期刊
JOURNAL OF BIOTECHNOLOGY
卷 117, 期 3, 页码 277-286出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2005.02.003
关键词
amino acids; tissue plasminogen activator; ammonia/ammonium; Chinese hamster ovary cells; glycosylation
Ammonium is a toxic and inhibitory byproduct of mammalian cell metabolism. At the end of a typical recombinant protein production campaign, the ammonium concentration can be as high as 10 mM, mainly due to glutantine metabolism. Intracellular pH (pH(i)) levels are sensitive to ammonium, which negatively impacts both cell growth and recombinant protein productivity. Ammonium also negatively affects the recombinant protein glycosylation profile, thus altering quality. Many strategies have been adopted to reduce ammonium accumulation, with limited results. This study investigated the addition of amino acids to the growth media for Chinese hamster ovary (CHO) cell cultures as a means of mitigating the negative effects of ammonium. Threonine, proline, and glycine additions improved CHO cell growth and recombinant protein levels. Further, the threonine, proline, and glycine additions positively impacted important metabolic parameters, including glucose consumption, lactate production, glutamine utilization, and final ammonium levels. Additionally, threonine, proline, and glycine increased the level of alpha(2,3)-linked sialic acid, galactose-beta(1,4)-N-acetylglucosamine, and alpha(2,6)-linked sialic acid residues on the recombinant tissue plasminogen activator (t-PA). Thus, threonine, proline, and glycine can be used to mitigate some of the toxic effects of ammonium on cell growth, recombinant protein productivity, and protein quality. (c) 2005 Elsevier B.V All rights reserved.
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