Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant dnase in batch culture with increased sialic acid

Abstract

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.

Production of Highly Sialylated Monoclonal Antibodies

“…In the field of mammalian cell engineering, a variety of approaches were developed. Antisense RNA and short interfering RNA were used in CHO cell lines to knock-down genes encoding for sialidases, leading to a significant increase in total amount of sialic acid in recombinant proteins [39,41]. Smaller but still significant improvement was achieved by the overexpression of CMP-sialic acid transporter, either alone [42] or in combination with CMP-sialic acid substrate [43].…”

“…Sialylation is likely to be limited after a certain time in batch culture due to a combination of adverse factors such as, but not limited to, ammonia accumulation [37,38], ST6Gal-I plasmid loss upon successive cell division and sialidases release in medium [39]. As Mab titre increases over time in the culture medium, the harvest time point selected must provide an acceptable compromise between high Mab yield and high sialylation.…”

Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells

“…RNAi technology (Brummelkamp et al, 2002;Xia et al, 2002) prompts an alternative strategy to improve protein sialylation. It has been reported that, when cytosolic sialidase (Neu2) in CHO cells was knocked down by RNAi, sialic acid content of glycoprotein expressed from the targeted cells was improved (Ferrari et al, 1998;Ngantung et al, 2006). However, the protein sialylation was only improved when cells were in the death phase, but not in the stationary phase.…”

“…Prior to this study, only one sialidase, the cytosolic sialidase (Neu2) was identified in CHO cells (Burg and MEthing, 2001a,b;Gramer and Goochee, 1993). When gene expression of CHO Neu2 was reduced by RNAi, sialic acid content of recombinant glycoprotein was improved only when cells were in the death phase (Ferrari et al, 1998;Ngantung et al, 2006). In this study, we identified and well characterized the other two sialidases in CHO cells.…”

Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems

“…For instance, dual-regulated control vectors (Prati et al, 2002) have been successfully used to investigate critical cell-cycle regulatory pathways and consequently to facilitate control of Chinese hamster ovary (CHO) cell proliferation (Fux et al, 2001). Additionally, antisense RNA expression has been used to enhance the production of recombinant therapeutic glycoproteins in CHO cells (Ferrari et al, 1998). In addition to experimental set-up to validate the optimal results obtained in this work, we propose to use antisense technology to modulate activities of enzymes that are not critical for hepatocyte specific function.…”