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"Method for Producing Injectable Formulations of Blood-Derived Protein Materials, and Materials Obtained Using Said Method" in Patent Application...

May 20, 2014



"Method for Producing Injectable Formulations of Blood-Derived Protein Materials, and Materials Obtained Using Said Method" in Patent Application Approval Process

By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Segura Ruiz, Alvaro (San Jose, CR); Vargas Arroyo, Mariangela (San Jose, CR); Leon Montero, Guillermo (Alajuela, CR); Villalta Arrieta, Mauren (San Jose, CR); Herrera Vega, Maria (Cartago, CR); Angulo Ugalde, Yamileth (San Jose, CR), filed on April 8, 2011, was made available online on May 8, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Universidad De Costa Rica).

This patent application is assigned to Universidad De Costa Rica.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The present invention relates to the field of purification of therapeutic proteins, particularly to a method for the production of injectable formulations of blood-derived protein products such as immunoglobulin, and/or albumin.

"Products derived from blood plasma such as human albumin, human immunoglobulin and heterologous antivenoms are important drugs in the treatment of various diseases, accidents and injuries. Due to the present increased demand for these products, it is important to improve the efficiency of production methods thereof, to adequately supply this demand, and thus prevent a global shortage of these drugs in the short term.

"To obtain protein products derived from blood, particularly plasma, there are several methods well known in the prior art. Among them we may find the following:

"Cohn's technique for plasma fractionation (cold alcohol fractionating) is the most common method used by the plasma-derived biological products industry (see Cohn E J, Strong L E, Hughes W L, Mulford D J, Ashworth J N, Melin M. Taylor HL 1946. 'Preparation and properties of serum and plasma proteins. IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissue and fluids', Journal of the American Chemical Society, 68: 459-475). To improve those aspects related to the production cost and performance of the original technique, several researchers have proposed modifications employing less reactive or suppress steps in the process (see Wink J., Hidalgo J., Seeberg V., Johnson FE 1957. 'Preparation and properties of a heat-treated human plasma protein fraction.' Vox Sanguinis 2: 174-186; Kistler, P Nitschmann, H. 1962. 'Large Scale Production of Human plasma Fractions', Vox Sanguinis 7: 414-424 Schneider, W., Wolter, D., McCarty, L. 1976, 'Alternatives for Plasma Fractionation.' Vox Sanguinis, (31) 2: 141-151). Moreover, other modifications suggest the incorporation of purification techniques based on the use of precipitating agents, one or more chromatographic steps or a combination thereof. Among the commonly used precipitating agents are ammonium sulfate, polyethylene glycol and caprylic acid.

"It has been reported the use of aqueous two-phase systems (ATPS) for the primary recovery and fractionating of compounds of interest with high commercial value. The ATPS are composed of mixtures of polymer-polymer, polymer-salt or salt-alcohol, and have been used in the primary recovery and partial purification of biological products such as proteins, genetic material, cells or organdies thereof, organic compounds such as fragrances and dyes, heavy metals, and certain drugs (see Benavides, J., Rito-Palomares, M. 2008. 'Review: Practical experiences from the development of two-phase Aqueous Processes for the recovery of high value biological products'. J Chem Technol Biotechnol 3:133-142; Huddleston, J., A. Veide, K Kohler, J. Flanagan, S-0. Enfors and A. Lyddiat. 1991. 'The molecular basis of partitioning in Aqueous two-phase systems'. Tibtech 9:381-388).

"U.S. Pat. No. 4,684,723 discloses a method for separating the alpha-1-proteinase inhibitor from other proteins and nucleic acids present in the plasma or culture medium using ATPS. Also, there is a patent application WO 2010/062244A1, which proposes the recovery and partial purification of therapeutic proteins, particularly monoclonal antibodies, in two stages of extraction in ATPS. In the first stage, the antibodies are partitioned towards the top phase, and in the second phase they precipitate in said phase. They are then recovered and re-dissolved, for further purification by chromatography. In another invention (U.S. Application No. 2010/0179252) the use of a multiphase system is proposed, comprising two types of polymers, one acidic and one etheric, and at least one salt for the separation of biomolecules, cells or particles. Additionally, patent application WO 2010/080062 presents a method for isolating biomolecules in ATPS polymer-salt, wherein the molecule of interest, for example a monoclonal antibody, is partitioned towards the phase that is not rich in the polymer. Additionally, there are studies on the conditions of partition, extraction and purification of antibodies in ATPS (see Andrews, BA, Nielsen, S., Asenjo, JA 2007, 'Partinioning and purification of monoclonal antibodies in Aqueous two-phase systems', Bioseparation 6 (5): 303-313; Azevedo, A., Rosa, P., Ferreira, F. Aires-Barros, M. 2007, 'Optimisation of Aqueous two-phase extraction of human antibodies', J. Biotechnology 132 (2): 209-217, and Rosa, P., Azevedo, A Sommerfeld, S., Mutter, M Aires-Barros, M., Backer, W. 2009, 'Application of Aqueous two-phase systems to antibody purification: A multi-phase approach', J. Biotechnology 139(4):306-313).

"Moreover, studies have been published on the partition of albumin in this type of systems with respect to variables such as pH, temperature, and type and concentration of polymer and salt. (see Gunduz, U. 2000. 'Partitioning of bovine serum albumin in an Aqueous two-phase system: optimization of partition coefficient'. Journal of Chromatography B: Biomedical Sciences and Applications, 743 (1-2): 259-262.; Farruggia, B., Nerli, B., Stang, G. 2003. 'Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in Aqueous two-phase systems.' Journal of Chromatography B. 798 (1): 25-33, Lu, Y., Yang, Y., Zhao, X., Xia, C. 2010 'Bovine serum albumin partitioning in polyethylene glycol (PEG) potassium citrate Aqueous two-phase systems.' Food and Bioproducts Processing. 88 (1): 40-46: Garza, M., Rito, M., Serna S., Benavides, J. 2010. 'Potential of Aqueous Two-Phase Systems constructed on Flexible devices: Human serum albumin as proof of concept'. Process Biochemistry 45 (7)1082-1087).

"The immunoglobulin purification technique by caprylic acid precipitation was first introduced by Steinbuch, M., Audran, R., 1969. 'The isolation of IgG from mammalian be With The aid of caprylic acid', Arch Biochem. Biophysics 134, 279-284. Subsequently, there were several methodologies based on this principle. For example, U.S. Pat. No. 4,164,495 (Hansen, 1979) uses protein precipitation with 1-8% v/v PEG and 0.1-5% v/v caprylic acid. In U.S. Pat. No. 5,075,425 (Kotitschke et al. 1991) contaminating proteins are precipitated with 2.5% caprylic acid, followed by adsorption on DEAE-Sephadex. Similarly, patent application WO2006064373 (Bloy et al., 2006) proposes the use of 2.5% caprylic acid to precipitate protein contaminants. Also, the U.S. Pat. No. 6,955,917 (Alred et al., 2003) proposes the use of a solution of 40% caprylate (15-50 mM, preferably 20 mM) for removing contaminating proteins and viral inactivation of the product, followed by a on exchange chromatography. This process is similar to that presented by the patent application WO200508293 (Romisch, et al, 2005), in which a solution of caprylate or heptoanate is applied for the same purpose.

"Finally, there are patents that propose the use of caprylic acid for other purposes. U.S. Pat. No. 5,164,487 (Kobe et al, 1992) in which caprylic acid at a concentration between 0.4 and 1.5%, is used to remove vasoactive substances and proteolytic enzymes, folio wed by ion exchange chromatography; and U.S. Patent 20070244305 (Parkinnen, 2007) presents contaminant protein precipitation with PEG, and caprylic acid is used as a step for the inactivation of viruses.

"Most methods proposed in these patents have yields around 60%, starting from an initial stage of cold alcohol fractionation.

"In the case of albumin, the differential thermal denaturation or selective thermo coagulation is a method used for its purification from plasma or a mixture of proteins containing it. U.S. Pat. No. 4,156,681 proposes heating the plasma at 68.degree. C. in ethanol and sodium caprylate, followed, by the addition of PEG to recover the precipitated albumin. Similarly, in U.S. Pat. No. 3,992,367 plasma is heated to 60.degree. C. and then the albumin is precipitated with ethanol. In another invention (U.S. Pat. No. 4,222,934), plasma is heated to 60.degree. C., PEG is added to remove the precipitate of denatured protein, and albumin is recovered by isoelectric precipitation. Moreover, in U.S. Pat. No. 4,177,188 the immunoglobulin is recovered prior to the thermal treatment.

"Therefore, there are processes which start from plasma, serum, some fraction of Cohn method or other starting material containing albumin and/or immunoglobulin for recovery thereof, and subsequently employing selective thermo coagulation and the caprylic acid precipitation to purify albumin and immunoglobulin respectively. However, to date the use of such purification techniques from fractions derived from an aqueous two-phase system (ATPS) has not been reported.

"The present invention overcomes the limitations of the prior art, since it presents a production line for obtaining injectable formulations of blood-derived protein products with reduced viral load for human use, an utmost important aspect that has not been described in the prior art with respect to obtaining immunoglobulin and albumin using ATPS."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "An object of the present invention is a method for the production of injectable formulations of blood-derived protein products with reduced viral load, which method comprises the steps of (FIGS. 1 and 4): a. fractionating the starting material in aqueous two-phase system by adding a polymer and at least one salt; b. adding phenol to the aqueous two-phase system as a first viral inactivation step; c. separating the upper and lower phases of aqueous two-phase system; d. purification of the products contained in the upper phase of the system by precipitation with a fatty acid; e. purification of the products contained in the lower phase of the system by thermo coagulation; f. removing of denatured protein precipitate formed during the purification steps of upper and lower phase of the two-phase system; g. increasing the purity of the purified products from the upper and lower phase by chromatography; h. nanofiltration of the products obtained in the previous step to remove viral particles; i. formulation, stabilization and sterilization of the products obtained.

"The method of the invention can be carried out starting from a material that can be selected from a group consisting of blood plasma, blood serum, a fraction obtained by Cohn's method or any other material containing blood-derived protein products, particularly albumin and/or immunoglobulin.

"Initially, the fragmentation of the starting material in a system is performed, comprising two aqueous phases. To do so, a polymer and a salt are added, wherein the selected polymer is polyethylene glycol with molecular weight between 1000-6000 Da, and preferably 3350 Da polyethylene glycol, which is used at a concentration in the range between 6 to 15% w/v, preferably between 6 and 9% w/v.

"The salt used in the fractionating may be monobasic potassium phosphate, dibasic potassium phosphate, monobasic sodium phosphate, dibasic sodium phosphate, ammonium sulfate and sodium citrate, preferably being used potassium phosphate monobasic and dibasic, at concentrations between 10 and 20 w/v, and preferably between 15 and 20% w/v.

"Additionally, a salt which is not involved in the formation of the two phases is employed, but which influences the partitioning of solutes in the system, preferably using sodium chloride at a concentration between 5 and 20 w/v, preferably between 12 and 15% w/v.

"This fractionation step is performed at a pH between 5.5 and 7.5, and preferably at a pH of about 6, and is performed at room temperature (20-25.degree. C.).

"As a first viral inactivation step, the method of the invention employs phenol between 0.05 and 0.3% v/v, in a preferred embodiment uses phenol at 0.25% v/v.

"After viral inactivation, we proceed to the separation of the upper and lower phases of the aqueous two-phase system using a combination of processes that can be selected from: rest and separation, rest and filtration, rest and decantation or simply centrifugation.

"The next step is the purification of the products contained in the upper phase of the ATPS obtained. This phase is rich in immunoglobulin, and fir its purification a caprylic acid precipitation is carried out, the latter at a concentration between 1 and 6% v/v, preferably at a concentration between 1.5 and 2% v/v.

"Thermo coagulation is also performed for the purification of the products that are in the bottom phase of the system, which is rich in albumin. This process is carried, out at a temperature between 60 and 70.degree. C., preferably at 65.degree. C. This operation is carried out in the presence of sodium caprylate at 0.012 M and 9% ethanol v/v.

"To increase the purity of the products obtained in the upper phase of the system, a chromatographic step is employed comprising ion exchange chromatography, affinity chromatography or hydrophobic exchange chromatography. The final purification stage of the products obtained in the lower phase of the system is carried out using ion exchange chromatography.

"The removal of viral particles from the products obtained as a result of the process of the present invention is carried out by nanofiltration, employing a 20 nm excluding filter. The products can be stabilized with agents such as sucrose and sodium caprylate to a solution of immunoglobulin and albumin, respectively. The immunoglobulin formulation can be maintained in solution or lyophilized. In the case of albumin a final step of pasteurization for 10 hours at 60.degree. C. is included. Finally, the obtained products are sterilized through a 0.22 mm exclusion membrane.

"Other objects of the present invention are the products obtained by the previously described method. The albumin as well as the immunoglobulin obtained through the preferred embodiments of the invention are injectable quality solution products, having a reduced viral load in accordance with the established specifications (WHO, 2010. Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins, WHO, 2004. Guidelines on viral inactivation and removal Routines Intended to ECOG the viral safety of human blood plasma products).

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

"FIG. 1. Flowchart representing the method of the invention for obtaining protein products derived from virus-free hyper immune equine plasma. It shows the value of yield and purity of the products obtained at each stage of the process. Operations with asterisk show viral inactivation or removal steps. Key: Igs=immunoglobulin, AV=antivenom.

"FIG. 2. Gel filtration of samples of the proposed method for producing antivenom from hyperimmune equine plasma. Superdex 200 10/300 GL, column was used, the elution was performed with a 150 mM NaCl buffer. 20 mM Tris-HCl, pH 7.5. A. hyperimmune equine plasma B. Resuspended ATPS top phase. C. Filtrate obtained from caprylic acid precipitation.

"FIG. 3. Gel filtration of samples of the proposed method for obtaining equine albumin. Superdex 200 10/300 GL column was used, elution was performed with a 150 mM NaCl butler, 20 mM Tris-HCl pH 7.5. A. hyperimmune equine plasma. B. ATPS lower phase. C. Cation exchange chromatography of the filtrate obtained after thermo coagulation.

"FIG. 4. Flowchart representing the method of the invention for obtaining virus-free protein products derived, from human plasma for intravenous use. It shows the yield and purity of the products obtained at each stage of the process. Operations with asterisk show viral inactivation or removal steps. Key: Igs=Immunoglobulin.

"FIG. 5. Gel filtration of samples of the proposed method for obtaining, gammaglobulin from human plasma. Superdex 200 10/300 GL column was used, elution was performed with a 150 mM NaCl buffer, 20 mM Tris-HCl, pH 7.5. A. Human plasma. B. Resuspended ATPS top phase. C. Anion exchange chromatography of the filtrate obtained after caprylic acid precipitation.

"FIG. 6. Gel filtration of samples of the proposed method for obtaining human albumin. Superdex 200 10/300 GL column was used, elution was performed with a 150 mM NaCl butler, 20 mM Tris-HCl, pH 7.5. A. Human plasma. B. ATPS lower phase. C. Cation exchange chromatography of lower phase."

URL and more information on this patent application, see: Segura Ruiz, Alvaro; Vargas Arroyo, Mariangela; Leon Montero, Guillermo; Villalta Arrieta, Mauren; Herrera Vega, Maria; Angulo Ugalde, Yamileth. Method for Producing Injectable Formulations of Blood-Derived Protein Materials, and Materials Obtained Using Said Method. Filed April 8, 2011 and posted May 8, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2003&p=41&f=G&l=50&d=PG01&S1=20140501.PD.&OS=PD/20140501&RS=PD/20140501

Keywords for this news article include: Antibodies, Anions, Alkenes, Phenols, Therapy, Alcohols, Peptides, Polyenes, Chemistry, Immunology, Amino Acids, Hydrocarbons, Legal Issues, Ethanolamines, Serum Albumin, Blood Proteins, Immunoproteins, Immunoglobulins, Serum Globulins, Ammonium Sulfate, Phosphoric Acids, Sodium Phosphate, Organic Chemicals.

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Source: Life Science Weekly