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EP-4737558-A2 - USE OF PERFUSION SEED CULTURES TO IMPROVE BIOPHARMACEUTICAL FED-BATCH PRODUCTION CAPACITY AND PRODUCT QUALITY

EP4737558A2EP 4737558 A2EP4737558 A2EP 4737558A2EP-4737558-A2

Abstract

Methods of improving the efficiency of production of a protein product of interest in mammalian cell culture are presented. In particular, the methods result in an increase in the quantity of a protein product produced, or decreases protein product production time in a manufacturing-scale bioreactor cell culture. The disclosed methods comprise: (a) culturing the N-1 bioreactor culture to high viable cell densities; and (b) seeding the production bioreactor culture at high viable cell seeding densities.

Inventors

  • YANG, William C.
  • LU, Jiuyi
  • HUANG, Yao-ming
  • YUAN, HANG
  • KSHIRSAGAR, Rashmi
  • RYLL, THOMAS

Assignees

  • Biogen MA Inc.

Dates

Publication Date
20260506
Application Date
20141219

Claims (15)

  1. A method of producing a protein of interest, comprising: (a) culturing mammalian cells comprising a gene that encodes the protein of interest in a N-1 culture vessel to achieve a cell density of at least 25 x 10 6 viable cells/ml; (b) inoculating a N culture vessel at a seeding density of at least 8.5 x 10 6 viable cells/ml with cells obtained from step (a); and (c) culturing the cells in the N culture vessel under conditions that allow production of the protein of interest, wherein the culture in step (a) is supplemented with nutrients at a level determined based on the viable cell density, and wherein waste products are removed during step (a) and/or the culture in the N-1 culture vessel of step (a) is perfused.
  2. The method of claim 1, wherein, during step (a), the viable cell density of the culture is periodically determined, optionally wherein the viable cell density of the culture in step (a) is determined 1-10 times per second.
  3. The method of claim 1 or claim 2, wherein the nutrients comprise amino acids, zinc, iron, and copper.
  4. The method of any one of claims 1-3, wherein the waste removal is performed by filtration, centrifugation, inclined cell settler, alternating tangential flow, or tangential flow.
  5. The method of any one of claims 1-4, wherein a cell density of at least 30 x 10 6 viable cells/ml, at least 35 x 10 6 viable cells/ml, at least 40 x 10 6 viable cells/ml, at least 50 x 10 6 viable cells/ml, or at least 60 x 10 6 viable cells/ml is achieved during step (a).
  6. The method of any one of claims 1-5, wherein the mammalian cells are CHO cells, HEK-293 cells, VERO cells, NS0 cells, PER.C6 cells, Sp2/0 cells, BHK cells, MDCK cells, MDBK cells, or COS cells.
  7. The method of any one of claims 1-6, wherein the culture in N-1 culture vessel of step (a) is perfused and the perfusion rate is 0.01 nL/cell/ day to 0.2 nL/cell/day.
  8. The method of any one of claims 1-7, further comprising the step of collecting the protein of interest produced by the cells in the N culture vessel.
  9. The method of any one of claims 1-8, wherein: (i) the culture of step (c) is performed in fed-batch mode; or (ii) the culture in N culture vessel of step (c) is perfused.
  10. The method of any one of claims 1-9, wherein the protein of interest is an antibody, fusion protein, alpha-synuclein, BART, Lingo, aBDCA2, anti-CD40L, STX-100, Tweak, daclizumab, pegylated interferon, interferon, etanercept, infliximab, trastuzumab, adalimumab, bevacizumab, Tysabri, Avonex, Rituxan, ocrelizumab, obinutuzumab or anything that binds to CD20.
  11. The method of any one of claims 1-10, wherein the N culture vessel is inoculated at a seeding density of at least 8.5 x 10 6 viable cells/ml, at least 10 x 10 6 viable cells/ml, 15 x 10 6 viable cells/ml, 20 x 10 6 viable cells/ml, 25 x 10 6 viable cells/ml or 30 x 10 6 viable cells/ml.
  12. The method of any one of claims 1-11, wherein: (i) the culture of step (a) is maintained for 3 days to 8 days; and/or (ii) the culture of step (c) is maintained for 8 days to 23 days.
  13. The method of any one of claims 1-12, wherein (a) the volume of the N-1 culture vessel is between 50 liters to 20,000 liters, between 50 liters to 10,000 liters, between 100 liters to 10,000 liters, or between 100 liters to 4,000 liters; and/or (b) the volume of the N culture vessel is between 200 liters to 20,000 liters, between 200 liters to 10,000 liters, between 1000 liters to 10,000 liters, or between 1000 liters to 5,000 liters.
  14. The method of any one of claims 1-13, wherein lactate levels are maintained below 15 mM, below 10 mM, below 1 mM or below 0.1 mM in the culture of step (a).
  15. The method of any one of claims 1-14, wherein pH of the culture of step (a) is maintained between 6.8 to 7.4 or between 6.9 to 7.3.

Description

Field of the Invention The present invention relates to methods of improving the efficiency of production of a bioproduct of interest in a mammalian cell culture. In particular, the methods increase the quantity of a bioproduct produced, and/or decrease bioproduct production time in a manufacturing-scale production bioreactor cell culture. The disclosed methods comprise: (a) culturing the cells in a N-1 seed train (growth stage) bioreactor cell culture to obtain high viable cell densities; and (b) seeding the production bioreactor culture at high viable cell seeding densities. BACKGROUND OF THE INVENTION Background Art The production bioreactor is one of the bottlenecks in bioproduct manufacturing. Traditional batch or fed-batch production processes consist of an unproductive growth phase, when cell mass accumulates, followed by a more productive stationary phase, when the majority of the bioproduct is generated. The unproductive growth phase lengthens the duration and lowers the volumetric productivity of the production process, which in turn leads to inefficient production bioreactor utilization and reduces the output rate. Improvements in volumetric productivity and production bioreactor usage have been seen by shifting the growth phase from the production stage bioreactor into the N-1 seed train stage (growth stage) bioreactor. (Pohlscheidt et al., Biotechnology Progress, 29(1), 222-9 (2013); Padawer et al., Biotechnology Progress, published online (2013). It is, however, difficult to obtain high viable cell densities in N-1 seed train stage. Traditional batch or fed-batch N-1 seed train cultures cannot sustain high viable cell densities. Thus, there is a need in the art for improved methods for culturing cells in a bioreactor. BRIEF SUMMARY OF THE INVENTION The present invention is related to methods for obtaining high viable cell density in the N-1 seed train stage by nutrient supplementation and/or waste product removal and/or by using a perfusion cell culture process in the N-1 seed train bioreactor. A high viable cell density in the N-1 seed train bioreactor allows for a higher viable cell seeding density in the production bioreactor. By the invention, the high-seed production bioreactor delivers the same or higher titer of the product in a shorter culture duration, leading to an increased N-1 seed train bioreactor occupancy per run and reduced production bioreactor occupancy per run. The shorter production duration and higher volumetric productivity can yield more production batches per run and lead to increased manufacturing capacity with few changes to the batch or fed-batch production bioreactor equipment. In some embodiments, the invention is related to a method of producing a protein, comprising: (a) culturing mammalian cells comprising a gene that encodes the protein of interest in a N-1 culture vessel to achieve a cell density of at least 25 x 106 viable cells/ml; (b) inoculating a N culture vessel at a seeding density of at least 8.5 x 106 viable cells/ml with cells obtained from step (a); and (c) culturing the cells in the N culture vessel under conditions that allow production of the protein of interest. In certain embodiments, during step (a), (i) the viable cell density of the culture is periodically determined; and (ii) the culture is supplemented with nutrients at a level determined based on the viable cell density. In some embodiments, the nutrients comprise amino acids, zinc, iron, and copper. In some embodiments, zinc is added to the cell culture of step (a) in a cumulative amount of between about 1.8 µM to about 1 mM. In some embodiments, iron is added to the cell culture of step (a) in a cumulative amount of between about 0.1 µM to about 10 mM. In some embodiments, copper is added to the cell culture of step (a) in a cumulative amount of between 0.008 µM to about 1 mM. In some embodiments, amino acids are added to the cell culture of step (a) in a cumulative amount of about 50 mM to about 2 M. In some embodiments, the invention is related to a method of producing a protein of interest, comprising: (a) culturing mammalian cells comprising a gene that encodes the protein of interest in a first N-1 culture vessel to achieve a cell density of at least 25 x 106 viable cells/ml; (b) inoculating a N culture vessel at a seeding density of at least 8.5 x 106 viable cells/ml with cells obtained from step (a); and (c) culturing the cells in the N culture vessel under conditions that allow production of the protein of interest, wherein the culture in step (a) is supplemented with nutrients at a level determined based on the viable cell density, and wherein the cumulative amount of amino acids added to the culture of step (a) is about 50 mM to about 2 M. In some embodiments, waste products are removed during step (a) of the present methods. In certain aspects, the waste removal is performed by filtration, centrifugation, an inclined cell settler, alternating tangential flow, tangential flow,