The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:

FIELD OF THE INVENTION

The present invention relates generally to the use of novel fermentation and chromatographic procedures separately and jointly for the production of recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein, in biologically active form from fluids, especially mammalian host cell culture supernatants.

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 biochemically 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.

ENBREL (Etanercept) is a recombinant fusion protein comprising the extracellular domain of the human tumor necrosis factor receptor superfamily, member IB (p75) and the Fc domain of human IgGl. ENBREL is an important scientific advance that in many people has been shown to reduce the signs and symptoms of rheumatoid arthritis, polyarticular-course juvenile rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis. TNF is a naturally occurring cytokine that is' involved in normal inflammatory and immune responses. It plays an important role in the inflammatory processes of rheumatoid arthritis (RA), polyarticular-course juvenile rheumatoid arthritis (JRA) and the resulting joint pathology. Elevated levels of TNF are found in the synovial fluid of RA patients.

OBJECTIVES OF THE INVENTION

The main object of the present invention is to use novel fermentation and chromatographic procedures for rapid and efficient recovery of recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein from cell culture supernatant

SUMMARY OF THE INVENTION

The present invention relates to the use of novel fermentation process for the overexpression of recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) - Human IgG Fc fusion protein in CHO cells.

The present invention also relates to the use of novel chromatographic procedures separately and jointly for the production of recombinant soluble Tumour Necrosis Factor Alfa receptor (TNFR) -Human IgG Fc fusion protein, in biologically active form from fluids, especially mammalian host cell culture supernatants.

LIST OF FIGURES

Figure 1: Nutrient consumption and lactate accumulation profile during fermentation run

Figure 2: Cell growth and viability profile during fermentation run

Figure 3: Expression profile of protein during fermentation run

Figure 4: Process chromatogram after affinity chromatography

Figure 5: Process chromatogram after Cation Exchange chromatography

Figure 6: Process chromatogram after Anion Exchange chromatography.

Figure 7: Electrophoretic pattern of Drug substance showing comparable molecular weight with RMP where Lane No. 1: Molecular weight Marker, Lane No. 2 : RMP and Lane No. 3 :
Formulated Drug Substance

Figure 8: Electrophoretic pattern of Drug substance showing comparable molecular weight with RMP where Lane No. 1: Molecular weight Marker, Lane No. 2 : RMP and Lane No. 3 :
Formulated Drug Substance

Figure 9: Western Blot Analysis of Drug substance showing comparable immuno-specificity
between RMP and drug substance where Lane No. 1: Molecular weight Marker, Lane No. 2: RMP
and Lane No. 3: Formulated Drug Substance

Figure 10: Isoelectric focussing of Drug substance when compared to RMP had demonstrated a
comparable pi range and charge variant profile where Lane No. 1: RMP and Lane No. 2:
Formulated Drug Substance

Figure 11: Comparable 2D Gel electrophoresis pattern of Drug substance with that of RMP

Figure 12: Protein A HPLC profile of Drug substance showed comparable retention time with that of RMP

Figure 13: Comparable hydrophobicity profile between Drug substance and RMP as depicted by
Reverse Phase HPLC profile

Figure 14: Size exclusion HPLC profile of Drug substance showed comparable similar
hydrodynamic radius

Figure 15: MALDI-TOF analysis of the intact molecule has determined the molecular mass of
RMP and Drug substance to be -145 kDa

Figure 16: The deconvolved mass spectral profile obtained by MALDI-TOF analysis of the tryptic
digests demonstrated a high degree of similarity between RMP & Drug substance

Figure 17: HPLC-based tryptic peptide mapping analysis has shown identical profiles between
RMP & Drug substance

Fig 18: Glycan analysis depicted above has demonstrated a comparable Glycan profile thus
indicating a high-degree of similarity between RMP & Drug substance

Figure 19: UV spectroscopy analysis has revealed a comparable primary structure profile between
RMP & Drug substance

Figure 20: CD spectroscopy analysis has revealed a comparable (a) secondary and (b) tertiary
structure profile between RMP & Drug substance

Figure 21: The in vitro cytotoxic effect of TNF has been extensively demonstrated using the
reporter cell line L929 that has been derived from a murine fibrosarcoma. The assay read outs
indicated that the degree of TNF-alpha induced cytotoxicity observed was inversely proportional
to increasing amount of RMP / Drug substance added. The potency value of the sample is
calculated using CFR 21/part 11 compliance with Parallel line assay (PLA) software.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved process for the cell culture manufacturing of Recombinant TNFR:Fc. In particular, the invention provides systems that help in the high cell density cell culture process, maintenance of high cell viability for a longer culture period. In addition the invention also helps in achieving proper glycosylation of Recombinant TNFR:Fc. The cell culture manufacturing process starts with seeding the bioreactor at a predefined cell density in chemically defined medium. The culture is fed in two stages, primary feeding which is designed to achieve high cell growth and, secondary feeding which is designed to maintain the higher cell viability and hyper glycosylation of the Recombinant TNFR:Fc. Furthermore the invention also relates to bioreactor operation procedure for the manufacturing of Recombinant TNFR:Fc.

This present invention relates to the rapid and efficient recovery of Recombinant TNFR:Fc from cell culture supernatant from Cell culture fluid by means of Affinity chromatography. These chromatographic steps are used for capture of recombinant TNFR:Fc. This separation involves in
selective binding of the desired compound to specific affinity resin and then elution with elution buffer. Culture supernatants are preferably concentrated and clarified before chromatographic treatment. TNFR:Fc containing eluent fractions are enriched with biologically active material, but they will be subjected to further processing by Anion exchange chromatographic steps. In this process the active materials are colleted in flow through. These processes are used for removal of process related impurities like host cell protein and host cell DNA. Furthermore, the invention also relates to the next chromatographic procedure where the flow-through from the previous step is further subjected to a Cation exchange chromatographic step. The active material is eluted with elution buffer containing salt. The present invention also relates to the recombinant TNFR:Fc recovery procedure involving serial application different chromatographic techniques as mentioned previously. All different steps, conditions and compositions are disclosed in the invention.

Example 1:

Before seeding the Bioreactor, it was assembled and sterilized by autoclaving at 121°C for 60
minutes. After sterilization, Bioreactor was charged with 7000ml of commercially available animal component free, chemically defined media Afterwards, the bioreactor was kept under positive pressure with air at a flow rate of 0.2 Litre per minute. The bioreactor was aerated over night for 100% air saturation. The d02 electrode was calibrated after stabilization of dissolved oxygen value. Sterile connection was created between the seed bottle and the seed port on the bioreactor head plate. The seed was then aseptically transferred to the bioreactor using peristaltic pump. The bioreactor was seeded with the density of 0.5 x 106 cells/mL. After seeding, the bioreactor was allowed to run at following pre-set parameters:

pH: 6.9 - 7.2
d02: 30 - 60% of air saturation
Temperature: 30 - 38°C.
Stir speed: 70-80RPM

The bioreactor was sampled at every 24/48 hours for in process quality control analysis. The
bioreactor process was a fed - batch process with feeding of different nutrients at definite culture stages. During first 120 hrs of culture time, the bioreactor was daily fed with 60mL of primary feed that comprise of glucose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors. Starting from 144 hrs of culture age the bioreactor was daily fed with lOOmL of secondary feed that comprise of Galactose and Mannose.

During first 120 hrs of culture age the bioreactor was operated at following pre-set and controlled parameters;

pH: 7.1+0.1
d02: 30 - 60% of air saturation Temperature: 36 - 37°C. Stir speed: 70-80RPM

From 120 to 288 hrs the bioreactor was operated at following parameters;

pH: 6.9 ±0.05
d02: 30 - 60% of air saturation
Temperature: 32 ± 0.5 °C.
Stir speed: 70-80RPM
From 288 hrs till the harvest, the bioreactor was operated at following parameters
pH: 6.9 ±0.05
d02: 30 - 60% of air saturation
Temperature: 31 ±0.5°C.
Stir speed: 70-80RPM
The bioreactor was harvested at a cell viability of 60 - 70%. The growth pattern, protein expression profile and nutrient consumptions are depicted (Fig 1-3).

Example 2:

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. The clarified harvest was diafilitered and buffer exchanged with Tris buffer pH 7.2 - 7.6 using a 30 kDa - TFF membrane at a Trans Membrane Pressure of 5-10 psi. Affinity chromatography was used in binding and elution mode with column of 30 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 solution. The protein of interest was eluted with citrate buffer (Fig 4). 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 Run 1 and Run 2 was pooled and buffer exchanged with Tris buffer pH 6.8 - 7.2 using a 30 kDa - TFF membrane at a Trans Membrane Pressure of 5-10 psi. Cation exchange chromatography in negative binding mode was carried out with column at an operational flow rate of 10 ml/min. 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 5). Thereafter, the flow through was filtered for virus removal using viral removal filter having an effective filtration area of 0.01 m2. Anion exchange chromatography was carried out with the nanofiltrate 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 6). The eluate was buffer exchanged and concentrated using a 30 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 containing (lOmg/mL Sucrose, 5.8mg/mL of Sodium chloride, 5.3mg/mL of L-Arginine hydrochloride, 2.6mg/mLSodium phosphate monobasic monohydrate and 0.9mg/mLsodium phosphate dibasic anhydrous) and adjusted to pH 6.3±0.2.

Example 3:

The formulated material was characterized as per the specifications set by product development specification. A 12% SDS PAGE under reducing condition was studied and showed single corresponding band at ~75 kDa, matches with RMP (Fig 7). A 8% non-reducing SDS-PAGE data is mentioned in which shows a single band corresponds to approximately 150 kDa matches with RMP (Fig 8). Western blot analysis showed a clear comparable immune-specificity with that of RMP for both reducing and non-reducing condition (Fig 9). An Isoelectric focusing was carried out to identify the isoelectric point of the target protein which matched with that of RMP (Fig 10). A 2-Dimensional gel electrophoresis profile of the purified drug substance matched with RMP (Fig 11). Protein A HPLC profile carried out during this step showed a retention time of 6.3 minutes in drug substance, which was very much comparable with the RMP (6.3 minutes) (Fig 12). RP-HPLC was performed to compare the hydrophobicity profile between the purified drug substance and the RMP. The result showed comparable RT (Fig 13). Size exclusion chromatography for determination of oligomeric status showed a retention time of 6.9 minutes (% of main peak is 100) for drug substance which was very much comparable with the RMP (6.9 minutes, % area of main peak 100 [Fig 14]). Intact molecular mass estimation performed by high-sensitivity MALDI-TOF MS analysis has revealed the molecular mass of purified recombinant Etanercept (TNFR:Fc) and that of RMP to be -145 kDa (Fig 15). Peptide mass fingerprinting of intact molecule by MALDI-TOF MS has demonstrated a high degree of similarity between drug substance with RMP (Fig 16). The results obtained in Peptide Mapping by HPLC showed a similar and corresponding profile to RMP (Fig 17). Identification of cleaved Glycans was carried out using by HPLC. The results have revealed a comparable glycosylation profile between the purified drug substance and RMP (Fig 18). UV spectral analysis has demonstrated comparable primary structure profile between the purified drug substance and RMP (Fig 19). CD spectral analysis has demonstrated comparable secondary and tertiary structure profile between the purified drug substance and RMP (Fig 20). Cytotoxicity rescue assay with L929 cell line showed the degree of TNF alpha induced cytotoxicity observed was inversely proportional to increasing amount of RMP and drug substance (Fig 21).

We claim:

1. A process for recovering tumor necrosis factor receptor Fc region (TNFR Fc),
comprising steps of:

a) contacting culture supernatant(s) with resin(s) for selective adsorption of compound(s);

b) eluting the adsorbed compound with eluant followed by enriching with biologically active material; and

c) subjecting the enriched product to Cation exchange chromatography to obtain the tumor necrosis factor receptor Fc region (TNFR Fc).

d) subjecting the enriched product to Anion exchange chromatography to obtain the tumor necrosis factor receptor Fc region (TNFR Fc).

e) subjecting the enriched product to combination of Anion and Cation exchange
chromatography to obtain the tumor necrosis factor receptor Fc region (TNFR Fc).

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

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

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

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

6. The process as claimed in claim 1, wherein the process removes host cell protein and host cell DNA from culture supernatant.

7. A cell culture manufacturing process for the manufacturing of recombinant tumor necrosis factor receptor Fc region (TNFR Fc).

8. A process of increasing glycosylation using predefined secondary feed

9. The process of using an additional column-washing step during the purification.