WO2011015920A2 - Procédé hautement efficace de purification et de production de trastuzumab de recombinaison - Google Patents

Procédé hautement efficace de purification et de production de trastuzumab de recombinaison Download PDF

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Publication number
WO2011015920A2
WO2011015920A2 PCT/IB2010/001897 IB2010001897W WO2011015920A2 WO 2011015920 A2 WO2011015920 A2 WO 2011015920A2 IB 2010001897 W IB2010001897 W IB 2010001897W WO 2011015920 A2 WO2011015920 A2 WO 2011015920A2
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WO
WIPO (PCT)
Prior art keywords
supernatant
recovery
monoclonal antibody
trastuzumab
recombinant
Prior art date
Application number
PCT/IB2010/001897
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English (en)
Other versions
WO2011015920A3 (fr
Inventor
Villoo Morawala Patell
Sami N. Guzder
Sunit Maity
Sunil Shekar
Original Assignee
Avesthagen Limited
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 Avesthagen Limited filed Critical Avesthagen Limited
Publication of WO2011015920A2 publication Critical patent/WO2011015920A2/fr
Publication of WO2011015920A3 publication Critical patent/WO2011015920A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the present invention relates to use of novel process of recovery of Recombinant Trastuzumab (Herceptin), a Monoclonal Antibody to human epidermal growth factor receptor-2 (HER-2), in biologically active form from fluids, includes but not limited to mammalian host cell culture supernatants by performing fermentation and chromatographic procedure separately and jointly.
  • the present invention principally pertains to a novel mode of recovering high yield of purified recombinant Trastuzumab by performing different chromatographic techniques, which can be very useful for the pharmaceutical industries as it furnishes the rapid and efficient recovery of Recombinant Trastuzumab.
  • Trastuzumab is a recombinant humanized IgG Ik monoclonal antibody glycoprotein that binds specifically to human epidermal growth factor receptor-2 (HER-2). Herceptin is designed to target and block the function of HER-2 protein over expression.
  • the antibody, Trastuzumab contains primarily human constant framework regions with the complementarity-determining regions (CDR) from a murine antibody with specificity for human HER-2.
  • CDR complementarity-determining regions
  • the humanized antibody against HER-2, Trastuzumab having an approximate molecular weight of 155 kilo-daltons is produced by a mammalian cell (Chinese Hamster Ovary) [CHO] suspension culture in a nutrient medium containing the antibiotic gentamicin. Gentamicin is not detectable in the final product.
  • Trastuzumab is packaged as a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous (IV) administration
  • Cancerous breast tissue cells that over-express the HER-2 gene produce extra protein receptors on the cell surface.
  • the higher density of receptors triggers the cell to divide and multiply at an accelerated rate, thus contributing to tumour growth.
  • Trastuzumab binds to numerous HER-2 receptor sites found on the cell surface, blocking the receptor sites and possibly preventing further growth by interrupting the growth signal.
  • US Patent Application number- 11/400,638 encompasses Trastuzumab as a single agent is indicated for the treatment of patients with metastatic breast cancer whose tumors over-express the HER-2 protein and who have received one or more chemotherapy regimens for their metastatic disease.
  • Trastuzumab in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors over-express the HER-2 protein and who have not received chemotherapy for their metastatic disease. This antibody should only be used in patients whose tumors have HER-2 protein over-expression.
  • the present invention principally relates to a new process of recovering higher yield of purified Recombinant Trastuzumab (Herceptin), a Monoclonal Antibody to human epidermal growth factor receptor-2 (HER-2) by performing bio- analytical techniques which includes but not limited to chromatographic techniques.
  • the recovery by the novel process can be very useful in commercial scale, especially in pharmaceutical industries as it furnishes the rapid and efficient recovery of Recombinant Trastuzumab.
  • the principal object of the present invention is to use of novel process of production and recovery by performing fermentation and chromatographic procedures for rapid and efficient recovery of recombinant Trastuzumab (Herceptin), a monoclonal antibody to human epidermal growth factor receptor-2 (HER-2), from cell culture supernatant.
  • the present invention leads to a novel process for recovering the maximum yield of the recombinant Trastuzumab (Herceptin).
  • the present invention relates to the use of novel process of recovery of Recombinant Trastuzumab (Herceptin), a Monoclonal Antibody to human epidermal growth factor receptor-2 (HER-2) by performing chromatographic procedures, separately and jointly, in biologically active form from fluids, especially mammalian host cell culture supernatants.
  • Herceptin Recombinant Trastuzumab
  • HER-2 Monoclonal Antibody to human epidermal growth factor receptor-2
  • the present invention also relates to the use of novel process of recovery by performing fermentation for the over-expression of Recombinant Trastuzumab (Herceptin), a Monoclonal Antibody to human epidermal growth factor receptor-2 (HER-2), protein in Chinese Hamster Ovary (CHO) cells.
  • Recombinant Trastuzumab Herceptin
  • HER-2 Monoclonal Antibody to human epidermal growth factor receptor-2
  • CHO Chinese Hamster Ovary
  • This present invention relates to the rapid, efficient and effective process of recovery of recombinant Monoclonal antibody to HER-2 from cell culture supernatant from Cell culture fluid by means of chromatography's techniques, especially from Affinity chromatography.
  • affinity chromatography is performed for capture of recombinant Monoclonal antibody. This process of separation involves in selective binding of the desired compound to specific affinity resin and then elution with elution buffer. Supernatants from culture are clarified before treatment of chromatography.
  • Monoclonal Antibody to HER-2 containing eluent fractions are enriched with biologically active material, but they will be subjected to further processing by Ion exchange chromatographic step.
  • the present invention also relates to the recovery procedure of recombinant Monoclonal Antibody to HER-2 involving serial application of different chromatographic techniques as mentioned previously, which can be really useful in the pharmaceutical preparations while doing antibodies purification. All different steps, conditions and compositions are disclosed in the present invention.
  • Clarification of the cell culture harvest was carried out by using a cellulose disposable filter with 650 - 1000 cm 2 effective filtration area and with an operating pressure of not more than 30 psi. The filtrate was checked for turbidity and target protein content. Affinity chromatography was used in binding and elution mode with column of 32 mm diameter for capturing; with Tris buffer pH 7.2 - 7.6 as equilibration buffer. After the sample is loaded on to the column, it is washed with equilibration buffer followed by 50 niM Tris-Cl, then with second wash with 50 mM Tris-Cl containing 100-400 mM NaCl pH 7-8 buffer solution.
  • the protein of interest was eluted with citrate buffer pH 3.0-3.8 (Fig 1).
  • the eluate was hold for 45 - 60 min at acidic pH at room temperature for virus inactivation and later neutralized.
  • the Protein A eluate fraction were pooled.
  • Anion exchange chromatography in negative binding mode was carried out at an operational flow rate of 140 cm/hr.
  • the column was equilibrated with Tris buffer pH 6.8 - 7.2. Protein of interest is collected in flow through. This step was used for the removal of process related impurities like leachate protein A, host cell DNA and host cell protein (Fig 2). Thereafter, the flow through was filtered for virus removal using viral removal filter having an effective filtration area of 0.01 m 2 .
  • the filtrate was buffer exchanged using a 50-kDa TFF membrane.
  • the buffer used for the diafiltration process is Tris buffer pH 6.8-7.2.
  • Cation exchange chromatography was carried out with the diafiltered protein solution after equilibrating the column with Tris buffer pH 6.8-7.2.
  • the protein of interest was eluted with elution buffer using NaCl salt gradient. This step was used for the removal of process related impurities like host cell DNA and host cell protein (Fig 3).
  • the eluate was buffer exchanged and concentrated using a 50-kDa TFF membrane at a Trans Membrane Pressure (TMP) of 5-10 psi.
  • TMP Trans Membrane Pressure
  • the buffer exchanged protein solution was filtered using 0.2 ⁇ m filter.
  • the drug substance was characterized as per the in house developed specifications.
  • the Drug Substance (Active Pharmaceutical Ingredient) was formulated using the ingredients mentioned below. They are 51 mM Sodium Phosphate buffer pH 6.2, 400 mg ⁇ , ⁇ -trehalose dihydrate, 9.9 mg L-histidine HCl, 6.4 mg L-histidine, and 1.8 mg polysorbate 20, USP, yields a solution containing 20-mg/mL trastuzumab.
  • the formulated material was characterized as per the specifications set by product development specification so as to meet the physico-chemical parameter comparable with the originator.
  • a 10% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS PAGE) under reducing condition was studied for the sample derived from the PAGE showed a clear corresponding band with Reference Medical Product (RMP) reflecting the fact that there is a comparable purity almost to homogeneity and integrity in terms of stability (Fig 4).
  • RMP Reference Medical Product
  • Protein A High Performance Liquid Chromatography (HPLC) profile carried out during this step with test molecule showed a clear single peak, as its retention time is very much comparable with that of the RMP. It also suggests that the test molecule in question is very specific and pure with respect to the RMP.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne l'utilisation d'un nouveau procédé de production et de récupération d'un anticorps monoclonal de recombinaison dirigé contre HER-2, sous une forme biologiquement active à partir de fluides, lequel procédé comprend, entre autres, des supernageants de culture provenant de cellules hôtes mammifères par mise en oeuvre d'une fermentation et d'une procédure chromatographique, séparément ou conjointement. La récupération selon le nouveau procédé peut être très utile dans l'industrie pharmaceutique car elle permet d'obtenir une récupération rapide et efficace du Trastuzumab de recombinaison.
PCT/IB2010/001897 2009-08-03 2010-08-02 Procédé hautement efficace de purification et de production de trastuzumab de recombinaison WO2011015920A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1841CH2009 2009-08-03
IN1841/CHE/2009 2009-08-03

Publications (2)

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WO2011015920A2 true WO2011015920A2 (fr) 2011-02-10
WO2011015920A3 WO2011015920A3 (fr) 2011-05-12

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013189544A1 (fr) * 2012-06-21 2013-12-27 Synthon B.V. Procédé de purification d'un anticorps
US20140017265A1 (en) * 2012-07-05 2014-01-16 Mersana Therapeutics, Inc. Terminally Modified Polymers and Conjugates Thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005016968A2 (fr) * 2003-07-28 2005-02-24 Genentech, Inc. Reduction de la lixiviation de la proteine a lors de la chromatographie d'affinite sur la proteine a
WO2005081711A2 (fr) * 2003-11-06 2005-09-09 Seattle Genetics, Inc. Composes de monomethylvaline capables de conjugaison aux ligands
WO2009108637A1 (fr) * 2008-02-25 2009-09-03 Prometheus Laboratories, Inc. Sélection d’un médicament pour le traitement du cancer du sein à partir des matrices d’anticorps

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005016968A2 (fr) * 2003-07-28 2005-02-24 Genentech, Inc. Reduction de la lixiviation de la proteine a lors de la chromatographie d'affinite sur la proteine a
WO2005081711A2 (fr) * 2003-11-06 2005-09-09 Seattle Genetics, Inc. Composes de monomethylvaline capables de conjugaison aux ligands
WO2009108637A1 (fr) * 2008-02-25 2009-09-03 Prometheus Laboratories, Inc. Sélection d’un médicament pour le traitement du cancer du sein à partir des matrices d’anticorps

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PAUL CARTER ET AL.: 'Humanization of an anti-p185HER2 antibody for human cancer therapy' PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE OF THE USA vol. 89, pages 4285 - 4289 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013189544A1 (fr) * 2012-06-21 2013-12-27 Synthon B.V. Procédé de purification d'un anticorps
KR20150027807A (ko) * 2012-06-21 2015-03-12 신톤 바이오파머슈티칼즈 비.브이. 항체의 정제 방법
US9845338B2 (en) 2012-06-21 2017-12-19 Synthon Biopharmaceuticals Bv Method of purifying an antibody
KR101941742B1 (ko) 2012-06-21 2019-01-23 신톤 바이오파머슈티칼즈 비.브이. 항체의 정제 방법
US20140017265A1 (en) * 2012-07-05 2014-01-16 Mersana Therapeutics, Inc. Terminally Modified Polymers and Conjugates Thereof

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WO2011015920A3 (fr) 2011-05-12

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