FIELD OF THE INVENTION

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.

BACKGROUND AND PRIOR ART OF THE INVENTION

In past, various media and methods were used for the cell culture manufacturing of recombinant glycoprotein or monoclonal antibody. Commonly employed bioreactor process includes; batch, semi fed-batch, fed-batch, perfusion and continuous fermentation. The ever-increasing demand of monoclonal antibody and other recombinant proteins in properly glycosyalted forms have increased the prospects of cell culture process development. In addition the regulatory hurdles imposed on the serum containing process has led to the development of cell culture process in a completely chemically defined environment.

Numerous techniques have in the past been applied in preparative separations of bio¬chemically significant materials. Commonly employed preparative separatory techniques include: ultrafiltration, column electrofocusing, flatbed electrofocusing, gel filtration, electrophoresis, isotachophoresis and various forms of chromatography. Among the commonly employed chromatoghraphic techniques are Ion exchange and adsorption chromatography. The extensive application of recombinant methodologies to large-scale purification and production of eukaryotic protein has increased the prospect of obtaining the molecule in required quantity using simplified purification procedures.

Cetuximab is a recombinant, human/mouse chimeric monoclonal antibody that binds specifically to the extracellular domain of the human epidermal growth factor receptor (EGFR). 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-1), 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-1, ciErbB-l) 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.

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. Moreover, 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. The novel process described in instant invention will be useful for rapid and efficient recovery of Recombinant Cetuximab on commercial scale in pharmaceutical industries.

OBJECTIVES OF THE INVENTION

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.

SUMMARY OF THE INVENTION

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.

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.

DETAILED DESCRIPTION OF THE INVENTION

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.

Figure 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.

Figure 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. (RMP: Reference Medicinal Product -Erbitux®)

Figure 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®)

DETAILED DESCRIPTION OF THE INVENTION

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. Generally, foremost, 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.

Example 1:

Clarification of the cell culture harvest was carried out by using a cellulose disposable
filter with 650 - 1000 cm2 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 m2. 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. The buffer exchanged protein solution was filtered using 0.2um 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.

Example 2:

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). 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). Protein A HPLC profile carried out during this step showed 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. This technique is used as one of the orthogonal methods for estimation of target protein (Fig 6).

CLAIMS

We claim:

1. A process for the recovery and purification of recombinant Monoclonal antibody to to EGFR comprising steps of: contacting culture supernatant(s) with resin(s) for selective adsorption of compound(s); eluting the adsorbed compound and subjecting the enriched product to series of separation techniques.

2. The process as claimed in claim 1, where elution of adsorbed compound is performed by affinity chromatography.

3. The process as claimed in claim 1, where monoclonal antibody to to EGFR containing eluent fractions are purified using ion exchange chromatography.

4. The process as claimed in claim 1, wherein supernatant is obtained from cell fermentation.

5. The process as claimed in claim 1, wherein said supernatant is mammalian host cell culture supernatant.

6. The process as claimed in claim 1, wherein said supernatant is cell culture-derived fluid.

7. The process as claimed in claim 1, wherein said supernatant is mammalian cell culture derived fluid

8. The process as claimed in claim 1, wherein the process buffer 20-100 mM Tris buffer pH 6.8-7.8 with 100-400 mM NaCl is used for the recovery of target protein from culture supernatant.

9. The process as claimed in claim 1, wherein the process buffer is particularly 40-60 mM Tris buffer pH 7.0-7.6 with 200-300mM NaCl is used for the recovery of target protein from culture supernatant

10. The process as claimed in claim 1, wherein said culture supernatant(s) are concentrated and clarified before contacting resins.

11. The process as claimed in claim 1, where eluent is held for 45 - 60 min at acidic pH at room temperature for virus inactivation.

12. The process as claimed in claim 1, where recombinant Monoclonal antibody to EGFR recovered and purified is Cetuximab.