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

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 10(5) cells to maximum cell density of 1.04 x 10(6) 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 10(6) cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 +/- 3.4 mug/mL, a 50% improvement.

Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENT™ CHO cell lines

“…Among the approaches that have been investigated, attention has been focused on modifying the ratios of each component or adding extra material as a supplement. The statistical design method, such as ''design of experiments'' (DOE), can be used to determine effective concentrations of each ingredient and better combinations among them with a minimized set of experiments (Parampalli et al 2007), and this approach has been widely used to develop commercial media. On the other hand, novel components can be found by screening libraries of natural extracts or rare materials.…”

Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody

“…Moreover, the toxicity of dextran sulfate the increase of the molecular weight (Dee et al 1997). Amino acids are considered as the most important factors for both cell growth and production because they can protect cells from nutrient depletion, elevated osmolarity, elevated pCO 2 (Parampalli et al 2007) and act as buffers of intracellular pH. Therefore, amino acids were usually added to the medium as critical factor.…”

Empirical Model for Optimizing Shea Butter Extraction in an Unbaffled Vessel Equipped with an Impeller

“…22 + a 12 x 1 x 2 + ε (3) According to Parampalli et al [16], the design of an experiment is an important aspect of response surface methodology (RSM). The accuracy of the approximation and the cost implication of constructing the response surface are largely dependent on the choice of the design of experimentations.…”