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

Authors

Ananth Parampalli, University of Nebraska - Lincoln
Kent M. Eskridge, University of Nebraska - LincolnFollow
Leonard Smith, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702, USAFollow
Michael M. Meagher, Department of Chemical Engineering, University of Nebraska-LincolnFollow
M. C. Mowry, University of Nebraska - Lincoln
Anuradha Subramanian, Department of chemical Engineering,University of Nebraska Lincoln.Follow

ORCID IDs

Kent M. Eskridge

Date of this Version

6-11-2007

Comments

Published in Cytotechnology (2007) 54:57–68. Copyright Springer Science+Business Media B.V. 2007. Used by permission.

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 x 10⁵ cells to maximum cell density of 1.04 x 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 (2x) EAA, 1.4 mM (0.5x) NEAA, 1x ITS supplement, 0.7x Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 x 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.