| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 28, 1998 |
| PATENT TITLE |
Manufacturing process for the production of purified transferrin |
| PATENT ABSTRACT | Method for obtaining highly purified transferrin from a partially purified plasma fraction containing transferrin in which the starting fraction is concentrated. Its ionic strength is reduced and then transferrin is adsorbed onto a chromatographic column. Following elution, the transferrin can be further processed through to packaging in final containers. The final purified transferrin product is sterile, at least 95% pure and is substantially free of enveloped and non-enveloped viruses |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 26, 1996 |
| PATENT REFERENCES CITED |
Harris, et al., Protein Purification Methods A Practical Approach, pp. 5-9, 126-148, 1989. Soliman, et al., A Medium Scale Method for the Purification of Human Serum Transferrin and IgG Globulin by Chromatographic Technique, U.A.R. J. Chem., vol. 14, No. 2, pp. 177-183, 1971. |
| PATENT CLAIMS |
We claim: 1. A method of purifying transferrin comprising the steps of a.) concentrating a partially purified transferrin preparation; b.) decreasing ionic strength of the transferrin preparation sufficient for subsequent adsorption of transferrin onto an ion exchange chromatographic medium; c.) treating the transferrin preparation prior to the following step (d) with an organic compound for inactivating enveloped viruses; d.) subjecting the preparation to an ion exchange chromatography medium, with an ionic strength sufficient to ensure that transferrin is adsorbed; e.) eluting transferrin from the ion exchange medium of step (d); f.) concentrating the resultant eluate; and g.) filtering the concentrated eluate for removing virus particles to obtain a sterile transferrin preparation substantially free of enveloped and non-enveloped viruses. 2. The method of claim 1 wherein the transferrin concentrated eluate from step (e) is saturated with iron. 3. The method of claim 2 wherein the ionic strength of the saturated eluate is decreased. 4. The method according to claim 1 wherein said partially purified transferrin preparation of step (a) is concentrated 5-fold. 5. The method according to claim 1 wherein the ionic strength is decreased by diafiltration. 6. The method according to claim 3 wherein the ionic strength is decreased by diafiltration. 7. The method according to claim 5 wherein the ionic strength of the transferrin preparation is decreased by diafiltration with 25 mM Tris buffer. 8. The method according to claim 6 wherein the ionic strength of the transferrin preparation is decreased by diafiltration with 25 mM Tris buffer. 9. The method according to claim 1 wherein the organic compound for inactivating viruses is an organic solvent detergent. 10. The method according to claim 1 wherein transferrin is eluted in step (d) using 0.04M NaCl, 25 mM Tris buffer. 11. The method according to claim 1, wherein said concentrating in step (a) is carried out by tangential flow ultrafiltration. 12. The method according to claim 1 where said eluate of claim 1 is concentrated to 10 mg/ml protein in step (e). 13. The method according to claim 2 wherein ferric chloride is used to saturate the transferrin preparation. 14. The method according to claim 1 wherein in step (g) the eluate is filtered using a 0.22 .mu.m filter to obtain a sterile transferrin preparation. 15. The method according to claim 1 wherein said filtering of the transferrin preparation in step (g) is carried out using a 15 nm filter to obtain a transferrin preparation that is substantially free of non-enveloped viruses. 16. A transferrin product comprising transferrin that is at least 95% pure, sterile and is substantially virus-free. 17. The transferrin product of claim 16, wherein the transferrin is sterile and substantially free of enveloped viruses. 18. The transferring product of claim 17, wherein the transferrin is sterile and substantially free of non-enveloped viruses. 19. A transferrin product comprising transferrin that is at least 95% pure, sterile and substantially free of enveloped and non-enveloped viruses. 20. The method according to claim 1, wherein said concentrating in step (f) is carried out by tangential flow ultrafiltration. 21. The method according to claim 1, wherein said concentrating in steps (a) and (f) is carried out by tangential flow ultrafiltration. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates to a process for the purification of transferrin resulting in a product adequate for medical uses. BACKGROUND OF THE INVENTION Transferrin is an iron-binding glycoprotein having a molecular weight of 76-81 kD. Iron-free transferrin is referred to as "apo-transferrin," whereas iron-saturated transferrin is referred to as "holo-transferrin." Both apo-transferrin and holo-transferrin are used for therapeutic purposes. In blood plasma, transferrin acts as an iron-transport protein that safely sequesters trivalent iron as it is delivered to the cell cytoplasm via cell surface transferrin receptors. Central nervous system (CNS) neurons are deficient in transferrin receptors, while tumor cells commonly have an excess. A conjugate of transferrin and a protein or a small molecule may provide a useful treatment for CNS tumors by selective delivery to the targeted cells. Such proteins may include toxins. Such molecules may includes chemotherapeutic agents, radioisotopes and nucleic acids. For example, U.S. Pat. No. 5,352,447 discloses a conjugate of transferrin and diphtheria toxin for the treatment of intracranial lesions. Several other medical applications also involve the use of purified transferrin products. For instance, transferrin products are used as cell growth factors in serum-free cell culture media. Purified transferrin products may also be used as reagents in diagnostic kits. Therefore, there is a continuing need for highly purified and sterile transferrin products that are free of enveloped and non-enveloped viruses. Moreover, there is the need for the ability to make these highly purified products on an industrial scale in a simple and cost effective manner. Fraction IV is often considered a waste product of the Cohn fractionation process. However, it is also known that Fraction IV contains a range of useful plasma proteins including albumin, alpha-1 proteinase inhibitor (also known as alpha-1 antitrypsin) and transferrin. Therefore, Fraction IV of the Cohn fractionation process (specifically Fractions IV.sub.1 and IV.sub.4) can serve as a source of a transferrin-containing liquid preparation in the present invention. Methods of obtaining partially purified transferrin fractions are known in the art. For example, U.S. Pat. No. 3,850,903 discloses a method of preparing a protein concentrate that is rich in albumin, transferrin and other .alpha.- and .beta.-globulin molecules. Specifically, the '903 patent discloses a Cohn IV.sub.4 precipitate that is treated with calcium phosphate followed by selective precipitation with block copolymers of ethylene oxide and polyoxypropylene polymer. As the final product contains a variety of plasma proteins, the '903 disclosure does not provide a method for obtaining highly purified transferrin product per se. U.S. Pat. No. 4,841,026 describes a method of preparing a sterile, virally inactivated, non-toxic transferrin preparation for use in cell culture systems. The method disclosed therein comprises the saturation of a Cohn fraction containing transferrin with an excess of iron followed by the removal of free iron radicals and unwanted proteins via filtration and ion exchange chromatography. To obtain the final transferrin product, the disclosed process requires a pasteurization period of 10 hours at 60.degree. C. U.S. Pat. No. 5,041,537 discloses a method of preparing biologically active transferrin, comprising precipitation of .gamma.-globulin from the Cohn fraction IV, ultrafiltration and adjustment of ionic/protein concentration followed by treatment with specific detergents. The fraction is then subjected to ion-exchange chromatography wherein all of the proteins except the transferrin are adsorbed, i.e., the transferrin is contained in the flow through fraction. The transferrin-containing fraction is subsequently concentrated and filtered sterile. Methods of inactivating viruses associated with plasma proteins are also known in the art. For example, it is known in the art that plasma proteins can be sterilized using (1) heat-inactivation in the wet and dry states, (2) detergents, and (3) a combination of treatment with .beta.-propiolactone and ultraviolet radiation. However, plasma proteins are heat-sensitive and therefore must be heat-inactivated in conjunction with stabilizers. Certain stabilizers may act to prevent complete sterilization. Accordingly, sterilization procedures using chemicals and/or radiation are considered preferable for applications requiring complete sterilization. Moreover, it is possible that the heat treatment of transferrin containing preparations will result in the formation of aggregates, thereby potentially hindering the purification and ultimate utilization of the transferrin product. It is also known that viruses can be enveloped or non-enveloped and that viral inactivation methods may be effective for one type of virus, but not another. For example, U.S. Pat. No. 4,540,573 ("the '573 patent") discloses a general method of obtaining substantially virus-free biologically active protein derivatives that are substantially free of enveloped viruses yet are not denatured by the virus inactivation process. Specifically, a method is disclosed wherein a protein containing composition, e.g. whole blood, is contacted with an organic solvent detergent ("OSD" process) to inactivate enveloped viruses contained therein. The OSD method has the advantage of inactivating hepatitis viruses present in the protein-containing composition without substantial denaturation of the proteins therein. The '573 patent does not disclose the purification of a transferrin-containing fraction. SUMMARY OF THE INVENTION It is an objective of this invention to provide a method of producing transferrin products with a high yield and high purity. The method is simple and economical for industrial-scale production of high purity transferrin products. It is a further objective of this invention to provide a method of preparing highly purified transferrin products that are substantially free of enveloped and non-enveloped viral contaminants. It is a further objective of this invention to provide a process wherein the transferrin product can be purified and sterilized prior to placement in a final container. The present invention relates to a method of purifying a transferrin-containing liquid preparation wherein the method comprises starting with a partially purified transferrin liquid preparation. The partially purified transferrin-containing fraction is then concentrated and the ionic strength thereof is lowered. The transferrin-containing fraction is chemically treated to inactivate enveloped viruses. Thereafter, the transferrin-containing fraction is applied to an ion exchange medium in which the transferrin is adsorbed. A fraction comprising transferrin is then eluted from the ion exchange column. In preferred embodiments, the transferrin is saturated with iron and nanofiltered to remove non-enveloped viruses and obtain a sterile product prior to packaging and, if a dry product is desired, the transferrin is lyophilized in a final container. The lyophilized product can be re-constituted at a later time. DETAILED DESCRIPTION OF THE INVENTION The starting protein fraction of the present invention is a transferrin-containing protein fraction which has been partially purified, as above-described. Preferably, it is a partially purified Cohn Fraction IV.sub.1 and IV.sub.4. The required and optimal process steps of the present invention are as follows: 1. Concentration The starting material derived from fractions IV.sub.1 and IV.sub.4 comprising a partially purified liquid transferrin preparation is first concentrated prior to further processing. In a preferred embodiment, the partially purified liquid transferrin preparation is concentrated by tangential flow ultra filtration, such as available from FILTRON. The partially purified liquid transferrin preparation is concentrated to between 15 mg/ml to 40 mg/ml transferrin or preferably 30 mg/ml. This step can be an optional step when the starting material is of this level of concentration. 2. Diafiltration After concentrating the partially purified liquid transferrin preparation, the ionic strength of the preparation is decreased to less than 5 mS. Ionic strength is reduced sufficiently so that the transferrin will be adsorbed onto a chromatographic column used during the later chromatography step. In a preferred embodiment, the ionic strength is lowered by diafiltration, for example using a 22.5 mM to 27.5 mM Tris buffer to a conductivity of about 2 mS. In the most preferred embodiment, the ionic strength is lowered using 25 mM Tris buffer, pH 7.5. 3. Chemical Viral-inactivation Treatment The liquid transferrin preparation is treated with chemicals to inactivate enveloped viruses at a point prior to ion exchange chromatography. In a preferred embodiment, the preparation is treated with known organic solvents with or without surfactants to inactivate viruses. In a more preferred embodiment, the preparation is treated with tri-n-butyl phosphate organic solvent, most preferably, the preparation is treated with 0.3.+-.0.015% tri-n-butyl phosphate and 1.0.+-.0.05% Polysorbate 80 to inactivate viruses. 4. Ion Exchange Chromatography The preparation is then subjected to ion exchange chromatography wherein the transferrin is adsorbed onto an anion exchange resin. In a preferred embodiment, the anion exchange chromatography resin is DEAE-650 (Toso Hass) or any other DEAE resin (DEAE=diethylaminoethyl). In the most preferred embodiment, the anion exchange chromatography resin is DEAE-650. In a preferred embodiment, the transferrin is eluted with a buffer containing NaCl, such as 30 to 40 mM NaCl in 25 mM Tris buffer. In the most preferred embodiment, the transferrin is eluted with 40 mM NaCl, 25 mM Tris buffer. 5. Second Concentration The transferrin-containing eluate is then concentrated. In a preferred embodiment, the eluate is concentrated by tangential flow ultra filtration, as in step (1). In a more preferred embodiment, the eluate is concentrated to 9-11 mg/ml of transferrin. In the most preferred embodiment, the eluate is concentrated to about 10 mg/ml of transferrin, of which the total protein content is 95% transferrin, preferably at least 99% transferrin, as described in the product specification in Table 1. 6. Conversion The transferrin-containing concentrate can then be saturated with iron (conversion from apo-transferrin to holo-transferrin). In a preferred embodiment, the concentrate is saturated with iron by either ferric chloride or ferrous chloride. In a more preferred embodiment, the concentrate is saturated with iron by the addition of ferric chloride so that iron/Tf=1.4 .mu.g/mg to 5.0 .mu.g/mg in the presence of 10 mM to 50 mM sodium bicarbonate, with mixing for 2 to 16 hours. In the most preferred embodiment, the concentrate is saturated with iron by the addition of ferric chloride so that Iron/Tf=3.0 .mu.g/mg in the presence of 10 mM sodium bicarbonate, with mixing for 2 hours. 7. Second Diafiltration The ionic strength of the saturated product is decreased to less than 5 mS. In a preferred embodiment, the ionic strength of the saturated product is decreased by diafiltration. In a more preferred embodiment, the ionic strength is decreased using 22.5 mM to 27.5 mM Tris buffer. In the most preferred embodiment, the ionic strength is decreased using 25 mM Tris buffer. 8. Sterile Filtration The saturated product can be further sterilized by filtration. In a preferred embodiment, the saturated product is filtered through a filter that is not more than 0.22 .mu.m. In the most preferred embodiment, the saturated product is filtered through a 0.22 .mu.m filter. 9. Vital Filtration The saturated product is then nanofiltered to remove viruses. In a preferred embodiment, the saturated product is filtered through a 15 nm or smaller filter. At this time, in the most preferred embodiment, the saturated product is filtered through a 15 nm filter. 10. Lyophilization The final bulk product may be filled in a final container and lyophilized. As will be recognized by those skilled in the art, the presently disclosed method provides a simple and economical method of producing highly purified transferrin. The method requires only two major pieces of equipment and three buffers. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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