A new Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins.
Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the alpha2,6-sialyltransferase (alpha2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both alpha2,6- and alpha2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing alpha2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the alpha2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-gamma and the tissue inhibitor of metalloproteinases-1. Interferon-gamma purified from the universal host carried 40.4% alpha2,6- and 59.6% alpha2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-gamma produced by normal CHO cells.
Bragonzi
A
A
DIBIT, Department of Biological and Technological Research, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milan, Italy.
Distefano
G
G
Buckberry
L D
LD
Acerbis
G
G
Foglieni
C
C
Lamotte
D
D
Campi
G
G
Marc
A
A
Soria
M R
MR
Jenkins
N
N
Monaco
L
L
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D018076:DNA, Complementary; D006023:Glycoproteins; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D007371:Interferon-gamma; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase