Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid.
Under some cell culture conditions, recombinant glycoprotein therapeutics expressed in Chinese hamster ovary (CHO) cells lose sialic acid during the course of the culture (Sliwkowski et al., 1992; Munzert et al., 1996). A soluble sialidase of CHO cell origin degrades the expressed recombinant protein and has been shown to be released into the culture fluid as the viability of the cells decreases. To reduce the levels of the sialidase and to prevent desialylation of recombinant protein, a CHO cell line has been developed that constitutively expresses sialidase antisense RNA. Several antisense expression vectors were prepared using different regions of the sialidase gene. Co-transfection of the antisense constructs with a vector conferring puromycin resistance gave rise to over 40 puromycin resistant clones that were screened for sialidase activity. A 5' 474 bp coding segment of the sialidase cDNA, in the inverted orientation in an SV 40-based expression vector, gave maximal reduction of the sialidase activity to about 40% wild-type values. To test if this level of sialidase would lead to increased sialic acid content of an expressed recombinant protein, the 474 antisense clone was employed as a host for expression of human DNase as a model glycoprotein. The sialic acid content of the DNase produced in the antisense cultures was compared with material made in the wild-type parental cell line. About 20-37% increase in sialic acid content, or 0.6-1.1 mole of additional sialic acid out of a total of 3.0 mole on the product, was found on the DNase made in the antisense cell lines.
Ferrari
J
J
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Gunson
J
J
Lofgren
J
J
Krummen
L
L
Warner
T G
TG
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D006224:Cricetinae; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D016372:RNA, Antisense; D011994:Recombinant Proteins; D003851:Deoxyribonucleases; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid