EP0448605A1 - Use of basic amino acids to solubilize immunoglobulins - Google Patents
Use of basic amino acids to solubilize immunoglobulinsInfo
- Publication number
- EP0448605A1 EP0448605A1 EP19900900689 EP90900689A EP0448605A1 EP 0448605 A1 EP0448605 A1 EP 0448605A1 EP 19900900689 EP19900900689 EP 19900900689 EP 90900689 A EP90900689 A EP 90900689A EP 0448605 A1 EP0448605 A1 EP 0448605A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- immunoglobulin
- amino acids
- histidine
- basic amino
- related protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39591—Stabilisation, fragmentation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Definitions
- the invention relates to the production and purification of proteins and to maintaining their solubil ⁇ ity during such production and in storage and formulation.
- it concerns use of basic amino acids to solubilize therapeutically and diagnostically important proteins, especially immunoglobulins, and especially those produced in cell culture.
- Purification and efficient storage of bulk product or vialed final material may also require that volumes be limited to workable quantities. This too requires a practical concentration level of at least 2 mg/ml in order to prevent substantial losses due to precipitation when handling reasonable volumes.
- the present invention resides in the discovery that immunoglobulins, in particular, do not consistently maintain solubility in aqueous media at concentrations greater than 2 mg/ml, that this low level of solubility causes losses in processing, and that the solubility of immunoglobulins can be enhanced by the inclusion in the solution of an effective concentration of basic a ino acids.
- Patent 4,568, 544, assigned to Asahi claims the use of lysine or arginine to increase tPA solubility.
- EP Application 242,653, assigned to Eisai describes tPA solubilization by N-methyl- glucosamine.
- EP Application 218,112, assigned to Eisai generally discloses the use of a partial gelatin hydrolysate cross-linked with diisocyanate for tPA solubilization, optionally with the addition of a basic amino acid.
- Japanese Application 62/153,224, assigned to Green Cross describes solubilization of plasminogen with sugar alcohols (such as mannitol) or a basic amino acid.
- German Patent Application 3,623,747 discloses the use of cholic acid/glycine or cholic acid/taurine conjugates for this purpose.
- U.S. Patent 4,511,502 to Genentech describes the solubilizing effect of urea and guanidinium chloride (GnHCl) on recombinantly produced proteins precipitated as refractile bodies.
- GnHCl urea and guanidinium chloride
- Kurisaki, J. et al, J Biochem (1977) 1:443-449 used GnHCl to solubilize apolipoproteins.
- Japanese Application 83/38597 describes the use of covalent conjugation of free amino sulfhydryl groups to succinyl glycine to effect solubilization of proteins.
- solubilization techniques which preserve the structure by employing nondenaturing and conformation conserving solubilization aids are specific for individual proteins, such as plasminogen, interferon and tPA.
- the present invention provides a workable procedure for immunoglobulins, in particular, and especially for IgG,.
- the invention is directed to a method to solubilize immunoglobulins and related proteins in aqueous medium by including an effective amount of one or more basic amino acids or their salts into the mixture.
- One or more basic amino acids and/or their salts may first be dissolved in aqueous solution and this solution used to dissolve and process the immunoglobulin, or the three components—water, immunoglobulin or related protein and solubilizing amino acid preparation—can be simultaneously mixed.
- the solubilizing amino acid preparation can be added either in solid or dissolved form to a suspension of the immunoglobulin or related protein.
- the invention is directed to compositions of matter which comprise the solubilized immunoglobulin or related protein in the presence of ef ⁇ fective amounts of the solubilizing amino acid(s).
- Figure 1 shows a flow diagram for purification of IgG-. from cell culture.
- immunoglobulins and related proteins refers to proteins which are incapable of stable solubilization in water at levels higher than 2.5 mg/ml. Solubility levels higher than this, which the protein does not normally tolerate, can be obtained using the method of the invention.
- immunoglobulins and related proteins for purposes of this application in ⁇ cludes any protein, most typically an immunoglobulin, but other proteins as well, which has a solubility in water when a solution of the material is agitated, of less than 2.5 mg/ml.
- Immunoglobulins and related proteins can be solubilized, according to the invention method, by inclu ⁇ sion of an effective amount of one or more basic amino acids and/or their salts.
- the method was developed in connection with the production of monoclonal antibodies by large scale cell culture of their secreting hybridomas; however, it is, of course, applicable to immunoglobulins however prepared.
- Polyclonal immunoglobulins can be, for example, isolated from serum and the method of the inven ⁇ tion is applicable to these polyclonal mixtures.
- Monoclonal antibodies can be prepared using cell culture methods involving a variety of techniques including perfusion, static maintenance reactor production, shake flasks, roller bottles, and a variety of cell culture techniques known and to be developed in the art. The method of production is not a part of the invention, but provides the problem that is the candidate for solution when high concentrations of immunoglobulins are produced or are desired. Production of IgG, presents an especially difficult case.
- Suitable related proteins which are subjects for the invention method include cellular receptors such as T4.
- various processes may be used to purify or isolate particular immunoglobulin or related protein fractions.
- Such techniques include chromatography of various types including gel filtration, ion exchange, affinity chromatography, high performance liquid chromatography, and so forth. In conducting these procedures, tractable volumes must be used, and high solubility levels maintained. Again, the particular methods do not form part of the invention, but rather cre ⁇ ate the setting in which the method of the invention is useful.
- Antibodies of various types are sometimes used therapeutically, for example, for passive immunization, when conjugated to a toxin for therapeutic purposes, or when conjugated to label for localization of tissues.
- Formulations for administration of immunoglobulin prepara ⁇ tions must also maintain high solubility levels of the components in order to restrict the volume of fluid administered to a tolerable level.
- Related proteins are also used therapeutically and have similar restrictions on solubility levels.
- solubility levels at a minimum of 3-5 mg/ml are required.
- Many immunoglobulins and other proteins, such as cellular receptors do not achieve this solubility level in water solution and require larger volumes than desirable to maintain solubility.
- immunoglobulins of the IgG class and, more particularly, those of the IgG, subclass, and T4 receptor proteins are difficult to maintain in solution. It appears also that without addi ⁇ tion of solubilizing amino acid according to the method of the invention, recoveries of immunoglobulins are diminished, often to a level below that which is economi ⁇ cally acceptable.
- the solubility enhancing components of the invention are basic amino acids and/or their salts.
- Basic amino acids are herein defined as those which, when dissolved in water at reasonable concentrations— e.g., 0.1-1M—result in a pH of solution of greater than 7.
- acids can be used to titrate the high pH back to neutrality (i.e., pH 6.5-7.5) if desired.
- the natively encoded amino acids histidine, arginine, and lysine are preferred, but any basic amino acid, such as, for example, hydroxylysine, ornithine, citrulline, and 2,4-diamino- butyric acid are also included.
- the pH of the solution to be used in solubilization can be adjusted, regardless of the form of the amino originally used. Therefore, either the free amino acids or their salts can be employed.
- the salts of the amino acids applicable in the invention are the water soluble salts, including the in ⁇ organic salts such as the sodium, potassium or ammonium salts or the salts of organic bases such as ethanolamine or triethylamine. Also included are the acid addition salts of the amino functions of the basic amino acid such as the inorganic acid salts, for example, the hydro- chlorides or sulfdtes, or the organic acid salts, such as acetates or citrates. If the solubilization is intended for formulation of therapeutic compositions, of course, pharmaceutically acceptable salts must be used.
- the solubilizing amino acid preparation must itself be soluble and must be consistent with a pH of the resulting solution in the range of 3-10, preferably 6.5-7.5, if for administration to patients. Additional buffer may be included in the mixture to maintain the pH, if necessary, however the basic amino acid itself, with proper pH adjustment, behaves as a buffer. These limitations also place restrictions on the nature and extent of neutraliza ⁇ tion achieved by particular salts of the amino acids in the preparation, as is understood by. the ordinary skilled practitioner.
- the effective concentration of the solubilizing amino acid preparation depends on the protein to be solubilized and the nature of the solubilizing prepara- tion. In general the degree of solubilization is in ⁇ dependent of the basic amino acid concentration once an effective threshold level is achieved. This level can readily be established for a particular combination by a simple optimization experiment. For example, for IgG, which has an innate solubility of less than 0.5 mg/ml in water, histidine at a level of 50 mM or greater is effec ⁇ tive to permit a solubility of greater than 2.0 mg/ml. To permit flexibility in manipulation, a histidine concentra ⁇ tion range of 0.15-0.2 M is preferred.
- T4 recep- tor solutions having more than 2.2 mg/ml T4 concentra ⁇ tions are unstable to agitation, but with the addition with 50 mM-150 mM histidine, the solution remains stable to concentrations of T4 up to 17 mg/ml.
- the solubility levels of T4 in completely unagitated solutions can be up to about 8 mg/ml; however, agitation comparable to that involved in normal handling reduces the level to about 2 mg/ml. Indeed, in one specific instance, shipment of a solution of T4 with a concentration of 2.2 mg/ml was found to contain precipitate upon delivery. The method to effect solubilization will depend on the situation encountered. If employed to solubilize a formulation, the amino acid preparation may first be dis ⁇ solved in the aqueous medium and the immunoglobulin or other protein added to the solution either in solid form or dispersed in a small amount of aqueous fluid.
- solid amino acids or a concentrated solu- tion thereof can be added to a suspension of the immunoglobulin or other protein, or all three of the components may be simultaneously mixed.
- the pH may be adjusted by titration at any step.
- the amino acid prepara ⁇ tion may be added to the harvested medium or the purifica ⁇ tion buffers directly to maintain solubility in the purification process steps and to improve overall re- covery.
- the final solubilized prepara ⁇ tion comprises an immunoglobulin or other related protein at a concentration of at least 3 mg/ml and the appropriate effective concentration of amino acid, wherein the pH of the composition is adjusted to the desired value by titration, if necessary.
- IgG was mixed to a suspension of 2-10 mg/ml in buffer (50 MM Na 2 HP0 4 + 200 mM NaCl; pH 7.0) with and without 200 mM histidine.
- Example 3 Purification of T4 Receptor A.
- samples of purified sT4 dissolved T4 were eluted through an S-Sepharose Fast Flow column with a resulting concentration of 6.23 mg/ml of sT4 in a buffer solution of 0.5 M NaCl and 50 mM sodium phosphate, pH 7.
- 150 mM histidine was added to one sample and samples both with and without added histidine were agitated on a Fisher Vortex Genie 2.
- the sample without histidine visibly precipitated and remained cloudy for over 24 hours.
- the sample with histidine remained in solution and clear for at least 24 hours.
Abstract
Procédé permettant de dissoudre en des concentrations pratiques des immunoglobulines dans un milieu aqueux, qui consiste à utiliser des préparations de dissolution à base d'acides aminés fondamentaux et/ou de leurs sels. Ainsi, l'histidine, la lysine, l'arginine et des mélanges de celles-ci en des concentrations appropriées sont capables d'augmenter la solubilité de protéines relativement insolubles et des immunoglobulines en général, notamment des IgG, et plus particulièrement des IgG3, et des T4.A process for dissolving immunoglobulins in practical concentrations in an aqueous medium, which consists in using dissolution preparations based on fundamental amino acids and / or their salts. Thus, histidine, lysine, arginine and mixtures thereof in appropriate concentrations are capable of increasing the solubility of relatively insoluble proteins and immunoglobulins in general, in particular IgG, and more particularly IgG3, and T4.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US28515488A | 1988-12-15 | 1988-12-15 | |
US285154 | 1988-12-15 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0448605A1 true EP0448605A1 (en) | 1991-10-02 |
EP0448605A4 EP0448605A4 (en) | 1991-11-21 |
Family
ID=23092978
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900900689 Withdrawn EP0448605A4 (en) | 1988-12-15 | 1989-12-06 | Use of basic amino acids to solubilize immunoglobulins |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0448605A4 (en) |
JP (1) | JPH04502914A (en) |
AU (1) | AU4803890A (en) |
CA (1) | CA2005119A1 (en) |
WO (1) | WO1990006764A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5180820A (en) * | 1989-08-30 | 1993-01-19 | Barde Yves Alain | Brain-derived neurotrophic factor |
PT100580A (en) * | 1991-06-12 | 1993-09-30 | Regeneron Pharma | PRODUCTION AND RECOVERY OF RECOMBINANT NEUROTROPHINS |
US5389529A (en) * | 1991-06-12 | 1995-02-14 | Regeneron Pharmaceuticals, Inc. | Modified lamβ signal sequence and processes for producing recombinant neurotrophins |
US6267958B1 (en) | 1995-07-27 | 2001-07-31 | Genentech, Inc. | Protein formulation |
JP5485489B2 (en) | 2000-08-11 | 2014-05-07 | 中外製薬株式会社 | Antibody-containing stabilized preparation |
CN1292655C (en) * | 2001-11-08 | 2007-01-03 | 蛋白质设计实验室股份有限公司 | Stable liquid pharmaceutical formulation of IgG antibodies |
CN1671741A (en) * | 2002-06-21 | 2005-09-21 | 拜奥根Idec公司 | Buffered formulations for concentrating antibodies and methods of use thereof |
US8080642B2 (en) | 2003-05-16 | 2011-12-20 | Vical Incorporated | Severe acute respiratory syndrome DNA compositions and methods of use |
JO3000B1 (en) * | 2004-10-20 | 2016-09-05 | Genentech Inc | Antibody Formulations. |
ES2959479T3 (en) | 2010-09-17 | 2024-02-26 | Takeda Pharmaceuticals Co | Stabilization of immunoglobulins through aqueous formulation with histidine at weakly acidic to neutral pH |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0035616A1 (en) * | 1980-02-14 | 1981-09-16 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Production of immunoglobulin having a high monomer content |
JPS58206531A (en) * | 1982-05-27 | 1983-12-01 | Teijin Ltd | Igm composition having low anticomplementary value |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4362661A (en) * | 1979-08-09 | 1982-12-07 | Teijin Limited | Immunoglobulin composition having a high monomer content, and process for production thereof |
DE3584902D1 (en) * | 1984-02-29 | 1992-01-30 | Asahi Chemical Ind | AQUEOUS SOLUTION OF AN INCREASED CONCENTRATION OF TISSUE PLASMINOGEN ACTIVATOR AND PRODUCTION METHOD. |
JPS60243028A (en) * | 1984-04-28 | 1985-12-03 | Kyowa Hakko Kogyo Co Ltd | Solubilization of interferon |
US4597966A (en) * | 1985-01-09 | 1986-07-01 | Ortho Diagnostic Systems, Inc. | Histidine stabilized immunoglobulin and method of preparation |
JPH0672105B2 (en) * | 1985-10-02 | 1994-09-14 | 持田製薬株式会社 | Thrombolytic agent and manufacturing method thereof |
-
1989
- 1989-12-06 EP EP19900900689 patent/EP0448605A4/en not_active Withdrawn
- 1989-12-06 WO PCT/US1989/005516 patent/WO1990006764A1/en not_active Application Discontinuation
- 1989-12-06 JP JP2501391A patent/JPH04502914A/en active Pending
- 1989-12-06 AU AU48038/90A patent/AU4803890A/en not_active Abandoned
- 1989-12-11 CA CA002005119A patent/CA2005119A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0035616A1 (en) * | 1980-02-14 | 1981-09-16 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Production of immunoglobulin having a high monomer content |
JPS58206531A (en) * | 1982-05-27 | 1983-12-01 | Teijin Ltd | Igm composition having low anticomplementary value |
Non-Patent Citations (2)
Title |
---|
PATENT ABSTRACTS OF JAPAN vol. 8, no. 46 (C-212)(1483) February 29, 1984 & JP-A-58 206 531 (TEIJIN K.K.) December 1, 1983 * |
See also references of WO9006764A1 * |
Also Published As
Publication number | Publication date |
---|---|
AU4803890A (en) | 1990-07-10 |
JPH04502914A (en) | 1992-05-28 |
CA2005119A1 (en) | 1990-06-15 |
WO1990006764A1 (en) | 1990-06-28 |
EP0448605A4 (en) | 1991-11-21 |
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Legal Events
Date | Code | Title | Description |
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PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
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17P | Request for examination filed |
Effective date: 19910617 |
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AK | Designated contracting states |
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A4 | Supplementary search report drawn up and despatched |
Effective date: 19910930 |
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AK | Designated contracting states |
Kind code of ref document: A4 Designated state(s): AT BE CH DE ES FR GB IT LI LU NL SE |
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RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: PRIOR, GARANCE Inventor name: TIPTON, BARBARA Inventor name: MOELLERING, BILL Inventor name: PRIOR, CHRISTOPHER, P. |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: CENTOCOR INC. |
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17Q | First examination report despatched |
Effective date: 19920915 |
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STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
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18D | Application deemed to be withdrawn |
Effective date: 19930326 |