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 Bevacizumab, a monoclonal antibody to VEGF (Vascular Endothelial Growth Factor), in biologically active form from fluids, especially mammalian host cell culture supematants.
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. Cancer of the colon and rectum (CRC) constitutes a major public health problem and is more prevalent in Western countries where the incidence is nearly double that of developing countries. There, it affects about one of twenty humans and ranks second amongst the most common malignancies in both men and women, with about 334,000 new cases diagnosed every year, distributed almost evenly between the sexes. Deaths fi^om cancers of the colon and rectum rank second (189,000) after limg cancer. Approximately 30% of all patients with CRC have metastatic disease at diagnosis, and 50% of early-stage patients will eventually develop metastatic or advanced disease. The prognosis for this patient population is poor. In Europe, 5-year

relative survival for patients diagnosed with cancer of the colon or rectum dtiring 1985-1989 was 48% for patients with colon cancer and 44% for patients with cancer of the rectum. Despite the recent addition of new therapeutic agents, efficacy remains unsatisfactory. Until the mid-1990s the only available drug, with limited activity in mCRC, was 5-fluorouracil (5FU). The recent introduction of two new cytotoxic drugs, irinotecan and oxaliplatin, in addition to 5FU resulted in significant progress in the treatment of mCRC.
In 1997, Scientists at Genentech reported the humanization of the mouse anti-VEGF monoclonal antibody (Mab A.4.6.1122). By site-directed mutagenesis of a human antibody framework, the residues involved in the six complementarity- determining regions, and also several framework residues, were changed to murine counterparts. The humanized anti-VEGF monoclonal antibody (rhuMab VEGF; bevacizumab; Avastin) bound VEGF with affinity very similar to that of the original antibody (Kd -0.5 nM).
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 Bevacizumab, a monoclonal antibody to VEGF (Vascular Endothelial Growth Factor), from cell culture supernatant
SUMMARY OF THE INVENTION
The present invention relates to the use of novel fermentation process for the overexpression of Recombinant Bevacizumab, a monoclonal antibody to VEGF (Vascular Endothelial Growth Factor), protein in CHO cells.
The present invention also relates to the use of novel chromatographic procedures separately and jointly for the production of Recombinant Bevacizumab, a monoclonal antibody to VEGF (Vascular Endothelial Growth Factor), protein, in biologically active form from fluids, especially mammalian host cell culture supematants. .
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 Anion 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 : 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 9 : lEX HPLC profile of Drug substance showed comparable retention time with that
of RMP
Figure 10 : Protein A HPLC profile of Drug substance showed comparable retention time
with that of RMP
Figure 11 : Size exclusion HPLC profile of Drug substance showed comparable similar
hydrodynamic radius
Figure 12 : MALDI-TOF analysis of the intact molecule has determined the molecular mass
of RMP and Drug substance to be ~ 149 kDa
Figure 13 : HPLC-based tryptic peptide mapping analysis has shown identical profiles
between RMP & Drug substance
Figure 14 : Glycan analysis depicted above has demonstrated a comparable Glycan profile
thus indicating a high-degree of similarity between RMP & Drug substance
Figure 15 : The in vitro efficacy has been extensively demonstrated using the reporter cell
line HUVEC. The API was bioactive on HUVEC (Human Umbilical Vein Endothelial Cells)
based cell proliferation assay with a relative potency of 1.36.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improved process for the cell culture manufacturing of Recombinant Monoclonal antibody to VEGF. 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 Monoclonal antibody to VEGF. 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 proper glycosylation of the Recombinant Monoclonal antibody to VEGF. Furthermore the invention

also relates to bioreactor operation procedure for the manufacturing of Recombinant Monoclonal antibody to VEGF.
This present invention relates to the rapid and efficient recovery of Recombinant Monoclonal antibody to VEGF from cell culture supernatant from Cell culture fluid by means of Affinity chromatography. This chromatographic step is used for capture of recombinant Monoclonal antibody to VEGF. This separation involves in selective binding of the desired compound to specific affinity resin and then elution with elution buffer. Culture supematants are clarified before chromatographic treatment. Monoclonal antibody to VEGF containing eluent fractions are enriched with biologically active material, but they will be subjected to fiirther processing by Anion exchange chromatographic step. 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 Monoclonal antibody to VEGF 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 10* 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. Starting from 24 hrs of culture age to 96 hrs of culture time, the bioreactor was

daily fed with 200mL of primary feed that comprise of glucose, lipids, amino acids, vitamins,
trace elements, cholesterol and growth factors. Starting fi-om 120 hrs of culture age the
bioreactor was daily fed with lOOmL of secondary feed that comprise of Galactose, Non
essential Amino Acids, Trace Elements and Vitamin mixture. 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 hrs till the harvest, 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
The bioreactor was harvested at a cell viability of 75 - 85%. The growth pattern, protein expression profile and nutrient consumptions are depicted (Figure 1,2,3).
Example 2:
Clarification of the cell culture harvest was carried out by using a cellulose disposable filter with 650 - 1000 cm^ 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 solution. The protein of interest was eluted with citrate buffer (Figure 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 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. (Figure 5). Thereafter, the flow through was filtered for virus removal using viral removal filter having an effective filtration area of 0.01 m^ 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 (Figure 6). 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 0.2^m filter. The drug substance was characterized as per the specifications. The Drug Substance (Active Pharmaceutical Ingredient) was formulated using formulation buffer containing 51 mM Sodium Phosphate buffer pH 6.2, 60 mg/ml Trehalose dihydrate and 0.04 % Polysorbate 20. (Each ml contains 25 mg/ml Bevacizumab, 60 mg a, a-trehalose dihydrate, 0.4 mg Polysorbate-20, 5.8 mg sodium phosphate monobasic monohydrate and 1.2 mg sodium phosphate dibasic anhydrous).
Example 3:
The formulated material was characterized as per the specifications set by product development specification..A 10% SDS PAGE under reducing condition was studied for the sample derived fi"om the PAGE showed a clear corresponding band with RMP (Figure 7). Western blot analysis clearly showed a clear corresponding band with RMP (Figure 8). Ion exchange HPLC profile showed a retention time of 12.42 minutes (% are of main peak 81.04) for test molecule which was very much comparable with the RMP (12.49 minutes, % are of main peak 87.87 ) (Figure 9). Protein A HPLC profile carried out during this step showed a retention time of 6.61 minutes in test molecule which was very much comparable with the RMP (6.62 minutes) (Figure 10). Size exclusion chromatography for determination of oligomeric status showed a retention time of 8.59 minutes (% are of main peak 98.94) for test molecule which was very much comparable with the RMP (8.59 minutes, % are of main peak 97.66) (Figure 11). Intact molecular mass estimation performed by high-sensitivity MALDI-TOF MS analysis has revealed the molecular mass of purified Bevacizumab to be 149 kDa (Figure 12). The results obtained Peptide Mapping by HPLC showed a similar and corresponding profile to RMP (Figure 13). Glycan profiling and Deglycosylation analysis using N-Glycanase by HPLC has revealed a comparable glycosylation profile between the purified product tested and RMP (Figure 14). The Relative potency analysis by HUVEC based cell proliferation assay showed a potency of 1.359 in comparison to RMP (Figure 15).

We claim:
1. A process for recovering recombinant Monoclonal antibody to VEGF
comprising steps of:
a) contacting culture supematant(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 recombinant Monoclonal antibody to VEGF.
d) subjecting the enriched product to Anion exchange chromatography to obtain the recombinant Monoclonal antibody to VEGF.
e) subjecting the enriched product to combination of Anion and Cation exchange
chromatography to obtain the recombinant Monoclonal antibody to VEGF.
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 supematant(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 recombinant Monoclonal antibody to VEGF
8. The process of using an additional column-washing step during the purification.
9. A process of achieving proper antibody glycosylation using a predefined secondary feed.
10. The process as claimed in claim 11, where secondary feed can be comprised of Crabohydrates.
11. The process as claimed in claim 11, where secondary feed can be comprised of Galactose
12. The process as claimed in claim 11, where secondary feed can be comprised of 1-2 Molar Galactose

13. The process as claimed in claim 11, where secondary feed can be comprised of 25-
75% of 1-2 Molar Galactose
14. The process as claimed in claim 11, where secondary feed can be comprised of 25-
75% of 1-2 Molar Galactose and Non Essential Amino Acids
15. The process as claimed in claim 11, where secondary feed can be comprised of 25-
75% of 1-2 Molar Galactose and 5-25% of Non Essential Amino Acids
16. The process as claimed in claim 11, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and 5-25% of Non Essential Amino Acids along with Trace Elements
17. The process as claimed in claim 11, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and 5-25% of Non Essential Amino Acids along with 5-20% of Trace Elements
18. The process as claimed in claim 11, where secondary feed can be comprised of 25-
75% of 1-2 Molar Galactose, 5-25% of Non Essential Amino Acids, 5-20% of Trace
Elements and Vitamin mixture
19. The process as claimed in claim 11, where secondary feed can be comprised of 25-
75% of 1-2 Molar Galactose, 5-25% of Non Essential Amino Acids, 5-20% of Trace
Elements and 5-25% of Vitamin mixture