WO2006137815A2 - Procede d'epuration de melanges d'immunoglobuline et d'albumine - Google Patents

Procede d'epuration de melanges d'immunoglobuline et d'albumine Download PDF

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Publication number
WO2006137815A2
WO2006137815A2 PCT/US2004/019674 US2004019674W WO2006137815A2 WO 2006137815 A2 WO2006137815 A2 WO 2006137815A2 US 2004019674 W US2004019674 W US 2004019674W WO 2006137815 A2 WO2006137815 A2 WO 2006137815A2
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WIPO (PCT)
Prior art keywords
albumin
igg
composition
proteins
components
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Ceased
Application number
PCT/US2004/019674
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English (en)
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WO2006137815A3 (fr
Inventor
Juan Felipe De Leon
Domingo Antonio Murature.
Hilda Lidia Nieres
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PURE PROTEIN PLUS Inc
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PURE PROTEIN PLUS Inc
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Priority to PCT/US2004/019674 priority Critical patent/WO2006137815A2/fr
Publication of WO2006137815A2 publication Critical patent/WO2006137815A2/fr
Anticipated expiration legal-status Critical
Publication of WO2006137815A3 publication Critical patent/WO2006137815A3/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA

Definitions

  • This invention relates to a method and to the product obtained thereby. More specifically, this invention is directed to a method for producing essentially purified protein fractions from a heterogeneous mixture, wherein the mixture includes proteinacious materials that are both endogenous to biological fluids, (such as plasma) and exogenous to such fluids (e.g. pathogens such as viruses and their by-products).
  • the method of this invention enables the concurrent attainment of highly purified albumin and human immunoglobulin (IgG) fractions directly from plasma, without prior fractionation of the plasma, so as to produce higher yields of therapeutically active proteins than attainable by previous methods.
  • the albumin attainable in accordance with the method of this invention is useful, for example, in so called “replacement solutions” to compensate for blood losses.
  • the immunoglobulin fraction attainable in accordance with the method of this invention is effective in the prevention and treatment of a number of infectious diseases, including those associated with Staphylococci, Streptococci, CoIi, Pseudomonas, Herpes zoster and pyocyaneus septicemias.
  • albumin and human immunoglobulin are generally obtained from the plasma fraction of whole blood.
  • IgG human immunoglobulin
  • albumin and IgG are structurally similar to other plasma components, further complicating its isolation and recovery. Comparable difficulties are encountered with the plasma IgG fraction. Accordingly, methods for the isolation and recovery of albumin and IgG from human plasma have generally required some form of stabilization of such materials prior to subjecting them to purification methods relying upon elevated temperatures for denaturation of the proteins associated with infections disease states. Moreover, because of the dissimilar chemistry and structure of albumin and IgG, it has not been previously practical to attempt to isolate and recover each of these materials concurrently, thus, necessitating multiple and often complex fractionation processes.
  • US 4,440,679 discloses a method for the pasteurization of fluid compositions containing thermally sensitive, therapeutically active proteins.
  • a protein solution is rendered heat stable, during pasteurization or heating at a temperature of about 60 to 75°C, by mixing with heat-stabilizing or pasteurization-stabilizing amounts of a polyol.
  • polyol is defined by Fernandes as a substance with more than one hydroxyl group (- OH), and includes polyhydric alcohols and carbohydrates, such as sugars.
  • the polyols that are preferred for use in his method are water miscible, physiologically compatible with the protein, and have a low molecular weight, i.e., a molecular weight less than about 5000.
  • These polyhydric alcohols include both simple sugars and polyhydric alcohols.
  • Typical examples of sugars that may be used in the Fernandes method are mono-, di-, and trisaccharides such as arabinose, glucose, galactose, fructose, ribose, mannose, rhamnose, sucrose, maltose, raffinose, melezitose, and so forth.
  • Exemplary of polyhydric alcohols or reduced sugars, included within the purview of the Fernandes invention are erythritol, ribitol, sylitol, sorbitol, mannitol, etc.
  • the organic carboxylic acid suitable for use in his method includes a carboxylic acid, such as caprylic acid, mandelic acid or citric acid, and the physiologically acceptable one such as an alkali metal such as sodium or potassium, or an alkali earth metal such as calcium, etc.
  • This organic carboxylic acid is used in the Gion method at a final concentration of 3-10 mM, preferably 3-5 mM.
  • US 6,365,395 discloses a process for selectively removing protein aggregates and virus particles from a protein solution in a two-step filtration process, so as to avoid premature plugging of the filtration membranes used in such separations.
  • a protein solution is filtered by tangential flow filtration through a cellulosic ultra filtrate membrane at a transmembrane pressure of between about 1 and about 10 psi to produce a first permeate and a retentate stream.
  • Water or aqueous buffer is added to the retentate stream to form an essentially constant volume retentate stream.
  • the first permeate is filtered through a second ultra filtraiion membrane to retain virus particles at a retention level of at least 3 LRV and to allow passage there through of a protein aggregate free and virus free protein solution.
  • the above and related objects are achieved by providing a method for the concurrent purification and isolation of albumin and an additional protein, such as IgG, from a fluid composition containing up to about 15%, by weight, of blood plasma proteins obtained from a plasma source that has not undergone prior fractionation, [add discussion of use of different source fluids including partially fractionated plasma source]
  • the concurrent purification and isolation of a plasma source includes the recovery of albumin and some other plasma component that is associated with albumin.
  • the albumin appears to enhance the stability of the recovered components by providing a physiological supplement to the isolated materials once it has been removed from its native environment.
  • This preference for concurrent purification albumin along with one or more proteins that associate with albumin permits recovery of relatively unstable plasma components that ordinarily could not be recovered alone, or if capable of independent recovery, were either only obtained in relatively low yields or suffered loss of therapeutic shortly after such independent recovery.
  • the composition is adjusted to a pH of from about 4.5 to about 7.5 with a physiologically acceptable buffer.
  • the composition is then further modified by the addition, relative to said IgG, of a thermal stabilization effective amount of a polyhydric alcohol, wherein the molar concentration of the alcohol within the fluid composition is at concentration in the range of from about 0.1 to about 0.5M; and, by the addition, relative to albumin, of a thermal stabilization effective amount of a carboxylic acid having from about 3 to 10 carbon atoms, or a physiological acceptable said of said carboxylic acid, wherein said acid and/or said acid salt is present within the fluid composition, relative to the total concentration of albumin, at weight ratio in the range of from about 0.005:1 to aboutn 0.020:1
  • the resulting fluid composition is thereafter heated to and maintained at a temperature in the range of from about 55 to 75 0 C for a period sufficient to effect essentially complete denaturation of proteins other than the albumin and the IgG components of said composition. Generally, heating the fluid composition for at least about 10 hours is sufficient of this purpose. Thereafter, the fluid composition is cooled and the soluble components thereof, the albumin and IgG, isolated from the solids and suspended matter by a combination of filters designed to initially remove the solids and suspended matter from the fluid composition, and thereafter through an array of tangential flow filtration membranes capable of isolation of soluble protein components of said composition by molecular weight.
  • the source of the soluble proteins sought to be recovery by the method of this invention can be any animal source, or, crizsly, derivative from a culture wherein an organism is engineered to produce soluble proteins of the type found in biological fluids of animals and/or human. Under some circumstances, it may be appropriate to subject the source of soluble proteins to preprocessing for neutralization or removal of a component thereof that is either highly infectious or which simply causes unacceptable interference in the recovery process. For simplicity of understanding ease of explanation, the method of this invention is herein further described within the context of purification and isolation of IgG and albumin from a human plasma.
  • the buffers which can be used in the method of this invention, to adjust and maintain the pH of the fluid composition include any of the well-known buffering system that is thermally stable within the temperature ranges (-55 to 75 0 C) contemplated by the method of this invention; and, which is otherwise physiologically compatible (does not denature proteins) with the objects of this invention.
  • the readily available buffers which satisfy such requirements include the traditional family of phosphate buffers (e.g. TRIS) and natural buffering agents that are sufficiently thermally stable in the processing environment of the method of this invention.
  • Stabilizers suitable for use in the method of this invention include the physiologically compatible polyhydric alcohols (herein also "polyols") which are known and available for the thermal stabilization of IgG, such as are described in Fernandes US 4,440,679, which is herein incorporated by reference in its entirety.
  • polyol as used herein is inclusive of a substance with more than one hydroxyl group (--OH) and includes polyhydric alcohols and carbohydrates such as sugars. It is preferred that the polyol be water miscible, physiologically compatible with the protein, and have a low molecular weight, i.e., a molecular weight less than about 5000.
  • Higher molecular weight polyols e.g., polysaccharides such as dextrin, starch, glycogen, cellulose, pentosans, pectin, hemicellulose, and the like, are not preferred for use in the present method because they are generally water immiscible and are difficult to separate from the protein composition after pasteurization has been completed.
  • Typical examples of sugars that may be employed in our method are mono-, di-, and trisaccharides such as arabinose, glucose, galactose, fructose, ribose, mannose, rhamnose, sucrose, maltose, raffinose, melezitose, and so forth.
  • polyhydric alcohols or reduced sugars included within the purview of the invention are erythritol, ribitol, sylitol, sorbitol, mannitol, etc. Also within the compass of the invention are mixtures of polyols and substances that produce a polyol in the presence of water or heat such as hydrates, actonides, or the like.
  • compositions that are suitable for the thermal stabilization of the albumin protein, within the processing environment of this invention include the physiological carboxylic acids, and their physiological acceptable salts, having from 5 to 10 carbon atoms, such as described in Gion US 4,754,019, which is herein incorporated by reference in its entirety.
  • These organic carboxylic acid suitable for use in this invention include the carboxylic acids such as caprylic acid, mandelic acid or citric acid is preferred.
  • the is physiological acceptable salts of such acids include the alkali metal salts, such as sodium or potassium, or an alkali earth metal such as calcium, etc.
  • the process of this invention is directed to the concurrent purification and isolation of dissimilar proteins from a common composition
  • the compounds selected for the stabilization of, for example, the IgG and, for example, the albumin must be compatible with one another within the processing environment of this invention.
  • the selection of stabilizing agents may require some minimum evaluation to insure that each is free from interference or interaction v/ith the other, and thereby free from compromise of the stabilization of the protein component for which it was intended.
  • the method of this invention is suitable for the concurrent purification and isolation of diverse protein fractions from a composition containing a biological fluid.
  • purification is intended to include the thermal inactivation and/or denaturation of endogenous and exogenous materials that can be present in a biological fluid, which if allowed to remain in tact could evoke an immune response or cause and infection in the recipient of the products sought to be recovered from the composition.
  • isolation is intended to include the physical separation of the soluble components of the composition remaining after removal of particulate matter, and that such isolation is based upon discrimination of products on the basis of molecular weight.
  • biological fluid is intended to include a protein containing liquid that is obtained from a natural or synthetic source wherein the protein components of interest are soluble, along with other endogenous and exogenous materials, in such fluid.
  • a biological fluid is obtained and the relative concentration of the proiein fraction therein confirmed by simple light scattering or other common analytical techniques.
  • the biological fluid comprises the plasma fraction of whole blood
  • the protein concentration therein is well-known, and no such confirmation necessary.
  • sufficient buffer and isotonic saline are added to provide a composition containing from about 1 to about 7%, by weight, protein.
  • the pH is adjusted to within a range of from 4.5 to about 7.5, and preferably to a pH of 6.5.
  • the protein stabilizers are then added to this buffered composition.
  • a polyhydric alcohol stabilizer is added to the composition in an amount in the range of from about 5 to about 7% weight based upon the volume of the composition.
  • a suitable carboxylic acid stabilizer is added to the composition, relative to total albumin, in a weight ratio relative to total protein in the composition, in the range of from 0.005:1 to about 0.020:1.
  • the stabilizers selected for use in this method must be compatible with one another and not otherwise diminish the stability of the proteins sought to be recovered.
  • the method of this invention can be used in conjunction with other purification processes which desire to avoid the traditional alcohol fractionation processing preliminary to purification and isolation of the proteins of interest.
  • the composition Upon completion of addition of the stabilizers to the composition, the composition is heated to a temperature in the range of from about 55 to 75 0 C, and maintained at such temperature for an interval sufficient to effect essentially completed denaturation of unstablized endogenous and exogenous proteins that may also be present in the composition. Generally, maintaining the composition at this denaturation temperature for at least about 10 hours is sufficient to accomplish the intended purpose.
  • the composition is then removed from the source of heating, and allowed to cool to room temperature. After the composition is cooled its is filtered to remove the precipitant that was formed during heating.
  • the manner of removal of such particulate matter can be any common filtration technique intended for separation of particulate matter from the liquid phase of the composition.
  • the soluble composition of the composition are further processed to isolate the suspended matter from the soluble proteins. This can include the passage of the composition through one or a series of membranes of graduated porosity until only the soluble proteins remains in the composition.
  • the soluble components of the composition are now isolated from one another by passage through a series of molecular sieves and thereby the dissolve proteins recovered, based upon their molecular weight. For example, in a purified composition containing
  • the IgG and albumin can be isolated from one another by tangential flow filtration through a membrane having a pore size of approximately 300,000 Daltons.
  • the permeate recovered in this manner is further subjected to tangential flow filtration through a second membrane having a pore size of approximately 10,000 Daltons.
  • the permeate recovered at this juncture is subject to High Pressure Liquid Chromatographic analysis. Such analysis indicates that the only protein fractions present in the composition are the monomeric IgG, dimeric IgG and albumin.
  • the yield attained is greater than ninety percent (90%).
  • Example I (Illustration of separation of IgG and albumin, as a mixture, from bovine plasma)
  • a sample of plasma was analyzed for total proteins - 5.5 weight percent total proteins and, electrophoresis performed to confirm protein composition - albumin 3.0 weight percent; IgG 1.4 weight percent (Tizard, I.R., 2000) and other endogenous proteins (Dus Santos MJ. et al, 2000). Separation Isolation Of IgG and albumin Fraction: - To the approximately 2800 mis of recovered plasma was added 5 weight percent sorbitol and octanoic acid 0.08 mmol, for each gram of albumin. This plasma solution was thereafter heated to 60°C for 10 hours. The resultant mixture comprises a cloudy liquid having particles (e.g. denatured protein) in suspension.
  • particles e.g. denatured protein

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention a trait à un procédé d'épuration d'isolation simultanées d'albumine et d'immunoglobuline à partir d'une composition contenant jusqu'à 15 % en poids de protéines de plasma sanguin obtenues à partir d'une source de plasma qui n'a pas été précédemment soumis à un fractionnement. Dans ce procédé, la composition est tamponnée et ensuite modifiée par l'ajout, par rapport à l'IgG, d'une quantité efficace de stabilisation thermique d'un alcool polyhydrique, et, par l'ajout, par rapport à l'albumine, d'une quantité efficace de stabilisation thermique d'un acide carboxylique (par exemple, de l'acide carboxylique ayant environ 3 à 10 atomes de carbone), ou un sel physiologiquement acceptable dudit acide. La composition de fluide obtenue est ensuite chauffée et maintenue à une température dans une plage suffisante pour effectuer une dénaturation sensiblement complète des protéines, autres que les constituants d'albumine et d'IgG de ladite composition. Ensuite, la composition de fluide est refroidie et les constituants de celle-ci, l'albumine et l'IgG, isolés des matières solides et de la matière suspendue par une combinaison de filtres destinée à l'élimination initiale des matières solides et suspendues de la composition de fluide, et ensuite à travers un réseau de membranes de filtration à écoulement tangentiel capables d'isoler les constituants de protéines solubles de ladite composition par le poids moléculaire.
PCT/US2004/019674 2004-07-29 2004-07-29 Procede d'epuration de melanges d'immunoglobuline et d'albumine Ceased WO2006137815A2 (fr)

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PCT/US2004/019674 WO2006137815A2 (fr) 2004-07-29 2004-07-29 Procede d'epuration de melanges d'immunoglobuline et d'albumine

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PCT/US2004/019674 WO2006137815A2 (fr) 2004-07-29 2004-07-29 Procede d'epuration de melanges d'immunoglobuline et d'albumine

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109096364A (zh) * 2018-07-12 2018-12-28 安陆恩彼饲料有限公司 一种血浆中功能性蛋白质的分离纯化方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4156681A (en) * 1974-03-28 1979-05-29 Plasmesco Ag Process for isolating albumin from blood
US4440679A (en) * 1980-03-05 1984-04-03 Cutter Laboratories, Inc. Pasteurized therapeutically active protein compositions
JPS62294621A (ja) * 1986-06-13 1987-12-22 Green Cross Corp:The アルブミンの回収方法
US6365395B1 (en) * 2000-11-03 2002-04-02 Millipore Corporation Process for removing protein aggregates and virus from a protein solution

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109096364A (zh) * 2018-07-12 2018-12-28 安陆恩彼饲料有限公司 一种血浆中功能性蛋白质的分离纯化方法

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