Abstract:
The invention relates to an integrated scheme for fractionation and purification of plasma products (human albumin, intravenous immunoglobulin (IVIG), clotting factor VIII and clotting factor IX) by sequential chromatography and virus reduction steps. The therapeutically administrable protein IVIG has purity levels exceeding 98%, aggregates and dimers at less than 0.2%, Fc function of &gt;90% and anti-complementary activity of less than 0.5 CH 50  per mg of Ig. The distribution of IgG isomers is comparable to the ranges seen in normal plasma. Human albumin for therapeutic use, purified by this integrated scheme has an electrophoretic purity of close to 100%, with monomers exceeding 98%. The levels of aluminium and pre-kallikrein activator are below the detection limit for the respective tests. The Factor IX preparations have a specific activity of ≧200 IU/mg. The impurity levels of Factor-II, Factor VII, Factor X are at least 10-fold lesser (≦0.5% instead of 5%) and the heparin impurity of ≦0.01 IU (against 0.5 IU limit for this impurity) is 50-fold lesser the specified pharmacopoeial limits. 
     The purification carried out by an all-chromatography scheme, avoids the use of ethanol precipitation in the entire manufacturing process of the said four plasma products. The invention describes an integrated process for purifying four different proteins from human plasma to high therapeutic grade purity levels, with a potential to purify more therapeutic proteins from a given plasma sample by incorporating additional chromatography steps in the sequence.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The invention relates to the methods to manufacture through fractionation and purification of human albumin, intravenous immunoglobulins, clotting factor VIII and clotting factor IX, from human plasma by sequential chromatography steps to produce highly pure, virus-free and therapeutically administrable proteins. The purification is carried out by an all-chromatography process that avoids the use of ethanol precipitation. 
         [0003]    2. Background Art 
         [0004]    The first reported plasma fractionation process for therapeutic purposes was developed about 60 years ago by Cohn and co-workers (J. Am. Chem. Soc., 68, 459-475, 1946; J. Am. Soc. 72, 465-474, 1950). Since then, plasma fractionation industry has grown mani-fold and is now one of the largest industry segments in global therapeutic protein manufacture (J. Curling, Integrating new technology into blood plasma fractionation (BioPharm September 2002, 16-25; Adil Denizli, Plasma Fractionation: conventional and chromatographic methods for albumin purification J. Biol. Chem., 39(4), 315, 341, 2011). 
         [0005]    Plasma protein fractionation is approximately an 11.8 billion dollar industry (Plasma Product Biotechnology meet 2011, Dr. Jean-Francois Prost, “Will plasma products inevitably be replaced by a new generation of therapeutics?”) supplying products to more than a million patients each year. It is estimated that more than 500 metric tons of human serum albumin and more than 40 tons of intravenous immunoglobulin are produced annually from more than 22 million liters of source and recovered plasma. Plasma contains about 60 g/L of protein, of which about 57 grams (not including processing losses) are used for many therapeutic products. Plasma-derived products, such as IVIG and albumin, are not expected to be manufactured by recombinant means. IVIG demand is now the primary driver of the plasma collection market, with demand growing 6-8% annually. 
         [0006]    According to a market report published by Robert, P. in Pharmaceuticals Policy and Law 11, 359-367, 2009, the demand for plasma in the 1950s and 60s was driven by the demand for human albumin. In the late 1960s, when Factor VIII concentrates became available, the global plasma requirement was driven by the growing demand for clotting factors. The IVIG usage has grown significantly in different medical areas such as neurology, rheumatology, nephrology, dermatology, oncology and infectious diseases, as well as allergy and immunology, in addition to the continuing increase of the number of patients with primary immune deficiencies. Based on this usage pattern, which characterizes the markets in most industrialized countries, the volume of IVIG was forecast to grow from about 82.3 metric tons in 2008 to about 107.9 tons by 2012, corresponding to an annual growth rate of 7%—the rate observed in the past ten years. According to this report, the demand of IVIG beyond 2012, is expected to grow depending upon the results of the Alzheimer&#39;s disease trials and the possible approval of IVIG for this new indication. 
         [0007]    In 2010, there were a total of 78 fractionation plants operating in the world, with 25 of them in China, 26 in Europe and 8 in United States (Worldwide supply and demand of plasma and plasma-derived medicines, Patrick Robert, Iranian J. of Blood and Cancer, Vol. 3, No. 3, 111-116, 2011). The global fractionation capacity was estimated at about 48.4 million liters, producing mainly albumin, polyvalent IVIG, Factor VIII, Factor IX and Hyperimmunes. Hemophiliacs are the target population for players interested in plasma protein R&amp;D. India needs about 900,000 liters of plasma derived proteins per year. India has been meeting this requirement so far by importing these proteins as the plasma fractionation industry is still at a nascent stage, one of the major hindrances being the existence of adequate infrastructure for plasma collection (Farrugia A., Plasma for Fractionation: Safety and Quality Issues. Haemophilia 10, 2004: 334-340). 
         [0008]    Although the number of plasma fractionators are growing and global capacities are increasing, so is the demand for IVIG growing, which is projected at about 7% to 13% annually between 2012 and 2015. To meet this demand, more raw plasma will need to be dedicated to immunoglobulin purification, along with improvements in the process that will increase the overall IVIG yield. This growing requirement for IVIG will limit the availability of plasma for the manufacture of new plasma-derived blood products unless their manufacture is integrated into the existing manufacturing processes for plasma-derived products such as immunoglobulins and albumin. Presently, although plasma based therapeutics are safe, efficacious and available, the cost of therapy remains a barrier for access to the drugs. This can be addressed only if human plasma, a uniquely rich starting material for therapeutic proteins, can be processed to prepare several more therapeutic proteins besides the conventional albumin, IVIG and clotting factor concentrates. This also requires the use of more discerning technologies of bioprocess operations like chromatography and membrane separations together with the most recent and proven methods of viral inactivation. 
         [0009]    Plasma being a scarce and exclusive commodity, there is a continuous need to upgrade the fractionation processes so as to maximally utilize this valuable resource. By continuously refining and upgrading the existing processes and improving process efficiencies, the market availability status and the quality of the end products can be improved. The fractionation industry which presently uses either a Cohn or a modified Cohn&#39;s method of ethanol precipitation in some cases may use chromatography based purification in the subsequent steps. Cohn&#39;s fractionation limits the number of proteins that can be purified by ethanol precipitation as only the more abundant proteins from plasma can be precipitated by this method. The less abundant therapeutic proteins with good therapeutic potential need to be processed using a different strategy. It&#39;s also now accepted in the plasma fractionation industry that although Cohn&#39;s method permits fractionation of large volumes of plasma at low cost, the quality of the product obtained by chromatography is superior (Braz. J. Med. Biol. Res., 31:1383-1388, 1998). Several companies have now moved to usage of a combination of both methods, where they have ethanol precipitation followed by chromatography. The inherent disadvantage in this method is the possibility of protein denaturation and/or aggregation during the addition of ethanol (Braz. J Med Biol Res, 31: 1375-1381, 1998). Protein losses may also occur in the supernatant after ethanol precipitation thereby decreasing the overall recovery of major proteins and almost a total loss of the minor proteins. The concentration of different proteins in the plasma varies over a very wide range, from less than 1 microgram per ml to 40 grams per litre. To develop separation and purification processes that can ensure production of several of these high and low level proteins from the same starting volume of plasma, is a challenge for the separation specialists. 
         [0010]    There are several patents in the prior art that disclose a process for fractionation and/or purification of plasma proteins to therapeutic grade purity, that involved the use of ethanol for initial precipitation, followed by chromatography steps with few technologies involving only chromatographic steps or only ethanol precipitation steps. 
         [0011]    Preparation of a high-purity human factor IX concentrate and other plasma proteins and their therapeutic use with chromatography process were discussed in U.S. Pat. No. 5,457,181. U.S. Pat. No. 4,371,520 discloses a process for preparing immunoglobulin suitable for intravenous injection, comprising treatment of an acid a plasma or combination of fractions I, II and III, combination of fractions II and III, fraction II, or fraction III obtained from plasma by Cohn&#39;s cold alcohol fractionation and chromatography methods. Process for the isolation and/or fractionation of peptide, 5 polypeptides or protein solutions were discussed in WO200763129, involves both chromatographic steps and ethanol precipitation steps. Patent application WO200623831 relates to chromatographic methods for recovering highly purified proteins sequentially from biological samples. U.S. Pat. No. 5,679,776 discloses a simple chromatographic process for purifying Factor VIII from total plasma. However, none of the above referred patents disclose a process that can simultaneously isolate and purify several proteins from the same starting volume from human plasma to therapeutic grade proteins by avoiding the use of ethanol precipitation step anywhere in the process. 
         [0012]    Another patent, U.S. Pat. No. 7,041,798 discloses a method for the chromatographic fractionation and ethanol precipitation of plasma and serum preparations, relates to the fractionation of plasma or serum into albumin and immunoglobulin by hydrophobic interaction chromatography. The rate limiting step is the processing time associated with starting material which can be challenging at large scale. 
         [0013]    U.S. Pat. No. 4,639,513 discloses a method for producing intravenously injectable immunoglobulin G (IgG) comprising a particulate separation step, an ion exchange separation step and an affinity separation step, ethanol precipitation, Additionally useful high purity by-products such as prothrombin complex, transferrin and albumin recovery were published. However, these methods often employ a separation medium that can be designed to selectively adhere either the protein of interest or the impurities. 
         [0014]    Patents U.S. Pat. No. 4,877,866 and DE 3640513C2 disclose the method appropriate for industrial-scale production and multistage purification of plasma that contains immunoglobulin G accompanied by treatment with ultrafiltration and ethanol precipitation. However, a need exists for an efficient process for purifying an immune serum globulin fraction from a crude plasma protein fraction. 
         [0015]    The patent application WO1994029334 discloses chromatographic and ethanol precipitation steps to produce therapeutic quality of the IVIG. However, the claimed method fails to take the subsequent purification of albumin into account. 
         [0016]    Although a number of chromatography based processes have been described in research publications for the production of IVIG without the use of ethanol precipitation, but at the industrial scale, an all-chromatography process scheme has just begun gaining acceptance due to the better quality of the final IVIG product (lesser protein denaturation and aggregation) and better yields (Lontos, J., Chromatographic purification of immunoglobulins at CSL bioplasma; a manufacturing perspective. Plasma Product Biotech meet, http://www.bo-conf.com/ppb05/present/ppt.htm 2005; Bertolini, J., Davies, J., Wu, J., Coppola, G., Purification of Immunoglobulins. 1998, WO 98/05686; Ultra-high yield intravenous immune globulin preparation, Zurlo, E. J., Curtin, D. D., Louderback, A. L., US patent application US20070049733A1). 
         [0017]    Several patents have explored the use of chromatographic methods for the final purification steps particularly ion exchange chromatography. A few patents have also tried to replace the ethanol fractionation of plasma with caprylate or citrate precipitation steps followed by chromatography based clean-up steps for purification. In spite of these advancements, it is glaringly apparent how Cohn&#39;s fractions II and III continue to play an important role as the starting material for IVIG purification in most industrial processes. This is clearly evident in a patent being granted (U.S. Pat. No. 6,893,639 B2) to an ethanol based plasma fractionation process at sub-zero temperatures, as recent as 2005 (the initial Cohn&#39;s patent for ethanol based plasma fractionation U.S. Pat. No. 2,390,074, was granted in 1945). Cohn himself had illustrated the problems of protein denaturation as an undesirable outcome of ethanol precipitation. 
         [0018]    Similar issues are applicable for human albumin as well. The industrial processes for purifying albumin start with Fraction V of ethanol fractionation of plasma by the Cohn&#39;s method (J. Am. Chem. Soc. 1946, 68, 459-475, Cohn et al.). Although ethanol fraction supernatants of fraction IV and even supernatants of fractions II &amp; III have been used by several manufacturers for albumin purification, followed by chromatography steps, a complete all-chromatography process has not been the norm in the industry for albumin purification (U.S. Pat. No. 5,346,992 and U.S. Pat. No. 4,288,154). Chromatography techniques for albumin purification in research publications and books have been cited since the 1980s (Curling J. M., Methods of plasma protein fractionation, Ed. J. Curling, Acad. Press, 77-91 (1980); Saint-Blancard J., Novel Trisacryl ion exchangers (Nouveaux echangeurs d&#39;ions Trisacryl), Ann. Pharm. Fr. 39, 403-409 (1981). Patents that claim to purify albumin by chromatography are also seen to use supernatant IV or precipitate V of the Cohn&#39;s fraction (U.S. Pat. No. 5,677,424; U.S. Pat. No. 4,675,384; U.S. Pat. No. 4,228,154; U.S. Pat. No. 8,088,416) as the starting material. 
         [0019]    U.S. Pat. No. 5,061,789 discloses a method for isolating blood-clotting factor IX by first adsorbing it onto a matrix derivatised with alpha hydroxylamine groups, eluting the factor and adsorbing it onto a column matrix of sulphated carbohydrates to collect a pure form of Factor IX at high yields. U.S. Pat. No. 5,138,034 discloses a process for isolation of the Factor IX by a sequence of steps involving ethanol precipitation, followed by treatment with an anion exchanger and affinity chromatography. U.S. Pat. No. 5,286,849 discloses the process for purification of Factor IX from an impure protein fraction by the addition of a solvent-detergent solution to inactivate any viral contaminants and purify the Factor IX by chromatography on a sulphated polysaccharide resin. The purified factor IX by the above method is claimed to have a specific activity of at least 85 units/mg. U.S. Pat. No. 5,457,181 discloses a method for preparing a high purity Factor IX concentrate from the supernatant fraction of cryoprecipitated human plasma. A pre-purification is done with DEAE Sephadex chromatography. The resulting Factor IX has a specific activity of at least 0.5 IU/mg of protein. The purification method comprises at least two successive chromatography steps. The first step is a DEAE Sepharose chromatography followed by an affinity on heparin sepharose. The elution buffer is a citrate buffer at pH 7.4 adjusted with 0.45M NaCl and supplemented with arginine as a stabiliser for Factor IX activity. U.S. Pat. No. 5,614,500 discloses a method for preparing pharmaceutical compositions comprising the active, highly purified and concentrated Factor IX proteins are provided. The Factor IX proteins are recovered from plasma or recombinant cell culture sources by an immunoaffinity chromatography procedure in the presence of a chelating agent and in the absence of an exogenous non-chelating protease inhibitor. U.S. Pat. No. 5,639,857 discloses a method for recovering active, highly purified and concentrated Vit-K dependent proteins from plasma, concentrate or mixtures of proteins produced by recombinant DNA technology. U.S. Pat. No. 5,714,583 discloses a process for purification of FIX by following the steps of anion exchange resin, heparin or heparin like resin followed by a hydroxyl apatite resin. The third eluate can be applied to an immobilised metal-affinity resin to get a fourth eluate that contains purified Factor IX. This process would apply to plasma derived as well as recombinant Factor IX. U.S. Pat. No. 6,063,909 discloses a novel method of protecting blood coagulation Factor IX from proteases during purification or storage was disclosed. Factor IX is stabilized in solution against activation to factor IX or against degradation to peptides or conformation by the addition of one or more soluble organic or inorganic salts like sodium sulphate, potassium chloride, sodium chloride and/or other salts in the range of 0.7M to 3M, to the Factor IX containing solution. U.S. Pat. No. 6,258,938 discloses a method to isolate the blood clotting Factor IX by using an antibody affinity column that specifically reacts with ligand stabilized protein to be isolated. The antibody to Factor IX is immobilized on to a solid support and a mixture containing the protein in the presence of the ligand is brought in contact with the antibody bound to the column resin. Elution is done under mild conditions to release the protein without the ligand. This gave a protein with a specific activity of at least 125-150 units/mg of protein. They claim that the stability of the purified Factor IX can be improved by the removal of or decreasing the amount of plasma hyaluronan binding protease that may get co-purified with factor IX according to the invention described in US 20040106779A1. The invention describes the method to decrease the ratio of Factor IX to PHBP by contacting it with an ion exchange column and eluting the protein with 0.35M to 0.4M salt to separate protease from factor IX. 
         [0020]    EP617049 and U.S. Pat. No. 5,378,365 discloses a process for the purification of factor IX, factor X and factor II from human plasma or fractions by chromatographic methods. 
         [0021]    Alcohol precipitation and chromatography methods were used in U.S. Pat. No. 4,093,608, which have disclosed a process for separating coagulation factor VIII from human fresh pooled plasma and purifying it. However, the process is expensive and also lacks specificity. 
         [0022]    U.S. Pat. No. 5,245,014 discloses a method for isolating biological compounds such as proteins, especially coagulation factor VIII in high yield and almost free of other proteins, from body chromatographic and alcohol precipitation methods. However, purity of the obtained factor VIII seems to be a challenge. 
         [0023]    U.S. Pat. No. 4,822,872, this invention discloses a method of purifying an anti-hemophilic factor (AHF) comprising of a cryoprecipitation of F-VIII and adsorption of the F-VIII protein on a water insoluble carrier. But, this is a complex process involving the preparation of custom-made resins that is not easy for industrial scale manufacturing. Other patents that discuss isolation and/or purification processes include WO2007136327; US20010051708. 
         [0024]    There was hence a requirement, to have an integrated sequential chromatography procedure using regular industrial chromatography resins, to manufacture several products from a given volume of plasma, which ensures maximum utilization of a scarce and precious resource like human plasma and thereby better revenues for the company manufacturing these products. 
         [0025]    The present invention discloses an approach to overcome the challenge of utilization of a scarce and precious resource like human plasma, by using a sequential chromatography procedure that can help purify the four proteins Albumin, IgG, and Clotting factors (F-VIII and F-IX), with a potential to purify more proteins from the same starting volume of plasma. This will in turn reduce the cost and improve the affordability of these life-saving medicines for the patients. 
       SUMMARY OF THE INVENTION 
       [0026]    The present invention is an improved process for manufacturing plasma proteins using an all-chromatography process. The integrated process scheme simultaneously purifies plasma proteins like Albumin, IgG and clotting factors (F-VIII and F-IX), from the same starting volume, thereby reducing the cost, improving affordability and ensuring a more economical process. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0027]      FIG. 1  is an illustration of an integrated process scheme for simultaneous purification of four different plasma products without the use of an ethanol precipitation step. 
           [0028]      FIG. 2  is a representation of Zone Electrophoresis of IgG and Albumin. 
           [0029]      FIG. 3A  represents the High-performance liquid chromatography (HPLC) analysis of IVIG. 
           [0030]      FIG. 3B  represents the HPLC analysis of Albumin. 
       
    
    
     DESCRIPTION OF TABLES 
       [0031]    Table 1: Test for impurities in Factor IX preparation 
         [0032]    Table 2: Summary of exemplary test results for purified IVIG 
         [0033]    Table 3: IgG subclass distribution for different batches 
         [0034]    Table 4: IgG—Process or source related impurities for different batches 
         [0035]    Table 5: Levels of Pre-kallilkrein activator in albumin preparations in two representative batches 
         [0036]    Table 6: Summary of exemplary Quality Control tests for impurities in purified Albumin preparation 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0037]    Integration of the processes is important to maximally utilize the plasma and recover as many therapeutic products from it as possible with limited resources. Certain combinations of chromatography steps leading to the purification of the individual proteins, starting from the plasma raw material may be available in literature. The present invention discloses an integrated process that involves identification of different products that could be extracted at a particular stage of the process to derive the final product of therapeutic grade purity in minimal steps from the intermediate product. This process involves analysis of the identified intermediates and the suitability of a fraction to be used as starting material to obtain the desired final product. 
         [0038]    The present invention has the advantages of minimal duplication of equipment and maximal usage of the facility while purifying the desired products in parallel from a said starting volume of plasma and not sequentially, where purification of product 2 begins after the purification of product 1. In normal purification schemes, the buffer salts are optimised for individual steps for a given product purification scheme. In the present invention, the suitability of the same buffers across the four product purification schemes laterally was also studied and an exercise at minimisation was attempted. This overlap of types of buffers also improves efficiency and economy of the process, thereby minimising the number of salts to be procured for the manufacture of the four products. 
         [0039]    The invention is schematically described in  FIG. 1 . The purification scheme shows the preferred embodiment of the sequence of chromatography steps to be followed starting from blood plasma which can be fresh frozen, source plasma, recovered plasma or other variants of liquid or thawed plasma. The characteristic feature is the absence of ethanol precipitation and the simultaneous purification of several plasma proteins to the desired therapeutic grade quality, using an all-chromatography based process. 
         [0040]    Human plasma is first set to a chromatographic method from which three major fractions of the plasma products are obtained. The said three major fractions are again subjected to product specific sequential chromatographic steps to purify the desired IVIG, albumin and coagulation factors IX and VIII. 
         [0041]    Obtaining the 3 Major Fractions Involves a Five Step Process Starting from Blood Plasma. 
         [0000]    1. Blood plasma can be fresh frozen, source plasma, recovered plasma or other variants of liquid or thawed plasma. The fresh frozen or source plasma is collected by a process called plasmapheresis.
       Plasmapheresis involves separation of blood into cellular and other components by any of the standard procedures or by the use of a specialized automated plasma collection system (like Haemonetics PCS-2 or other similar equipments). The automated collection system uses sterile disposable sets in a self-contained automated process that separates the plasma from the cellular blood components that are then returned to the donor.
 
2. The plasma collected by plasmapheresis is stored frozen below −20° C., preferably below −40° C. in sterile collection bottles.
 
3. Production is initiated by thawing the frozen plasma in the bottles and pooling the liquid plasma in an appropriate collection vessel.
 
4. The thawed plasma fraction after filtration is loaded onto a gel filtration column.
       
 
         [0043]    Gel Filtration Column Set Up:
       The column is packed with any of the matrices like Cellufine, Sepharose, Sephacryl or any other commercial brands and packed to a height of in the range of 30 to 60 cm.   The column is run in a buffer composed of phosphate, citrate or other similar buffer salts that give a pH between 6.0 and 7.5. The buffer salt molarity does not exceed 0.2M, preferably less than 150 mM.   Additives like NaCl and other salts are added in suitable quantities to preserve the activity of sensitive proteins.   The column is loaded with around 50-150 liters of thawed plasma.
 
5. This results in three major fractions (fraction 1, fraction 2 and fraction 3)
       
 
         [0048]    Isolation and Purification of Factor VIII from Fraction-1: 
       Purification of Factor VIII Involves 3 Steps— 
       [0049]    1. The fraction I collected is further processed using a series of chromatography steps for purifying coagulation Factor VIII using an anion exchange resin packed in a column, the resin is chosen from one of the commercially available anion exchangers like DEAE, Q or related resins. 
         [0050]    Anion Exchange Column Setup:
       Salts like acetate, citrate or related salts are used for the equilibration buffer in the molarity range of 0.005M to 0.1M containing salts like NaCl and other salts in the range of 0.01M to 0.15M at pH in the range of 5.5 to 8.0.   This column is washed with buffer and eluted using an elution buffer in the concentration range of 0.05M to 0.5M containing sodium chloride more specifically in the range of 0.05M to 0.15M. To this buffer of pH 5 to 8.0, sodium chloride is added to a final elution concentration in the range of 0.25M to 2.5M.
 
2. The anion exchange column step is followed by a solvent-detergent (S/D) treatment for a specified time of less than 12 hrs at room temperature or around 24° C., for virus inactivation. The S/D treated sample is loaded onto a cation exchange column to remove the viral inactivation reagents.
       
 
         [0053]    Cation Exchange Column Setup:
       The said cation exchange column comprising SP (sulpho propyl) or CM (caboxymethyl) or related resins is eluted with sodium acetate buffer containing 0.1M to 1.5M sodium chloride. The eluate from this column can be optionally loaded onto a gel filtration column containing separation media in the range for high molecular weight separations   The column can be run using the final formulation buffer. This helps in getting rid of any minor protein impurities in the FVIII sample and facilitates exchange of protein into the final formulation buffer.
 
3. This is subjected to a second virus removal step by nanofiltration using commercially available molecular size cut-off filters followed by a third viral inactivation step after filling the liquid into the vials, freeze drying and then subjecting it to heat treatment at 80° C. for 72 hrs.
       
 
       Production of IgG, Albumin and Coagulation Factor IX: 
       [0056]    1. One of the said 3 fractions (Fraction 3) collected from the initial gel filtration separation of plasma is further fractionated by loading onto an anion exchange column like DEAE or Q Sepharose, more preferably DEAE or any similar weak anion exchangers.
 
2. This fraction 3 is allowed to pass through the above anion exchange column, which results in the binding of Factor-IX, which is later eluted and the remaining two proteins IgG and Albumin are recovered in the unbound fraction. Factor-IX is further purified by a two-step chromatography procedure.
 
3. The unbound (flow through) fraction containing IgG and albumin are further processed by dedicated chromatography steps to get therapeutic grade purity levels of IgG and albumin.
 
       Anion Exchange Column Equilibration: 
       [0000]    
       
         
           
             The anion exchange column like DEAE is equilibrated with acetate, citrate or any other anionic buffer salt of molarity 0.01M to 0.15M in the pH range 6.5 to 8.0.
 
4. The flow through fraction that has albumin, IgG desalted by diafiltration or passing through a chromatography column
 
5. The euglobulins are removed by precipitation and this sample is used as the starting material for the purification of IgG and Albumin.
 
           
         
       
     
       Desalting: 
       [0058]    Desalting of the second lot of the sample is achieved by Sephadex-G25 chromatography using any anionic salt buffer like sodium acetate buffer in the required pH range of 6.5 to 8.0; or by other methods like diafiltration on a membrane filtration set-up can also be employed.
 
6. The desalted and concentrated sample is subjected to low pH precipitation for the removal of euglobulins.
 
7. The euglobulin precipitation step is carried out in the conductivity range of 0.5 mS to 5 mS, more preferably in the range of 0.6 mS to 4 mS and preferably in the pH range of 4 to 7. The plasma sample under these conditions is held at a temperature between 2 to 20 degrees centigrade, for a time of 2 hrs to 16 hrs. The euglobulin pellet obtained after this step has a weight of about 20-45 gm/L of plasma.
 
8. The supernatant sample after removal of euglobulin pellet is again loaded on an ion exchange column, more preferably a second anion exchange column like DEAE, Q or equivalent to be able to separate the albumin and IgG from the load sample.
 
Column setup: The column has a height between 5 and 25 cm and the column is equilibrated with a buffer made from salts like acetate, citrate or phosphate at concentration ranges from 5 mM to100 mM in the pH range of 4.0 to 7.0, more preferably in the range of 4.5 to 6.5. The elution is carried out with buffers preferably of the same salt in the concentration range of 50 mM to 200 mM, in the pH range of 4.0 to 7.0.
 
9. Elution peak fraction obtained contains albumin as the major protein, while the flow through fraction contains IgG and thus these two different fractions are processed separately for further purification.
 
         [0059]    Purification of IgG: 
         [0000]    1. The flow through fraction containing IgG, from anion exchange column, is loaded onto another anion exchange column (Q, DEAE, TMAE or any other anionic resins from commercial vendors) to further purify the IgG from the other plasma proteins that are present in the sample. 
       Column Properties: 
       [0000]    
       
         
           
             A column of height 5 cm to 20 cm is packed, more preferably between 6 cm and 18 cm, with the chosen anionic exchange resin. The column is equilibrated with a buffer of acetate, citrate, phosphate or any other suitable anionic salt with a molarity in the range of 5 mM to 100 mM, more preferably in the range of 10 mM to 60 mM, and of pH in the range of 5 to 8, more preferably between 5.5 and 7.5.
 
2. The IgG containing fraction is again collected as the flow through fraction from this column. The sample is subjected to low pH treatment for 12-16 hrs, followed by a solvent-detergent (S/D) treatment for virus inactivation of enveloped viruses at 30° C., more preferably at 25° C. to 32° C., for 4 to 16 hrs.
 
3. To remove the S/D chemicals, the sample is loaded on a cation exchange column and the eluate containing IgG is collected and processed for virus removal.
 
4. This sample is passed through a nanofilter to remove viruses and a series of ultra-filtration/diafiltration steps are carried out to diafilter and concentrate the sample. This sample is formulated, sterile filtered and filled in vials.
 
5. The solvent-detergent solution addition is done at concentrations required for viral inactivation. The final IgG sample obtained by this process has the required purity, efficacy and safety profile required for intravenous administration.
 
6. The bulk solution is formulated as a 5% IgG solution containing 10% D-maltose in water in the pH range of 5.1 to 6.0.
 
           
         
       
     
         [0061]    The manufacturing process of IgG described above doesn&#39;t compromise on the biological activity of the IgG molecules. They are highly pure, functionally intact with normal IgG sub-class distribution and effector functions. The preparations are also safe with regard to pathogen safety and product and process related impurities. The protein composition, by Zone electrophoresis, expected as per pharmacopoeial guidelines is IgG &gt;96% and other contaminant proteins &lt;4%. But the protein composition of the present invention, is IgG=100% as per Zone electrophoresis with 0% contaminant proteins. The distribution of isoforms IgG1 (63% to 69%), IgG2 (23% to 31%), IgG3 (2.9% to 5.8%), IgG4 (1.4% to 2.9%) is exactly within the specified limits for each form, matching the plasma distribution of isoforms. Some of the comparator products tested have one or more isoforms outside the specification range. These results were tablated in Table 3 (IgG Subclass distribution for different Batches). Levels of process related contaminants are several fold lower than the specifications in the monographs and when compared to market comparators. For instance, prekallikrein activator is 7.4 IU/ml against a limit of &lt;35 IU/ml activated coagulation factors has &gt;400 sec clotting time against a limit of &gt;150 sec, IgA is 0.5 mg against a limit of &lt;4 mg/L and IgM is 0.000009 mg/mL against a limit of &lt;0.1 mg/mL. These results were tabulated in table 4 (IgG—Process or source related impurities for different batches). The IgG preparation described herein has an average index of Fc function of 80%, which is significantly higher than the limit set forth in the pharmacopoeial monograph. This demonstrates that the disclosed process for IgG preparation in the present invention retains the effector functions and is biologically active. 
         [0062]    Purification of Albumin: 
         [0000]    1. The eluate from the second anion exchange column (like DEAE) containing albumin, is loaded on an ion exchange column having groups like SP, CM or equivalent. 
       Column Equilibration: 
       [0000]    
       
         
           
             Column is equilibrated with a suitable buffer that is a salt of a weak acid-strong base like (Na or K salts of acetate, citrate or phosphate) at a concentration of 10 mM to 100 mM in the pH range of 4 to 6. The column is eluted with the same buffer containing NaCl in the range of 25 mM to 0.2M, in the same pH range.
 
2. Virus inactivation is carried out by caprylate addition and low pH treatment in a solution containing caprylate, at pH 4.5 for 10-12 hours.
 
3. After the said treatment, the albumin sample is concentrated and subjected to a heat treatment step. Heat treatment is carried out in the temperature range of 45° C. and 65° C., more preferably 50° C.-60° C., for a period of 1 hr to 10 hrs, more preferably 2 hrs to 6 hrs and then subjected to filtration.
 
4. The filtrate obtained is diafiltered and concentrated before the addition of formulation excipients. The final formulated sample also contains 16 mM each of N-acetyl tryptophanate and sodium caprylate. This is pasteurized at 60 degrees C for 10-12 hours.
 
5. This formulated bulk solution is filled aseptically in vials, stoppered, sealed and then subjected to a second round of pasteurization at conditions similar to the initial pasteurization.
 
           
         
       
     
         [0064]    In the present invention, the process for the isolation and purification albumin allows albumin to be purified to near homogeneity and predominantly in the monomeric form, with aggregate levels not exceeding 0.5%. This is ten-fold lower than that stated in the pharmacopoeia, where the specification limit for aggregates is expected to be not greater than 5%. The albumin preparation is purified to near homogeneity that is apparent from detection of only one principal band of 100% albumin in the electropherogram when investigated using Zone Electrophoresis method. The limit specified in the monograph allows not more than 5% of bands other than the principal band. According to this invention, there are no additional protein bands and the principal albumin band makes up 100% of the protein present in the sample. Very low levels of prekallikrein activator contaminant in albumin preparation were observed with prekallikrein activator being about fifteen-fold lower than the pharmacopoeial limit as determined by the assay described in the E. P. monograph. The limit set for this impurity is &lt;35 IU/ml and the said albumin preparation contains prekallikrein contaminant in the range of 1-3 IU/mL. The albumin solution also has very low levels of metal ions like aluminium (below LLD 10 ppb) and potassium (below LLD of 0.00002 mmol per gm of protein) as determined by atomic absorption spectrometry. The pharmacopoeial limit is set to not exceed 200 ppb for Aluminium and ≦0.05 mmol of Potassium per gm of protein. These results were tabulated in table 6 (Summary of a few major QC tests for impurities in purified Albumin preparation). These results show that the said albumin preparation is very pure and devoid of other plasma proteins and process related contaminants, much lower than the limits specified by regulatory guidelines. 
         [0065]    Purification of Factor IX: 
         [0000]    1. The purification of Factor IX is achieved from the eluate fractions of the first anion exchange column.
 
2. The column is eluted using buffers containing sodium chloride in the concentration range of 0.06M to 0.3M at the same pH.
 
3. The eluate collected is concentrated on a tangential flow filtration set-up to a reduced volume so that viral inactivation can be accomplished.
 
4. This eluate is subjected to solvent-detergent (S/D) treatment under standard conditions frequently used for inactivating enveloped viruses.
 
5. Further purification of Factor IX, from additives and other protein impurities is achieved by loading on an affinity or metal chelate chromatography column to specifically bind the F-IX protein.
 
       Column Equilibration: 
       [0000]    
       
         
           
             The anion exchange column like DEAE or equivalent is equilibrated with anionic buffers in the molarity range of 0.01M to 0.15M in the pH range of 6.5 to 8.0 and eluted with sodium chloride in the range of 0.06M to 0.3M at the same pH. The affinity column is equilibrated with an anionic buffer in the molarity range of 0.01M to 0.1M and contained a divalent cation in the pH range of 5 to 9. This column is eluted with the same buffer containing sodium chloride from 0.05M to 1.5M.
 
6. The eluate from the affinity column is loaded onto a gel filtration chromatography column packed with resins like Sephadex, Superose, Superdex or Sephacryl in the separation range of 10 KD to 600 KD, equilibrated with buffers containing sodium salts of citrate or acetate in the molarity range of 5 mM to 100 mM and a pH in the range of 6.0 to 8.0. The peak fraction containing Factor-IX is collected and subjected to concentration step.
 
7. The factor IX protein sample is concentrated using ultra filtration and nanofiltered to remove viruses. Finally, this sample is sterile filtered and filled in vials for lyophilization.
 
           
         
       
     
         [0067]    The chromatography based process for the production of Factor IX gives a preparation of F-IX with specific activity in the range of ≧200 IU/mg. This preparation also has impurity levels many fold lesser than the specifications of the products in the market and as per the limits specified by the regulatory authorities. The main contaminants in factor IX are Factor-II (the level in our preparation is around 0.798%, against the regulatory limit of &lt;5%), Factor VII (the level in our preparation is around 0.09%, against the regulatory limit of &lt;5%), Factor X (the level in our preparation is around 0.3%, against the regulatory limit of &lt;5%). The contaminating levels of Heparin in the Factor IX preparation is around 0.0095 IU/IU of Factor IX against the regulatory specified limit of not to exceed 0.5 IU of Heparin per IU of Factor IX. These results are formulated in Table 1 (Test for impurities in Factor IX preparation). 
         [0068]    The present invention is not limited to the products specified in the embodiments, but has the potential to purify several other plasma products by incorporating minor variations in the wash and elution conditions. By circumventing the ethanol precipitation steps mentioned in the Cohn&#39;s method of fractionation, the purification scheme of the present invention saves on the use of several thousands of liters of ethanol, a reagent that requires explosion-proof manufacturing facilities, affects protein quality (more aggregates, denaturants), activity (lower potency) and yield (lower recoveries). The quality control tests on the plasma products purified by this scheme show that the products fulfill the QC specifications exceedingly well.