EP2368123A1 - Contrôle lors de la fabrication, dans un procédé de production d'epo - Google Patents

Contrôle lors de la fabrication, dans un procédé de production d'epo

Info

Publication number
EP2368123A1
EP2368123A1 EP09764266A EP09764266A EP2368123A1 EP 2368123 A1 EP2368123 A1 EP 2368123A1 EP 09764266 A EP09764266 A EP 09764266A EP 09764266 A EP09764266 A EP 09764266A EP 2368123 A1 EP2368123 A1 EP 2368123A1
Authority
EP
European Patent Office
Prior art keywords
erythropoietin
membrane
antibody
bound
acid
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Withdrawn
Application number
EP09764266A
Other languages
German (de)
English (en)
Inventor
Franz-Rudolf Kunz
Florian Glaser
Wolfgang Wienand
Rudolf Hanko
Wilfried Eul
Dietmar Reichert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evonik Operations GmbH
Original Assignee
Evonik Degussa GmbH
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 Evonik Degussa GmbH filed Critical Evonik Degussa GmbH
Publication of EP2368123A1 publication Critical patent/EP2368123A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/746—Erythropoetin

Definitions

  • the present invention relates to a method for the detection of erythropoietin, in particular for Inreakontrolle of culture supernatants of erythropoietin-producing eukaryotic cells in the context of the fermentative production process.
  • the method is characterized in particular by the fact that the isoform composition of the produced erythropoietin can be determined directly using a special isoelectric focusing (IEF).
  • IEF isoelectric focusing
  • the quality of the synthesis crude product can be assessed during or immediately after the end of the fermentation, and the subsequent purification process can thus be controlled. This is particularly advantageous in the context of the use of perfusion reactors.
  • Erythropoietin is a glycoprotein having a molecular weight of about 34 to 39 kDa. It consists of an unbranched polypeptide chain with 165 amino acids and an O-glycosidic (Ser 126) and three N-glycosidic (Asn 24, Asn 38 and Asn 83) bonded sugar side chains (carbohydrate moiety).
  • the side chains are composed of the monosaccharides mannose, galactose, fucose, N-acetylglycosamine, N-acetylgalactosamine and N-acetylneuraminic acid.
  • the erythropoietin can occur in various isoforms.
  • erythropoietin Variance in the molecular weight of erythropoietin is due to the heterogeneity of sugar chains that are terminally linked to neuraminic acid derivatives. Through different lengths and branching of the chains, a multitude of "sugar branches" can be constructed, which effect the expression of all isoforms of an EPO molecule.
  • EPO is mainly produced in the kidneys and stimulates the growth factor as the formation of erythrocytes in the bone marrow.
  • the damaged kidneys produce too little or no EPO at all, with the result that too few erythrocytes emerge from the stem cells of the bone marrow.
  • This renal anemia can be treated by administering EPO in physiological amounts that stimulate the formation of erythrocytes in the bone marrow.
  • the EPO used for administration can either be obtained from human urine or generated by genetic engineering methods. Since EPO is present in trace amounts in the human body, the isolation of EPO from the natural source for therapeutic applications is virtually impossible. Therefore, genetic engineering methods provide the only economic opportunity to produce this substance in larger quantities.
  • erythropoietin takes place by genetic engineering, above all in so-called CHO cells (Chinese hamster ovary), three basically different methods being used for the cultivation of the eukaryotic host cells (described, inter alia, in EP-AO 148) 605 and EP-A-205,564, BioProcess International 2004, 46; Gorenflo et al., Biotech Bioeng., 2002, 80, 438 and WO9501214).
  • Cultivation introduced into the bioreactor. Nutrients are not added until the end of cultivation and cells are removed from the fermenter, only oxygen is supplied. When one or more substrates are consumed, the process is stopped and the products are harvested from the fermentation supernatant.
  • the second known cultivation method is the continuous process in which fresh medium is constantly fed and taken to the same extent fermenter content.
  • it comes to a constant supply of nutrients, while removing or diluting undesirable metabolic products such as the growth-inhibiting substances ammonium and lactate. Therefore, with this method, higher cell densities can be achieved and over be maintained for a comparatively long period of time.
  • a special case of continuous process management is provided by so-called dialysis reactors in which high molecular weight substances such as proteins are retained in the fermenter, while low molecular weight substances such as substrates can be added or the main waste products ammonium and lactate can be removed from the system.
  • dialysis reactors in which high molecular weight substances such as proteins are retained in the fermenter, while low molecular weight substances such as substrates can be added or the main waste products ammonium and lactate can be removed from the system.
  • perfusion reactors in the microbial production of chemical compounds and proteins with various cell restraint systems is well known and also described for EPO.
  • the third possible method is the fed-batch fermentation, in which the culture is started with a fraction of the total fermenter volume and after a short growth phase fresh substrate is added. This enables higher cell densities and longer process times than in the batch process.
  • Another advantage of this method is that the metabolism of the cells can be influenced by the amount of feed, which can lead to a lower production of waste substances. Compared to the continuous process, the product of the cells is accumulated here in the fermenter over a relatively long period of time and thus higher product concentrations are achieved, which facilitates the subsequent work-up.
  • a sample containing erythropoietin is subjected to separation in a polyacrylamide gel, for example, an isoelectric focusing, and the proteins contained are separated by applying an electric field.
  • the electrophoresis is followed by a so-called immunoblot (immunoimprint or immuno-transfer), in which the proteins are transferred to a membrane.
  • immunoblot immunoimprint or immuno-transfer
  • an image of the individual proteins is obtained on the surface of the membrane.
  • the membranes used have the advantage that the proteins are mainly strongly fixed by hydrophobic interactions and the membranes otherwise behave chemically neutral. Because the EPO molecules are located on the membrane surface, they are readily accessible to antibodies that are used to visualize the erythropoietin.
  • isoelectric focusing the isoforms of erythropoietin can be separated.
  • the membrane is first incubated with a monoclonal anti-EPO antibody which specifically binds to all existing EPO molecules. Other proteins do not react with the antibody. Monoclonal antibodies not bound to EPO can be washed off the membrane because the remainder of the membrane surface has previously been blocked with a non-specific protein.
  • the binding of the antibody to EPO is reversible because it is based on non-covalent interactions, and may be e.g. reversed by pH changes.
  • the antibodies bound to erythropoietin are transferred to a second membrane: in an acidic environment, the antibody changes the conformation of its binding domain and upon application of an electric field, the monoclonal antibody dissociates from the EPO molecule and travels in the electric field in the direction Cathode, where it is bound to a second membrane.
  • the EPO molecules as well as other non-specific proteins remain on the first membrane, since the binding to the first membrane is not affected by pH fluctuations. This is a re-image of the EPO band or the EPO bands obtained. However, there are no erythropoietin molecules on the second membrane, but the specific monoclonal antibodies that previously bound to the erythropoietin molecules fixed on the first membrane.
  • the visualization of the antibody band (s) is carried out by a second antibody (secondary antibody) which reacts with the anti-EPO monoclonal antibody.
  • This secondary antibody is coupled to specific enzymes (e.g., alkaline phosphatase or peroxidase) that catalyze substrate conversion that undergoes a color reaction.
  • the need for the second transfer is to reduce the nonspecific signals since the enzyme-labeled secondary antibody can not undergo nonspecific reactions to other components of the sample on the first membrane.
  • a signal amplification and a concomitant increase in sensitivity by multiple formation of the antibody i5 enzyme conjugates to the first antibody is conceivable.
  • a disadvantage of the double blot method is the significantly increased material and time required.
  • Erythropoietin known and familiar to the expert, but it is either the detection of erythropoietin described above the significantly more complex doping
  • the object of the present invention is to provide a simplified and improved method for the detection of erythropoietin, which serves the in-process control, wherein the method makes it possible to characterize the culture supernatants from the EPO fermentation processes so that only selected and suitable evaluated fermentation solutions are supplied to each extensive cleaning processes.
  • the present method should be superior to the prior art methods from the economical point of view.
  • the technical object is achieved by a method for determining the isoform composition of erythropoietin comprising the following steps: a) isoelectric focusing of a sample containing erythropoietin in a gel over a pH range whose lower limit is from 2.5 to 3.5 and the upper limit thereof is from 5 to 8, wherein the erythropoietin-containing sample is from a culture supernatant of erythropoietin-producing eukaryotic cells; b) transferring the proteins contained in the gel and separated on a membrane; c) Detection of erythropoietin bound to the membrane by specific antibodies.
  • first antibodies directed against erythropoietin are bound to the erythropoietin bound to the membrane in step c), whereby the binding of the first antibodies to the erythropoietin bound to the membrane is detected, while the first antibody is detected on the erythropoietin bound erythropoietin bound to the membrane. It is preferred that the detection of the binding of the first antibody to the erythropoietin bound to the membrane by second antibodies, which are directed against the first antibody.
  • first antibodies directed against erythropoietin bind to the erythropoietin bound to the membrane, and second antibodies directed against the first antibodies bind to the first antibodies bound to erythropoietin.
  • the isoform composition of the fermentation supernatants are determined directly, an advantageous and provided a simplified method which can be used to control the fermentation process and to decide on the selection of the culture supernatants to be purified.
  • the method is simplified to such an extent that the detection of the erythropoietin can already take place after a single protein transfer (blot).
  • an enzyme preferably alkaline phosphatase
  • the second antibody which produces a color reaction by catalytically reacting a substrate.
  • two antibody solutions on the membrane for colohmetic EPO detection of which the second antibody contains an alkaline phosphatase, so that then a substrate of this enzyme can be used as a staining reagent.
  • anti-EPO mouse and anti-mouse IgG in combination with BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitrotetrazolium blue).
  • the solution used in the washing steps following incubation of the membrane with the first antibody directed against erythropoietin, an organic acid in an aqueous medium.
  • This unspecifically bound antibodies are removed from the membrane again and possibly still existing free binding sites on the blotting membrane blocked. Overall, this significantly increases the selectivity of the bands.
  • the solution contains 0.1 to 1, 5 wt .-%, preferably 0.5 to 1 wt .-%, particularly preferably 0.6 to 0.8 wt .-% of the organic acid.
  • the organic acid is mono-, di- or tricarboxylic acids (such as, for example, acetic acid, propionic acid, lactic acid, succinic acid, ascorbic acid, adipic acid or citric acid), particularly preferably acetic acid.
  • the incubation with the first antibody solution is carried out in duplicate and a four-step washing procedure is performed therebetween.
  • TBST Tris-Buffered-Saline-Tween
  • TBS Tris-Buffered-Saline
  • Dilute aqueous acetic acid and very particularly preferably dilute aqueous acetic acid in the concentration range between 0.5% and 1% are particularly preferably used for this purpose.
  • the membrane used is a polyvinylidene fluoride membrane.
  • a membrane is used for blotting, which is suitable for binding proteins.
  • Particularly preferred is a microporous polyvinylidene fluoride (PVDF) membrane, and most preferred is the Immobilon-P blotting membrane from Millipore.
  • PVDF microporous polyvinylidene fluoride
  • polyacrylamide gels are used for the isoelectric focusing, which are mounted on an inert Sufo- Ne.
  • IEF utilizes standardized polyacrylamide gels immobilized on an inert support sheet, e.g. made of polyester.
  • gels whose pH gradient is formed by free carrier ampholytes in the electric field are particularly preferred. Very particular preference is given to Blank PreNets from Serva.
  • Gels ampholines are used, which adjust a pH range of pH 3 to pH 6 during the isoelectric focusing in the gel. Very particular preference is given to Servalyt TM 3-6 from Serva.
  • the sample containing erythropoietin is from a culture supernatant of erythropoietin-producing eukaryotic cells maintained in perfusion reactors.
  • the erythropoietin-containing sample is desalted before isoelectric focusing and optionally concentrated.
  • the determination of the isoform composition of the erythropoietin takes place during the fermentation.
  • an ELISA test is used for determining the EPO content of the culture supernatants.
  • the EPO ELISA test from Roche Diagnostics.
  • the reference material used is a commercially available Erypo®
  • the invention further provides a method for the in vitro control of culture supernatants resulting from the fermentation of erythropoietin-producing eukaryotic cells, comprising the following steps:
  • the method is characterized in particular by the fact that the isoform composition of the fermentation supernatants can be determined directly with a special isoelectric focusing (IEF). Together with the data from the EPO content determination (preferably determined by means of ELISA), the EPO quality in the crude product can be directly assessed during or immediately after the end of the fermentation, thus controlling the purification process.
  • IEF isoelectric focusing
  • FIG. 1 shows a blot of various EPO fractions (fractioni to
  • Fraction 5 on a membrane after isoelectric focusing and development.
  • the sample is from a perfusion fermentation of an erythropoietin-producing CHO cell line over a period of 47 days.
  • EPO is produced by fermentation in CHO cells.
  • the fermentation is carried out according to standard methods, as described in the patent and scientific literature for eukaryotic, in particular CHO cells.
  • Cultivation takes place in the perfusion reactor in culture medium which is free from animal components.
  • the harvest takes place continuously over a period of up to 50 days.
  • Each fermentation solution to be analyzed is desalted and concentrated prior to isoelectric focusing.
  • a total EPO content of about 14 mg / L is determined by ELISA test
  • 15 mL of the sample with the Ultrazentrifugationskit and a molecular weight CutOff of 1 OkDa by centrifugation 60 min at 4000 g and 60 min at 14000 g) to 200 .mu.l concentrated and 12 .mu.l of the concentrate with 28 .mu.l ultrapure water and 10 .mu.L Ethanol mixed and stored for 60 min at -20 0 C.
  • the sample solution is centrifuged in a refrigerated centrifuge (20 min, 16100 g, 0 0 C) and the LJ supernatant of the solution for isoelectric focusing used (IEF sample solution).
  • Isoelectric focusing starts with a prefocussing of the
  • the preparation of the Immobilon P blotting membrane (Millipore) is carried out. Thereafter, the gel is blotted onto the membrane under the following conditions: 50 V constant for 50 min in blotting buffer (1X Tris / Glycine with 20% methanol, Bio-Rad). Immediately after protein transfer, three washes are sequentially performed first in methanol and then twice in water for 30 seconds each. The membrane is then placed in blocking solution (5% skim milk powder solution (Bio-Rad) in IxTBS buffer (Bio-Rad)) and incubated at room temperature for 60 min with gentle shaking.
  • blocking solution 5% skim milk powder solution (Bio-Rad) in IxTBS buffer (Bio-Rad)
  • the membrane is treated with the second antibody solution (1% BSA (Sigma) in 30 ml of IXTBS buffer (Bio-Rad) with 60 ⁇ l of 500 mM sodium azide solution of 35 ⁇ l of anti-mouse IgG (Sigma) - lo is added).
  • the second antibody solution 1% BSA (Sigma) in 30 ml of IXTBS buffer (Bio-Rad) with 60 ⁇ l of 500 mM sodium azide solution of 35 ⁇ l of anti-mouse IgG (Sigma) - lo is added).
  • EPO ELISA test from Roche Diagnostics GmbH (photometric enzyme-linked immunosorbent assay for the quantitative in vitro determination of erythropoietin in human serum / plasma for research purposes using antibody precoated microtiter plates).
  • EPO contents are determined in the following ranges: Fraction 1 (fermentation until day 5): approx. 80 mg / L

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé de détermination de la composition d'isoformes de l'érythropoïétine, comprenant les étapes suivantes : a) focalisation isoélectrique d'un échantillon contenant de l'érytropoïétine dans un gel, dans un domaine de pH dont la limite inférieure se situe entre 2,5 et 3,5 et dont la limite supérieure se situe entre 5 et 8, l'échantillon contenant l'érythropoïétine provenant d'un surnageant de culture de cellules eucaryotiques produisant de l'érythropoïétine; b) transfert, sur une membrane, de la protéine contenue dans le gel et séparée; c) caractérisation de l'érytropoïétine liée à la membrane, par des anticorps spécifiques; et mise en oeuvre d'un procédé de contrôle, lors de la fabrication, de surnageants de culture de cellules eucaryotiques produisant de l'érythropoïétine, dans le cadre d'un processus de production par fermentation.
EP09764266A 2008-12-16 2009-12-07 Contrôle lors de la fabrication, dans un procédé de production d'epo Withdrawn EP2368123A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008054716A DE102008054716A1 (de) 2008-12-16 2008-12-16 Inprozesskontrolle in einem Verfahren zur Herstellung von EPO
PCT/EP2009/066517 WO2010076125A1 (fr) 2008-12-16 2009-12-07 Contrôle lors de la fabrication, dans un procédé de production d'epo

Publications (1)

Publication Number Publication Date
EP2368123A1 true EP2368123A1 (fr) 2011-09-28

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Family Applications (1)

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EP09764266A Withdrawn EP2368123A1 (fr) 2008-12-16 2009-12-07 Contrôle lors de la fabrication, dans un procédé de production d'epo

Country Status (10)

Country Link
US (1) US20120021441A1 (fr)
EP (1) EP2368123A1 (fr)
JP (1) JP2012512406A (fr)
CN (1) CN102317791A (fr)
BR (1) BRPI0923039A2 (fr)
CA (1) CA2747320A1 (fr)
DE (1) DE102008054716A1 (fr)
IL (1) IL213544A0 (fr)
SG (1) SG172203A1 (fr)
WO (1) WO2010076125A1 (fr)

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* Cited by examiner, † Cited by third party
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US9201548B2 (en) * 2012-05-03 2015-12-01 Texas Instruments Incorporated Material-discerning proximity sensing
CN110196336A (zh) * 2019-06-04 2019-09-03 迪瑞医疗科技股份有限公司 促红细胞生成素化学发光免疫检测试剂盒及其制备方法

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WO2003080852A1 (fr) * 2002-03-26 2003-10-02 Lek Pharmaceutical And Chemical Company D.D. Procede de preparation du profil de glyco-isoformesd'erythropoietine recherche

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IL77081A (en) 1984-12-04 1999-10-28 Genetics Inst AND sequence encoding human erythropoietin, a process for its preparation and a pharmacological preparation of human erythropoietin
IL79176A (en) 1985-06-20 1992-06-21 Kirin Amgen Inc Process for the recovery of erythropoietin from a fluid
US4954437A (en) 1986-09-15 1990-09-04 Integrated Genetics, Inc. Cell encoding recombinant human erythropoietin
KR100263845B1 (ko) 1989-10-13 2000-08-16 스튜어트 엘.왓트 에리트로포이에틴 동형체와 그의 제조방법 및 그를 포함하는제약학적 조성물
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WO2003045996A1 (fr) * 2001-11-28 2003-06-05 Sandoz Gmbh Purification chromatographique d'erythropoietine humaine recombinee
WO2003080852A1 (fr) * 2002-03-26 2003-10-02 Lek Pharmaceutical And Chemical Company D.D. Procede de preparation du profil de glyco-isoformesd'erythropoietine recherche

Non-Patent Citations (1)

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Title
See also references of WO2010076125A1 *

Also Published As

Publication number Publication date
WO2010076125A1 (fr) 2010-07-08
BRPI0923039A2 (pt) 2015-12-15
IL213544A0 (en) 2011-07-31
JP2012512406A (ja) 2012-05-31
US20120021441A1 (en) 2012-01-26
CA2747320A1 (fr) 2010-07-08
DE102008054716A1 (de) 2010-06-17
CN102317791A (zh) 2012-01-11
SG172203A1 (en) 2011-07-28

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Effective date: 20140305