Cytotechnology

, Volume 54, Issue 1, pp 57–68 | Cite as

Developement of serum-free media in CHO-DG44 cells using a central composite statistical design

  • Ananth Parampalli
  • Kent Eskridge
  • Leonard Smith
  • Michael M. Meagher
  • Mark C. Mowry
  • Anuradha Subramanian
Original Research

Abstract

A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM’s F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM’s F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 × 105 cells to maximum cell density of 1.04 × 106 cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2×) EAA, 1.4 mM (0.5×) NEAA, 1× ITS supplement, 0.7× Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 × 106 cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 ± 3.4 μg/mL, a 50% improvement.

Keywords

Response surface method Media optimization 

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Ananth Parampalli
    • 1
  • Kent Eskridge
    • 3
  • Leonard Smith
    • 2
  • Michael M. Meagher
    • 1
  • Mark C. Mowry
    • 1
  • Anuradha Subramanian
    • 1
  1. 1.Department of Chemical EngineeringUniversity of NebraskaLincolnUSA
  2. 2.Toxicology and Aerobiology DivisionU.S. Army Medical Research Institute of Infectious DiseasesFrederickUSA
  3. 3.Department of StatisticsUniversity of NebraskaLincolnUSA

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