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      Role of environmental conditions on the expression levels, glycoform pattern and levels of sialyltransferase for hFSH produced by recombinant CHO cells

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      Cytotechnology
      Springer Nature

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          Abstract

          A recombinant CHO cell line in which the expression of human follicle stimulating hormone (hFSH) was under the control of the beta actin promoter was maintained in steady state perfusion cultures on a protein free medium. The level of expression of the hFSH was controlled by varying the steady state level of dissolved oxygen (10-90% of air saturation) and of sodium butyrate (0-1.5mM). Under these conditions, the specific productivity of hFSH (qFSH) varied from 0.7 to 4.8 ng hFSH/10(6) cells/h. As the specific productivity of hFSH increased, there was a shift in the FSH isoforms to the lower pI fractions, corresponding to increased sialic acid content. As the specific productivity of hFSH increased, shifting the isoform distribution towards the lower pI isoforms, that the sialyltransferase enzymic activity also increased.

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          Immunological and biological potencies of the different molecular species of gonadotrophins.

          Pituitary gonadotrophins (follicle-stimulating hormone, FSH; luteinizing hormone, LH) exist in different molecular forms within the anterior pituitary gland and serum of several non-mammalian and mammalian species, including man. The number and relative abundance of each gonadotrophin species will depend on the specific technique utilized for their isolation, the tissue source and the physiological status of the donor. Intracellular FSH and LH from glands of rodents (hamsters and rats) and primates exhibit charge heterogeneity and therefore may be separated into several forms or iso-hormones by isoelectric focusing (IEF). These FSH and LH species differ from each other not only in their isoelectric point (pI) but also in their relative abundance, receptor binding activity, biological activity and plasma half-life. Almost all gonadotrophin species isolated from pituitary extracts have also been detected in vitro and in vivo as secreted forms. Less basic rodent LH and FSH forms exhibit low receptor binding and in-vitro biological activities; a similar trend is found in LH and FSH species isolated from glands of monkeys and humans. However, these relatively acidic isohormones have longer circulatory half-lives and higher in-vivo biological activities than less negatively charged forms. The overall pattern of charge heterogeneity of gonadotrophins varies according to the specific endocrine status of the donor. Sex steroid hormones (mainly oestrogens) and gonadotrophin-releasing hormone seem to act in concert at the pituitary level to influence the physicochemical and functional characteristics of gonadotrophins and therefore their biological expression at the target cell. The effects of these factors appear to be mediated through the incorporation of specific carbohydrate residues and/or degree of terminal sugar sulphation at co-post-translational levels. The first result of these complex interactions between the gonad and the hypothalamic-pituitary unit is the production and secretion of various types of gonadotrophin molecules in proportions according with the physiological requirements of the subject at a given time, to perform specific actions upon gonadal maturation and/or function.
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            Comparison of N-linked oligosaccharides of recombinant human tissue kallikrein produced by Chinese hamster ovary cells on microcarrier beads and in serum-free suspension culture.

            Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.
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              Environmental Effects on Protein Glycosylation

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                Author and article information

                Journal
                Cytotechnology
                Cytotechnology
                Springer Nature
                0920-9069
                1573-0778
                1994
                1994
                : 15
                : 1-3
                : 217-221
                Article
                10.1007/BF00762396
                7765934
                abdb1ecf-387f-4a30-ae14-4d6af1fb4f13
                © 1994
                History

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