MXPA98009535A - Methods for the terminal sterilization of biologi products - Google Patents

Abstract

The invention relates to methods for sterilizing biologically active products, particularly prophylactic therapeutic products and the compositions obtained therefrom. The methods include obtaining a dry sample containing an amount of trehalose sufficient to give the product heat stability and exposing the dry sample to heating conditions at a temperature, and for a sufficient time, to substantially inactivate viruses, essentially enveloped viruses without lipids . The drying methods include both environmental drying conditions and lyophilization. The heating conditions include any known in the art and cover a wide range of temperatures and heating times. The compositions obtained contain stable products and do not contain measurable infectious viruses, particularly parvovir

Description

METHODS FOR TERMI NAL STERILIZATION OF BIOLOGICAL PRODUCTS ICO TECHNICAL FIELD The invention relates to the field of sterilization of products derived from blood and other biological sources. The invention involves heating biologically active products in the presence of trehalose for a time and under conditions sufficient to kill viruses, particularly parvoviruses. ICA ANTECE DENTE TECHNOLOGY The complete removal of viruses and other contaminants for biologically active products is essential for the production and use of a wide variety of therapeutic and prophylactic products. A number of methods are currently being used. Primarily, these are dry heat treatment, chromatography, solvent-detergent (SD) treatment and pasteurization. These methods also suffer from drawbacks and none has succeeded in removing all known viruses. They can also be viruses that have not yet been characterized as not being inactivated by these methods. For review, see Cuthbertson et al., (1991) Blood Separation and Plasma Fractionation, Wiley-Liss, I nc. p. 385-435; Mozen (1993) J. Clin. Apheresis 8: 126-130; Ingerslev (1994) Haemostasis 24: 31 1 -323; Dorner et al., (1993) Virological Safety Aspects of Plasma Derivatives, Brown, ed. , Dev. Biol. Stand. , Basel, Karger, vol. 81, pgs. 137-143; Mannucci (1993) Vox Sang 64: 197-203; and Hamman et al., (1994) Vox Sang, 67: 72-77. A wide variety of products with therapeutic utility are derived from biological sources such as plasma and cell lines. The majority of the plasma used for fractionation in the United States was obtained by plasmapheresis in collection centers distributed throughout the country. The centers provide plasma to commercial fractionators in the United States and Europe. Approximately 9 million liters of plasma are recovered each year from approximately 13 million donations. The Red Cross adds approximately 800,000 liters to this number. Human plasma products can be classified into several groups: albumin products; the immunoglobulins; cold insoluble globulins; the coagulation products; and proetase inhibitors. Albumin products, also called fraction V products, are mainly used to restore colloidal osmotic pressure under shock conditions such as burns, or hemorrhagic shock where fluid loss is a major problem. Immunoglobulins, or "gamma globulins" are isolated from fraction II and contain a mixture of antibodies representative of the plasma combination source. A number of hyperimmune glubulins used for passive immunization are isolated from the donor plasma at high levels of protective antibody. Cold insoluble globulins include fibrinogen and von Willebrand factor. The coagulation products include the antihemophilic factor VI I I and the factor IX complex used for replacement therapy in hemophilia A and B, respectively. An active form of the factor IX complex, called the anti-inhibitor coagulant complex, is prepared and used to treat patients with an inhibitor of factor VI H. Protease inhibitors include a1 proteinase inhibitor, also known as anti-trypsin a1 which is used to treat a congenital deficiency. Antithrombin I I I is an inhibitor that is also congenitally deficient which leads to thrombotic complications. Other body fluids are the source of therapeutic products. For example, erythropoietin was previously purified from the blood or urine of patients with aplastic anemia. Patent of E. U.A. Do not . 4,677, 195. The high purity albumin of human placenta was also obtained. Grandgeorge and Véron (1993) Virological Safety Aspects of Plasma Derivatives, Brown, ed. Dev. Biol. Stand. Basel, Karger, vol. 81, pgs. 237-244. The production of recombinant proteins in the milk of transgenic animals is now a commercial reality. Numerous therapeutic products are now obtained from cell cultures expressing recombinant proteins. Cell cultures are routinely grown in the presence of animal or human serum. The products are obtained from cells or from cell culture supernatant and, therefore, they may contain viruses, either from the medium or in the cells themselves. These products obtained include, but are not limited to, colony stimulation factors, monoclonal antibodies and derivatives thereof, growth factors such as erythropoietin, interleukins. Growth factors alone represent a multi-billion dollar industry. For review, see, Erickson (1991) Sci. Am., Feb. 1991 p. 126-127. Although the risk of viral contamination of proteins derived from cell culture is much lower than that associated with plasma products, as long as there is a risk of viral contamination when treated with cells. For this reason, products such as monoclonal antibodies are subjected to heat treatment in order to inactivate viruses. In addition, it is common practice to add human serum albumin (HSA) to stabilize recombinant protein formulations. Major viruses that develop in blood of clinical interest include hepatitis B and C viruses and the retrovi rus of VI H and HTLV (human T cell leukemia virus). With respect to blood derivatives. VLTH I and I I and cytomegalovirus (CMV) appear to be associated with cells and therefore do not present a risk in products without cells. As new viruses are discovered, the inactivation protocols are changed to adapt them. For example, the discovery that the human immunodeficiency virus (VI H) survived the normal process of factor VII, necessitated a protocol change requiring the addition of ASH to stabilize the product under the new, more severe conditions. Mozen (1993). Methods for inactivating HIV and hepatitis viruses in plasma fractions are known. As described above, heating at 60 ° C for 10 hours in the presence of ASH inactivates VI H. It was found that hepatitis that is not A, nor B (H NAN B) is inactivated in Factor VII and IX preparations by heating at 80 ° C for 72 hours in the dry freezing state. A study group of the U K Haemophilia Center, Directors on Surveillance of Virus Transmission by Concentrates (1988) Lancet Oct. 8, p. 814-816. In recent years, few transfusion-transmissible diseases have been identified, which, although not common from a public health perspective, have both real and potential transfusion impacts to use plasma and plasma derivatives as well as cellular products. These include parvovirus transmission (B 19). This etiologic agent seems to be resistant to the current methods used for viral inactivation. Sherwood (1993) Brown, ed. Virological Safety Aspects of Plasma Derivatives, Dev. Biol. Stand. Basel, Karger vol. 81, pgs. 25-33. These virus inactivation methods currently in use can also cause changes in the biological activity of the biological products obtained. The immunogenicity of the products is of special interest where the sterilization treatment can induce the cleavage and / or aggregation of proteins. For example, it has been found that factor VI I I concentrates evidence exhibiting FV M I activation, with more than one higher potency of one stage and two steps, faster FXa generation and increased lower molecular weight polypeptide contents. Viral inactivation procedures can also induce change in components that are not F VI I i and these may be partially responsible for the immunosuppressive activity of some of these concentrates Barrowcliffe (1993) Virological Safety Aspects of Plasma Derivatives Brown, ed. Dev. Biol. Stand. Basel, Karger, vol. 81, pgs. 125-135. Notorious changes in immune system functions both in vitro and ex vivo have been found in patients frequently exposed to biologically derived products. In VI H negative patients, changes include decreased numbers and functions of immune competent cells as assessed by their response to the stimulus and in terms of markers of their cellular change. These changes probably occur when chronic viral disease is present. further, denatured allogeneic protein impurities from factor concentrates and other contaminants may also be responsible for immunosuppression. See, Ingerslev (1994) for review. The human parvovirus is a newly discovered agent who was given the code number B 19. Cossart et al., (1975) Lancet 1: 72-73. It is a very small single-stranded DNA virus (24 nm) with a very simple protein coating, but no external lipid coat. It causes a temporary viremia of 1-2 weeks but it can achieve extraordinarily high virus titers of at least 101 2 particles per ml. Although parvovirus usually causes a relatively minor disease that is often not clinically apparent, producing a rash similar to moderate rubella known as the fifth disease or infectious erythema, it can also cause more severe reactions. Parvovirus infects bone marrow support cells and this can lead to a severe life-threatening condition in patients with pre-existing secondary anemia. The aplastic crisis as a result of acute interruption of hemopoiesis can occur in patients with congenital haemolytic anemias and immuno-deficiency states. Parvovirus also causes hydrops fetalis in pregnant women. Therefore, parvovirus represents a danger of infection for those patients who receive therapeutic agents derived from plasma, particularly in those patients with hemostatic disorders. It is of interest that this virus is transmitted by some concentrates despite the use of robust virucidal methods and chromatographic removal, not only because of the risk of parvovirus transmission, but because it can exist due to other existing viruses with the same characteristics. A study in children with hemolytic disorders found that parvovirus is rapidly infectious in plasma derivatives that have not been heat treated. In one study, a small group of children (N = 9) treated with heat treated factor VI I I concentrate were not infected with parvovirs. Williams and others (1990) Vox Sang. 58: 177-181. However, others have found that heat treated products transmit parvovirus infection. Corsi et al. (1988) J. Med. Virol. 25: 151-153. Unlike hepatitis and VI H, the parvovirus was not tested for plasma donations in individuals and therefore is present in the combined plasma. As mentioned before, several treatments have been proposed or are in use to inactivate viruses in biologically derived therapeutics. For review, see Soumela (1993) Trans. Med. Rev. VI L42-57. The final product has been obtained by a combination of division steps and inactivation steps, both of which serve to reduce viral load. There are several heat treatments currently in use. Heating in solution is commonly used for albumin products. In some way this is known as pasteurization. The viruses are inactivated by heating the liquid samples for at least 10 hours at 60 ° C in the presence of a small amount of stabilizer such as caprylate or tryptophanate. This method is not suitable for other products, however, most proteins are denatured under these conditions. It has been shown that pasteurization inactivates a broad spectrum of viruses including VI H, VH B, HCV, HAV, VS H, poliovirus CMV, mumps virus, measles virus and rubella virus. Nowak et al. (1993) Virological Safety Aspects of Plasma Derivatives, Brown, ed. , Dev. Biol. Stand. Basel, Karger, vol. 81, pgs. 169-176; and Soumela (1993). In these studies, parvovirus was not tested. In addition, the use of pasteurized coagulation factors has been associated with the formation of neoantigens, Ingerslev (1994). The heating of the dry products was carried out when labile proteins dried by freezing tolerated temperatures of up to 68 ° C. Previous methods that included heating at 60 ° C were used to inactivate hepatitis virus. See, for example, the Patent of E. U.A. No. 4,456, 590. However, these conditions were insufficient to inactivate HIV as evidenced by the transmission of the virus through purified coagulation factors. It was also found that products treated at 68 ° C for 72 hours are unsafe. Soumela (1993). More recently, higher temperatures and longer heating times, such as 80 ° C for 72 hours have been used to ensure the inactivation of hepatitis viruses and VI H. See, for example, Knevelman and others (1994) Vox Sang 69: 89-95. However, the most labile biological assets, especially biopharmaceuticals, do not survive exposure to such extreme temperature / time conditions. Another drawback of this method is the unpredictable result that counts with respect to inactivation of parvovirus. Santagostino et al. (1994) Lancet 343: 798; and Yee et al. (1995) Lancet 345: 794. The inactivation of Solvent / Detergent (SD) virus is supported by an alteration of the membranes of viruses that have lipid shells. Viruses become non-infectious either by structural alteration or destruction of the cell receptor recognition site. Although most human pathogenic viruses have a lipid envelope, parvovirus and HAV are not inactivated by this method. For review, see Wieding et al. (1993) Ann. Hematol. 67: 259-266. The SD method is in use in several countries. Horowitz et al. (1993) Virological Safety Aspects of Plasma Derivatives; Brown, ed. Dev. Biol. Stand Basel, Karger, vol. 81, pgs. 147-161. In a related method, viruses have been used to inactivate lipid-covered viruses. Isaacs et al. (1994) Ann. NY Acad. Sci. 724: 457-464. A number of other methods have been developed or are under development. For example, chilled plasma sterilization was carried out by exposing plasma to a combination of α-propiolactone and UV light. However, this method reduces the activity of labile proteins. Several chemical treatments have been proposed including the use of psoralens and UVA, BPD-MA and light, although this can be limited by the inactivation of virus coated with lipids. Caprylate and sodium chloride have also been found to be virucidal viruses. Various methods of physical separation including affinity chromatography have been tested; fractionation with cold ethanol, fine pore membranes, and perfluorocarbon emulsions. Lawrence (1993) Virological Safety Aspects of Plasma Derivatives, Brown, ed., Dev. Biol. Stand. Basel, Karger, vol. 81, pgs. 191-197; Burnouf (1993) id., Pgs. 199-209; Teh (1993) Vox Sang. 65: 251-257; Lebing and others (1994) Vox Sang 67: 1 17-124; DiScípio (1994) Prot. Exp. Purif. 5: 178-186; Morgenthaler and Omar (1993) Virological Safety Aspects of Plasma Derivatives, Brown, ed. , vol. 81 P. 185-190; Erickson (1992) Sci. Am. September p. 163-164; and McCreath et al. (1993) J. Chromatog. 629: 201-213. The determination of successful virus activation during the manufacture of a plasma protein requires that three prerequisites be met. First, the manufacturing process should be downloaded as accurately as possible. Second, the relevant test viruses should be selected for the washing experiments. Third, the resulting samples should be analyzed appropriately for infectious viruses. The process of such a test is described in detail for example by Hilfenhaus et al. (1993) Brown, ed. Virological Safety Aspects of Plasma Derivatives Dev. Biol. Stand. Basel Karger vol. 81 pages. 1 17-123. These guidelines have been followed in the present. Trehalose, (aD-glucopyranosyl-aD-glucopyranoside), is a non-reducing disaccharide present in nature which was initially found to be associated with the prevention of damage by drying in certain plants and animals that can be dried without damage and can be revived when rehydrate. Trehalose is commercially available in the dihydrate form. It has been shown that trehalose is useful to prevent the denaturing of proteins, viruses and food products during desiccation. See the Patents of E. U.A. Nos. 4, 891, 319; 5, 149.653; 5, 026, 566; Blakeley et al. (1990) Lancet 336: 854-855; Roser (July 1991) Trends in Foods Sci. And Tech. 166- 169; Colaco et al. (1992) Biotechnol. Internet. 345-350; Roser (1991) BioPharm. 4: 47-53; Colaco and others (1992) Bio / Tech. 10: 1007-101 1; and Roser and others (May 1993) New Scientist, p. 25-28. Trehalose dihydrate is available in crystalline formulations to the degree of good manufacturing processes (BPM). A method for forming an anhydrous dessicant form of trehalose is described in EP Patent Publication no. 600 730. This method involves heating a trehalose syrup in the presence of a seed crystal and recovering the anhydrous trehalose. Trehalose is widely found in such species of diverse animals and plants such as bacteria, yeast, fungi, insects and invertebrates. In many insects, it is the main sugar in the blood. The only main source for man is the diet in foods such as mushrooms and yeast products. Madsarovova-Nohejlova (1973) Gastroenterol 65: 130-133. Trehalose was described for use in a peritoneal dialysis system in the U. U.A. Patent. No. 4, 879,280 wherein it is mentioned as one of several disaccharides as a replacement for the prior art system utilizing glucose. Trehalose is mentioned for the use of a dialysis system as a disaccharide that will not be easily separated to glucose and, therefore, prevents the elevation of the blood glucose level. Trehalose has been described as being suitable for use in parenteral formulations mainly because it can be sterilized by autoclaving without obtaining the brown color associated with conventional parenteral formulations. Japanese Patent No. 6-70718. Trehalose is a common component of the human diet and information is available in your metabolism. After oral ingestion, trehalose is not absorbed intact through the gastrointestinal tract, since only monosaccharides can pass through the intestinal epithelium. Ravich and Bayless (1993) Clin. Gast 12-335-356. Trehalose is metabolized by the enzyme trehalose in two glucose molecules. Sacktor (1968) Proc. Nati Acad. Sci. USA 60: 1007-1014. Trehalose is a normal constituent of most mammalian bodies, including humans, and has been identified in human serum, lymphocytes and the liver, but is mainly located in the hairy part of the intestinal tract and the proximal renal tubules. Belfiore et al. (1973) Clin. Chem. 19: 447-452; Eze (1989) Biochem, Genet. 27: 487-495; Yoshida et al. (1993) Clin. Chim. Acta 215: 123-124; and Kramers and Catovsky (1978) Brit. J. Haematol. 38: 4453-461. Trehalose is a protein bound to the membrane of the intestinal tract of humans and animals. Bergoz et al. (1981) Digestion 22: 108-112; Riby and Galand (1985) Comp. Biochem. Physiol. 82B: 821-827; and Chen et al. (1987) Biochem. J. 247: 715-724. All references cited herein are incorporated by reference. DESCRIPTION OF THE INVENTION The invention relates to methods for stabilizing products, particularly therapeutic products, derived from biological sources and the compositions obtained by them. The methods include drying the product in the presence of an amount of trehalose sufficient for heat stability to the product and exposing the dried sample to the heating conditions at a temperature and for a time sufficient to substantially inactivate the viruses. Preferably, the heating conditions are sufficient to inactivate the encapsulated viruses without lipids. The drying methods are any known in the art including both environmental drying conditions including spray and vacuum drying and lyophilization. The heating conditions cover a wide range of temperature combinations and heating time. The invention also encompasses the compositions obtained by the methods. These compositions contain stable biological products and do not contain detectable infectious viruses, particularly parvovirus. BEST MODE FOR CARRYING OUT THE INVENTION As described here in detail, there are numerous methods of terminal sterilization of biological products derived from blood. These methods are well known in the art, as exemplified by the references cited herein and do not need to be described in detail. Although strict purification methods such as antibody affinity chromatography can result in virus-free biological products, none of the commercially availamethods has been found to return to products consistently free of infectious viruses, particularly viruses encapsulated without lipids. Furthermore, increasing the severity of the sterilization conditions in order to return to a product free of infectious viruses has the drawbacks of decreasing the activity and / or increasing the immunogenicity of the product. In addition to blood products, there are numerous biologically active products that can benefit from the sterilization methods provided herein. These include, but are not limited to, recombinantly produced proteins, isolated natural proteins, antibodies, enzymes, cytokines and growth factors, as well as pharmaceutically active molecules such as analgesics, anesthetics, anti-hemetics, antibiotics, chemotherapeutic agents, hormones, vitamins. and steroids. Also, for use in the claimed methods, any substances that are introduced aseptically into an individual are suita These include, but are not limited to, drugs, antibiotics, imaging agents, diagnostic reagents. Most important, in the case where the product to be administered is labile, such as cephalosporins, therapeutic antibodies and erythropoietin, the invention provides sta dry, sterile compositions that can be rehydrated just before use. Trehalose is suitafor injecta infusiagents, etc. , in that it breaks down into two molecules of glucose when exposed to trehalose in the bloodstream. Glucose can cause a minor temporary increase in blood sugar levels, but it is of little clinical interest. The present invention encompasses methods of terminal sterilization of products that need to be administered aseptically to an individual. The steps of the method include obtaining a dry sample containing the product and an amount of α-D-glucopyranosyl-α-D-glucyranoside (trehalose) sufficient to render the product substantially stato heat; and heating the dried sample to a temperature for a time sufficient to substantially inactivate the viruses, preferably under heating conditions that inactivate virus encapsulated without lipids. The dry sample can also contain suitable pH buffer solutions, adjuvants, etc. Preferably in an amount that produces a suitable concentration upon rehydration. The product can be derived from a variety of sources. Preferably, the products are derived from any biological source, including, but not limited to, blood, plasma, serum, placenta, milk, urine, cell cultures, and cell culture supernatants. Additionally, the product can be derived synthetically, either by chemical or enzymatic synthesis or by the use of recombinant DNA techniques. The methods of preparing these sources and methods of isolating the products are well known in the art. Normally, products isolated or derived from blood, plasma and serum, include, but are not limited to, albumin products, immunoglobulins, coagulation products and protease inhibitors. Albumin products, include but are not limited to; ASH, insoluble globulins and fibrinogens. Immunoglobulins include, but are not limited to, antibodies against tetanus, pertussis, hepatitis B, Rho (D), Zoster varicella, and rabies. The coagulation products include, but are not limited to, antihemophilic factor VIII, factor IX complex and factor IX activating complex. Proetase inhibitors include, but are not limited to, a-1 protease inhibitor, a-1 antitrypsin, and antithrombin III. Other sources of these products are available, for example, you can get albumin from placental sources. Where the biological source is cell culture or cell culture supernatant of the product includes, but is not limited to, colony stimulation factors, monoclonal antibodies and derivatives thereof, and growth factors. Normal growth factors include, but are not limited to, naturally derived and recombinant erythropoietin, cytokines and interleukins. When the product is an agent that needs to be administered aseptically, the products include, but are not limited to, analgesics, anesthetics, chemotherapeutic agents, hormones and vaccines. Analgesics include, but are not limited to, morphine, benzocaine, pethidine and Demerol, anesthetics include, but are not limited to, bupivicaine, atracurium and vecuronium. Chemotherapeutic agents include, but are not limited to, radioisotopes, vinca alkaloids such as vinblastine, vincristine and vindesine sulfates, adriamycin, bleomycin sulfate, Carboplatin, cisplatin, cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Duanorubicin hydrochloride, Doxorubicin hydrochloride. , Etoposide, fluorouracil, mechloro-lamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaza hydrochloride, streptozotocin, taxol, thioguanine and uracil mustard. Suitable hormones include, but are not limited to estrogen, testosterone, progesterone and synthetic analogs thereof. Normal vaccines include antigen subunit vaccines, both single and multiple, as well as dead bacteria and viral preparations and cancer antigens. Normal antibiotics include, but are not limited to, cephalosporins and aminoglycosides. The method includes a first step to obtain a dry sample. A variety of methods can be used to dry the sample. These include, but are not limited to, air drying, vacuum drying, spray drying and freeze drying. These methods are exemplified in detail in the examples presented herein and are well known in the art. See, for example, Patents of the U.S.A. Nos. 4,891,319; 5,026,566; and 5,149,653. Samples are normally prepared in solution or suspension and include the product, a sufficient amount of trehalose to give heat stability to the product and any other normal additive such as suitable pH buffer solutions, adjuvants, etc. Typically, trehalose is present in an amount of 1-50% by weight of the solution. However, trehalose may be more dilute or concentrated. If it is less diluted, the drying times will be prohibitive, and if it is more concentrated, the solution can become viscous. It will be necessary to empirically determine the exact initial concentrations of product, trehalose and any additive, but these are well within the experience of one skilled in the art given the examples provided herein. Preferably, the concentration of product and trehalose is such that less than about 30% loss of activity of the product occurs after drying. More preferably, it is less than 15% loss of activity and more preferably, less than 10% loss. Once the dry sample has been obtained, it is subjected to heating conditions at a temperature and duration sufficient to inactivate the viruses. Normally, the dry sample is subjected to heating at 80 ° C for 72 hours to inactivate the lipid encapsulated viruses and 90 ° C for 72 hours to further inactivate the encapsulated viruses without lipids. The optical combination of heat and duration of heating will be determined empirically. Said determination is within the experience of someone in the art given the examples provided herein. Normally, optimal conditions are determined by punching a test sample with the virus that will be inactivated or with a virus that has similar physical characteristics. Normally, it is considered that a loss of log10 from the titration of four minced viruses is "inactivation" while a log10 drop in titration of 4 is lost, it is the requirement of regulatory authority for the inactivation / removal procedure. The method results in stability to the substantial heat of the product. Preferably, the method results in less than about 30% loss in product activity. More preferably, the method results in less than about 15% loss in activity of the product. More preferably, the method results in less than about 10% loss in product activity. The method provides dry samples with high stability and is capable of being stored for long periods of time. This storage stability is related to the residual moisture of the sterilized product. Preferably, the method produces a dry sample having a residual moisture content of less than about 4%. More preferably, the content of residual moisture is less than about 2%. More preferably, the residual humidity is about 0.8-1.0%. Residual moisture can be measured by a variety of methods, including, but not limited to, differential thermal analysis, thermogravimetric methods or Karl Fisher coulometric titration. The scale of heating temperature and times varies widely, the optimum time for a particular sample can be determined empirically damaging the knowledge in the field and the examples provided herein. The results presented herein indicate that heating to about 80 ° C for about 72 hours is effective, as is heating to 90 ° C for about 20 hours. Longer periods and / or higher temperatures can be used with potential concomitant loss in product activity. Preferably, the method results in a reduction of four times log10 in ineffectiveness of contaminating viruses. A number of methods are known for making this determination. Many of these are cited before and a number of others are known in the art. Since lipid-encapsulated viruses are less resistant to heat treatment than encapsulated viruses without lipids, a determination of infectious quality loss of an encapsulated virus without lipids, indicates that all viruses encapsulated without lipids also have to be inactivated. Virus encapsulated without lipids include, but are not limited to, hepatitis A virus and parvovirus. Preferably, the viruses encapsulated without lipids and parvoviruses.

The invention also encompasses compositions obtained by the methods of the claimed invention. The compositions are free of detectable ineffective viruses and are extremely stable to storage. In addition, reduced denaturation to chemical degradation of therapeutic products during the process results in a decrease in the incidence of immune reactions to product receptors. The compositions preferably are in the form of a single dose, especially for drugs such as analgesics and chemotherapeutics. Simple dosage forms can be produced by taking aliquots of the initial solution or suspension in suitable containers and processing the containers separately. Preferably, said taking of notes and processing is automatic. Alternatively, the material can be processed in batches of more than one dose and the dried product can be divided into single doses. The invention therefore encompasses single dosage forms of the claimed composition. For products such as ASH, in the form of batches. Large batches can be processed to be commercially supplied in bulk. The invention, therefore, also encompasses bulk forms of the claimed composition. The following examples are provided to illustrate, but not to limit, the claimed invention. Example 1 Comparison of the effect of different sugars on parvovirus ineffectiveness and alkaline phosphatase activity A stock solution of 1 mg / ml alkaline phosphatase in a 50% trehalose solution formed in 25 mM pH H EPES buffer solution containing 50 mg / ml. mM of ammonium bicarbonate and 2% of ASH was stung with 106 5 of TCI D 50 / ml of canine parvovirus. Aliquots of 250 μl of the minced formulation were vacuum dried or freeze-dried in 3 ml Wheaton glass pharmaceutical flasks using a FTS dryer or Labconco freeze dryer. For vacuum drying, the shelf temperature was initially set at 30 ° C while the vacuum was reduced in one form in steps to 30 mTorr, when the shelf temperature was increased to 60 ° C and drying was carried out during 12- 16 additional hours, for freeze drying, the samples were frozen by reducing the shelf temperature from -40 ° C to 5 ° C per minute and the bottles were allowed to freeze completely for 1 hour before the vacuum was reduced to 10 mTorr and the samples were dried at -40 ° C for 40 hours. The shelf temperature was then raised to -20 ° C to 0.05 ° C per minute and the samples were dried at + 20 ° C for 3 hours and finally the shelf temperature was raised to + 40 ° C at 0.05 ° C per minute and the samples were dried at + 40 ° C for an additional 2 hours. The bottles were capped under vacuum and the samples were kept at 4 ° C as dry controls. For terminal sterilization, the bottles were treated at 80 ° C for 72 hours or 90 ° C for 20 hours in a Heraeus drying oven. The reduction of Log10 in parvovirus activity and percentage of reduction in alkaline phosphatase activity was evaluated. The parvovirus was analyzed by titration on cell culture and the end point was determined by agglutination of porcine red blood cells. In summary, the dried samples were reconstituted in their original volume using sterile distilled water and a 10-fold dilution series prepared in Eagle Minimum Essential Maintenance culture medium (EMEM). A suspension of A72 cells in EMEM containing 5% fetal bovine serum was prepared by trypsinization of a confluent flask of cells and 1 ml of cell suspension containing 2-5x103 cells / ml was taken in aliquots in the wells of a plate of 24-well cell culture. 100 μñ of each dilution of the viral sample was inoculated into four wells by replicating the cell culture plate and the plates were incubated for 14 days at 37 ° C in a 5% CO2 atmosphere. After 14 days, the cell culture medium for each well and tested for hemagglutinin activity using 1% porcine red blood cells. The viral titration as logio TCID 50 / ml was calculated using the Karber formula for quantification of the end point in the analysis of virus ineffectiveness, namely Karber formula = -log of dilution interval- (sum of positive tests) -0.5. To analyze alkaline phosphatase colometrically using the Sigma Fast ™ alkaline phosphatase assay commercial reagent. Briefly, a series of dilutions was prepared for the samples and a normal alkaline phosphatase preparation (1 mg / ml) and 100 μl of substrate was added to 100 μl of sample in an EIA plate and incubated in the dark for 30 days. minutes Color development was read at 405 nm using a multiple "tek" titration sweep interspersed with a delta soft-plate reading package and the activity of the samples was calculated for absorbance values corresponding to the linear section of the normal curve . The dry controls were assigned to 100% activity and percentage reduction in activity of the treated samples were calculated from these values. For all virus analyzes, the log10 reduction in the titration of the treated samples was calculated by the subtraction of log-, or TCI D 50 / ml of virus recovered from log 10 TC ID 50 / ml of values of the dry controls . The results are shown in Table 1 . In Table 1, >; means that the virus was below the level of detection of the analysis, RIT means "reduction in titration", ASH is for human serum albumin, * means log10 of TCI D 50 / ml of parvovirus in dry control = 6.5, ** means alkaline phosphatase activity in dry control = 100%, G PS is for glucopyranosyl sorbitol and n. r. unrealized. The abbreviations are the same through the examples section and the tables.

Table 1 Example 2 Terminal sterilization to eliminate the infectivity of parvovirus without loss of biological activity by vacuum drying in trehalose; effect of temperature and time.

A stock solution of 1 mg / ml alkaline phosphatase in a 50% trehalose solution formed in 25 mM pH buffer HEPES containing 50 mM ammonium bicarbonate and 2% ASH was minced with 1065 TCID 50 / ml parvovirus of canine and dried under vacuum in an FTS dryer. 250 μl of the formulation was dried in 3 ml bottles in the FTS dryer using a manual program that achieved operating parameters of 30 mTorr vacuum at 60 ° C shelf temperature. The bottles were capped under vacuum and the samples were kept at 4 ° C as dry controls. For terminal sterilization, the bottles were treated at 80 ° C for 72 hours or 90 ° C for 144 hours in a Heraeus drying oven. The reduction of log10 in parvovirus activity and percentage of reaction in alkaline phosphatase activity were evaluated. The parvovirus was analyzed by cell culture titration followed by agglutination of red blood cells from portions as described in Example 1. Viral titration as log10 TCID 50 / ml was calculated using the Karber formula as described in Example 1. The alkaline phosphatase was analyzed colometrically, using a commercial reagent, Sigma Fast ™, alkaline phosphatase subtracted analysis was analyzed as described in Example 1. A summary of the results obtained is shown in Table 2 describing the reduction of log10 in parvovirus titre and percentage reduction in alkaline phosphatase activity following terminal sterilization at 80 ° C for 72 hours or at 90 ° C for 144 hours. In Table 2, * logio means TCID 50 / ml parvovirus in dry control = 6.5 ** means alkaline phosphatase activity in dry control = 100% and > is below the detection limit of the analysis. Table 2 Example 3 Terminal sterilization to eliminate infectivity of covered and uncovered viruses without loss of biological activity by vacuum drying in trehalose. In this experiment, the same formulations were chopped as in Example 1 with three different virus preparations: poliovirus; parvoviruses (RNA not covered and DNA containing virus respectively); and measles virus (covered DNA virus). The reduction of log 10 in virus titration and percentage of reduction in alkaline phosphatase activity were evaluated as previously or previously described. The polioviruses were analyzed using a cytopathic analysis of cells. In summary, the dried samples were reconstituted in their original volume using sterile distilled water and a series of 10-fold dilutions prepared in EMEM, and dilutions were inoculated into 100μ in five wells by replicating a 96-well cell culture plate containing one confluent monolayer of Vero cells. These plates were incubated for 7 days and the cytopathic effect induced by virus was classified by the inspection of the wells using light microscopy. The viral titration, such as log? 0 TCID 50 / ml of the recovered virus, was again calculated using the Karber formula for quantification of the final point in the analysis of the effectiveness of the virus. For all virus analyzes, the log10 reduction in the titration of the treated samples was calculated by subtracting log10 TCID 50 / ml virus recovered from log10 TCID values 50 / ml of dry controls. The infectivity of the measles virus was determined using a plaque analysis. In summary, the dried samples were reconstituted in their original volume using sterile distilled water and a 10-fold dilution series prepared in EMEM. 200 μl of each dilution were incubated in wells in duplicate in a 6-well plate containing a confluent monolayer and Vero cells. After adsorption of the virus to the cells for 1 hour at 37 ° C, 2 ml of superior medium (EMEM containing 1% carboxymethyl cellulose and 5% fetal bovine serum) was added to each well. The plates were then incubated for 7 days at 37 ° C in a 5% CO2 atmosphere. The virus induced by the formation of layers in the monolayers of cells was visualized by crystal violet tinsión. The plates were counted and the recovered virus was quantified by the calculation of plaque formation units per ml., Namely, PFU / ml = number of plates x dilution factor x 5. For all virus analyzes the reduction of log10 in the titration of the treated samples was calculated by subtracting log10PFU / ml of virus recovered from the log10PFU / ml values of the dry controls. The results obtained are presented in Table 3 showing the reduction of log10 in poliovirus, measles virus and parvovirus titration and percentage of reduction in alkaline phosphatase activity after terminal sterilization at 80 ° C for 72 hours or 90 ° C for 20 hours. In Table 3, * means logio TCID 50 / ml poliovirus in dry control = 4.5; ** equals log10 PFU / ml of measles virus in dry control = 5.10; *** equals log10 TCID 50 / ml parvovirus in dry control = 6.5; **** is equal to the activity of alkaline phosphatase in dry control = 100%; and > The same is for values below the detection limit of the analysis. Table 3 Example 4 Terminal sterilization to remove parvovirus from the blood product without loss of biological activity by drying in trehalose Fibrinogen (Fraction 1, Type 1-S, Sigma Chemical Company) was dissolved in 10% solution and 25% trehalose or sucrose containing 10% sodium citrate and 15% sodium chloride and the solutions were centrifuged to remove any insoluble material and the protein concentrations were adjusted to a final fibrinogen concentration of 5 mg / ml. The mother fibrinogen solutions were chopped with 1065 TCID 50 / ml canine parvovirus and 12 ml aliquots of the fibrinogen solution were dispensed into 5 ml bottles of Wheaton pharmaceutical glass and the samples were vacuum dried in a FTS dryer or dried by freezing in a Labconco freezing dryer. For vacuum drying, the shelves of the dryer were pre-cooled to 10 ° C and the vacuum was reduced to 30,000 mTorr for 2 minutes, 20,000 mTorr for 2 minutes and 10,000 mTorr for 20 minutes. The vacuum was then raised to 30,000 mTorr for 5 minutes and then reduced to 30 mTorr and the samples were dried overnight at a shelf temperature of 60 ° C. For freeze drying, the samples were frozen at 5 ° C / min. at -40 ° C, they were kept at -40 ° C for 16 hours and then the shelf temperature was raised to -35 ° C and the samples were dried under a vacuum of 10 mTorr for 80 hours. The shelf temperature was raised to 25 ° C and the samples were dried at a vacuum of 10 mTorr for an additional 5 hours. All the bottles were sealed under vacuum and the samples were terminally sterilized by heat sterilization in a Heraeus oven at 90 ° C for 20 or 48 hours. The flasks were reconstituted and the total soluble protein and the coagulable protein were determined. The coagulation analysis for fibrinogens was a modification of the thrombin coagulation analysis of the National Institute of Biological Standards. In summary, the fibrinogen and normal samples were coagulated by the addition of thrombin to the fibrinogen solution and the protein concentration in the coagulum measured by coagulum solubilization in 7M urea and quantifying the absorbance at 280 nm.

By adding 3 ml of water, all preparations were easily reconstituted separately from the fibrinogen / sucrose preparations that were exposed at 90 ° C / 20 hours. Freeze-dried preparations of fibrinogen / sucrose that were exposed at 90 ° C / 20 hours showed a different brown coloration.

Protein analyzes showed that most of the protein was dissolved by reconstitution, (approximately 90%), except in the case of flasks at 90 ° C / 20 hours of fibrinogen / sucrose that showed very little soluble protein. Coagulation analyzes showed that the soluble protein, (approximately 95%) was coagulated by the addition of thrombin. A summary of the results obtained is shown in Table 4 which describes coagulable fibrinogen and the log10 reduction in parvovirus titration. Table 4 Although the above invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is outlined by the appended claims.

Claims (58)

1. R EIVI N D ICAC ION ES 1. A method for terminal sterilization of biologically active product for sterile administration comprising the steps of: (a) obtaining a dry sample comprising the product and an amount of α-D-glucopyranosyl-α-D-glucopyranoside (trehalose) sufficient to return the product substantially stable to heat; and (b) heating the dried sample to a temperature and for a sufficient time to substantially inactivate the infectious virus.
2. 2. The method according to claim 1, wherein the product is derived from the blood and is selected from the group consisting of albumin products, immunoglobulins, coagulation products and protease inhibitors.
3. 3. The method according to claim 2, wherein the albumin products are selected from the group consisting of: ASH, cold soluble globulin and fibrinogen.
4. 4. The method according to claim 2, wherein the immunoglobulins are selected from the group consisting of antibodies against tetanus, pertussis, hepatitis, herpes, varicella zoster, lentivi rus and rabies.
5. 5. The method according to claim 2, wherein the coagulation products are selected from the group consisting of anti-hemophilic factor Vi, factor IX complex; and activated factor IX complex.
6. 6. The method according to claim 2, wherein the protease inhibitors are selected from the group consisting of, a-1 protease inhibitors, and antithrombin III.
7. The method according to claim 1, wherein the biologically active product is obtained from a biological source selected from the group consisting of blood, plasma, serum, placenta, milk, urine, cell cultures and cell culture supernatant.
8. The method according to claim 7, wherein the biological source is cell culture or cell culture supernatant and the product is selected from the group consisting of colony stimulation factors, monoclonal antibodies and derivatives thereof and factors of increase.
9. The method according to claim 7, wherein the biological source is cell culture or cell culture supernatant and the product is recombinant.
10. The method according to claim 8, wherein the growth factors are selected from the group consisting of erythropoietin, cytokines and interleukins.
11. The method according to claim 1, wherein the biologically active product is an analgesic.
12. The method according to claim 11, wherein the analgesic is selected from the group consisting of morphine, benzocaine, pethidine and Demerol.
13. 13. The method according to claim 1, wherein the biologically active product is an anesthetic.
14. 14. The method according to claim 13, wherein the anesthetic is selected from the group consisting of bupivicaine, atracurium and vecuronium. 5.
15. The method according to claim 1, wherein the biologically active product is a chemotherapeutic agent.
16. The method according to claim 15, wherein the chemotherapeutic agent is selected from the group consisting of, radioisotopes, vinca alkaloids, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine, Decarbazine, Dactinomycin, Duanorubicin hydrochloride , Doxorubicin hydrochloride, Etoposide, fluorouracil, mechlorolamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaza hydrochloride, streptozotocin, taxol, thioguanine and uracil mustard.
17. 17. The method according to claim 1, wherein the biologically active product is a hormone.
18. 18. The method according to claim 17, wherein the hormone is selected from the group consisting of estrogen, testosterone, progesterone and synthetic analogs thereof.
19. 19. The method according to claim 1, where the biologically active product is a vaccine.
20. The method accordto claim 19, wherein the vaccine is selected from the group consistof both se and multiple antigen subunit vaccines and killed bacteria, as well as viral preparations and cancer antigens. twenty-one .
21. The method accordto claim 1, wherein the dry sample is obtained by the method selected from the group consistof air dry vacuum dry spray dryand freeze dry
22. 22. The method accordto claim 1, wherein the substantial heat stability results in less than about 30% loss in product activity.
23. 23. The method accordto claim 22, wherein the stability results in less than about 15% loss in product activity.
24. The method accordto claim 23, wherein the stability results in less than about 10% loss in activity of the product.
25. 25. The method accordto claim 1, wherein the dry sample has a residual moisture content of less than about 4%.
26. 26. The method accordto claim 25, wherein the residual moisture content is less than about 2%
27. 27. The method accordto claim 26, wherein the residual moisture content is less than about 1%.
28. 28. The method accordto claim 1, wherein the heattemperature is about 80 ° C and the heatduration is about at least 72 hours.
29. 29. The method accordto claim 1, wherein the heattemperature is about 90 ° C and the heatduration is about at least 20 hours.
30. 30. The method accordto claim 1, wherein the substantial inactivation of the infectious virus results in approximately 104 times the reduction in infectivity of the viruses.
31. 31. The method accordto claim 1, wherein the inactivation results in 104 fold reduction in infectivity of virus encapsulated without lipids.
32. 32. The method accordto claim 1, wherein the lipid-free encapsulated viruses are selected from the group consistof hepatitis A virus and parvovirus.
33. 33. The method accordto claim 32, wherein the virus encapsulated without lipids is parvovirus.
34. 34. A composition that is obtained accordto the method of claim 1.
35. 35. The composition accordto claim 34, wherein the product is derived from the blood and is selected from the group consistof albumin products, immunoglobulins. , coagulation products and protease inhibitors.
36. 36. The composition according to claim 35, wherein the albumin products are selected from the group consisting of, ASH, cold soluble globulin and fibrinogen.
37. 37. The composition according to claim 35, wherein the immunoglobulins are selected from the group consisting of antibodies against tetanus, pertussis, hepatitis B, Rho (D), varicella Zoster and rabies.
38. 38. The composition according to claim 35, wherein the coagulation products are selected from the group consisting of antihemophilic factor VIII, factor IX complex, and activated factor IX complex.
39. 39. The composition according to claim 35, wherein the protease inhibitors are selected from the group consisting of, a-1 protease inhibitor, and antithrombin III.
40. 40. The composition according to claim 35, wherein the biologically activated product is obtained from a biological source selected from the group consisting of blood, plasma, serum, placenta, milk, urine, cell cultures and cell culture supernatant.
41. 41. The composition according to claim 40, wherein the biological source is a cell culture or cell culture supernatant and the product is selected from the group consisting of colony stimulation factors, monoclonal antibodies and derivatives thereof. , and growth factors.
42. 42. The composition according to claim 41, wherein the biological source is cell culture or supernatant of cell cultures and the product is recombinant.
43. 43. The composition according to claim 41, wherein the growth factors are selected from the group consisting of erythropoietin, cytokines and interleukins.
44. 44. The composition according to claim 34, wherein the biologically active product is an analgesic.
45. 45. The composition according to claim 44, wherein the analgesic is selected from the group consisting of morphine, benzocaine, pethidine and Demerol.
46. 46. The composition according to claim 34, wherein the biologically active product is an anesthetic.
47. 47. The composition according to claim 46, wherein the anesthetic is selected from the group consisting of bupivicaine, atracurium and vecuronium.
48. 48. The composition according to claim 34, wherein the biologically active product is a chemotherapeutic agent.
49. 49. The composition according to claim 48, wherein the chemotherapeutic agent is selected from the group consisting of, radioisotopes, vinca alkaloids, adriamycin, bleomycin sulfate, Carboplatin, cisplatin, cyclophosphamide, Cytarabine, Decarbazine, Dactinomycin, Duanorubicin hydrochloride. , Doxorubicin hydrochloride, Etoposite, fluorouracil, mechlorolarmine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaza hydrochloride, streptozotocin, taxol, thioguanine and uracil mustard.
50. 50. The composition according to claim 34, wherein the biologically active product is a hormone.
51. 51 The composition according to claim 50, wherein the hormone is selected from the group consisting of, estrogen, testosterone, progesterone and synthetic analogues thereof.
52. 52. The composition according to claim 34, wherein the biologically active product is a vaccine.
53. 53. The composition according to claim 52, wherein the vaccine is selected from the group consisting of both simple and multiple antigen subunit vaccines and killed bacteria and viral preparations.
54. 54. The composition according to claim 34, wherein the dry sample has a residual moisture content of less than about 4%.
55. 55. The composition according to claim 54, wherein the residual moisture content is less than about 2%.
56. 56. The composition according to claim 55, wherein the residual moisture content is less than about 1%.
57. 57. The composition according to claim 34, wherein the virus encapsulated without lipids is selected from the group consisting of hepatitis A virus and parvovirus.
58. 58. The composition according to claim 57, wherein the virus encapsulated without lipids is parvovi rus. RESU MEN The invention relates to methods for sterilizing biologically active products, particularly to therapeutic or prophylactic products and to the compositions obtained therefrom. The methods include obtaining a dry sample containing an amount of trehalose sufficient to give the product heat stability and exposing the dry sample to heating conditions at a temperature, and for a sufficient time, to substantially inactivate a virus, especially virus encapsulated without lipids . The drying methods include both environmental drying conditions and lyophilization. The heating conditions include any known in the art and cover a wide range of temperatures and heating times. The compositions obtained contain stable products and do not contain measurable infectious viruses, particularly parvoviruses.