Sustained High‐Yield Production of Recombinant Proteins in Transiently Transfected COS‐7 Cells Grown on Trimethylamine‐Coated (Hillex) Microcarrier Beads

Abstract

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.

Attachment and growth of human embryonic stem cells on microcarriers

“…Examples include the expansion of cells for large-scale production of growth factors (Knibbs et al, 2003), vaccines (Frazatti-Gallina et al, 2004), and antibodies (Voigt and Zintl, 1999). We have shown previously that human fibroblast feeders can be expanded in stirred suspension culture on polystyrene microcarrier particles , which compliments feeder-dependent hESC expansion.…”

Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues

“…Increases in production and quality have been achieved by optimizing the culture media and process parameters or by metabolic engineering of production cell lines for a higher growth rate, enhanced survival, desired glycosylation profiles, and increased specific productivity (Al-Rubeai, 1998;Baker et al, 2001;Carvalhal et al, 2003;Chen et al, 2001;deZengotita et al, 2000;Fox et al, 2004;Geserick et al, 2000;Kaufmann et al, 2001;Knibbs et al, 2003;Mastrangelo et al, 2000;Meents et al, 2002a,b;Mueller et al, 1999;Umana et al, 1999;Wu et al, 2004;Wurm, 2004). Most of today's available biotechnologically relevant cell lines were derived from their host tissues and engineered and/or selected for indefinite attachment-dependent growth or proliferation in suspension and serum-free media (Wurm, 2004).…”

Better and faster: improvements and optimization for mammalian recombinant protein production

“…Cell growth and protein yields may also be made more robust by the use of carefully controlled growth environments (such as bioreactors) or by providing microenviroments via the use of micro or macrocarriers (Cytodex or Cytopore from GE Lifesciences or Fibra-Cel from New Brunswick) which shield cells from harmful shear forces and can increase yields 2–5 fold [19–21]. Hypothermic shifts (37 ° C to 33 ° C) have also been found to be advantageous for increasing cell specific productivity (e.g., at least two-fold for a variety of test proteins) and in particular for CHO cells [20, 22].…”