Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells.

Culture media design is central to the optimization of monoclonal antibody (mAb) production. Although general strategies do not currently exist for optimization of culture media, the combined use of statistical design and analysis of experiments and strategies based on simple material balances can facilitate culture media design. In this study, we evaluate the effect of selected amino acids on the growth rate and monoclonal antibody production of a Chinese hamster ovary DG-44 (CHO-DG44) cell line. These amino acids were selected based on their relative mass fraction in the specific mAb produced in this study, their consumption rate during bioreactor experiments, and also through a literature review. A Plackett-Burman statistical design was conducted to minimize the number of experiments needed to obtain statistically relevant information. The effect of this set of amino acids was evaluated during exponential cell culture (considering viable cell concentration and the specific growth rate as main output variables) and during the high cell-density stage (considering mAb final concentration and specific productivity as relevant output variables). For this particular cell line, leucine (Leu) and arginine (Arg) had the highest negative and positive effects on cell viability, respectively; Leu and threonine (Thr) had the highest negative effect on growth rate, and valine (Val) and Arg demonstrated the highest positive impact on mAb final concentration. Results suggest the pertinence of a two-stage strategy for amino acid supplementation, with a mixture optimized for cell growth and a different amino acid mixture for mAb production at high density.

Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway.

Rapid modulation of ligand binding affinity ("activation") is a central property of the integrin cell adhesion receptors. Using a screen for suppressors of integrin activation, we identified the small GTP-binding protein, H-Ras, and its effector kinase, Raf-1, as negative regulators of integrin activation. H-Ras inhibited the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression was not associated with integrin phosphorylation and was independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlated with activation of the ERK MAP kinase pathway. Thus, regulation of integrin affinity state is a novel, transcription-independent function of a Ras-linked MAP kinase pathway that may mediate a negative feedback loop in integrin function.

Functional coupling of the delta-, mu-, and kappa-opioid receptors to mitogen-activated protein kinase and arachidonate release in Chinese hamster ovary cells.

To examine whether the mitogen-activated protein kinase (MAPK) cascade and phospholipase A2 (PLA2) are involved in the signal transduction mechanism of the opioid receptor, the delta-, mu-, and kappa-opioid receptors were stably expressed from cDNA in Chinese hamster ovary cells. Activation of the delta-, mu-, and kappa-receptors by agonists induced a rapid and transient increase in MAPK activity accompanied by reduced electrophoretic mobility of the 42-kDa isoform of MAPK (p42), probably owing to phosphorylation. The opioid receptor-mediated increase in MAPK activity was suppressed not only by pretreatment with genistein, a tyrosine protein kinase inhibitor, but also by prolonged exposure to phorbol 12-myristate 13-acetate and pretreatment with GF 109203X, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of PKC as well as tyrosine protein kinase. Furthermore, stimulation of the delta-, mu-, and kappa-receptors with opioid agonists in the presence of A23187, a calcium ionophore, resulted in an increase in arachidonate release, suggesting that PLA2 is activated by the opioid receptors when the intracellular Ca2+ concentration is elevated. Both MAPK activation and increase in arachidonate release mediated by the opioid receptors were abolished by pretreatment with pertussis toxin, suggesting that these responses are mediated by Gi or Go types of GTP-binding regulatory proteins.

Viable AC-2, a new adult bovine serum- and colostrum-based supplement for the culture of mammalian cells.

In this study we have shown that Viable AC-2, a medium based on the ultrafiltrate fraction of bovine colostrum and adult bovine serum, can be used successfully as a fetal bovine serum (FBS) substitute in the culture of several anchorage-dependent and independent cell lines. Of the 15 cell lines cultured in 8% Viable AC-2 in microplates, 10 reached the maximum cell density of 65%-123% of that in 10% FBS, 4 cell lines reached maximum cell density of 35%-65% of that in 10% FBS and only one cell line, a human osteosarcoma G-292, grew slowly in Viable AC-2. In a small-scale suspension culture, 8%-15% Viable AC-2 supports the growth of Chinese hamster ovary cells (CHO-K1) on microcarriers in spinner flasks significantly better than 10% FBS. Shionogi mouse mammary tumour cell line (S115) transfected with human alpha 2-adrenergic receptor subtype C2 was used as a model to study recombinant protein production in Viable AC-2-supplemented medium. The results showed that in cell culture flasks and in an ACUSYST-R bioreactor, the alpha 2-C2 receptor expression level per mg of total protein was similar in both Viable AC-2 and FBS.

Binding of botulinum type B neurotoxin to Chinese hamster ovary cells transfected with rat synaptotagmin II cDNA.

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a. In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.