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WO1997003092A1 - Procede pour eliminer le polyethyleneglycol d'une solution de proteines ou de peptides - Google Patents

Procede pour eliminer le polyethyleneglycol d'une solution de proteines ou de peptides Download PDF

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Publication number
WO1997003092A1
WO1997003092A1 PCT/DK1996/000314 DK9600314W WO9703092A1 WO 1997003092 A1 WO1997003092 A1 WO 1997003092A1 DK 9600314 W DK9600314 W DK 9600314W WO 9703092 A1 WO9703092 A1 WO 9703092A1
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WO
WIPO (PCT)
Prior art keywords
protein
solution
peptide
peg
process according
Prior art date
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Ceased
Application number
PCT/DK1996/000314
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English (en)
Inventor
Per Kaersgaard
Søren Knud CARLSEN
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Hemasure AS
Original Assignee
Hemasure AS
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 Hemasure AS filed Critical Hemasure AS
Publication of WO1997003092A1 publication Critical patent/WO1997003092A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Definitions

  • a process for removal of polyethylene glycol from a protein or peptide solution A process for removal of polyethylene glycol from a protein or peptide solution.
  • the present invention relates to a method for the removal of polyethylene glycol (hereinafter designated PEG) from a protein or peptide solution.
  • PEG polyethylene glycol
  • Hask ⁇ et al. (Haematologia 14 (2), 199-206 (1981)) describes a method for fractionation of plasma proteins with PEG-4000 and examine the effects of pH, ionic strenght and temperature.
  • a method of protein fractionation suitable for large scale production is described by Bj ⁇ rling (Vox Sang. 49, 240-243 (1985)).
  • the method take advantage of the high protein- binding capacity of ion exchangers under special conditions in the presence of PEG.
  • Danish patent application No. 646/84 describes a method for manufacturing of a high purity concentrate of Factor VLU by fractionation with PEG in at least two steps, in which one of the steps is a precipitation with PEG in the presence of a salting-in agent.
  • US patent specification No. 4,164,495 describes a method for recovering IgG in a pure and anticomplementary activity free condition by fractionated precipitation of blood plasma with PEG in the presence of caprylic acid.
  • European patent application having publication number 123,375 describes manufacturing of a dry ⁇ -globulin preparation capable of intravenous injection by fractionating human plasma with PEG.
  • the method provides a ⁇ -globulin preparation with improved water solubility and stability against increase of anticomplementary activity and decrease of antibody titer.
  • the protein fractions obtained by the PEG fractionation methods generally contains residual PEG and it may, uierefore, be desirable to remove or reduce the content of PEG, especially if the purified protein is used as a drug in mammals.
  • the removal of contaminating PEG from a protein fraction may be carried out according to known methods of protein fractionation such as precipitation of the proteins with another precipitation agent or adsorbtion of the proteins on a chromatography column.
  • Precipitation with another precipitation agent ethanol, ammonium sulfate a.o.
  • introduces other substances that subsequently has to be removed, and adsorbtion of the proteins to a chromatography column may give rise to high concentrations of elution substances after elution of the proteins from the chromatography material.
  • European patent application having publication number 123,375 describes a method for removal of PEG from a solution of ⁇ -globulin by treatment with a nonpolar styrene divinylbenzene copolymer.
  • the adsorbtion treatment is carried out, batchwise or by a column method, by contacting an aqueous solution of ⁇ -globulin contaminated with PEG to remove the PEG from the aqueous solution by adsorbtion of PEG to the synthetic copolymer.
  • PEG may also be removed by diafiltration, as described in European patent application having publication number 270,025, but some proteins tend to aggregate at the ultrafiltration membrane, building up a secondary and denser membrane, that reduces the permeability of PEG.
  • the diafiltration time will, therefore, be very long, especially if the PEG is of a high molecular weight, such as PEG-3000 or PEG-4000.
  • PEG can be removed from a protein or peptide solution, which process comprises adsorbtion of PEG to activated carbon.
  • This absorbtion can be done by filtration of the protein or peptide solution through a filter containing activated carbon.
  • PEG may also be removed by adding activated carbon to the PEG containing protein or peptide solution, and then removing the activated carbon by centrifugation or filtration, or by leading the solution through a column with activated carbon.
  • This invention relates to a process for removing PEG from a protein or peptide solution.
  • the PEG containing solution may have been obtained by a process by which PEG is used in a provisional purification of the protein or peptide in question.
  • PEG is removed from this protein or peptide solution by adsorbing the PEG to activated carbon.
  • Polyethylene glycol or poly(ethylene oxide) or polyoxyethylene or simply PEG is a water soluble polymer with the general chemical formula: HOCH 2 CH 2 (CH 2 CH 2 O) n CH 2 CH 2 OH, wherein n is an integer.
  • polyethylene glycols have designations containing a figure, which figure is the approximate average molecular weight.
  • PEG-3000 or Macrogol 3000 signify heterogeneous mixtures having nominal average molecular weights of 3000.
  • PEG 400 and PEG 4000 signify heterogeneous mixtures having nominal average molecular weights of 400 and 4000, respectively.
  • PEG is a well-known agent for fractional precipitation which stems primarily from its benign chemical properties. Unlike ethanol and other organic precipitating agents, PEG has little tendency to denature or otherwise interact with proteins even when present at high concentrations and elevated temperamres.
  • the process of this invention is performed by contacting the PEG containing protein or peptide solution with activated carbon and, thereafter, removing the activated carbon.
  • the removal of the activated carbon is to be performed in such a way that the resulting protein or peptide solution has a substantially lower content of PEG, compared with the content of protein or peptide, than the content of PEG in the parent protein solution.
  • the protein or peptide solution from which PEG is to be removed is preferably an aqueuos protein or peptide solution containing PEG.
  • Activated carbon or activated charcoal is commercial available as a non-soluble porous powder or granular with grains of different sizes or mesh. Activated carbon is videly used in the chemical and pharmaceutical industry for decolorisation or organic contaminant retention. Activated carbon filters are commercial available as filter sheets, filter pads, or filter cartrigdes in which the finely pulverised activated carbon is bonded to a non-migrating fibre matrix made by e.g. cellulose.
  • the protein or peptide to be purified may be any protein or peptide obtained from plants, animals, or microbes including proteins or peptides obtained by recombinant technology.
  • the protein is especially a plasma protein selected from the group consisting of albumin, immunoglobulins, coagulation factors, antithrombin HI, protein C, protein S, complement factors, transferrin, haptoglobin, ⁇ 2 -macroglobulin, and ⁇ ,-antitrypsin.
  • the protein is selected from immuglobulins and albumin.
  • the activated carbon is added to the PEG-containing protein or peptide solution batchwise, and after stirring for a period of time that is sufficient for adsorbing the PEG, the activated carbon is separated from the solution by methods known per se such as centrifugation, sedimitation, or filtration.
  • the removed activated carbon may subsequently be washed and the washing solution may be added to the purified, more protein or peptide containing solution, to increase the recovery of, for example, a valuable protein or peptide in the purified solution.
  • granular activated carbon is packed into a column.
  • the PEG-containing protein or peptide solution is then led through the column and the effluent is collected.
  • the column may subsequently be washed and the washing solution may be added to the purified more protein or peptide containing solution, to increase the recovery of, for example, a valuable protein or peptide in the purified solution.
  • the PEG-containing protein or peptide solution is filtered through an activated carbon filter with a flow rate that permits the adsorbtion of the PEG to the activated carbon in the filter.
  • the removal of the PEG by filtration may be combined with the removal of other contaminating substances, with a decolorization, or with a clarification of the solution by the activated carbon filter.
  • the filtration may be performed by techniques known per se and with known equipments usually obtainable from the manufacturer of the filters.
  • the filter may subsequently be washed and the washing solution may be added to the purified more protein or peptide containing solution, to increase the recovery of e.g. a valuable protein or peptide in the treated solution.
  • Another aspect of this invention is a process for purifying a PEG containing solution of a protein or peptide, which process comprises precipitating the protein or peptide with PEG, resolubilizing the precipitated protein or peptide and treating the resolubilised protein or peptide solution with activated carbon.
  • the desired protein or peptide is extracted from the purified protein or peptide solution in a manner known per se, for example, by lyophilization.
  • the PEG content was determined by means of spectrophotometric measurements of the extinction value at 535 nm after formation of a complex of with barium iodide: 1000 ⁇ l of 0.5 M perchloric acid was added to 200 ⁇ l of test solution, in duplo standard solutions (10 - 75 ⁇ g/ml), and blind sample (destilled water) in centrifuge glasses and mixed thoroughly. After at least 15 minutes, the solutions were centrifuged for 15 minutes at 3000 ⁇ m. To 10 800 ⁇ l of the supernatants were added 200 ⁇ l of a 5 % barium chloride solution. For each 30 seconds 100 ⁇ l of iodine-R (0.1 M) was added to all test tubes and mixed thoroughly. After exactly 20 minutes the extinction value at 535 nm were measured towards the blind sample.
  • IgG was measured by radial immuno diffusion towards rabbit anti-human IgG (A-424, 15 DAKO, Denmark).
  • Amount of activated carbon PEG mg/ml IgG, mg/ml added, g/25 ml
  • Example 3 A 20 % solution of human serum albumin (HSA) (ALBUMIN Novo Nordisk, Gentofte, Denmark) was diluted until a concentration of 3% HSA and 2.55 g/litre of PEG-3000 was added. The pH value was adjusted to 5.0. 830 ml of this solution was then filtered through an AKS-4 activated carbon filter from Seitz (Seitz-Filter-Werke, Bad Kreuznach, Germany). The filter area was 143 cm 2 . Samples of 100 ml of the filtered solution was collected and PEG was determined similarly as described in example 1. The data are given in table 3.
  • HSA human serum albumin
  • the concentration of PEG was determined to 2600 ⁇ g/ml. After filtration of 300 ml of the PEG-containing HSA-solution, more than 90 % of PEG was removed, corresponding to an adsorbtion of about 5 mg PEG/cm 2 of filter-area. No adso ⁇ tion of HSA to the filter was observed.
  • PEG was measured similarly as described in example 1 and HSA was measured by rocket immuno electrophoresis towards rabbit anti-Human Albumin (A001 , DAKO, Denmark).
  • Example 4 Resolubilized crude-IgG was obtained from the routine production of Nordimmun ® .
  • the crude-IgG was obtained by precipitation with PEG-3000, and the precipitate, therefore contained residual amounts of PEG-3000.
  • Crude-IgG consist of the human plasma proteins IgG, IgA, IgM, albumin, haptoglobin, and ⁇ 2 -macroglobulin.
  • Samples of resolubilized crude-IgG were then filtered through an In line 90 filter housing (filter area 64 cm 2 ) from CUNO equiped with either Zeta plus activated carbon R 33 SLP (CUNO), or Zeta plus activated carbon R 53 SLP (CUNO).
  • the filters were of pharmaceutical grade.
  • a sample of resolubilized crude-IgG was centrifugated and filtered through one of the filters. 50 ml samples of filtrate were collected and PEG and IgG were determined analogous as described in example 1 (table 4).
  • the concentration of PEG and IgG were 3.91 mg/ml and 86.6 mg/ml before filtration, respectively.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Water Supply & Treatment (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Le polyéthylène est utilisé pour la précipitation fractionnaire des protéines et des peptides. Les fractions de protéines et de peptides obtenues selon les méthodes de fractionnement par PEG contiennent généralement des résidus de PEG. La présente invention concerne un procédé permettant d'éliminer d'une solution de protéines ou de peptides le PEG contaminant, lequel procédé consiste à adsorber le PEG contenu dans la solution sur un carbone activé.
PCT/DK1996/000314 1995-07-13 1996-07-10 Procede pour eliminer le polyethyleneglycol d'une solution de proteines ou de peptides Ceased WO1997003092A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DK0823/95 1995-07-13
DK82395 1995-07-13
DK97095 1995-09-04
DK0970/95 1995-09-04

Publications (1)

Publication Number Publication Date
WO1997003092A1 true WO1997003092A1 (fr) 1997-01-30

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Application Number Title Priority Date Filing Date
PCT/DK1996/000314 Ceased WO1997003092A1 (fr) 1995-07-13 1996-07-10 Procede pour eliminer le polyethyleneglycol d'une solution de proteines ou de peptides

Country Status (1)

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WO (1) WO1997003092A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012062998A1 (fr) 2010-11-08 2012-05-18 Institut National Polytechnique De Lorraine Procede de purification d'un melange visqueux contenant un ou plusieurs contaminants
WO2017045617A1 (fr) * 2015-09-17 2017-03-23 中国人民解放军军事医学科学院野战输血研究所 Procédé de séparation et de purification d'α2-macroglobuline à partir d'un sédiment de constituant iv de cohn
US9910041B2 (en) 2013-07-12 2018-03-06 Emd Millipore Corporation Method of determining virus removal from a sample containing a target protein using activated carbon
EP3643722A1 (fr) * 2012-08-07 2020-04-29 Kyowa Kirin Co., Ltd. Procédé de purification de protéine
US11472836B2 (en) * 2013-02-26 2022-10-18 Emd Millipore Corporation Selective removal of a protein from a mixture of proteins using activated carbon by adjusting solution conditions

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3966702A (en) * 1973-07-16 1976-06-29 Ralston Purina Company Process for the production of oilseed isolates
US4164495A (en) * 1976-04-06 1979-08-14 Nordisk Insulinlaboratorium Method of recovering immunoglobulin using a polyol and an alkanoic acid
US4404285A (en) * 1981-06-24 1983-09-13 Amf Incorporated Process for preparing a zero standard serum

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3966702A (en) * 1973-07-16 1976-06-29 Ralston Purina Company Process for the production of oilseed isolates
US4164495A (en) * 1976-04-06 1979-08-14 Nordisk Insulinlaboratorium Method of recovering immunoglobulin using a polyol and an alkanoic acid
US4404285A (en) * 1981-06-24 1983-09-13 Amf Incorporated Process for preparing a zero standard serum

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Volume 163, 1987, LIONEL MARCUS et al., "Effect of Particle Size of Activated Charcoal on Separation of Triton X-100 from Protein, Liver Cytosol and Lipoxygenase Extracts", pages 112-116. *
CUNO Microfiltration Products, GM 20.1 E, issued 20.07.87, pages 3-4. *
DIALOG INFORMATION SERVICES, File 5, Biosis, Dialog Accession No. 6494608, Biosis Accession No. 85095129, OGINO K. et al., "Removal of Humic Substance Dissolved in Water I"; & J. COLLOID INTERFACE SCI. 121 (1), 1988, 161-169. *
VOX SANG, Volume 23, 1972, A. POLSON et al., "Fractionation of Plasma with Polyethylene Glycol", pages 107-118. *
ZHURNAL MIKROBIOLOGII EPIDEMIOLOGII I IMMUNOBIOLOGII, Volume 4, 1984, IGUDIN L.I. et al., "Polyethylene Glycols and Their Use in Biology and Medicine", pages 17-21. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012062998A1 (fr) 2010-11-08 2012-05-18 Institut National Polytechnique De Lorraine Procede de purification d'un melange visqueux contenant un ou plusieurs contaminants
EP3643722A1 (fr) * 2012-08-07 2020-04-29 Kyowa Kirin Co., Ltd. Procédé de purification de protéine
US11472836B2 (en) * 2013-02-26 2022-10-18 Emd Millipore Corporation Selective removal of a protein from a mixture of proteins using activated carbon by adjusting solution conditions
US9910041B2 (en) 2013-07-12 2018-03-06 Emd Millipore Corporation Method of determining virus removal from a sample containing a target protein using activated carbon
WO2017045617A1 (fr) * 2015-09-17 2017-03-23 中国人民解放军军事医学科学院野战输血研究所 Procédé de séparation et de purification d'α2-macroglobuline à partir d'un sédiment de constituant iv de cohn

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