WO2011015921A1 - Procédé hautement efficace de purification et de production de cetuximab de recombinaison - Google Patents

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

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Publication number
WO2011015921A1
WO2011015921A1 PCT/IB2010/001898 IB2010001898W WO2011015921A1 WO 2011015921 A1 WO2011015921 A1 WO 2011015921A1 IB 2010001898 W IB2010001898 W IB 2010001898W WO 2011015921 A1 WO2011015921 A1 WO 2011015921A1
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WO
WIPO (PCT)
Prior art keywords
supernatant
egfr
cetuximab
recombinant
recovery
Prior art date
Application number
PCT/IB2010/001898
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English (en)
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 WO2011015921A1 publication Critical patent/WO2011015921A1/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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production

Definitions

  • the present invention relates to the use of novel fermentation and chromatographic procedures separately and jointly for the production of Recombinant Cetuximab, a monoclonal antibody to human epidermal growth factor receptor (EGFR), in biologically active form from fluids, includes but not limited to mammalian host cell culture supernatants.
  • the present invention pertains principally to a novel mode of recovering high yield of purified recombinant Cetuximab by performing different chromatographic techniques, which can be very useful for the pharmaceutical industries as it furnishes the rapid and efficient recovery of Recombinant Cetuximab.
  • Cetuximab is composed of the Fv-regions of a murine anti-EGFR antibody with human IgGl heavy and kappa light chain constant regions.
  • the antibody has an approximate molecular weight of 152 kDa and is produced in mammalian (murine myeloma) cell culture.
  • Cetuximab is supplied as a sterile, clear, colorless liquid of pH 7.0 to 7.4 for IV use.
  • the EGFR is a transmembrane glycoprotein that is a member of a subfamily of type 1 -receptor tyrosine kinases including EGFR (HER-I), HER-2, HER-3 and HER-4.
  • EGFR is constitutively expressed in many normal epithelial tissues, including the skin and hair follicle. Over-expression of EGFR is detected in many human cancers including those of the colon and the rectum.
  • Cetuximab binds specifically to the epidermal growth factor receptor (EGFR, HER-I, c-ErbB-1) on both normal and tumor cells, and competitively inhibits the binding of EGF and other ligands. Binding of Cetuximab to EGFR blocks phosphorylation and activation of receptor-associated kinases, resulting in inhibition of cell growth, induction of apoptosis and decreased MMP and VEGF production.
  • EGFR epidermal growth factor receptor
  • Cetuximab used in combination with irinotecan, is indicated for the treatment of EGFR-expressing, metastatic colorectal carcinoma in patients who are refractory to irinotecan-based chemotherapy.
  • This antibody administered as a single agent, is indicated for the treatment of EGFR-expressing, metastatic colorectal carcinoma in patients who are intolerant to irinotecan-based chemotherapy.
  • Cetuximab has also been approved for treatment with concomitant radiotherapy in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN).
  • the present invention primarily relates to a new process of recovering higher yield of Recombinant Cetuximab, a monoclonal antibody to human epidermal growth factor receptor (EGFR), by performing bio-analytical techniques especially different chromatographic techniques.
  • EGFR human epidermal growth factor receptor
  • the principal object of the present invention is to use novel fermentation and chromatographic procedures for rapid and efficient recovery of Recombinant Cetuximab, a monoclonal antibody to human epidermal growth factor receptor (EGFR), from cell culture supernatant.
  • Cetuximab a monoclonal antibody to human epidermal growth factor receptor (EGFR)
  • the present invention relates to the use of novel chromatographic procedures separately and jointly for the production of Recombinant Cetuximab, a monoclonal antibody to human epidermal growth factor receptor (EGFR), protein, in biologically active form from fluids, especially mammalian host cell culture supernatants.
  • Cetuximab a monoclonal antibody to human epidermal growth factor receptor (EGFR), protein
  • the present invention also relates to the use of novel fermentation process for the overexpression of Recombinant Cetuximab, a monoclonal antibody to human epidermal growth factor receptor (EGFR), protein in Chinese Hamster Ovary CHO cells.
  • Cetuximab a monoclonal antibody to human epidermal growth factor receptor (EGFR)
  • EGFR epidermal growth factor receptor
  • Figure 1 Process chromatogram of affinity chromatography: Chromatographic profile of the first step of recovery of the target protein from the affinity chromatography. This is the purification chromatogram from a supernatant containing Cetuximab as explained in example 1 (Supernatant derived from a harvested media containing Cetuximab after clarification). The blue line represents the UV absorbance at 280nm. Majority of the other protein comes out in the flow through as can be observed in the figure.
  • FIG. 2 Process chromatogram of anion chromatography: Chromatographic profile of the second step of recovery of the target protein from the anion exchange chromatography.
  • This figure shows a chromatogram of purification from the eluent of the affinity chromatography as explained in the example 1 (Start material on to this column was the buffer exchanged neutralized elute containing the Cetuximab from the affinity column). It is clear from the figure that the majority of the load has come out in the flow through mode. A minor peak in the end may be very minute amount of the Cetuxiumab has bound, as it is clear from the recovery that the majority of it has come out in follow-through.
  • FIG 3 Process chromatogram of cation chromatography: Chromatographic profile of the final step of recovery of the target protein from the cation exchange chromatography. This is a chromatogram from step 3 of a process according to the invention and it is more specifically a polishing step as explained in the example 1.
  • Figure 4 Electrophoretic pattern of Drug substance: showing comparable molecular weight with RMP where Lane No. 1: Molecular weight Marker, Lane No. 2: RMP (Reference Medicinal Product) and Lane No. 3: Formulated Drug Substance. Electrophoretic profile reveals the purity and identity of the target protein with the Originator. The electrophoretic diagram depicts the stability of the drug substance and also the purity of the process close to homogeneity and comparability of the drug substance with that of the Originator. (RMP: Reference Medicinal Product -Erbitux®)
  • Figure 5 Western blot analysis: Test material and RMP are transferred on to the membrane and probed with Goat anti human IgG ALP Conjugate ( 1 : 1000 dilutions) Antibody. Drug substance showing clear corresponding signal with with RMP where Lane No. 1: Molecular weight Marker, Lane No. 2: RMP and Lane No. 3: Formulated Drug Substance. Showing the signal indicating the specificity and the identity with respect to the originator molecule.
  • FIG. 6 Protein A HPLC Profile of Drug substance showing comparable molecular weight with RMP: This profile shows similar retention time in the protein A column. This is one of the orthogonal methods for estimation of target protein (recombinant Trastuzumab).
  • This figure is an analytical protein A column chromatogram of the purified drug substance containing inflximab as it was compared with that of the originator. The extra peak seen at the beginning of the chromatograms is from the buffer as it can be observed in both the chromatogram (innovators as well as the Cetuximab purified product). This method not only gives the protein estimation but also the purity of the product with respect to the innovator's molecule.
  • RMP Reference Medicinal Product -Erbitux®
  • This present invention relates to the rapid, efficient and effective recovery of Recombinant Monoclonal antibody to EGFR from cell culture supernatant from Cell culture fluid by means chromatography's techniques, especially from Affinity chromatography.
  • affinity chromatography is performed for capture of recombinant Monoclonal antibody to EGFR. This process of separation involves in selective binding of the desired compound to specific affinity resin and then elution with elution buffer. The culture supernatants are clarified before chromatographic treatment.
  • Monoclonal antibodies to EGFR containing eluent fractions are enriched with biologically active material, but they will be subjected to further processing by Ion exchange chromatographic step. These chromatographic processes are used for removal of process related impurities like host cell protein and host cell DNA.
  • the present invention also relates to the recombinant Monoclonal antibody to EGFR recovery procedure involving serial application of different chromatographic techniques as mentioned previously. 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 mM Tris-Cl, 250 mM NaCl pH 7.4 buffer solutions. The protein of interest was eluted with citrate buffer (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 of Runl and Run 2 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 specifications.
  • the Drug Substance (Active Pharmaceutical Ingredient) was formulated using formulation buffer solution containing 8.48 mg/mL sodium chloride, 1.88 mg/mL sodium phosphate dibasic heptahydrate, 0.41 mg/mL sodium phosphate monobasic monohydrate, yielding a concentration of 2 mg/mL of cetuximab.
  • 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
  • Western blot analysis clearly showed a clear corresponding band with RMP It is clearly indicating that the product specificity and identity with respect to the originator product (Fig 5).

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

Abstract

La présente invention concerne l'utilisation de nouveaux procédés de fermentation et chromatographiques séparément ou conjointement pour la production et la récupération d'un anticorps monoclonal de recombinaison dirigé contre EGFR, sous une forme biologiquement active à partir de fluides, comprenant entre autres des supernageants provenant de culture de cellules hôtes mammifères. La récupération selon le nouveau procédé peut être très utile dans l'industrie pharmaceutique.
PCT/IB2010/001898 2009-08-03 2010-08-02 Procédé hautement efficace de purification et de production de cetuximab de recombinaison WO2011015921A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1843CH2009 2009-08-03
IN1843/CHE/2009 2009-08-03

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WO2011015921A1 true WO2011015921A1 (fr) 2011-02-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8513390B2 (en) 2009-01-12 2013-08-20 Cytomx Therapeutics, Inc. Modified antibody compositions, methods of making and using thereof
CN107793469A (zh) * 2017-10-16 2018-03-13 上海药明生物技术有限公司 一种去除宿主细胞蛋白含量的亲和纯化工艺

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056847A2 (fr) * 2002-12-16 2004-07-08 Genmab A/S Anticorps monoclonaux humains contre le recepteur de facteur de croissance epidermique (egfr)
US20080255027A1 (en) * 2006-12-21 2008-10-16 Wilson Moya Purification of proteins
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
WO2004056847A2 (fr) * 2002-12-16 2004-07-08 Genmab A/S Anticorps monoclonaux humains contre le recepteur de facteur de croissance epidermique (egfr)
US20080255027A1 (en) * 2006-12-21 2008-10-16 Wilson Moya Purification of proteins
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

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8513390B2 (en) 2009-01-12 2013-08-20 Cytomx Therapeutics, Inc. Modified antibody compositions, methods of making and using thereof
US8563269B2 (en) 2009-01-12 2013-10-22 Cytomx Therapeutics, Inc. Modified antibody compositions, methods of making and using thereof
US9453078B2 (en) 2009-01-12 2016-09-27 Cytomx Therapeutics, Inc. Modified antibody compositions, methods of making and using thereof
US10059762B2 (en) 2009-01-12 2018-08-28 Cytomx Therapeutics, Inc. Anti-EGFR activatable antibodies
US10118961B2 (en) 2009-01-12 2018-11-06 Cytomx Therapeutics, Inc. Modified antibody containing the cleavable peptide with the amino acid sequence TGRGPSWV
US10875913B2 (en) 2009-01-12 2020-12-29 Cytomx Therapeutics, Inc. Methods of treatment using activatable anti-EGFR antibodies
CN107793469A (zh) * 2017-10-16 2018-03-13 上海药明生物技术有限公司 一种去除宿主细胞蛋白含量的亲和纯化工艺
CN107793469B (zh) * 2017-10-16 2021-07-30 上海药明生物技术有限公司 一种去除宿主细胞蛋白含量的亲和纯化工艺

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