Enhancing recombinant protein yield and quality using novel CHO GT cells in high density fed batch cultures

Impact of Dynamic Online Fed-Batch Strategies on Metabolism, Productivity and N-Glycosylation Quality in CHO Cell Cultures Danny Chee Furng Wong,1,2 Kathy Tin Kam Wong,1 Lin Tang Goh,1 Chew Kiat Heng,2 Miranda Gek Sim Yap1 Bioprocessing Technology Institute, Agency for Science and Technology Research (A* STAR), 20 Biopolis Way, #06-01, Centros, Singapore 138668; telephone: 65 6478 8880; fax: 65 6478 9561; e-mail: miranda_yap@ bti.a-star.edu.sg Department of Pediatrics, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 Received March 2004; accepted 25 August 2004 Published online December 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20317 Abstract: As we pursue the means to improve yields to meet growing therapy demands, it is important to examine the impact of process control on glycosylation patterns to ensure product efficacy and consistency. In this study, we describe a dynamic on-line fed-batch strategy based on low glutamine/glucose concentrations and its impact on cellular metabolism and, more importantly, the productivity and N-glycosylation quality of a model recombinant glycoprotein, interferon gamma (IFN-g). We found that low glutamine fed-batch strategy enabled up to 10-fold improvement in IFN-g yields, which can be attributed to reduced specific productivity of ammonia and lactate. Furthermore, the low glutamine concentration (0.3 mM) used in this fed-batch strategy could maintain both the N-glycosylation macro- and microheterogeneity of IFN-g. However, very low glutamine (95%). Table V. Microheterogeneity if IFN-g glycans on Asn97 harvested during high viability (>95%). Fed-batch Glutamine (mM) Asn25 ID High mannose Hybrid Complex (bi-antennary) Complex (tri-antennary) Complex (tetra-antennary) Batch 0.1 0.3 Fed-batch (mM) Glutamine/ glucose (mM) 0.5 0.3/0.35 0.3/0.70 M04 M05 M07 + + + M08 + + + + H02 + H04 + H03-F + + + H06-F + + C03-F + + + + C04-F + + + + C07-F + + + + C10-F + + + + C13-F + + + + C21-F + + + C05-F + + C08-F +++++ +++ +++ +++ C14-F + + + + C22-F + C27-F + + + C09-F + + + C16-F + + + + + + + + + + + + + + Asn97 High mannose Hybrid Complex (bi-antennary) + + +++ + +++++ + Complex (tri-antennary) + + Approximate quantification was obtained using relative peak intensity of mass spectometry (+: – 30%, +++: 30 – 60%, +++++: 60 – 90%). properties of the biotherapeutic is the sialylation of an N-glycan. It is therefore particularly important to ensure that process development not only improves yield but maintains high degree of sialylation as well. We found that typically IFN-g from batch cultures contained an average of 2.8 mol sialic acid / mol IFN-g (Fig. 8B). IFN-g harvested from 0.5 mM glutamine setpoint fed-batch during high viability has a comparable sialic acid content of 2.9 mol sialic acid / mole IFN-g; however, with further glutamine limitation, sialic acid content decreased significantly. At 0.3 mM glutamine, sialic acid content decreased by 17%, while 0.1 mM glutamine setpoint fedbatch decrease by 23% when compared to batch culture. The addition of glucose control also did not improve sialylation, but rather decreased sialylation, especially at lower glucose control. The observed decrease in sialylation in fed-batch could be due to a multiplicity of factors, but since the IFN-g was harvested at high viability it is unlikely that released cytosolic sialidase is responsible for the reduced sialylation. Sialylation decrease could either be due to impaired sialyltransferases activity, low concentrations of substrate, or nucleotide-sugar donor, CMP-NeuAc. Nyberg et al. (1999) found that glutamine limitation can limit the formation of UDP-GlcNAc by limiting amino sugar formation. UDP-GlcNAc is essential for the formation of N-acetylmannosamine (ManNAc), a direct precursor of 174 Glutamine Complex (tetra-antennary) ID Batch 0.1 0.3 Glutamine/ glucose 0.5 0.3/0.35 0.3/0.70 M01 + M02 + + + + + M04 + M05 + M07 + M08 + + + + + H02 + + + H03 + + + + H06 + + + + + C01 + + + + + C02 + + + + + C03 + + + C04 + + + + + C07 +++ +++ +++++ +++ +++++ C10 + + + + + C13 +++ +++ +++ +++ +++ C21 + + + + + C05 + + + C08 + +++ +++ + C11 + + + + C14 + + + + C15 + + + + + C22 + + + + + C09 + + C16 + + + + + C20 + + + C23 + + + + + + + + + + + + + +++++ + +++ + + + + + Approximate quantification was obtained using relative peak intensity of mass spectrometry: (+: – 30%, +++: 30 – 60%, +++++: 60 – 90%). CMP-NeuAc (Pels Rijcken et al., 1995). However, earlier on we hypothesized that the use of dynamic feeding could maintain nucleotide sugar concentrations since glycosylation site-occupancy is not affected by glucose or glutamine limitation. Since sialylation is the final terminal step of N-glycosylation, it would probably be more sensitive to substrate depletion. Another obvious cause of sialylation decrease could be a decreased number of complex species as well as molecular weight of complex glycans observed with glucose/glutamine limitation (Tables IV, V). Therefore, less complex type glycans are available for sialylation and, at the same time, the lower molecular masses meant that most of these complex glycans lack the necessary sugar chain extension for sialylation to proceed. Despite the lowered sialic acid content of low glutamine or glucose setpoint fed-batch, its high IFN-g yield makes it an attractive process for further development. Precursor feeding strategies could very well be effective in increasing its sialic acid content. The addition of 20 mM of ManNAc to CHO cells culture has been shown to be effective in improving sialylation by increasing intracellular CMPNeuAc availability (Gu and Wang, 1998). BIOTECHNOLOGY AND BIOENGINEERING, VOL. 89, NO. 2, JANUARY 20, 2005 Impact of Culture Viability on N-glycosylation Quality glycosidases could be degrading the glycans leading to shorter glycans of lower molecular weight. Losses in high molecular weight glycan species appears to be lower in batch and in 0.3/0.35 mM glutamine/glucose fed-batch where IFN-g yields were low. This suggests that degradation is not as significant with low IFN-g yields. When culture viability decreased, we found that IFN-g sialic acid content decreased as well. IFN-g molecules harvested at lower viability tend to have lower sialic acid content compared to those harvested at higher viability (Fig. 8B). Approximately 10– 20% decrease in mol sialic acid/mol IFN-g could be detected with viability drop. Sialic acid loss did not appear to be an issue where IFN-g yields were low, as seen by the relatively unchanged sialic acid content for batch and 0.3/0.35 mM glutamine/glucose setpoint fed-batch. Gramer and Goochee (1993) identified sialidase activity in CHO cell supernatant that has optimum activity at pH 5.5 but still has significant activity at pH 7.0. Since the pH of the reactor is controlled at 7.15, intracellular sialidase released into culture supernatant would still have significant desialylation activity. Previous work by Gu and Wang (1998) and Goldman et al. (1998) showed that an increase in sialidase activity followed viability loss closely in perfusion and stirred-tank CHO cell culture and sialylation was stable until the onset of cell death and lysis. We too have found that sialidase activity in culture supernatant increases with viability loss (data not shown). Although viability of fed-batch cultures typically dropped at around 96– 120 h, IFN-g production yields can still be improved by –10 times (Fig. 7A). However, degradative enzymes released during cell lysis could have detrimental effects on glycoprotein quality. We found that glycan siteoccupancy of IFN-g molecules harvested at low viability had very similar distribution to that of those harvested at high viability (Fig. 8A). This showed that harvesting at low viability had little impact on the macroheterogeneity of IFN-g. However, when the microheterogeneity of IFN-g glycans were examined, it was found that glycan species from low viability-harvested IFN-g tend to have lower molecular weight (Tables VI, VII). Generally, there were decreases in higher molecular weight glycan species coupled with increases in low molecular weight glycan species detected on Asn25 and Asn97 in both batch and fed-batch cultures. However, major glycan species, C08-F on Asn25 (Table IV) and C07 on Asn97 (Table V), was maintained despite viability drops. If this was due to synthesis efficiency defects, the previously high molecular weight glycans detectable at high viability, which have already been synthesized, should still be detectable at low viability. Since these high molecular weight species could no longer be detected, they were probably degraded. This suggested that extracellular Table VI. Microheterogeneity of IFN-g glycans on Asn25 harvested during low viability (70 – 80%). Fed-batch (mM) Glutamine/ glucose Glutamine Asn25 High mannose Hybrid Complex (bi-antennary) Complex (tri-antennary) Complex (tetra-antennary) ID M04 M05 M07 M08 H02 H04 H03-F H06-F C03-F C04-F C07-F C10-F C13-F C21-F C05-F C08-F C14-F C22-F C27-F C09-F C16-F Batch 0.1 0.3 + + + + 0.5 + + 0.3/0.35 + + 0.3/0.70 + + + + + + + + + + +++ + + + + + + + +++++ + +++++ + + + + + + + + + + +++++ + + + + + + +++++ + +++++ + + + Approximate quantification was obtained using relative peak intensity of mass spectrometry: (+: – 30%, +++: 30 – 60%, +++++: 60 – 90%). WONG ET AL.: DYNAMIC ONLINE FED-BATCH STRATEGIES 175 Table VII. Microheterogeneity of INF-g glycans on Asn97 harvested during low viability (70 – 80%). Fed-batch (mM) Glutamine/ glucose Glutamine Asn97 ID High mannose Hybrid Complex (bi-antennary) Complex (tri-antennary) Complex (tetra-antennary) M01 M02 M04 M05 M07 M08 H02 H03 H06 C01 C02 C03 C04 C07 C10 C13 C21 C05 C08 C11 C14 C15 C22 C09 C16 C20 C23 Batch 0.1 + + + 0.3 0.5 0.3/0.35 0.3/0.70 + + + + + + + + + + + + + + + + + + + +++ + + + + +++ +++ +++ + + + + +++ + + + + + + +++ +++ + + + + + + + + + + + + +++ + +++ + + + + + + + + + + + + + + + + + + +++ +++ + + + + + + + + + + + +++ + +++++ + +++ + + + + + + + +++++ + +++ + + + + + + + + + Approximate quantification was obtained using relative peak intensity of mass spectrometry: (+: – 30%, +++: 30 – 60%, +++++: 60 – 90%). These findings suggest that release of intracellular sialidase during cell death contributes significantly to sialic acid removal from sialylated N-glycans harvested. To prevent this, sialidase inhibitors can be added prior to cell lysis to prevent loss of sialic acid significantly (Gramer and Goochee, 1993; Gramer et al., 1995; Gu and Wang, 1998). This method, however, is not ideal for bioprocesses, as it involves the addition of extra chemicals during a process. Simpler alternatives to prevent sialic acid loss include optimization of media to prolong viability or termination of the culture prior to cell lysis especially if limited yield increase does not justify prolonging culture life. CONCLUSIONS Experimental data presented here demonstrate that dynamic glutamine or glutamine/glucose controls are effective strategies for enhancing cellular metabolism by decreasing metabolite waste production. This ultimately leads to higher viable cell density and prolonged viability, causing significant increases in glycoprotein productivity and yield. Feeding volumes recorded during glutamine 176 setpoint fed-batch could also be utilized for profile feeding, thereby removing the need for complicated on-line control systems. However, these strategies influence glycoprotein quality significantly, especially in terms of N-glycan microheterogeneity distribution and sialylation degree. It is obvious that the cell culture variables that affect glycosylation are as varied as they are complex. There is a need to consider possible extracellular factors that can influence enzyme activity or substrate availability as well as possible extracellular modification by cytolysis-associated glycosidases during process development for glycoprotein production. 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WONG ET AL.: DYNAMIC ONLINE FED-BATCH STRATEGIES 177 [...]... dynamic glutamine or glutamine/glucose controls are effective strategies for enhancing cellular metabolism by decreasing metabolite waste production This ultimately leads to higher viable cell density and prolonged viability, causing significant increases in glycoprotein productivity and yield Feeding volumes recorded during glutamine 176 setpoint fed- batch could also be utilized for profile feeding, thereby... NH, James DC, Ison AP, Bull AT, Jenkins N 1995 N-glycans of recombinant human interferon-gamma change during batch culture of Chinese hamster ovary cells Biotechnol Bioeng 48:639 – 648 Hooker AD, Green NH, Baines AJ, Bull AT, Jenkins N, Strange PG, James DC 1999 Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells Biotechnol Bioeng 63:559 –... Metabolic control of recombinant protein N-glycan processing in NS0 and CHO cells Biotechnol Bioeng 73:188 – 202 Cruz HJ, Moreira JL, Carrondo MJT 1999 Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells Biotechnol Bioeng 66:104 – 108 Europa AF, Gambhir A, Fu PC, Hu WS 2000 Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells Biotechnol... Precursor feeding strategies could very well be effective in increasing its sialic acid content The addition of 20 mM of ManNAc to CHO cells culture has been shown to be effective in improving sialylation by increasing intracellular CMPNeuAc availability (Gu and Wang, 1998) BIOTECHNOLOGY AND BIOENGINEERING, VOL 89, NO 2, JANUARY 20, 2005 Impact of Culture Viability on N-glycosylation Quality glycosidases... and cell yields on serum in cultures of mammalian cells Biotechnol Bioeng 28:1376 – 1389 Goldman MH, James DC, Rendall M, Ison AP, Hoare M, Bull AT 1998 Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells Biotechnol Bioeng 60:596 – 607 Goochee CF, Monica T 1990 Environmental effects on protein glycosylation Biotechnology... Effects of ammonium and lactate on growth and metabolism of CHO cell culture Biotechnol Prog 13:688 – 691 Lee YY, Yap MGS, Hu WS, Wong KTY 2003 Low-glutamine fed- batch cultures of 293-HEK serum-free suspension cells for adenovirus production Biotechnol Prog 19:501 – 509 Ljunggren J, Haggstrom L 1994 Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures Biotechnol... 1992 Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-g Biotechnol Bioeng 39:327 – 335 Hooker AD, James DC 1998 The glycosylation heterogeneity of recombinant human IFN-gamma J Interferon Cytokine Res 18:287 – 295 Hooker AD, James DC 2000 Analysis of glycoprotein heterogeneity by capillary electrophoresis and mass spectrometry Mol Biotechnol 14:241... glycan species coupled with increases in low molecular weight glycan species detected on Asn25 and Asn97 in both batch and fed- batch cultures However, major glycan species, C08-F on Asn25 (Table IV) and C07 on Asn97 (Table V), was maintained despite viability drops If this was due to synthesis efficiency defects, the previously high molecular weight glycans detectable at high viability, which have already... Viability on N-glycosylation Quality glycosidases could be degrading the glycans leading to shorter glycans of lower molecular weight Losses in high molecular weight glycan species appears to be lower in batch and in 0.3/0.35 mM glutamine/glucose fed- batch where IFN-g yields were low This suggests that degradation is not as significant with low IFN-g yields When culture viability decreased, we found that IFN-g... WL 1982 Gycolysis, glutaminolysis and cell proliferation Cell Biol Int Rep 6:635 – 649 Nyberg GB, Balcarcel RR, Follstad BD, Stephanapoulous G, Wang DIC 1999 Metabolic effects on recombinant interferon-g glycosylation in continuous culture of Chinese hamster ovary cells Biotechnol Bioeng 62:336 – 347 Parekh RB 1991 Mammalian cell gene expression: protein glycosylation Curr Opin Biotechnol 2:730 – 734 . site- occupied IFN-g in batch and fed- batch cultures. B: Sialic acid content of maximum IFN-g harvested during high viability, > 95% ( . ) and low viability, 70– 80% (D) in batch and fed- batch cultures. viability, causing significant increases in glycoprotein productiv- ity and yield. Feeding volumes recorded during glutamine setpoint fed- batch could also be utilized for profile feeding, thereby removing. glucose setpoint coupled with glutamine profile feeding (data points represent the averages of two runs). Figure 7. Recombinant human IFN-g production in CHO cells during batch and fed- batch cultures.