US10806783B2 (en) 2014-04-11 2020-10-20 Stabilitech Biopharma Ltd Vaccine compositions
US11098344B2 (en) 2014-06-18 2021-08-24 Luminex Corporation Methods for generating stabilized lyophilized materials
US9738923B2 (en) 2014-06-18 2017-08-22 Luminex Corporation Methods for generating stabilized lyophilized materials
US10144954B2 (en) 2014-06-18 2018-12-04 Luminex Corporation Methods for generating stabilized lyophilized materials
US11306155B2 (en) 2014-07-16 2022-04-19 Sanofi Biotechnology Methods for treating patients with heterozygous familial hypercholesterolemia (heFH) with an anti-PCSK9 antibody
US10544232B2 (en) 2014-07-16 2020-01-28 Sanofi Biotechnology Methods for treating patients with heterozygous familial hypercholesterolemia (heFH) with an anti-PCSK9 antibody
US10501523B2 (en) 2014-07-18 2019-12-10 Sanofi IL-8 level based method of predicting the outcome of colon cancer treatment
US11208461B2 (en) 2014-07-18 2021-12-28 Sanofi Method for predicting the outcome of a treatment with aflibercept of a patient suspected to suffer from a cancer
CZ306818B6 (en) * 2015-01-29 2017-07-26 Masarykova Univerzita A method of suppressing freeze-induced protein denaturation when freezing and/or lyophilizing biologically active substances
US10772956B2 (en) 2015-08-18 2020-09-15 Regeneron Pharmaceuticals, Inc. Methods for reducing or eliminating the need for lipoprotein apheresis in patients with hyperlipidemia by administering alirocumab
US11904017B2 (en) 2015-08-18 2024-02-20 Regeneron Pharmaceuticals, Inc. Methods for reducing or eliminating the need for lipoprotein apheresis in patients with hyperlipidemia by administering alirocumab
US11154418B2 (en) 2015-10-19 2021-10-26 Zeltiq Aesthetics, Inc. Vascular treatment systems, cooling devices, and methods for cooling vascular structures
US10765552B2 (en) 2016-02-18 2020-09-08 Zeltiq Aesthetics, Inc. Cooling cup applicators with contoured heads and liner assemblies
US11076879B2 (en) 2017-04-26 2021-08-03 Zeltiq Aesthetics, Inc. Shallow surface cryotherapy applicators and related technology
US10980871B2 (en) 2017-05-08 2021-04-20 Iosbio Ltd Vaccine compositions
US11446175B2 (en) 2018-07-31 2022-09-20 Zeltiq Aesthetics, Inc. Methods, devices, and systems for improving skin characteristics
CN110699346A (en) * 2019-11-13 2020-01-17 河北省微生物研究所 Preparation method of glucose oxidase freeze-dried powder

Also Published As

Publication number Publication date
AU2309692A (en) 1993-02-11

Similar Documents

Publication Publication Date Title
WO1993000807A1 (en) Method for stabilization of biomaterials
AU2001268057B2 (en) Preservation and storage medium for biological materials
US6890512B2 (en) Methods of preventing aggregation of various substances upon rehydration or thawing and compositions obtained thereby
US5792643A (en) Methods for preserving recombinant retroviruses
Izutsu et al. Decreased protein-stabilizing effects of cryoprotectants due to crystallization
Carpenter et al. Modes of stabilization of a protein by organic solutes during desiccation
Lee Spray-drying of proteins
EP0871476B1 (en) Dried blood factor composition comprising trehalose
AU2009200127B2 (en) Preservation of bioactive materials by freeze dried foam
EP1374827B1 (en) Method for preparing stabilised reaction mixtures, which are totally or partially dried, comprising at least one enzyme, reaction mixtures and kits containing said mixtures
AU622978B2 (en) Storage of materials as solution in glassy, amorphous composition containing hydrophilic carrier
CA2576519C (en) Il-1 antagonist formulations
AU732274B2 (en) Methods and compositions for producing dried, storage-stable platelets
US20030022333A1 (en) Long-term shelf preservation by vitrification
AU2001268057A1 (en) Preservation and storage medium for biological materials
AU650678B2 (en) Stabilization (using gelatin from a cold water fish) of enzymes in diagnostic controls
JP2002542815A (en) Preservation method of virus and mycoplasma
CA2458794C (en) Plasticized hydrophilic glasses for improved stabilization of biological agents
EP0816509A2 (en) Thermostabilization, osmoprotection, and protection against desiccation of enzymes, cell components and cells by mannosyl-glycerate
US20010046487A1 (en) Methods for loading platelets, stabilizing platelets for dry storage and compositions obtained thereby
Hartmanis et al. Solubilization of a membrane-bound diol dehydratase with retention of EPR g= 2.02 signal by using 2-(N-cyclohexylamino) ethanesulfonic acid buffer.
Carpenter et al. Long-term preservation of dried phosphofructokinase by sugars and sugar/zinc mixtures
Gibson et al. The stabilisation of analytical enzymes using polyelectrolytes and sugar derivatives
AU700666B2 (en) Isoenzyme calibrator/control products
Boudrant et al. Continuous proteolysis with a stabilized protease. I. Chemical stabilization of an alkaline protease

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CS DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO PL RO RU SD SE US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE BF BJ CF CG CI CM GA GN ML MR SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 1992914862

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1992914862

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase