Aberrant Metabolic Sialylation of Recombinant Proteins Expressed in Chinese Hamster Ovary Cells in High Productivity Cultures

189

149

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

Section: Effect Of Temperaturesupporting

confidence: 65%

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo‐Fc expressing CHO cells cultivated in controlled batch bioreactors

Trummer

Fauland

Seidinger

et al. 2006

189

149

“…Indeed, our data support the earlier hypothesis that changes in glycoprotein sialylation caused by SB are due to metabolic increases in CMP-sialic acid pools via lysosomal salvage pathways, thus by-passing de novo synthesis (Santell et al, 1999).…”

Section: Discussionsupporting

confidence: 90%

“…In contrast, others observed that 1.5 mM SB increased sialylation of follicle stimulating hormone (Chotigeat et al, 1994). Some have suggested that changes in sialylation may be due to SB's ability to effect the recycling of sialic acids from existing glycoproteins, thus by-passing de novo biosynthesis (Santell et al, 1999). Clearly the effect of SB on glycosylation is complex.…”

Section: Introductionmentioning

confidence: 97%

Sodium butyrate alters erythropoietin glycosylation via multiple mechanisms

Crowell

Qin

Grampp

et al. 2007

Recombinant human erythropoietin (rHuEPO) produced in a human kidney fibrosarcoma cell line, HT1080, was used as a model to study the effects of sodium butyrate (SB) on protein glycosylation. Treatment with 2 mM SB resulted in complex changes with respect to sugar nucleotide pools including an increase in UDP-Gal and a decrease in UDP-GlcNac. In addition, polylactosamine structures present on rHuEPO increased after SB treatment. To determine if these phenotypic changes correlated with changes in mRNA abundance, we profiled mRNA levels over a 24-h period in the presence or absence of SB using oligonucleotide microarrays. By filtering our data through a functional glycomics gene list associated with the processes of glycan degradation, glycan synthesis, and sugar nucleotide synthesis and transport we identified 26 genes with significantly altered mRNA levels. We were able to correlate the changes in message in six of these genes with measurable phenotypic changes within our system including: neu1, b3gnt6, siat4b, b3gnt1, slc17a5, and galt. Interestingly, for the two genes: cmas and gale, our measurable phenotypic changes did not correlate with changes in mRNA expression. These data demonstrate both the utility and pit falls of coupling biochemical analysis with high throughput oligonucleotide microarrays to predict how changes in cell culture environments will impact glycoprotein oligosaccharide content.

“…Santell et al (1999) reported lower sialic acid content with a 4-fold increase in specific productivity. Chotigeat et al (1994) reported a 6.9-fold increase in specific productivity with SB as well as increases in both sialic acid and sialyltransferase activity.…”

Section: Discussionmentioning

confidence: 93%

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

Borys

Dalal

Abu‐Absi

et al. 2010

CHO cells express glycoproteins containing both the N-acetylneuraminic acid (Neu5Ac) and minor amounts of the N-glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well-known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50-62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early-temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO(2) with this product showed that the Neu5Gc levels were 46% lower at high pCO(2) conditions (140 mmHg) compared to moderate pCO(2) levels (20-80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics.