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 Rituximab, a monoclonal antibody to CD20, 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.

Lymphomas are cancers of the lymphatic system - the body's blood-filtering tissues that help to Figureht infection and disease. Like other cancers, lymphomas occur when cells divide in uncontrolled manner in which growth control is lost. Consecutively, lymphatic cells may overcrowd, invade, destroy lymphoid tissues, and metastasize (spread) to other organs.

There are two types of lymphomas: "Hodgkin's Disease" (HD) and Non-Hodgkin's Lymphoma (NHL). NHL is a heterogeneous disease, which causes approximately 50,000 new cases and almost 25,000 deaths every year, in North America alone. The incidence of these malignancies has been increasing steadily over the past several decades in North American and Western European countries at an annual rate of 4%. NHLs are among the leading types of cancers and causes of cancer- related mortality, accounting for -3% of cancer deaths in the US and UK in both men and women.

NHL occurs more often in patients between the ages of 40 and 70. A number of factors, including congenital and acquired immunodeficiency states, and infectious, physical, and chemical agents, have been associated with an increased risk for NHL. Infectious agents, such as viral infections (e.g., Epstein-Barr virus, HIV, human T-cell leukemia virus), and bacterial infections (e.g., Helicobacter pylori) may be associated with the development of NHL.

NHLs are much less predictable than HD, and they are more likely to spread to areas beyond the lymph nodes.

Histologically, -90% of NHLs are of B-cell (10% of T-cell) origin, the most prevalent worldwide being diffuse large (33%) and follicular (22%) types. Most aggressive form of NHL is diffuse large B-cell type, 50% of which present in an advanced stage. Majority (67%) of patients who present with a disseminated disease (i.e. advanced stage) at the time of diagnosis, require systemic therapy.

Thus far, the standard chemotherapy, including single-agent chlorambucil, cyclophosphamide or fludarabine, or combination regimens (e.g. cyclophosphamide, vincristine and prednisone [CVP] or cyclophosphamide, doxorubicin, vincristine and prednisone [CHOP]), have not proven curative. Up to 75% of patients achieve remission (i.e. complete response [CR]), but most relapse after a median of 2 years and only about 20% remain disease tree for >10 years. In patients with early or non bulky localized disease stage, the 5-year failure-free and overall survival (OS) rates of 80-90% and 60-70%, respectively, have been reported with 3-4 cycles of combination chemotherapy followed by involved-field radiotherapy. However, for those with disease in advanced stages, the standard first line treatment, which over the past two decades comprised CHOP chemotherapy regimen, yielded limited success; CR rates have been reported in 50-60% and 5-year OS in only 35^45% of patients.

Monoclonal antibody-based therapies gained importance to overcome the pit fall in the chemotherapy. Monoclonal antibodies were first described in 1975. Developments in the field of recombinant DNA led to advances in the generation of chimeric antibodies and humanized antibodies.

The Rituximab is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Rituximab has a binding affinity for the CD20 antigen of approximately 8.0 nM. Rituximab action appears to be mediated via three potential humoral and cell-mediated effector mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity. Recent studies have shown that CDC appears to be the predominant mechanism by which Rituximab exerts its therapeutic effect.

In 1997, the US Food and Drug Administration (FDA) approved rituximab, a monoclonal antibody for the treatment of low grade non- Hodgkin's B cell lymphomas. Since then, it has been used in over 500 000 patients with generally excellent tolerability.

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 Rituximab, a monoclonal antibody to CD20, from cell culture supernatant

SUMMARY OF THE INVENTION

The present invention relates to the use of novel fermentation process for the over expression of Recombinant Rituximab, a monoclonal antibody to CD20, protein in CHO cells. The present invention also relates to the use of novel chromatographic procedures separately and jointly for the production of Recombinant Rituximab, a monoclonal antibody to CD20, protein, in biologically active form from fluids, especially mammalian host cell culture supernatants.

DESCRIPTION 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 (RMP: Reference Medicinal Product - RituxanTM Mabthera®)

Figure 8 : Western Blot Analysis of Drug substance showing comparable immuno- specificity between RMP and drug substance where Lane No. 1 : RMP and Lane No. 2 :
Formulated Drug Substance

Figure 9 : IEX HPLC profile of formulated Drug substance showed comparable retention time with that of RMP

Figure 10 : Protein A HPLC profile of formulated Drug substance showed comparable retention time with that of RMP

Figure 11 : Size exclusion HPLC profile of formulated Drug substance showed comparable similar hydrodynamic radius

Figure 12 : MALDI-TOF analysis of the intact molecule has determined the molecular mass of RMP and formulated Drug substance to be -148 kDa

Figure 13 : HPLC-based tryptic peptide mapping analysis has shown identical profiles
between RMP & formulated Drug substance

Figure 14 : Glycan analysis depicted above has demonstrated a comparable Glycan profile thus indicating a high-degree of similarity between RMP & formulated Drug substance

Figure 15 : The WIL2-S cell line derived from human B-lymphoblasts and known to express the CD 20 antigen has been used in this experiment. A fixed number of WIL2-S cells per well are exposed to varying amounts of RMP / DP in the presence or absence of a constant amount of complement. The assay read outs done at 530excitationand 590emmision nM indicated that the degree of cytotoxicity observed was directly proportional to increasing amount of RMP / DP added. The potency value of the sample is calculated using CFR 21/part 11compliance with Parallel line assay software with a relative potency of 1.075.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved process for the cell culture manufacturing of Recombinant Monoclonal antibody to Cd20. 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 CD20. 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 CD20. Furthermore the invention also relates to bioreactor operation procedure for the manufacturing of Recombinant Monoclonal antibody to CD20.

This present invention relates to the rapid and efficient recovery of Recombinant Monoclonal antibody to CD20 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 CD20. This separation involves in selective binding of the desired compound to specific affinity resin and then elution with elution buffer. Culture supernatants are clarified before chromatographic treatment. Monoclonal antibody to CD20 containing eluants fractions are enriched with biologically active material, but they will be subjected to further 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 CD20 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 0.35 - 0.4 x 106 cells/mL. After seeding, the bioreactor was allowed to run at following pre-set parameters:

pH: 6.9 - 7.2
dO2: 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 0 hrs of culture age to 72 hrs of culture time, the bioreactor was daily fed with 80mL of primary feed that comprise of glucose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors. Starting from 96 hrs of culture age the bioreactor was daily fed with 100mL of secondary feed that comprise of Glucose, Galactose, and Non essential Amino Acids. During first 120 hrs of culture age the bioreactor was operated at following pre-set and controlled parameters;

pH: 7.1 ±0.1
dO2: 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
dO2: 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 nutrient consumptions, growth pattern and protein expression profile are depicted (Figure 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. 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-C1, 250 mM NaC1 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 was loaded on to next column of anion. 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 0.001 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 NaC1 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 0.2nm filter. The drug substance was characterized as per the specifications. The Drug Substance (Active Pharmaceutical Ingredient) was formulated using formulation buffer containing (9mg/mL of Sodium chloride, 7.35mg/mL of Sodium citrate dihydrate, 0.7mg/mL of Polysorbate 80) and adjusted to pH 6.5. (Each ml contains 10 mg/ml rituximab,)

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 from 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 that the test molecule which was very much comparable with the RMP (Figure 9). Protein A HPLC profile carried out during this step showed a retention time of 6.63 minutes in test molecule which was very much comparable with the RMP (6.65 minutes) (Figure 10). Size exclusion chromatography for determination of oligomeric status showed a retention time of 8.18 minutes (% are of main peak 100) for test molecule which was very much comparable with the RMP (8.17 minutes, % are of main peak 100 ) (Figure 11). Intact molecular mass estimation performed by high-sensitivity MALDI-TOF MS analysis has revealed the molecular mass of purified Rituximab to be 148 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 WIL2-S based cytotoxicity assay showed a potency of 1.075 in comparison to RMP (Figure 15).

1. A process for recovering recombinant Monoclonal antibody to CD20 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 recombinant Monoclonal antibody to CD20.

d) subjecting the enriched product to Anion exchange chromatography to obtain the recombinant Monoclonal antibody to CD20.

e) subjecting the enriched product to combination of Anion and Cation exchange chromatography to obtain the recombinant Monoclonal antibody to CD20.

2. The process as claimed in claim 1, wherein said supernatant is host cell culture, not limited to mammalian cells or supernatant comprising of cell culture derived fluid,

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

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

5. A cell culture manufacturing process for the manufacturing of recombinant recombinant Monoclonal antibody to CD20

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

7. A process of achieving proper antibody glycosylation using a predefined secondary feed.

8. The process as claimed in claim 1, where secondary feed can be comprised of Crabohydrates.

9. The process as claimed in claim 1, where secondary feed can be comprised of Glucose

10. The process as claimed in claim 1, where secondary feed can be comprised of 0.5-1.0 Molar Glucose.

11. The process as claimed in claim 1, where secondary feed can be comprised of 25-100% of 0.5-1.0 Molar Glucose.

12. The process as claimed in claim 1, where secondary feed can be comprised of Galactose.

13. The process as claimed in claim 1, where secondary feed can be comprised of 0.25-1.0 Molar Galactose

14. The process as claimed in claim 1, where secondary feed can be comprised of 25-75% of 0.25-1.0 Molar Galactose

15. The process as claimed in claim 1, where secondary feed can be comprised of 25-100% of 0.5-1.0 Molar Glucose and 25 - 75% of 0.25-1.0 Moiar Galactose

16. The process as claimed in claim 1, where secondary feed can be comprised of 25-100% of 0.5-1.0 Molar Glucose and 25 - 75% of 0.25-1.0 Molar Galactose and Non Essential Amino Acids

17. The process as claimed in claim 1, where secondary feed can be comprised of 25-100% of 0.5-1.0 Molar Glucose and 25 - 75% of 0.25-1.0 Molar Galactose and 5-25% of Non Essential Amino Acids.

18. The molecule obtained from the process as claimed in claim 1, wherein the molecule contains single N-linked Glycosylated structure within Fc domain.

19. The molecule as claimed in claim 18, wherein the glycosylated moiety consists of a bi-antennary structure terminating with either zero (GO) (40-60%), one (G1) (35-55%) or two (G2) (<15%) residues of galactose.