Development of a method to quantify gene expression levels for glycosylation pathway genes in chinese hamster ovary cell cultures.

Changes in protein glycosylation owing to changes in environmental conditions are not well understood. To better understand these relationships, methods to quantify controlling factors are needed. Because enzymes are translated from genes, the ability to quantify gene expression levels for glycosylation-related enzymes would be advantageous. We developed quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays to monitor gene expression in Chinese hamster ovary (CHO) cells for five terminal glycosylation genes. The five enzymes were sialidase, a putative alpha2,3-sialyltransferase, beta1,4-galactosyltransferase, cytosine monophosphate-sialic acid transporter, and uracil diphosphate-galactosyl transporter. Four of these CHO cell genes were publicly available from GenBank; however, the alpha2,3-sialyltransferase gene for Cricetulus griseus (CHO cell species) was not available and, therefore, was sequenced as a part of this work. The qRT-PCR primers and probes (based on the TaqMan chemistry) were designed and validated for these five genes. The gene expression profiles were obtained for CHO cells producing the recombinant interleukin-4/13 cytokine trap molecule in batch reactors.

Temperature effects on product-quality-related enzymes in batch CHO cell cultures producing recombinant tPA.

Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors. Previous studies have demonstrated that enzymes, such as proteases and sialidases, accumulate in batch bioreactors. These enzymes are known to be detrimental to the quality of recombinant glycoproteins. Bioreactor temperature has been used to control cell growth and recombinant protein production rates. However, the effect of culture temperature on the production of proteases and sialidases has not been investigated. In this study, Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 degrees C over 8 days and with a constant temperature of 37 degrees C. Analysis of extracellular protease activity indicated that two major proteases were present (50 and 69 kDa). The 50 kDa protease activity decreased similarly with time for both culture conditions. The 69 kDa protease activity increased with time for both culture conditions. The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase. Intracellular sialidase activity was present in both cultures. The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells were inoculated into fresh medium. The initial peak in intracellular sialidase activity was followed by a first-order decay. The intracellular sialidase activities for the two culture conditions were not significantly different. The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions. Thus, the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.

Gene-expression profiles for five key glycosylation genes for galactose-fed CHO cells expressing recombinant IL-4/13 cytokine trap.

Recombinant protein glycosylation profiles have been shown to affect the in-vivo half-life, and therefore the efficacy and economics, for many therapeutics. While much research has been conducted correlating the effects of various stimuli on recombinant protein glycosylation characteristics, relatively little work has examined glycosylation-related gene-expression profiles. In this study, the effects of galactose feeding on the gene-expression profiles for five key glycosylation-related genes were determined for Chinese hamster ovary cells producing a recombinant IL-4/13 cytokine trap fusion. The genes investigated were sialidase, a putative alpha2,3-sialyltransferase, CMP-sialic acid transporter, beta1,4-galactosyltransferase, and UDP-galactosyltransferase. Additionally, the sialic acid content (sialylation) of the recombinant protein was examined. The peak sialic acid content of the IL-4/13 cytokine trap fusion protein was observed to be similar for the control and galactose-fed cultures. The gene-expression profiles for four of the glycosylation genes were observed to be sensitive to the glucose concentration and not significantly different for the control and galactose-fed cultures prior to glucose depletion. However, the sialidase gene-expression profiles were different for the control and galactose-fed cultures. The sialidase gene-expression profile increased significantly for the galactose-fed cultures prior to glucose depletion, whereas for the control cultures, the sialidase gene-expression profiles did not increase until the late stationary phase. The intracellular sialidase enzyme activity decreased exponentially with time for the control cultures; however, for the galactose-fed cultures, the intracellular sialidase enzyme activity decreased initially and then remained relatively high compared to the control cultures. These results indicate that the galactose feeding may increase the potential for desialylation, which offsets any improvements in the sialylation rate due to increased substrate levels. Thus, galactose feeding is an unnecessary expense for the production of the IL-4/13 cytokine trap fusion protein in a batch process.