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US7291484: Processes for culturing E1-immortalized cells to increase product yields


Filing Information

Inventor(s) Christopher A. Yallop ·
Assignee(s) Crucell Holland B.V. ·
Attorney/Agent(s) TraskBritt, P.C. ·
Primary Examiner Celine Qian ·
Assistant Examiner Laura M Mitchell ·
Application Number US11259245
Filing date 10/26/2005
Issue date 11/06/2007
Prior Publication Data
Predicted expiration date 05/06/2024
U.S. Classifications 435/691  · 435/383  · 435/375  · 435/455  · 435/703  ·
International Classifications C12N1500  · C12N502  · C12N506  · C12P2108  · C12P2300  ·
Kind CodeB2
This application is a continuation of International Patent Application PCT/EP2004/050724, filed May 6, 2004, designating the United States of America, published, in English, as International Patent Publication WO 2004/099396 A1 on Nov. 18, 2004, which itself claims priority from International Patent Application Nos. PCT/EP03/50155 filed May 9, 2003, PCT/EP03/50390 filed Sep. 1, 2003, PCT/EP03/50940 filed Dec. 4, 2003, and PCT/EP04/050061 filed Jan. 30, 2004, the contents of the entirety of each of which are incorporated herein by this reference.
Foreign Priority WO20PCTEP0350155 - 05/09/2003 · WO20PCTEP0350390 - 09/01/2003 · WO20PCTEP0350940 - 12/04/2003 · WO20PCTEP0450061 - 01/30/2004 ·
24 Claims, 12 Drawings


The invention provides processes for culturing cells derived from embryonic retinoblast cells immortalized by adenovirus E1 sequences, preferably PER.C6® (human embryonic retina) cells, to improve product yields from such cells. Feed strategies for such cells and cultures with very high cell densities are provided, resulting in high yields of products, such as recombinant antibodies.

Independent Claims | See all claims (24)

  1. 1. A method for culturing cells, said cells being human embryonic retina (HER) cells immortalized by adenovirus E1 sequences and able to grow in suspension, the method comprising the steps of: a) determining at least once during the culturing of the cells the concentration of at least one medium component selected from the group consisting of glucose, glutamine, phosphate, leucine, serine, isoleucine, arginine, methionine and cystine, and b) adding components to the medium during the culturing of the cells at or prior to the depletion of at least one of the components of which the concentration was determined in step a), wherein the components added at least comprise glucose, glutamine, phosphate, leucine, serine, isoleucine, arginine, methionine and cystine, wherein components in step b) are added to an end concentration in mmoles/l of freshly added component per 10×106 cells/ml of between 4.0 and 8.0 for glucose, 0.47 and 0.93 for phosphate, 0.44 and 0.88 for leucine, 0.37 and 1.47 for serine, 0.33 and 0.67 for isoleucine, 0.31 and 0.61 for arginine, 0.15 and 0.31 for methionine, and 0.1 and 0.6 for cystine.
  2. 18. A method for producing a product in HER cells immortalized by adenovirus E1 sequences, said cells being in a culture medium, wherein said product is chosen from the group consisting of a recombinant protein, a virus, and a recombinant adenovirus with a deletion in the E1 region, the method comprising the step of adding at least glutamine, glucose, phosphate, leucine, serine, isoleucine, arginine, methionine and cystine to the cluture medium to an end concentration in mmoles/liter of freshly added component per 10×106 cells/ml of between 1.01 and 3.46 for glutamine, 4.0 and 8.0 for glucose, 0.47 and 0.93 for phosphate, 0.44 and 0.88 for leucine, 0.37 and 1.47 for serine, 0.33 and 0.67 for isoleucine, 0.31 and 0.61 for arginine, 0.15 and 0.31 for methionine, and 0.1 and 0.6 for cystine.
  3. 20. A method for producing a product in HER cells immortalized by adenovirus E1 sequences, wherein said cells are cultured in a culture medium, comprising the step of the following components to the culture medium in the following amounts per liter: 3.6-21.6 mmoles glucose, 6.8-40.9 mmoles glutamine, 0.40-2.4 mmoles leucine, 2.31-13.9 mmoles serine, 0.3-1.8 mmoles isoleucine, 0.28-1.66 mmoles arginine, 0.14-0.83 mmoles methionine, 0.15-0.9 mmoles cystine, 0.27-1.62 mmoles valine, 0.26-1.58 mmoles lysine, .18-1.08 mmoles threonine, 0.06-0.36 mmoles asparagine, 0.078-0.47 mmoles tyrosine, 0.06-0.36 mmoles histidine, 0.012-0.072 mmoles phenylalanine, 0.036-0.22 mmoles tryptophan, and 0.45-2.7 mmoles phosphate.

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Referenced By

Document NumberAssigneeInventorsIssue/Pub Date
US8008043 Crucell Holland B.V. Christopher A. Yallop Aug 2011
US8546123 --