Tina Etcheverry

Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.

Performance of small-scale CHO perfusion cultures using an acoustic cell filtration device for cell retention : Characterization of separation efficiency and impact of perfusion on product quality

Several small-scale Chinese hamster ovary (CHO) suspension cultures were grown in perfusion mode using a new acoustic filtration system. The separation performance was evaluated at different cell concentrations and perfusion rates for two different CHO cell lines. It was found that the separation performance depends inversely on the cell concentration and perfusion rate. High media flow rates as well as high cell concentrations resulted in a significant drop in the separation performance, which limited the maximal cell concentration achievable. However, packed cell volumes of 10% to 16% (corresponding to 3 to 6. 10(7) cells/mL) could be reached and were maintained without additional bleeding after shifting the temperature to 33 degrees C. Perfusion, up to 50 days, did not harm the cells and did not result in a loss of performance of the acoustic filter as often seen with other perfusion systems. Volumetric productivities in perfusion mode were 2- to 12-fold higher for two cell lines producing two different glycoproteins when compared to fed-batch or batch processes using the same cell lines. Product concentrations were in the range of 20% to 80% of batch or fed-batch culture, respectively. In addition, using the protease-sensitive product rhesus thrombopoietin, we could show that cultivation in perfusion mode drastically reduced proteolysis when compared to a batch culture without addition of protease inhibitors such as leupeptin.

Effect of Copper Sulfate on Performance of a Serum-Free CHO Cell Culture Process and the Level of Free Thiol in the Recombinant Antibody Expressed

A recombinant Chinese hamster ovary (CHO) cell line was used to express a humanized antibody. Product quality analysis of this humanized antibody showed the presence of free thiol, due to unpaired cysteine residues in the Fab region. Decreased potency of this thiol Fab made it critical to minimize the levels of free thiol. In an effort to do this, we evaluated the effect of copper sulfate addition to the cell culture production medium. As a component of the production medium, copper sulfate can act as an oxidizing agent, thereby facilitating disulfide bond formation. Four concentrations of copper sulfate were added at the beginning of 2‐L benchtop production cultures of the recombinant CHO cell line: 0, 5, 50, and 100 μM. We found that these copper sulfate additions had no effect on cell growth or antibody production. However, a slight dose‐dependent depression in culture viability was observed. Analysis of the purified antibody showed that either the 50 or 100 μM copper sulfate additions reduced the level of free thiol by more than 10‐fold.

Purification of insulin-like growth factor-I and related proteins using underivatized silica

Adsorption chromatography using underivatized porous glass can be an effective capture step for the purification of recombinant proteins. Classical desorption techniques using chaotropic agents or harsh chemical solvents often result in elution of inactive material and may not be economical at the process scale. More recently, elution schemes have used tetramethylammonium chloride (TMAC) to obtain biologically active material. A TMAC elution was shown to be effective in the initial purification steps for the recovery of recombinant human insulin-like growth factor-I (rhIGF-I) from an Escherichia coli fermentation broth. However, TMAC also elutes other, more hydrophobic, proteins that are difficult to remove in subsequent purification steps. This paper describes the capture of IGF-I from a crude fermentation broth and a more specific elution using a combination of ethanol and NaCl rather than TMAC. This elution also can be used with other proteins including an IGF-I binding protein (BP3) expressed in mammalian cell culture.

Factorial Design and Analysis for Rapid Development of an Antibody Production Process in CHO Cells

A stepwise factorial design and analysis approach was pursued for the development of a recombinant antibody process in CHO cells. The influence of eleven factors on cell growth and metabolism, viability, productivity and product quality were measured. Experiments were conducted in four groups to use the information from one experiment to assist in designing the next one. The methodology was successful. The product titers were improved significandy. By product accumulation was reduced leading to a robust process with higher final culture viability. Interactions between process variables were determined. Product quality was evaluated for selected cases. The only significant effect on product quality was in glycan composition which varied as a function of glutamine levels.

Alteration in Glycosylation: Controlling the Diversity of Recombinant Glycoproteins

N-linked Oligosaccharides on a recombinant protein play a critical role in the metabolic behavior of the molecule. The extent of glycosylation on the protein can determine clearance rates and the ultimate pharmacokinctic profile. The amount of terminal sialylation is particularly important in this respect. Changes in the glyeosylation pattern due to changes in culture conditions will be shown for a model glyeoprotein produced in CHO cells. The parameter determining the sialic acid content will be discussed together with a method to control for this type of heterogeneity.