WO2008068455A1 - Purification de protéines - Google Patents

Purification de protéines Download PDF

Info

Publication number
WO2008068455A1
WO2008068455A1 PCT/GB2007/004418 GB2007004418W WO2008068455A1 WO 2008068455 A1 WO2008068455 A1 WO 2008068455A1 GB 2007004418 W GB2007004418 W GB 2007004418W WO 2008068455 A1 WO2008068455 A1 WO 2008068455A1
Authority
WO
WIPO (PCT)
Prior art keywords
precipitate
polyethylene glycol
protein
zinc
process according
Prior art date
Application number
PCT/GB2007/004418
Other languages
English (en)
Inventor
Petra Eaglesfield
Emma Rosemary Waite
Anne Gillian Welch
Peter Reynolds Foster
Ronald Vance Mcintosh
Original Assignee
Common Services Agency
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Common Services Agency filed Critical Common Services Agency
Publication of WO2008068455A1 publication Critical patent/WO2008068455A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • 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

Definitions

  • the present invention relates to a process for the purification of a therapeutic protein, particularly a blood plasma protein, from a proteinaceous mixture of proteins by employing polyethylene glycol and zinc as precipitation agents.
  • Protein precipitation uses the difference in solubility of proteins in a mixture to separate the proteins.
  • the solubility behaviour of a protein is a unique property determined by its composition and conformation and the environment surrounding the molecule. The distribution and degree of hydrophilic and hydrophobic regions determines the degree to which a protein is soluble in an aqueous environment. Proteins are least soluble at their iso-electric point. The availability of solvent (e.g. water) is paramount in influencing the solubility of proteins. Interactions involved in solvation at the protein surface are susceptible to changes in pH, dielectric constant and ionic strength, temperature and surface tension increment, as well as interactions with specific substances which cause the solubility of a protein to change, e.g. by changing the net charge or conformation of the protein.
  • Possible precipitation agents include the use of neutral salts (such as ammonium sulphate, sodium sulphate), precipitation with water-miscible organic solvents (such as ethanol), non-ionic polymers (such as polyethylene glycol, polyvinylpyrrolidone, dextran, hydroxyethyl-starch), polyelectrolytes (such as polyethyleneimmine and carboxy-methycellulose), metal ions (including zinc, copper, barium, manganese and calcium) and other precipitants such as fatty acids (e.g. caprylic acid), amino acids (e.g. glycine), ionic polysaccharides (e.g. heparin) and dyes (e.g. Rivanol).
  • neutral salts such as ammonium sulphate, sodium sulphate
  • water-miscible organic solvents such as ethanol
  • non-ionic polymers such as polyethylene glycol, polyvinylpyrrolidone, dextran, hydroxyeth
  • Blood plasma is a source of therapeutic proteins and these are conventionally separated using the Cohn cold ethanol fractionation method (Colin EJ. et al, 1946 J. Am. Chem. Soc.68, 459-475) or similar procedures. This involves a series of precipitation steps which enable the mixture to be separated into its principal constituents, typically coagulation factors, albumin and immunoglobulins, (the latter two of which constitute about 46% and 13% by weight respectively of the total protein present in the plasma). Whilst the quality and purity of proteins produced by this method is excellent, the yield is often poor, typically being around 50% or less for immunoglobulins. This may be a disadvantage when valuable and possibly rare immunoglobulins are to be recovered from donor plasma.
  • immunoglobulins in high yield from people having high levels of certain specific immunoglobulins, such as anti-tetanus, anti-hepatitis, anti-D, (anti- rhesus), anti-rabies, anti-zoster etc.
  • certain specific immunoglobulins such as anti-tetanus, anti-hepatitis, anti-D, (anti- rhesus), anti-rabies, anti-zoster etc.
  • Zinc has also been suggested as a means for separating the protein components of human plasma.
  • albumin was precipitated using zinc acetate in R. A. Kekwick et al. "The Separation of Protein Fractions from Human Plasma with Ether", Medical Research Council Special Report Series No.286, p23-29.
  • this report showed that a concentration of 2OmM zinc acetate was optimal.
  • an ion exchange resin was required to remove the residual zinc. It is also reported in Robert B. Pennell, "Fractionation and Isolation of Purified Components by Precipitation Methods", The Plasma Proteins Academic Press 1960, Chapter 2, page 9-49, that immunoglobulins may be precipitated using 2OmM zinc at pH7.2 and 0°C.
  • the present invention provides a process for the purification of the therapeutic protein from a proteinaceous mixture thereof, which comprises; adding a polyethylene glycol to an aqueous liquid containing the proteinaceous mixture to produce a precipitate; separating the precipitate; resuspending the precipitate in aqueous liquid; adding zinc ions to the liquid resuspension to produce a zinc protein precipitate; and separating the zinc protein precipitate.
  • the present invention relies on one or more polyethylene glycol precipitation steps (in which the desired protein may be present in the precipitate or the supernatant), with a final step which generally involves dilution at low pH, followed by precipitation of the desired protein using zinc ions. It has been surprisingly found that much lower amounts of zinc are required to produce the zinc protein precipitate than in the absence of residual polyethylene glycol and that residual polyethylene glycol remains in the supernatant when the zinc precipitate is separated. Thus, the present process is surprisingly effective, in that residual polyethylene glycol is able to be removed employing small amounts of zinc ions, leading to a product which is very low in both residual polyethylene glycol and zinc.
  • the purity of plasma proteins (such as albumin and immunoglobulin) separated by the process is comparable to that produced by the conventional Cohn ethanol fractionation process, but the yield is significantly greater.
  • immunoglobulin yields of about 75-95% are possible using the process of the present invention compared to yields of around 50% using the conventional Cohn ethanol fractionation process.
  • the mechanism of polyethylene glycol (PEG) precipitation is believed to be based on volume exclusion, whereby the PEG molecule, in a random coil configuration, sterically excludes protein from the aqueous solvent, with solubility in PEG being strongly influenced by the molecular size of the protein (ie the larger the protein, the lower the concentration of PEG required for precipitation).
  • the mechanism of metal ion precipitation is different to that of PEG and is due to the ability of metal ions to change the iso-electric point of a protein by binding to specific sites on the protein molecule (e.g. zinc binding to the imadazole group of histidine residues).
  • the present invention demonstrates that effects of PEG and zinc on protein solubility can be combined to exploit these abilities in a synergistic manner.
  • PEG is generally regarded as non-denaturing and may stabilise protein formulations. Nonetheless, the presence of extraneous ingredients in pharmaceutical preparations is generally undesirable.
  • Polyethylene glycol has proved to be more difficult than other precipitants to remove from the final protein solution because of its molecular conformation. Chromatography, Ultrafiltration and liquid-liquid extraction have typically been employed for this purpose prior to the present invention.
  • the polyethylene glycol is generally PEG 4000 or 6000.
  • the polyethylene glycol is generally added at the various polyethylene glycol precipitation stages to achieve a concentration in the proteinaceous liquid of 1-25%, particularly 2-20% by volume. Typically solutions of 4%, 12% and 15% are used at different stages. The polyethylene glycol percentages are given on a v/v volume basis.
  • a typical fractionation process involves the following steps.
  • the precipitate In the case of blood plasma, the precipitate generally contains alpha-, beta- or gammaglobulins. Much of the albumin is left in the supernatant waste.
  • a second step involving a 2-7% polyethylene glycol (typically 4% or 6% polyethylene glycol) solution at pH4.7 results in a supernatant containing the desired protein (gamma-globulins).
  • gamma-globulins the desired protein
  • most of the remaining albumin is in the precipitate, and the immunoglobulins are in the supernatant.
  • a 6-18% polyethylene glycol (typically 12% polyethylene glycol) solution at pH8.0 leads to a protein-containing precipitate.
  • the precipitate purifies the immunoglobulin to greater than 90%, and the final traces of albumin are in the supernatant waste.
  • Zinc ions are added to concentrate the product protein into the state of a zinc protein precipitate.
  • Zinc ions are present in a concentration of typically 1-1OmM, especially l-5mM and typically 2-3 mM zinc.
  • the pH is generally in the region 8-9 (especially 8.5).
  • the proteinaceous mixture is generally dissolved in a buffer, such as sodium acetate, and the pH is typically adjusted by addition of NaOH solution.
  • the precipitate is generally separated by centrifugation or filtration.
  • the zinc ions may be added in the form of any pharmaceutically acceptable zinc salt or complex which is soluble in the protein solution concerned under the chosen conditions.
  • Zinc chloride and zinc acetate are preferred zinc compounds.
  • the process can be carried out at room temperature, but is usually carried out at 2-8 0 C for clinical material to minimise the level of bioburden.
  • Using the purification process of the present invention it is possible to achieve polyethylene glycol residual values of less than 0.1% and residual zinc values of less than l.Oppm.
  • the invention is particularly applicable to the preparation of therapeutic proteins from natural or biosynthetic sources, including immunoglobulins, monoclonal antibodies, albumin etc and their derivatives. This is also applicable to the separation of synthetic peptides from cell culture and other production mixtures.
  • the invention is especially applicable where the concentration of total protein in the starting material is high (e.g. greater than about 10 g/L) where efficient technology with a greater capacity than chromatography for concentration and purification of proteins is needed to achieve cost effective production. It is also possible to separate immunoglobulins of different specificities.
  • the immunoglobulins may be from human, animal (e.g. cattle, horse, sheep) or a biosynthetic source.
  • the low levels of residual zinc in the final product are physiologically tolerated but may be further reduced by ultrafiltration.
  • Viruses may be eliminated by conventional techniques such as solvent/detergent treatment, acid treatment, heat treatment or virus filtration.
  • Figure 1 shows a PEG fractionation process according to the invention and used for the production of human IgG
  • Figure 2 shows the process used for the production of sheep IgG.
  • Example 1 PEG fractionation of human plasma
  • Immunoglobulin G is traditionally purified from human plasma by cold ethanol fractionation using a variation of the method described by Cohn referenced above.
  • the quality and purity of IgG obtained by this method is excellent, however the yield is poor, typically around 5g of IgG per litre of starting plasma (from an average initial IgG content of about 10g/L).
  • the PEG fractionation scheme of the present invention can be applied to the preparation of IgG from human plasma and offers a marked increase in yield compared with cold ethanol fractionation. PEG4000 was used.
  • the precipitate (3Og) was resuspended in 15 volumes of 2OmM sodium acetate / 2OmM salt buffer at pH 4.0 and the resuspended precipitate adjusted to pH 8.5 with 0.25M NaOH. 2OmL of a 5OmM zinc acetate solution was added to produce a 2mM concentration. After equilibration for 40 minutes the mixture was centrifuged and the precipitate (26g) retained. The IgG was resolubilised using 2OmM acetate / 2OmM salt buffer at pH 4.0 at a ratio of 10:1 (v/w) for analysis.
  • CAE Cellulose Acetate Electrophoresis
  • Table 1 Comparison of yield and purity for PEG / Zinc and ethanol fractionated IgG from human plasma (assuming 10g/L IgG in starting plasma)
  • the IgG may be further purified and any residual albumin and aggregates removed using the following protein purification methods:
  • the IgG fraction (ca. 25L) can then be further purified using ion-exchange chromatography, depth filtration, ultrafiltration / diafiltration followed by sterile and / or virus filtration and formulation. Fragments of IgG can be generated by digestion with pepsin or papain, and then purified using the techniques listed above.
  • Table 2 shows the yield of IgG at the zinc acetate precipitate stage of the process for 4 batches of anti-toxin and comparative data for Fraction II of human IgG (prepared by 2 different Methods).
  • the purity of the PEG-fractionated IgG is similar to cold ethanol fractionated IgG, but the yield is much higher.
  • Human Immunoglobulin is prepared by an adaptation of the Kistler and Nitschmann method of cold ethanol fractionation. Method 1 involved precipitation of Fraction I +III at 8% and Method 2 involved precipitation of Fraction I +III at 12% see PR Foster (2005) In Kirk-Othmer Encyclopaedia of Chemical Technology 5 th Ed. Volume II, "Plasma Fractionation”.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

On récupère une protéine thérapeutique, en particulier une immunoglobuline, avec un rendement de production élevé à partir d'un mélange protéique (par exemple à partir de plasma sanguin) en y ajoutant un polyéthylèneglycol pour produire un précipité, en séparant le précipité et en remettant celui-ci en suspension dans un liquide aqueux. On ajoute des ions du zinc à la nouvelle suspension liquide pour produire un précipité des protéines et de zinc et on sépare le précipité et on le traite pour récupérer la protéine thérapeutique.
PCT/GB2007/004418 2006-12-05 2007-11-20 Purification de protéines WO2008068455A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0624227.5 2006-12-05
GB0624227A GB0624227D0 (en) 2006-12-05 2006-12-05 Protein purification

Publications (1)

Publication Number Publication Date
WO2008068455A1 true WO2008068455A1 (fr) 2008-06-12

Family

ID=37671849

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/004418 WO2008068455A1 (fr) 2006-12-05 2007-11-20 Purification de protéines

Country Status (2)

Country Link
GB (1) GB0624227D0 (fr)
WO (1) WO2008068455A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010022293A3 (fr) * 2008-08-20 2010-08-19 Baxter International Inc. Procédés de traitement de compositions contenant des microparticules
EP2288358A1 (fr) * 2008-04-16 2011-03-02 Biogen Idec MA Inc. Procédé d'isolement de biomacromolécules à l'aide de polyalkylène glycol et de métaux de transition
US8323615B2 (en) 2008-08-20 2012-12-04 Baxter International Inc. Methods of processing multi-phasic dispersions
US8323685B2 (en) 2008-08-20 2012-12-04 Baxter International Inc. Methods of processing compositions containing microparticles
US20140288283A1 (en) * 2007-05-29 2014-09-25 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Truncated l1 protein of human papillomavirus type 6
WO2016012803A1 (fr) * 2014-07-25 2016-01-28 Bio Products Laboratory Limited Procédé amélioré pour la préparation de l'immunoglobuline g (igg)
US11739117B2 (en) * 2019-09-20 2023-08-29 Plasma Technologies, Llc Therapeutic protein compositions and methods

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046211A1 (fr) * 1997-04-17 1998-10-22 Amgen Inc. Gels d'alginate a liberation prolongee

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046211A1 (fr) * 1997-04-17 1998-10-22 Amgen Inc. Gels d'alginate a liberation prolongee

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
OSADA T ET AL: "Purification and characterization of alpha-macroglobulin and ovomacroglobulin of the green turtle (Chelonia mydas japonica).", JOURNAL OF BIOCHEMISTRY FEB 1988, vol. 103, no. 2, February 1988 (1988-02-01), pages 212 - 217, XP009097449, ISSN: 0021-924X *
ROTHSTEIN F: "Differential precipitation of proteins. Science and technology.", BIOPROCESS TECHNOLOGY 1994, vol. 18, 1994, pages 115 - 208, XP009097194, ISSN: 0888-7470 *
WICKERHAUSER M ET AL: "Large scale preparation of macroglobulins.", VOX SANGUINIS 1972 JUL-AUG, vol. 23, no. 1, July 1972 (1972-07-01), pages 119 - 125, XP009097174, ISSN: 0042-9007 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9745351B2 (en) * 2007-05-29 2017-08-29 Xiamen University Truncated L1 protein of human papillomavirus type 6
US20140288283A1 (en) * 2007-05-29 2014-09-25 Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. Truncated l1 protein of human papillomavirus type 6
EP2288358A4 (fr) * 2008-04-16 2012-12-26 Biogen Idec Inc Procédé d'isolement de biomacromolécules à l'aide de polyalkylène glycol et de métaux de transition
EP2288358A1 (fr) * 2008-04-16 2011-03-02 Biogen Idec MA Inc. Procédé d'isolement de biomacromolécules à l'aide de polyalkylène glycol et de métaux de transition
AU2009236653B2 (en) * 2008-04-16 2014-09-25 Biogen Ma Inc. Method of isolating biomacromolecules using polyalkylene glycol and transition metals
US8367427B2 (en) 2008-08-20 2013-02-05 Baxter International Inc. Methods of processing compositions containing microparticles
WO2010022293A3 (fr) * 2008-08-20 2010-08-19 Baxter International Inc. Procédés de traitement de compositions contenant des microparticules
US8323685B2 (en) 2008-08-20 2012-12-04 Baxter International Inc. Methods of processing compositions containing microparticles
US8323615B2 (en) 2008-08-20 2012-12-04 Baxter International Inc. Methods of processing multi-phasic dispersions
JP2012500799A (ja) * 2008-08-20 2012-01-12 バクスター・インターナショナル・インコーポレイテッド 微粒子を含む組成物を処理する方法
WO2016012803A1 (fr) * 2014-07-25 2016-01-28 Bio Products Laboratory Limited Procédé amélioré pour la préparation de l'immunoglobuline g (igg)
US11149079B2 (en) 2014-07-25 2021-10-19 Bio Products Laboratory Limited Process for the preparation of immunoglobulin G (IgG)
US11739117B2 (en) * 2019-09-20 2023-08-29 Plasma Technologies, Llc Therapeutic protein compositions and methods

Also Published As

Publication number Publication date
GB0624227D0 (en) 2007-01-10

Similar Documents

Publication Publication Date Title
JP3094167B2 (ja) 免疫血清グロブリンの精製方法
JP4445202B2 (ja) 治療的使用のためのヒト免疫グロブリン濃縮物の調製方法
US9708365B2 (en) Purification process for monoclonal antibodies
KR100501263B1 (ko) 정맥 주사용 면역글로불린의 제조 방법 및 그 외의 면역글로불린 제품
US5561115A (en) Low temperature albumin fractionation using sodium caprylate as a partitioning agent
US6835379B2 (en) Method of producing IgG
EP0893450A1 (fr) Procédé chromatographique de purification d'anticorps avec un haut rendement et rendant inactifs les viraux
WO2008068455A1 (fr) Purification de protéines
CN106459140B (zh) 用于纯化免疫球蛋白的方法
CN106414476B (zh) 用于纯化免疫球蛋白的方法
CA3080108C (fr) Procede ameliore de purification d'immunoglobuline
AU2001273907A1 (en) A method of producing IgG
JP2019513119A (ja) 血液系物質からタンパク質を抽出する方法
EP2636681A1 (fr) Procédé d' enrichissement d'IgA
AU2014286268B2 (en) Process
EP2817334A1 (fr) Précipitation de la fraction i-iv-1 d'immunoglobulines à partir de plasma
US4670544A (en) Process for the manufacture of the cold insoluble globulin and pharmaceutical preparation containing it
CA2832665C (fr) Procede de production de formulations injectables de produits proteiques hemoderives et produits obtenus suivant ce procede
CN115925781A (zh) 从血浆中提取蛋白质的方法
WO2001048016A1 (fr) Purification de fibrinogene present dans des proteases de plasma
AU2021414086A1 (en) Systems and Methods for Process Scale Isolation of Immunoglobulin G
CN115768789A (zh) 用于获得包含人血浆来源的免疫球蛋白m的组合物的方法
WO2007136327A1 (fr) Procédé de production d'igg
JP2017524701A (ja) 免疫グロブリンＧ（ＩｇＧ）を調製するための改良された方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07824633

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07824633

Country of ref document: EP

Kind code of ref document: A1