Differential Effect of Culture Temperature and Specific Growth Rate on CHO Cell Behavior in Chemostat Culture

Mauricio Vergara,Claudia Altamirano,María Rodríguez-Moyá,Nelson Osses,Silvana Becerra,Ramon Gonzalez,Julio Berrios,Juan Reyes

doi:10.1371/journal.pone.0093865

Abstract

Mild hypothermia condition in mammalian cell culture technology has been one of the main focuses of research for the development of breeding strategies to maximize productivity of these production systems. Despite the large number of studies that show positive effects of mild hypothermia on specific productivity of r-proteins, no experimental approach has addressed the indirect effect of lower temperatures on specific cell growth rate, nor how this condition possibly affects less specific productivity of r-proteins. To separately analyze the effects of mild hypothermia and specific growth rate on CHO cell metabolism and recombinant human tissue plasminogen activator productivity as a model system, high dilution rate (0.017 h−1) and low dilution rate (0.012 h−1) at two cultivation temperatures (37 and 33°C) were evaluated using chemostat culture. The results showed a positive effect on the specific productivity of r-protein with decreasing specific growth rate at 33°C. Differential effect was achieved by mild hypothermia on the specific productivity of r-protein, contrary to the evidence reported in batch culture. Interestingly, reduction of metabolism could not be associated with a decrease in culture temperature, but rather with a decrease in specific growth rate.

Highlights

The market of biopharmaceutical products and drugs based on recombinant proteins (r-proteins) is growing rapidly, with total sales reaching more than 138 billion dollars in 2010 [1]
Separate temperature reduction and specific growth rate effects were investigated in chemostat culture, once a steady state was reached
These results indicate that at low specific growth rates, the use of mild hypothermia conditions influence the productivity of the recombinant protein positively

Summary

Introduction

The market of biopharmaceutical products and drugs based on recombinant proteins (r-proteins) is growing rapidly, with total sales reaching more than 138 billion dollars in 2010 [1]. While in the past 20 years production systems have improved in terms of cell growth, from maximum cell concentrations of 1–2 to 10–15 million cells/ml, as well as in specific (10–20 to 50–90 pg/ cell/day) and volumetric (0.05–0.1 to 1–5 g/L) productivities [3], the demand for this growing market still requires further production capacity under stringent optimization schemes [4] In this aspect, considering that the productivity of r-proteins is directly proportional to the mass of viable cells, culture viability and longevity, different approaches have been investigated to optimize the production capacity of the cultures. Among the possible causes that could be involved in this phenomenon are: cell cycle arrest in G1 phase, considered as a more metabolically active phase [16,8,17,18,13]; reduced or delayed catabolism of carbon and energy sources [19,20]; increased levels of transcription and increased mRNA stability of r-proteins [21,8,10,22]; increase in folding capacity and expression of endoplasmic reticulum chaperones [23,24,25]

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