Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Abstract

Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells. The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin binding. In this report, the site occupancy of t-PA is shown to increase gradually over the course of batch and fedbatch CHO cultures. Additional cell culture factors, including butyrate and temperature, are also shown to influence the degree of glycosylation. In each of these cases, conditions with decreased growth rate correlate with increased site occupancy. Investigations using quinidine and thymidine to manipulate the cell cycle distribution of cultures further support this correlation between site occupancy and growth state. Comparison of the cell cycle distribution across the range of cell culture factors investigated shows a consistent relationship between site occupancy and the fraction of cells in the G 0 /G 1 phase of the cell cycle. These results support a correlation between growth state and site occupancy, which fundamentally differs from site occupancy trends previously observed and illustrates the importance of the growth profile of CHO cultures in producing consistently glycosylated recombinant glycoproteins.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…This effect was also found in the production of human tissue-type plasminogen activator. A decline in growth rate induced by the supplementation of butyrate resulted in an enhanced glycosylation site occupancy (Andersen et al, 2000).…”

“…Limitations in glucose and glutamine have been found to affect IFN-γ site-occupancy in CHO cultures (Hayter et al, 1992;Nyberg et al, 1999). In 2000, Andersen's group found that the site-occupancy of recombinant human t-PA was elevated gradually over the course of batch and fed-batch CHO cultures (Andersen et al, 2000). By controlling cell culture factors, including butyrate and temperature, they also showed the degree of site-occupancy that could be influenced (Andersen et al, 2000).…”

“…In 2000, Andersen's group found that the site-occupancy of recombinant human t-PA was elevated gradually over the course of batch and fed-batch CHO cultures (Andersen et al, 2000). By controlling cell culture factors, including butyrate and temperature, they also showed the degree of site-occupancy that could be influenced (Andersen et al, 2000). In addition, there was an important correlation between the growth profile of CHO cultures and site-occupancy, in which a decrease in cell growth led to an increase in site-occupancy (Wang et al, 1991).…”

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

“…As a result of various cell culture factors, glycosylation variations in mammalian cell bioprocesses can found in the glycan site (macroheterogeneity) and glycan profile (microheterogeneity). 16 Glycan structures that are produced from any cell are governed by a network of enzymes that do not always allow for individual reactions. This network is affected by multiple factors, including the availability of precursor, co-factors and enzyme activities levels, all of which give rise to the variable final glycan structure.…”

Optimization of the medium perfusion rate in a packed-bed bioreactor charged with CHO cells

“…It is well known that modification of the perfusion rate during a perfusion process, as well as modification of other bioprocess factors, can influence the recombinant protein quality and in particular its glycosylation pattern (Jenkins et al 1996;Andersen et al 2000). Glycosylation is usually recognized as an important function in the solubility, immunogenicity, and pharmacokinetic properties of human glycoproteins and those are the key parameters in the safety and clinical efficacy of a product (Goochee et al 1991).…”