Classifications

• • 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
  • A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving 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
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/16—Blood plasma; Blood serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
  • A61L2/0023—Heat
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

• CARBOXYLIC ACID SUCH AS CITRIC ACID FOR DESINFECTING OR ENHACING THE PRODUCTION OF BLOOD PRODUCTS SUCH AS PLASMA, CRYOPRECIPITATE OR/ AND PLATELET
• the present invention relates to an improved method for producing increased amounts of safe coagulation factor concentrates from blood plasma.
• the invention is also directed to enhancing the yield and purity of blood components and inhibiting the activation or denaturation of certain blood components, blood cells and plasma proteins, and to the removal of activated and denatured components, thereby improving the safety and efficacy of end products.
• clotting or "coagulation” factors from human blood.
• proteins that cause the clotting of blood to staunch bleeding from wounds, etc.
• Individuals with any of a series of genetic abnormalities affecting the proteins responsible for blood coagulation are afflicted with a disease (hemophilia) in which the blood fails to clot normally, subjecting the individual to the danger of uncontrolled bleeding. For many years, this condition has been treated by administering concentrates of the missing or defective proteins.
• Many clotting factors are synthesized in the liver so that victims of liver disease are also in need of augmentation of their clotting factors. Additionally, there are other important medical uses for clotting- related factors including the use of fibrin to produce "fibrin sealant" or "fibrin glue”.
• AIDS Abnormal Immuno Deficiency Syndrome
• HIV HIV
• tests to screen out AIDS-tainted blood have been improved, some infected blood does slip by.
• the danger of other blood-borne diseases, such as the various types of hepatitis and other, as yet unknown, infectious agents makes it desirable to reduce or eliminate virus and other disease organisms from plasma used to prepare clotting factors.
• One way of achieving this goal is to replace pooled plasma products with products from a single donor since with pooled products "one bad apple spoils the entire barrel".
• clotting factor concentrates from blood have not changed greatly over the last few decades.
• a concentrate of clotting factors is derived from pooled plasma by a cryoprecipitation step.
• Various additives such as ethanol or polyethylene glycol are usually added to enhance the efficiency of the cryoprecipitation step.
• the partially purified factors may be further purified by additional precipitation steps or by chromatographic methods, and most recently by methods using monoclonal antibodies.
• citrate trisodium citrate and other citric acid salts
• citrate to enhance the yield and purity of cryoprecipitate. Furthermore, added citrate can inhibit the activation or denaturation of blood components including blood cells and plasma proteins and/or facilitate the removal of the activated or denatured components and improves the safety and efficacy of end products.
• a method for reducing transfusion-associated disease and adverse effects in plasma and for enhancing the purity and safety of multiple derivative components of blood including blood cells and plasma there is the step of adding at least about 2% by weight of carboxylic acid salt or equivalent weight of carboxylic acid to the blood or plasma.
• the invention is directed to enhancing the production of other derivative blood components including blood cells and plasma proteins.
• the invention includes reducing transfusion-associated disease and adverse effects in plasma and for enhancing the purity and safety of multiple derivative components of blood including blood cells and plasma from plasma.
• the derivatives comprises at least one product from the group of Enriched Cryoprecipitate, Cryo-Depleted Plasma, Fibrinogen, Fibrin Glue or Sealant, vWF (von Willdebrand's factor), Fibronectin, Factor VIII, Prothrombin Complex, a Serpine, Albumin, and a Globulin from plasma.
• vWF von Willdebrand's factor
• Fibronectin Fibronectin
• Factor VIII Prothrombin Complex
• Serpine Albumin
• Globulin Globulin from plasma.
• a salt of citric acid or equivalent weight of citric acid
• the plasma may be collected into a blood bag containing the carboxylic acid or the carboxylic acid salt. This blood bag can be different from a bag or container used to collect whole blood.
• an amount of additional carboxylic acid or the salt thereof may be added directly to the bag used to collect the whole blood.
• citrate is used appropriately in the collection of blood, in the processing and transfer of blood in a separation of discrete blood components. Citrate is used in increased, namely, additional quantities over the level traditionally employed for anticoagulation in one or other collection or processing bag.
• FIGURE 1 is a graphic representation of the improvement in cryoprecipitate yield resulting from the present invention.
• FIGURE 2 is a representation of the blood and plasma fractionation scheme using the invention.
• the proteins may actually fall out of solution, i.e., form a precipitate, if the protein more readily interacts with itself or with other proteins than with water.
• This process may denature the proteins (make them irreversibly insoluble), so it is usual to freeze protein solutions rapidly and to a low temperature (i.e., -20°C. or lower) to minimize the formation of ice crystals and to prevent the growth of those crystals that do form. This is done to limit protein denaturation on ice crystal surfaces.
• ice crystals may cause "activation" of the prothrombin complex, resulting in spontaneous clot formation after the plasma is thawed.
• the first step in the typical procedure for producing plasma cryoprecipitate is to centrifuge whole blood to separate the plasma from the red blood cells. This procedure is well known in the art and is often accomplished in special centrifuges that hold individual blood bags so that the plasma/red cell separation occurs without even opening the blood bag. Following the centrifugation, it is common practice to express the supernatant plasma into a "satellite" blood bag for further processing. Once the plasma is separated from red and white blood cells, the typical procedure is to rapidly freeze the plasma and to then slowly thaw the frozen plasma at about 4°C, during which thawing the clotting factors and other proteins form a cryoprecipitate which can be readily harvested by filtration or centrifugation. This cryoprecipitate is not rendered irreversibly insoluble and can be readily redissolved in a low ionic strength buffer, or even water, as is well known in the art.
• Cryoprecipitation is generally believed to result when the removal of water from the immediate vicinity of the protein molecules causes the protein molecules to preferentially associate with each other rather than with water.
• This "removal" of water may represent changes in the solubility of the proteins with changes in temperature (i.e., water becomes less effective at dissolving the proteins).
• the process may also be accomplished or enhanced by using additives which "tie up" the water and cause it to interact with the proteins to a lesser degree.
• additive substances can be any of a number of hydrophilic materials such as ethanol, polyethylene glycol, heparin, Pluronic RTM polyol polymers and various "salts" such as ammonium sulfate or ammonium acetate.
• the "salting out" of proteins from solution is a classical biochemical procedure.
• These and other materials used to increase the yield of cryoprecipitate generally operate to decrease the effective activity of water in the mixture. That is, the water molecules preferentially interact with the added hydrophilic material instead of with the proteins. This permits the proteins to interact with each other and, therefore, precipitate from solution. Similarly, lowering the temperature also decreases the activity of water, allowing protein-protein interactions to predominate.
• the hydrophilic additives just mentioned have the advantage of being relatively inexpensive and easy to use. However, their use usually necessitates additional washing steps to ensure that the additives are not carried over into the final product. Some additives may also damage or denature the labile clotting factors one is seeking to purify.
• the present inventor has discovered that one of the agents frequently used as an anticoagulant in blood fractionation unexpectedly serves to enhance cryoprecipitate formation.
• Citrate trisodium citrate or similar salts as well as derivatives of other low molecular weight carboxylic acids such as isocitric acid
• Citrate is a fairly effective chelator of calcium ions.
• citrate By effectively lowering the calcium ion level, citrate inhibits a considerable variety of blood clotting pathways which depend on the presence of calcium ions. However, citrate has not been employed as an agent to enhance the preparation of cryoprecipitate proteins from plasma and other blood fractions.
• the following table shows the enhanced production of cryoprecipitate caused by increasing the level of trisodium citrate in plasma.
• citrate As the citrate is increased, the weight of recovered cryoprecipitate is increase.
• the cryoprecipitate When the cryoprecipitate is redissolved in a fixed quantity of buffer or water, the increasing amount of cryoprecipitate yields increasing amounts of Factor VIII and fibrinogen as compared to the original plasma.
• the precise reason for this increase in yield is not known. However, it seems reasonable to speculate that one action of citrate may be to inhibit the activation of clotting factors. Since many of these factors act as proteases when activated, activation naturally digests clotting proteins thus reducing the yield of these proteins. However, lack of inactivation does not seem sufficient to account for the entire increase in cryoprecipitate yield.
• citrate On the surface, one might not expect citrate to be more effective than any hydrophilic salt. In terms of salting proteins out of solution, one would expect various agents to operate based on their colligative properties. That is, one might expect equimolar concentrations of various agents to behave similarly. This does not appear to be the case with citrate and cryoprecipitate formation.
• trisodium citrate is almost 5 times greater than that of salt to get equal osmotic effects one must use about 2.5X (on a weight basis) as much trisodium citrate as sodium chloride. That is, for an accurate matching more citrate rather than more salt should have been used.
• control were sent to a clinical chemistry laboratory to determine the presence of various blood proteins including clotting factors. These results are shown in the following table.
• citrate-treated plasma was frozen and cryoprecipitate was collected in the usual manner.
• citrate cryoprecipitate it is preferred to add the citrate prior to freezing, but good results are achieved by adding the citrate during the thawing process.
• the volume of cryoprecipitate formed from the unit of plasma was approximately 20 ml— that is, 10% of the total volume.
• an analysis of the cryoprecipitate and the supernatant plasma showed that about 12 g (60%) of the citrate was concentrated in the cryoprecipitate with only 30% being left in the supernatant. This indicates that there is a strong interaction between the cryoprecipitate proteins and the citrate.
• citrate cryoprecipitate is almost insoluble in room temperature water. It is soluble, however, in room temperature saline buffer and most soluble when the buffer contains citrate.
• citrate cryoprecipitate contains essentially all of the Fibrinogen, Fibronectin, Factor VIII and von Willdebrand's factor found in a treated aliquot of plasma.
• the citrate cryoprecipitate may also contain other minor factors (like Factor XIII) not yet assayed in these experiments. What may be significant is what the citrate cryoprecipitate does not contain. It has significantly less of albumin, globulins and other minor proteins than "normal" cryoprecipitate. Experiments are going on to characterize these differences.
• citrate Although citrate appears to influence strongly the precipitation of the clotting factors, it does not appear to denature these proteins. Citrate at 2% by weight was added to an aliquot of plasma that was stored at room temperature for six days. Clotting factors and platelets were counted at the beginning and the end of the time period. As compared to control plasma, the addition of citrate did not appear to harm the clotting factors. There is actually some suggestion that the citrate may actually help preserve platelets. This would be consistent with the hypothesis that citrate inhibits some of the proteases.
• citrate appears to enhance the susceptibility of microorganisms to a variety of "disinfecting agents" including heat.
• sodium citrate was added to a typical bacterial growth broth. Twenty-five ml aliquots of the broth were spiked with 1 x 10 4 organisms of either Escherichia coli or Staphylococcus epidermidis. Samples of the broth were brought to 2% by weight trisodium citrate and then subjected to
• citrate and similar molecules act are not know. Multiple carboxyl groups appear important particularly in the case of cryoprecipitate. Oxalic and lactic acids are less effective. It seems possible that some type of charge interaction favors the precipitation of the' clotting factors. As mentioned above, there appears to be good data supporting the hypothesis that citrate preferentially cross-links the cryoprecipitate proteins. While chelating ability is clearly important for the well known anti-coagulation effects of citrate, chelation may not be central to the present invention as isocitrate is believed to be a poorer chelating agent than citrate. It may also be that the participation of many of the effective molecules in the tricarboxylic acid cycle may also be related to their effects — particularly those on bacterial growth. That is, it seems likely that the cryoprecipitate phenomenon and the bacterial growth phenomenon have separate explanations.
• the invention has mainly been described with regard to cryoprecipitate.
• the starting point is collected blood — here a bag of CPDA (citrate-phosphate-dextrose-adenine) treated blood.
• the extra citrate could be added to this first bag or could be added, for instance, at the stage of the second empty bag with that bag containing citrate at an effective concentration of greater than about 2% to about 10% by weight or by volume trisodium citrate.
• the citrate can even be added at the frees/thaw step. Because of this further addition of citrate, different new products are obtained.
• This citrate-related process has the following features that relate to FIGURE 2:
• the products and processes starting from the Second Empty Bag onwards show improvements in purity and safety over normally fractionated plasma.
• the enriched cryoprecipitate 1 is at a higher yield than in the prior art.
• the enriched cryoprecipitate 1 has fewer "contaminating" extraneous serum proteins as compared to "normal” cryoprecipitate.
• crude fibrin glue 4 is produced directly from enriched cryoprecipitate by addition of thrombin or prothrombin complex (Factors II, VII, IX, and X)
• the resulting glue is superior in strength to crude fibrin glue produced in the same manner from "normal cryoprecipitate.” It compares favorably, and may actually be superior to, highly refined fibrin glue.
• the product is safer because citrate inhibits bacterial growth and because citrate can facilitate a "pasteurization" step to further destroy pathogens.
• autologous fibrin glue becomes much more feasible.
• Autologous products are inherently safer.
• fibrinogen with enriched cryoprecipitate it is feasible to produce "refined" fibrin glue or sealant which is primarily pure fibrinogen plus thrombin (added during application) although Factor VIII and other ingredients may be included.
• the cryo-depleted plasma 3 is safer for the above mentioned reasons. It is also inherently safer because it contains far less fibrinogen than normally processed plasma. This means it is virtually impossible for this material to develop microclots due to activation of the prothrombin complex—such microclots can cause intravascular coagulation and related transfusion problems. Also, the elevated citrate level reduces the likelihood of activation of prothrombin complex .
• a second bag with the increased carboxylic acid and/or citrate derivative (or other use of higher levels of citrate) is a feature which prior to the present invention has never been part of a blood fractionation process or part of the production of blood components for clinical use. There are significant advantages to using increased citrate (or related carboxylic acids).
• Fibronectin 6 and Factor VIII 7 can be produced by standard chromatographic methods, but use of differential extraction simplifies the process.
• the enriched citrate cryoprecipitate 1 is mostly insoluble in water. It is soluble in normal saline and very soluble as saline to which citrate is added. Therefore, extraction with buffers having different amounts of citrate results in preferential solubility of the different products.
• Products 8, 9 & 10 can be produced from Single Donor or from Pooled Plasma (preferably in combination with Iodine disinfection — as detailed, for example, in U.S. Patent No. 6,045,787).
• Albumin & Globulins may be further fractionated to yield separate albumin and gamma globulin. Generally, well-known fractionation techniques are used. Anion exchange (DEAE) is used to purify prothrombin complex. DEAE/sephadex can be used in a single donor process both to purify prothrombin and to dehydrate. Serpines 9 are purified by standard methods. Yields are improved because of the protease inhibiting properties of the added citrate. Again, these products are safer because of the citrate, because of the iodine step, because single donor products can be readily prepared and because pasteurization is facilitated.
• Anion exchange (DEAE) is used to purify prothrombin complex. DEAE/sephadex can be used in a single donor process both to purify prothrombin and to dehydrate.
• Serpines 9 are purified by standard methods. Yields are improved because of the protease inhibiting properties of the added citrate. Again, these products are safer because of the

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Engineering & Computer Science (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Medicinal Chemistry (AREA)
• Epidemiology (AREA)
• Pharmacology & Pharmacy (AREA)
• Zoology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Environmental Sciences (AREA)
• Cell Biology (AREA)
• Molecular Biology (AREA)
• Dentistry (AREA)
• Communicable Diseases (AREA)
• Pest Control & Pesticides (AREA)
• Plant Pathology (AREA)
• Agronomy & Crop Science (AREA)
• Oncology (AREA)
• Biotechnology (AREA)
• Developmental Biology & Embryology (AREA)
• Immunology (AREA)
• Virology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Diabetes (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26959126

Family Applications (1)

Country Status (6)

Families Citing this family (7)

Family Cites Families (12)

• 2002
  • 2002-02-07 CA CA002438223A patent/CA2438223A1/en not_active Abandoned
  • 2002-02-07 EP EP02717409A patent/EP1363616A1/en not_active Withdrawn
  • 2002-02-07 AU AU2002248415A patent/AU2002248415B8/en not_active Ceased
  • 2002-02-07 MX MXPA03007069A patent/MXPA03007069A/es not_active Application Discontinuation
  • 2002-02-07 WO PCT/US2002/003996 patent/WO2002087560A1/en not_active Application Discontinuation
  • 2002-02-07 BR BR0207058-8A patent/BR0207058A/pt not_active IP Right Cessation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events