BR-112014014116-B1 - FLOCCULATION METHOD

Abstract

FLOCCULATION METHOD. The present invention relates to a method for harvesting recombinant proteins from mammalian cell culture fluid. The method allows the use of cationic polymers, nonionic polymers, and nonionic surfactants.

Inventors

• THOMAS M. MCNERNEY
• Krista Petty
• ANNE C. THOMAS
• Xiaoyang Zhao

Assignees

• AMGEN INC

Dates

Publication Date

20260317

Application Date

20121214

Priority Date

20111215

Claims (14)

1. 1. Mammalian cell culture harvesting method CHARACTERIZED in that it comprises: culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is achieved, adding a cationic polymer selected from a diallyldimethylammonium chloride or polydiallyldimethylammonium chloride polymer, and a non-ionic polymer selected from a polyethylene glycol and dextran, to the cell culture medium that initiates flocculation, mixing the cell culture medium during flocculation, allowing the flocculant to settle for initial sedimentation, and recovering from the clarified primary supernatant.
2. 2. Method according to claim 1, CHARACTERIZED in that the non-ionic polymer is selected from PEG 3000 and PEG 6000.
3. 3. Method according to claim 2, characterized in that the concentration of PEG 3000 is between 3% and 4.5%.
4. 4. Method according to claim 2, CHARACTERIZED in that the concentration of PEG 6000 is between 2.5% and 3.5%.
5. 5. Method, according to claim 1, CHARACTERIZED in that it further comprises the addition of a non-ionic surfactant to the cell culture medium, wherein preferably the non-ionic surfactant is Sapoin or Triton X-100.
6. 6. Method according to claim 5, CHARACTERIZED in that the non-ionic surfactant is Triton X-100, wherein preferably the concentration of Triton X-100 is 0.05% (w/v).
7. 7. Method according to claim 1, CHARACTERIZED in that it further comprises: washing the primary sedimentation flocculant, allowing the washed flocculant to sediment to a secondary sedimentation, and recovering the secondary clarified supernatant.
8. 8. Method according to claim 7, CHARACTERIZED in that the primary sedimentation flocculant is washed in a 9% sucrose solution.
9. 9. Method according to claim 1, characterized in that the cationic polymer, the non-ionic polymer, and optionally a non-ionic surfactant are added simultaneously.
10. 10. Method according to claim 1, CHARACTERIZED in that the cationic polymer is added first and mixed for at least 30 seconds, followed by the addition of the non-ionic polymer, and optionally a non-ionic surfactant.
11. 11. Method according to claim 1, CHARACTERIZED in that polydiallyldimethylammonium chloride is added at a concentration of 20 to 90 pg/total cell density.
12. 12. Method according to claim 1, CHARACTERIZED in that polydiallyldimethylammonium chloride is added at a concentration of 25 pg/total cell density, wherein mammalian cells originate from a diploid cell line.
13. 13. Method according to claim 1, CHARACTERIZED in that polydiallyldimethylammonium chloride is added between 43 pg/total cell density and 57 pg/total cell density, wherein the mammalian cells originate from a tetraploid cell line.
14. 14. Method according to claim 1, CHARACTERIZED in that the mammalian cell culture medium is between 36°C and 20°C, wherein preferably the mammalian cell culture medium is at or above 20°C.

Description

[01] This application claims the benefit of U.S. Provisional Application No. 61/576,303 filed on December 15, 2011, which is incorporated herein by reference. FIELD OF THE INVENTION [02] The present invention relates to a method for harvesting recombinant proteins from mammalian cell culture broth. The method allows the use of cationic polymers, non-ionic polymers and non-ionic surfactants. FUNDAMENTALS OF THE INVENTION [03] The clinical fabrication of therapeutic proteins is an expensive undertaking on a large scale. The demand for larger quantities of recombinant therapeutic proteins has driven advances in cell culture processing, resulting in dramatically increased product titration. High-titration cell culture processes are typically achieved by maintaining high densities of viable cells for extended periods of culture. A corresponding increase in biomass solids (viable and non-viable cells) and submicron cellular debris particles is also observed. The higher load of submicron cellular debris particles can challenge mammalian cell culture harvesting processes, making the harvesting process less effective at removing debris without a substantial loss of product yield. [04] Cationic polymeric flocculants are used for many applications ranging from drinking water purification, wastewater treatment, uses in the petroleum, mining and paper manufacturing industries, cosmetics and medical applications, as well as for encapsulating mammalian cells and enzymes and for flocculating microbial cell cultures. However, for use in a commercial-scale mammalian cell harvesting process, the long flocculation settling time can be problematic, resulting in a harvesting process that is time-consuming and less effective than standard harvesting practices. [05] There is a continuing need to improve mammalian cell culture harvesting methods, particularly commercial-scale methods. Any improvements that allow for faster recovery times and/or higher recovery can lead to cost reductions associated with the manufacture of protein therapies. The invention fulfills this need by providing a fast and efficient method for harvesting cell cultures. SUMMARY OF THE INVENTION [06] The present invention provides a method for harvesting mammalian cell culture comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is obtained, adding a cationic polymer and a non-ionic polymer to the cell culture that initiates flocculation, mixing the cell culture medium during flocculation, allowing the flocculating agent to sediment, and recovering the clarified supernatant. [07] The present invention also provides a method for harvesting mammalian cell culture comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is achieved, adding polydiallyldimethylammonium chloride and PEG 3000 to the cell culture medium which initiates flocculation, mixing the cell culture medium during flocculation, allowing the flocculating agent to sediment and recovering the clarified supernatant. [08] The present invention also provides a method for harvesting mammalian cell culture comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is achieved, adding polydiallyldimethylammonium chloride, PEG 3000 and Triton X-100 to the cell culture medium initiating flocculation, mixing the cell culture medium during flocculation, allowing the flocculant to sediment and recovering the clarified supernatant. [09] The present invention also provides a method for harvesting mammalian cell culture comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is achieved, adding a cationic polymer and a non-ionic polymer to the cell culture medium that initiates flocculation, mixing the cell culture medium during flocculation, allowing the flocculant to settle to a primary sedimentation, recovering the primary clarified supernatant, washing the primary sedimentation flocculant, allowing the washed flocculant to settle to a secondary sedimentation, and recovering the secondary clarified supernatant. [010] The present invention also provides a method for harvesting mammalian cell culture comprising culturing mammalian cells expressing a recombinant protein in a cell culture medium for a predetermined time or until a desired cell density and/or packed cell volume is achieved, adding a cationic polymer and a non-ionic polymer to the cell culture medium that initiates flocculation, mixing the cell culture medium during flocculation, allowing the flocculant to settle to a primary sedimentation, recovering the primary clarifi