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 novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, which has longer biological half-life compared to r-HuEPO.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.
Anemia is often an associated condition in patients with chronic kidney disease (CKD). There is a direct relationship between the severity of the anemia and the decline in renal function. This anemia is a source of significant morbidity causing symptoms such as lack of energy, breathlessness, dizziness, angina, and poor appetite and decreased exercise tolerance. The main cause of this anemia is a decreased production of erythropoietin, a naturally occurring hormone mainly produced by the kidney. Much of the impaired quality of life and morbidity suffered by patients with CKD may be a consequence of this anemia and it may have a major impact on their sense of well-being as well as impairing their ability to work and affecting

their social and sexual lives. In the past, iron and folate were the main treatments for this condition and blood transfusions, with their associated risks of transmission of infection and induction of cytotoxic antibodies, which could jeopardize a future renal transplant, were used sparingly. In 1983 the cloning of the human gene for erythropoietin was achieved, production of recombinant human erythropoietin (r-HuEPO) followed and by 1986 the efficacy of r-HuEPO treatment in dialysis patients was first demonstrated.
Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects. The aim of inventing this new molecular analogue of recombinant human erythropoietin (r-HuEPO) is to obtain a therapeutic with a longer biological half-life compared to r-HuEPO, allowing a reduction of the frequency of injections necessary to maintain a desired level of systemic haemoglobin and haematocrit. The chronic nature of CRF (unless a subject receives a kidney transplant) means that treatment may continue for a long part of the subject's life and multiple weekly injections of r-HuEPO can have a major impact on subjects and care givers.
The development of Darbepoetin alfa arose from the understandings that the higher isoforms (those with a greater number of sialic acid residues) of recombinant human EPO were more potent biologically in vivo as a result of a longer circulating half-life than the lower isomers (those with a lower number of sialic acid residues). Because the majority of sialic acid residues are attached to the three N-linked glycosylation chains of the EPO molecule, attempts were made to synthesize EPO analogues with a greater number of N-linked carbohydrate chains. This was achieved using site-directed mutagenesis mutagenesis to change the amino acid sequence at sites not directly involved in binding to the EPO receptor. Thus, five- amino acid substitutions were implemented (allowing Darbepoetin alfa to carry a maximum of 22 sialic acid residues, compared with recombinant or endogenous EPO, which support a maximum of 14 sialic acid residues). The additional N-linked carbohydrate chains increased the molecular weight of epoetin from 30.4 to 37.1 kD, and the carbohydrate contribution to the molecule correspondingly increased from 40% to approximately 52%.
OBJECTIVES OF THE INVENTION
The present invention relates generally to the use of novel chromatographic procedures separately and jointly for the production of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, in biologically active form from fluids, especially mammalian host cell culture supematants, a new molecular analogue of

recombinant human erythropoietin, which has longer biological half-life compared to r-HuEPO.
SUMMARY OF THE INVENTION
The present invention relates to the use of novel fermentation process for the overexpression of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, new molecular analogue of recombinant human erythropoietin, which has longer biological half-life compared to r-HuEPO in CHO cells. Summary of the present invention also includes use of novel chromatographic purification for rapid and efficient recovery of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, new molecular analogue of recombinant human erythropoietin, which has longer biological half-life compared to r-HuEPO.
DESCRIPTION OF FIGURES
Figure l.Chromatogram of Anion Exchange
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an improved process for the cell culture manufacturing of new molecular analogue of recombinant human erythropoietin. In particular, the invention provides system that helps in achieving proper glycosylation of new molecular analogue of recombinant human erythropoietin. In addition, the invention also helps in maintaining higher cell viability for a longer period of time. 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 the desired cell growth and, secondary feeding which is designed to maintain the higher cell viability and hyper glycosylation of the new molecular analogue of recombinant human erythropoietin. Furthermore the invention also relates to bioreactor operation procedure for the manufacturing of new molecular analogue of recombinant human erythropoietin.
This present invention relates to the rapid and efficient recovery of new molecular analogue of recombinant human erythropoietin from cell culture supernatant from Cell culture fluid by Ion exchange chromatography. The first step of chromatography is an Anion exchanger in

binding and elution mode. The elution is based on selective enrichment of Novel erythropoetin isoforms. Elution is carried out with Tris buffer containing lOOmM to 200 mM salt (pH 7-8). The set of isoforms eluted in this fraction contained the sialic acid residues approximately ranging from 14-18. The differential glycosylation pattern in various isoforms formed determines the effective separation which in turn is affected by the salt concentration in the elution buffer. The present invention specifically describes the separation of isoforms containing 14-18 sialic acid residues. The additional glycosylation will affect the biochemical and biological properties of this novel erythropoietin analogue. Due to additional glycosylation, this novel analogue will have a slower serum clearance and therefore a longer half-life. The longer serum half-life will increase the in vivo biological activity and also will allow the analogue to be administered less fi-equently.
Example 1:
Before seeding, the Bioreactor was assembled and sterilized by autoclaving at 121°C for 45
minutes. After sterilization, Bioreactor was charged with 7200ml 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 0.4 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. During first 72 hrs of culture time, the bioreactor was daily fed with 300mL of primary feed that comprise of glucose, galactose, mannose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors. Starting from 96 hrs of culture age the bioreactor was daily fed with 150mL of secondary feed that comprise of Galactose, trace Elements, Manganese Chloride 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: 6.8 ±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 70 - 80%.
Example 2:
The first step of chromatography is an Anion exchanger in binding and elution mode. The elution is based on selective enrichment of Novel erythropoetin isoforms. Elution is carried out with Tris buffer containing lOOmM to 200 mM salt (pH 7-8). The set of isoforms eluted in this fraction contained the sialic acid residues approximately ranging fi"om 14-18 (Figure. 1)

We claim:
1. A process for recovering a new molecular analogue of recombinant human erythropoietin, subjecting to anion exchange chromatography and eluted with Tris containing salt at a concentration between lOOmM and 200mM, to obtain the new molecular analogue of recombinant human erythropoietin, which has longer biological half-life compared to r-HuEPO
2. The process as claimed in claim 1, wherein said supernatant is cell culture derived fluid.
3. The process as claimed in claim 1, wherein said supernatant is a mammalian cell culture derived fluid.
4. The process as claimed in claim 1, wherein said culture supematant(s) are clarifled before contacting resins.
5. A cell culture manufacturing process for the manufacturing of recombinant a new molecular analogue of recombinant human erythropoietin, which has longer biological half-life compared to r-HuEPO.
6. A process of increasing protein glycosylation using a predefined secondary feed.
7. The process as claimed in claim 6, where secondary feed can be comprised of Carbohydrates.
8. The process as claimed in claim 6, where secondary feed can be comprised of Carbohydrates and Trace Elements.
9. The process as claimed in claim 6, where secondary feed can be comprised of Carbohydrates, Trace Elements and Manganese Chloride.
10. The process as claimed in claim 6, where secondary feed can be comprised of Galactose and Mannose
11. The process as claimed in claim 6, where secondary feed can be comprised of 1-2 Molar Galactose and Mannose
12. The process as claimed in claim 6, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and Mannose
13. The process as claimed in claim 6, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and Mannose along with Trace Elements
14. The process as claimed in claim 6, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and Mannose along with 0.01 - 1.0% Trace Elements and Manganese Chloride.
15. The process as claimed in claim 6, where secondary feed can be comprised of 25-75% of 1-2 Molar Galactose and Mannose along with 0.01 - 1.0% Trace Elements
and 0.02-5 micro molar Manganese Chloride.
16. A new molecular analogue of recombinant human erythropoietin.

17. A new molecular analogue of recombinant human erythropoietin containing more glycosylation sites compared to r-HuEPO.
18. A new molecular analogue of recombinant human erythropoietin as in claim 16, additional glycosylation site is an N-linked glycosylation site having an N-linked carbohydrate chain attached thereto.
19. A new molecular analogue of recombinant human erythropoietin as in claim 18, additional glycosylation site is an 0-linked glycosylation site having an 0-linked carbohydrate chain attached thereto.
20. A new molecular analogue of recombinant human erythropoietin as claimed in claim 18 and 19 and a combination thereof
21. A new molecular analogue of recombinant human erythropoietin, which has longer biological half-life compared to r-HuEPO.
22. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has longer biological half-life compared to r-HuEPO containing Sialic acid residues more than 14.
23. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has longer biological half-life compared to r-HuEPO containing Sialic acid residues less than 18.
24. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has longer biological half-life compared to r-HuEPO containing Sialic acid residues between 14-18.
25. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has in vivo biological activity of increasing haematocrit.
26. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has more in vivo biological activity of increasing haematocrit in comparison to r-HuEPO.
27. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has delayed serum clearance than r-HuEPO.
28. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing haemoglobin.
29. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing erythropoiesis
30. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing WBC count.
31. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing lymphocytes

32. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing all B-cell lineages.
33. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing all T-cell lineages.
34. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity of increasing all haematopoietic lineages.
35. A new molecular analogue of recombinant human erythropoietin as in claim 18,
which has novel in vivo biological activity of increasing all precursors of
haematopoietic and lymphocytic lineages.
36. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity involved in modulating cytokines required for erythropoiesis and subsequent maturation.
37. A new molecular analogue of recombinant human erythropoietin as in claim 18, which has novel in vivo biological activity involved in modulating cytokines required for lymphocytes and subsequent maturation.

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