AU2002248415B8 - Carboxylic Acid Such as Citric Acid for Disinfecting or Enhacing the Production of Blood Products Such as Plasma, Cryoprecipitate and/or Platelet - Google Patents

Description

00 CARBOXYLIC ACID SUCH AS CITRIC ACID FOR DISINFECTING OR ENHACING THE PRODUCTION

OF

BLOOD PRODUCTS SUCH AS PLASMA, CRYOPRECIPITATE OR/AND PLATELET Cct Related Applications o00 The present application is a continuation in part of US Patent C-i Application Serial No. 09/694,178 filed October 23, 2000, and US Patent SApplication Serial No. 09/778,681 filed on 7 February 2001 and US Patent cApplication Serial No. 60/278,496 filed 23 march 2001 and claims priority from the later two applications. All these applications are incorporated by reference herein.

Background of the Invention Area of the Art 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, blbod cells and plasma proteins, and to the removal of activated and denatured components, 'thereby improving the safety and efficacy of end products.

Description of the Prior Art There are a number of medical indications for administration of "clotting" or "coagulation" factors from human blood. These factors are proteins that cause the clotting of blood to staunch bleeding from wounds, WO 02/087560 PCT/US02/03996 2 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 clottingrelated factors including the use of fibrin to produce "fibrin sealant" or "fibrin glue".

While some of the clotting factors are currently produced through biotechnology, at this time there is still no cost effective method of artificially manufacturing all of these proteins or these proteins in sufficient quantities. Further, the "artificially produced" factors made by recombinant and related technologies tend to be more expensive. Many of the "minor" factors are not yet (and may never be) available from biotechnology sources and so must be purified from donated human blood. This is especially true in Third World countries where the biotechnology products are generally not available or affordable. Therefore, much of the supply of anti-hemophilia factor (AHF, also known as Factor VIII), and other blood clotting factors are prepared from pooled human plasma. A hemophiliac requires treatment for a whole lifetime. Victims of liver disease and other users of clotting factors may also require prolonged treatment. Therefore, these patients are exposed to blood p&oducts produced from the blood of a large number of donors.

The presence of AIDS (Acquired Immuno Deficiency Syndrome) virus or HIV in the blood supply means that hemophiliacs and other users of clotting factors have become infected with this terrible disease. Although tests to screen out AIDS-tainted blood have been improved, some infected blood does slip by. Even if the AIDS problem is solved, the danger of other WO 02/087560 PCT/US02/03996 3 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". However, even with the use of clotting factors derived from a single donor, there is still danger. Even though tests may show the donor is free of known disease, the donor may be incubating a disease that will later show on the tests, or the donor may harbor a yet unknown disease or a yet unknown strain of a known disease. These dangers have been lessened by use of plasma pre-treatments that inactivate disease organisms. Unfortunately, the best commonly used treatments either do not inactivate all types of disease organisms or damage the labile clotting factors during the process of inactivating disease organisms.

The basic methods for preparing clotting factor concentrates from blood have not changed greatly over the last few decades. Generally, 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. Following cryoprecipitation, the partially purified factors may be further purified by additional precipitation steps or by chromatographic methods, and most recently by methods using monoclonal antibodies. For additional information on the basic techniques of clotting factor purification and the history of the development of these methods, the reader is directed to U.S. Pat. Nos. 3,560,475, 3,631,018, 3,682,881, 4,069,216, and 4,305,871 and 5,770,704 by the present inventor, the contents of which are incorporated herein by reference, and the references cited therein.

WO 02/087560 PCT/US02/03996 4 Summary of the Invention It is an object of the present invention to enhance the yield and purity of cryoprecipitate; It is a further object of the present invention to inactivate and/or enhance the inactivation of disease organisms within plasma at the same time that cryoprecipitate production is enhanced and the cryoprecipitate is further purified.

It is a further object of the present invention to inhibit the activation or denaturation of blood components, including blood cells and plasma proteins, and/or to remove these activated or denatured components, thereby improving the safety and efficacy of the end product.

It is an additional object of the present invention to provide an improved method for blood f.ractionation.

Derivatives of simple carboxylic acids, particularly trisodium citrate and other citric acid salts (hereinafter "citrate") are shown to be unexpectedly effective agents for enhancing the production of blood clotting factors. It is believed that other small carboxylic acids, isocitric acid in particular, may show similar properties. However, to date most of the tests have been made with citric acid and its salts. Addition of citrate to plasma, especially at concentrations between 2 and 10 by weight, does not appreciably denature [abile proteins. However, in this concentration range citrate is effective in inactivating or inhibiting a variety of pathogenic microorganisms. Further, the added citrate potentiates or enhances the killing of microorganisms by heat treatment. That is, heating of the material to relatively low temperatures above 45 0 C) which do not denature proteins enhances the killing of microorganisms in the presence of citrate.

Most significantly, added citrate causes a dramatic increase in the weight of cryoprecipitate that can be produced from plasma by the usual procedures.

WO 02/087560 PCT/US02/03996 The majority of significant clotting factors are greatly concentrated in the resulting cryoprecipitate. The supernatant contains little if any of these clotting factors. It is apparent that increasing the amount of citrate in blood bags so that the final concentration will be at least 2% by weight results in plasma that can be used to produce improved platelet concentrates and enriched cryoprecipitate. The added citrate can help eliminate or suppress contaminating microorganisms and can itself be removed later by ion exchange or similar methods well known in the art.

Another aspect of the invention is the use of 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.

According to the invention there is provided 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. In this method, 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.

Moreover, 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, 6 00oo Prothrombin Complex, a Serpine, Albumin, and a Globulin from plasma. At least 2% by weight of a salt of citric acid (or equivalent weight of citric acid) is Sadded to the plasma. The plasma may be collected into a blood bag containing the carboxylic acid or the carboxylic acid salt. This blood bag can Mr 5 be different from a bag or container used to collect whole blood. Alternatively or additionally, an amount of additional carboxylic acid or the salt thereof may Sbe added directly to the bag used to collect the whole blood.

00oO In a further preferred form of the invention, citrate is used appropriately N in the collection of blood, in the processing and transfer of blood in a

(N

C 10 separation of discrete blood components. Citrate is used in increased, C namely, additional quantities over the level traditionally employed for anticoagulation in one or other collection or processing bag.

Definitions of the specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided a method for reducing transfusion-associated disease and adverse effects and for enhancing the purity and safety of multiple derivative components of blood including blood cells and plasma comprising the step of adding at least about 2% by weight of carboxylic acid or a salt of carboxylic acid to the blood or plasma.

According to a second embodiment of the invention, there is provided a method for reducing transfusion-associated disease and adverse effects in plasma by producing a cryo-depleted plasma comprising the steps of: adding at least about 2% by weight of carboxylic acid or a salt of the carboxylic acid to the plasma; forming a cryoprecipitate in the plasma; and separating the cryoprecipitate from the plasma thereby yielding cryodepleted plasma.

According to a third embodiment of the invention, there is provided a fibrin glue produced by a method for reducing transfusion-associated disease and adverse effects in plasma and for enhancing the purity and safety of 00 multiple derivative components of blood through fractionating plasma, 0 0said method comprising the steps of: adding at least about 2% by weight of carboxylic acid or a salt of the G carboxylic acid to the plasma; C 5 subjecting the plasma to a freeze/thaw step to produce an enriched cryoprecipitate and cryo-depleted plasma; and separating the enriched cryoprecipitate from the cryo-depleted plasma; 00oo wherein the enriched cryoprecipitate is used to produce the fibrin glue.

N Brief Description of the Figures 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.

Detailed Description of the Invention The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide enhanced production of plasma proteins along with inactivation of blood borne disease organisms.

WO 02/087560 PCT/US02/03996 7 The traditional method for producing clotting factors, as well as many of the presently used methods, operate because many plasma proteins responsible for clotting precipitate form cryoprecipitate) from solution at low temperatures when they are sufficiently concentrated. When a protein solution is frozen, ice crystals form and protein molecules, which are excluded from the crystals become increasingly concentrated. Cooling or freezing the water also lowers the chemical activity of the water. Depending on the particular proteins, 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. When the chemical activity of water is lowered such precipitation is favored. This process may denature the proteins (make them irreversibly insoluble), so it is usual to freeze protein solutions rapidly and to a low temperature -20 0 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. However, even when freezing is carried out with great care, 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 40C., 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 WO 02/087560 PCT/US02/03996 8 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 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. These 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 WO 02/087560 PCT/US02/03996 9 derivatives of other low molecular weight carboxylic acids such as isocitric acid) has unusually favorable properties when used in blood fractionation procedures at levels significantly higher than those traditionally used as an anticoagulant. Citrate is a fairly effective chelator of calcium ions. 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. As the citrate is increased, the weight of recovered cryoprecipitate is increase. 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.

Treatment Cryoprecipitate Factor VIII Fibrinogen control O.1g 120% 46 mg/dl 2% citrate 0.3 g 180% 53 mg/dl citrate 0.9 g 247% 105 mg/dl citrate 3.0 g 622% 152 mg/dl These data are graphically represented in Fig. 1. These results indicate that as the citrate concentration is increased the amount of recovered clotting factors increases linearly. However, at the highest concentration of citrate it would appear that there might be an increase in WO 02/087560 PCT/US02/03996 the precipitation of other proteins. It may be possible to adjust the citrate concentration to favor the precipitation of different proteins. Tests have shown that besides more than 95% of the Factor VIII and Fibrinogen, virtually all of the Fibronectin and the von Willdebrand's factor become concentrated in the citrate-enhanced cryoprecipitate. Additional experiments have been undertaken to determine if metalloproteins or other factors are preferentially concentrated in the citrate cryoprecipitate. Initial results do not show any other proteins as strongly concentrated as those already mentioned. However, there is some indication that ceruloplasmin and total T3 are somewhat concentrated in the cryoprecipitate.

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.

The following quantities of either salt (NaCI) or citrate (trisodium citrate) were added to 40 ml aliquots of fresh human plasma. After thorough mixing the samples were frozen overnight at -70 0 C and then completely thawed at 4 0 C. The samples were then centrifuged at 4000 RPM for 20 min to harvest the cryoprecipitate. An attempt was made to match the effective sodium concentration between the sodium chloride and sodium citrate on the basis that each molecule of trisodium citrate would provide three sodium ions whereas each molecule of sodium chloride would provide only a single sodium ion. This attempt at compensation was inaccurate because the matching should be done on a molar rather than a percent basis. However, this failed experiment points out the incredible superiority of sodium citrate over sodium chloride for producing cryoprecipitate. In the following table the citrate (trisodium citrate) or salt WO 02/087560 PCT/US02/03996 11 (sodium chloride) are shown, first as weight percentages and then as molarities. The third column shows the effective osmotic effect of the solutions, which is two times higher on a molar basis for citrate than for sodium chloride. This is because each molecule of sodium chloride releases only two particles (one sodium ion and one chloride ion) whereas each molecule of trisodium citrate releases four particles (three sodium ions and one citrate ion). Because the molecular weight of trisodium citrate is almost 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.

Weight Molarity Osmotic Effect Cryoprecipitafe Control 0.18g 2% citrate 0.07 0.28 0.52g citrate 0.17 0.68 1.3g citrate 0.34 1.36 3.6g 6% sodium chloride 1.02 2.04 0.12g sodium chloride 2.56 5.13 0.14g sodium chloride 5.13 10.26 0.15g These results show that the effect of citrate on cryoprecipitate production is not strongly related to the colligative or osmotic properties of the citrate. Sodium chloride seems not to enhance cryoprecipitate formation. Only at osmotic levels that are greatly above those of the maximal citrate concentration, cryoprecipitate formation begins to approach that of the control plasma. Further, the resulting cryoprecipitate does not appear as pure (that is, larger amounts of other non-clotting proteins are included).

Following the experiment, the supernatants and the original plasma (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.

WO 02/087560 PCT/US02/03996 Weight Fibrinogen (mg/dl) Factor VIII Albumin (g/dl) Control 287 24 3.2 2% citrate 216 6 3.1 citrate 114 <3 3.2g citrate <40 <3 3.2g 6% NaCI 298 30 3.1g NaCI 229 24 NaCI 97 9 2.9g As the amount of citrate is increased the levels of fibrinogen and Factor VIII in the supernatant decrease dramatically. At the same time, the level of albumin [the major plasma protein) is essentially unaffected. In other words, most of the clotting factors precipitate and are found in the cryoprecipitate, b but little or no albumin precipitates. In the case of sodium chloride, equimolar concentrations are much less effective at precipitating the clotting factors. One has to go up to 30% sodium chloride to see a significant precipitation of the clotting factors. However, at this level the albumin also begins to precipitate. Citrate is far more effective at selectively precipitating the clotting factors.

Further insight into the citrate effect is gleaned by analyzing the distribution of citrate in a typical cryoprecipitate experiment. For this experiment, one unit (about 200 ml) of plasma was brought to 10% wt/vol.

trisodium citrate. In all experiments pH measurements showed that natural buffering of the plasma prevented significant changes in pH. This citratetreated plasma was frozen and cryoprecipitate was collected in the usual manner. As an aside, in producing 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 mi-that is, 10% of the total volume. Surprisingly, an analysis of the cryoprecipitate and the supernatant plasma showed that about 12 g of the citrate was concentrated in the cryoprecipitate WO 02/087560 PCT/US02/03996 13 with only 30% being left in the supernatant. This indicates that there is a strong interaction between the cryoprecipitate proteins and the citrate.

Further, while normal cryoprecipitate can be redissolved in room temperature water or buffer, 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. One way of explaining these phenomena is to assume that the multiple negative charges on the citrate molecule are interacting with positive charges on the cryoprecipitate proteins to cross-link them. Added citrate "satisfies" these positive charges so that cross-linking is abolished. Because of the concentration of clotting proteins into the cryoprecipitate, it is tempting to theorize that all of the clotting proteins share some sort of positive charge motif that interacts with the citrate molecules.

In summary, compared to "normal" cryoprecipitate 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.

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 WO 02/087560 PCT/US02/03996 14 platelets. This would be consistent with the hypothesis that citrate inhibits some of the proteases.

2% Citrate Plasma Measurement Day 1 Day 6 Fibrinogen (mg/dl) 243 239 Factor II 104 91 Factor V 49 22 Factor VII 81 72 Factor VIII 103 92 Factor IX 106 Factor X 92 97 Platelets (10 3 /10- 6 L) 311 239 PT (sec) 14.1 16.2 PTT (sec) 31.4 47.9 Control Plasma Measurement Day 1 Day 6 Fibrinogen (mg/dl) 241 239 Factor II 103 93 Factor V 58 Factor VII 86 69 Factor VIII 100 89 Factor IX 106 92 Factor X 93 Platelets (x10 3 /10 6 L) 317 192 PT (sec) 13.7 19.5 PTT (sec) 32.5 50.2 Significantly, bacteriology experiments showed that 2% trisodium citrate strongly inhibits growth of Escherichia coli and completely inhibits the growth of Staphylococcus epidermidis. Growth of bacteria (primarily skin bacteria from inadequate surface disinfection) in platelet concentrates significantly lowers the useable life of platelet-rich solutions. Addition of citrate inhibits bacterial growth thereby potentially extending the life of such WO 02/087560 PCT/US02/03996 concentrates. As has been demonstrated above, addition of citrate does not damage the plasma constituents and actually significantly enhances the production of cryoprecipitate. Therefore, it is proposed to significantly increase the level of citrate in blood collection bags from the 0.4% currently used for anticoagulation to at least 2% trisodium citrate by weight. This level would inhibit or kill many contaminating microorganisms and would render the plasma more suitable for production of cryoprecipitate. It is also a simple matter to add trisodium citrate just before cryoprecipitate production where levels beyond 2% are needed.

Added citrate appears to enhance the susceptibility of microorganisms to a variety of "disinfecting agents" including heat. In one experiment 2%, sodium citrate was added to a typical bacterial growth broth. Twenty-five ml aliquots of the broth were spiked with 1 x 10' organisms of either Escherichia co/i or Staphylococcus epidermidis. Samples of the broth were brought to 2% by weight trisodium citrate and then subjected to "pasteurization" at 650C for either 5 or 10 min. after which the samples were plated on growth media and incubated. The 10 min citrate treatment caused total destruction of the bacteria. At 10 min the control bacteria were essentially unaffected. However, the 5 min treatment citrate did not kill all of the E. coli bacteria (approximately a 3-log kill). Staphylococcus epidermidis was more sensitive and was completely killed in the presence of citrate. Addition of citrate clearly enhances the ability of heat to kill microorganisms. Further, added citrate appears to stabilize labile proteins against heat denaturation. These results indicate that addition of increased citrate makes possible effective heat treatment of the plasma.

A problem with platelet concentrates and with plasma is the growth over time of bacteria that are originally present in very low numbers. Some of the contaminating bacteria apparently come from the skin surface when the blood is obtained by venipuncture. Further, there is growing evidence WO 02/087560 PCT/US02/03996 16 that blood is not completely aseptic. That is, there are normally a small number of bacteria circulating in the human bloodstream. Normal immunity prevents the overgrowth of these bacteria. To simulate this situation 10 ml samples of human plasma were inoculated at very low levels (10 organisms per ml) with the bacteria listed in the following table. Either normal plasma or plasma with 2% by weight of citrate was employed. The samples were incubated at room temperature for seven days with a sample plated on growth agar at each time point. Three different human plasmas were used, but all produced identical results. In the table "ng" "no growth" while indicates some bacterial growth and indicates more extensive growth.

a) Escherichia coli b) Klebsiella pneumoniae c) Staphylococcus epidermidis d) Staphylococcus aureus e) Pseudomonas fluorescens f) Yersinia enterocolitica g) Serratia marcescens Inoculum Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 N C N C N C N C N C N C N C a) ng ng ng ng ng ng ng ng ng ng ng ng b) ng ng ng ng ng ng ng ng ng ng ng ng d) ng ng ng ng ng ng ng ng ng ng ng ng e) ng ng ng ng ng ng ng ng ng ng ng ng f) ng ng ng ng ng ng ng ng ng ng ng ng g) ng ng ng ng g n g ng ng ng ng ng ng ng ng At days six and seven, the normal plasma showed growth of all of the inoculated bacteria species except for Serratia. On the other hand, none of the plasma samples containing citrate demonstrated any bacterial growth.

This indicates that 2% by weight citrate is able to inhibit strongly the growth of a wide range of bacteria. Combining these results with the favorable platelet results demonstrates that addition of 2% or more citrate WO 02/087560 PCT/US02/03996 17 to platelet concentrates can preserve the concentrates against bacterial growth without damaging the platelets. If there is any concern about excess citrate in the platelets, it can be readily removed by treatment with an anion exchange resin or similar material.

It was suspected that the failure to observe Serratia was due to the slow growth rate of this organism. Therefore, the experiment was repeated using Serratia marcescens and Staphylococcus epidermidis to inoculate plasma samples at the level of 100 organisms per ml. In this case the one day time point for Serratia showed 92 colonies while that for Staphylococcus showed 101 colonies for the normal plasma. Thus, no growth was observed in either case for the citrate-containing plasma.

The precise mechanism by which citrate and similar molecules act is 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. There are other characteristics, and blood components, and products that form the subject of the invention. These are illustrated in the WO 02/087560 PCT/US02/03996 18 attached FIGURE 2 that shows a Fractionation Scheme, and different blood components and plasma proteins, which are obtained from the system and to which the citrate technology is applied. 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 by weight or by volume trisodium citrate. As mentioned above, 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: 1. 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. If crude fibrin glue 4 is produced directly from enriched cryoprecipitate by addition of thrombin or prothrombin complex (Factors II, VII, IX, and 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. Further, because of the higher yield of enriched cryoprecipitate, and greater strength of the crude fibrin glue autologous fibrin glue becomes much more feasible. Autologous products are inherently safer. Further, because of the much greater yield of fibrinogen with enriched cryoprecipitate, it is feasible to produce "refined" fibrin glue or WO 02/087560 PCT/US02/03996 19 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).

2. Fibrinogen 3, Fibrin Glue 4, von Willdebrand's factor Fibronectin 6 and Factor VIII 7 can be produced by standard chromatographic methods, but use of differential extraction simplifies the process. As mentioned above, 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.

3. rroducts 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).

4. It is also possible to use iodine disinfection before the cryoprecipitate step. A co-pending application demonstrates that citrate enhances the iodine treatment. However, the columns used in the 00 referenced patent remove citrate. Therefore, after Iodine disinfection, 0 0additional citrate can be added at the second blood empty bag step but before the freeze/thaw procedure.

SAlbumin Globulins (product 10) 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 00 purify prothrombin and to dehydrate. Serpines 9 are purified by standard c methods. Yields are improved because of the protease inhibiting properties of C 10 the added citrate. Again, these products are safer because of the citrate, C because of the iodine step, because single donor products can be readily prepared and because pasteurization is facilitated.

The invention covers the process and products obtained by the process. The following claims are thus to be understood to include what is specifically illustrated and described above, what can be obviously substituted and also what incorporates the essential idea of the invention. The illustrated embodiment has been set forth only for the purposes of example and that should not be taken as limiting the invention. Therefore, it is to be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described herein.

The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

Claims (18)

1. 2. The method of claim 1, wherein the carboxylic acid is citric acid. (.i (.i
2. 3. The method of claim 2, wherein a trisodium salt of citric acid is added to the plasma.
3. 4. The method of any one of claims 1 to 3, wherein the plasma is a platelet concentrate. The method of any one of claims 1 to 4 further comprising the step of heating the plasma above 45 0C following the step of adding at least about 2% by weight of carboxylic acid or said salt of carboxylic acid.
4. 6. The method of any one of claims 1 to 5 further comprising the step of removing the carboxylic acid or the carboxylic acid salt by means of ion exchange chromatography.
5. 7. The method of any one of claims 1 to 6, wherein the plasma is placed into a blood bag containing the carboxylic acid or the carboxylic acid salt.
6. 8. The method of Claim 7, wherein the blood bag is a different from a bag or container used to collect whole blood.
7. 9. A method for reducing transfusion-associated disease and adverse effects in plasma by producing a cryo-depleted plasma comprising the steps of: adding at least about 2% by weight of carboxylic acid or a salt of the carboxylic acid to the plasma; forming a cryoprecipitate in the plasma; and separating the cryoprecipitate from the plasma thereby yielding cryo- depleted plasma. 00 ZZ The method of claim 9, wherein the carboxylic acid or salt of the carboxylic acid is added during the thawing portion of a freeze/thaw step.
8. 11. The method of claim 9 or claim 10 further comprising the step of fractionating the cryo-depleted plasma to produce at least one of prothrombin complex, serpine, albumin and globulin.
9. 12. The method of claim 9 or claim 10, wherein the plasma is subjected to an 00 iodine disinfection step.
10. 13. The method of any one of claims 9 to 12, wherein the carboxylic acid is Scitric acid.
11. 14. The method of claim 13, wherein a trisodium salt of citric acid is added to the plasma. The method of any one of claims 9 to 14, further comprising the step of heating the plasma above 45 °C following the step of adding.
12. 16. The method of any one of claims 9 to 15 further comprising the step of removing the carboxylic acid or the carboxylic acid salt by means of ion exchange chromatography.
13. 17. The method of any one of claims 9 to 16, wherein the step of adding is accomplished by placing the plasma into a blood bag containing the carboxylic acid or the carboxylic acid salt.
14. 18. The method of claim 17, wherein the blood bag is different from a bag or container used to collect whole blood.
15. 19. A fibrin glue produced by 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 through fractionating plasma, said method comprising the steps of: adding at least about 2% by weight of carboxylic acid or a salt of the carboxylic acid to the plasma; 00 23 O O subjecting the plasma to a freeze/thaw step to produce an enriched t cryoprecipitate and cryo-depleted plasma; and separating the enriched cryoprecipitate from the cryo-depleted plasma; 0 wherein the enriched cryoprecipitate is used to produce the fibrin glue.
16. 20. The fibrin glue of claim 19, wherein the fibrin glue further comprises at least one of prothrombin complex and thrombin. 00
17. 21. A cryo-depleted plasma when produced by the method of any one of N claims 9 to 18.
18. 22. The method as defined in claim 1 or claim 9 and substantially as described in the section entitled "Detailed Description of the Invention". Date: 3 March 2008