Effect of Bcl-xL Overexpression on Sialylation of Fc-Fusion Protein in Recombinant Chinese Hamster Ovary Cell Cultures Jong Hyun Lee Dept. of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea
Yeon-Gu Kim Biotechnology Process Engineering Center, KRIBB, Ochang 363-883, Republic of Korea
Gyun Min Lee Dept. of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea DOI 10.1002/btpr.2115 Published online June 26, 2015 in Wiley Online Library (wileyonlinelibrary.com)
The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl-xL overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc-fusion protein, which were derived from DUKX-B11 and DG44, respectively, were engineered to have regulated Bcl-xL overexpression using the Tet-off system. For both cell lines, Bcl-xL overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc-fusion protein titer increased by Bcl-xL overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl-xL overexpression, the sialylation of Fc-fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl-xL overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl-xL overexpression in rCHO cell culture increased Fc-fusion protein production and also reduced the impairment of sialylation of Fc-fusion protein by maintaining high viability durC 2015 American Institute of Chemical Engineers Biotechnol. Prog., ing batch cultures. V 31:1133–1137, 2015 Keywords: CHO cells, Fc-fusion protein, sialylation, apoptosis, Bcl-xL, inducible expression
Introduction Chinese hamster ovary (CHO) cells are one of the most popular hosts for therapeutic protein production. In the production of a therapeutic glycoprotein using recombinant CHO (rCHO) cells, sialylation, the attachment of sialic acid residues to a protein, is a critical factor in terms of biological functions such as solubility, thermal stability, biological activity, immunogenicity, and pharmacokinetics.1,2 Sialic acid, the terminal carbohydrate for N-linked complex glycan structures, generally increases the in vivo circulatory half-life of a glycoprotein because of its ability to prevent the degradation of glycoprotein by asialoglycoprotein receptors.3 Previously, it was suggested that the oligosaccharides of glycoproteins secreted by rCHO cells can potentially be impaired by sialidase released upon cell lysis during cell culture.4 In addition, a continuous loss of terminal sialic acids of interferon-c secreted by rCHO cells was observed coincident with the loss of
Additional Supporting Information may be found in the online version of this article. Correspondence concerning this article should be addressed to G. M. Lee at [email protected]. (or) Y.-G. Kim at [email protected]. C 2015 American Institute of Chemical Engineers V
cell viability in a batch culture, indicating that the decrease of sialylation after the loss of cell viability was owing to the extracellular accumulation of sialidase resulting from cell lysis.5 Therefore, to avoid the reduction in sialylation of glycoproteins by sialidase, it is important to minimize cell lysis by delaying the onset of cell death in rCHO cell cultures. During rCHO cell cultures, cell death is triggered by a variety of stresses including nutrient depletion, accumulated toxic byproducts, and shear stress.6 Bcl-xL, a member of the bcl-2 family, is a well-known antiapoptotic protein that inhibits the release of proapoptotic molecules from mitochondria. In rCHO cell culture, numerous reports have shown that Bcl-xL overexpression extended culture longevity by suppressing apoptotic cell death and resulted in improved glycoprotein production.7 However, the potential of Bcl-xL overexpression to prevent the reduction in the sialylation of glycoprotein, resulting from cell lysis in rCHO cell culture, has not been examined yet. In this study, we investigated the effect of Bcl-xL overexpression on the sialylation of glycoprotein in two rCHO cell lines producing the same Fc-fusion protein. The two cell lines were derived from DUKX-B11 and DG44, respectively. For Bcl-xL overexpression, we used the Tet-off system, a doxycycline-controlled expression system.8 1133
1134
Biotechnol. Prog., 2015, Vol. 31, No. 4
Figure 1. Batch cultures of Bcl-xL-off-DX and Bcl-xL-off-DG in the absence and the presence of doxycycline. (A) Inducible overexpression of Bcl-xL regulated by doxycycline during cultures of Bcl-xL-off-DX and (B) Bcl-xL-off-DG. (C) Culture profiles of cell growth, viability, and Fc-fusion protein production of Bcl-xL-off-DX and (D) Bcl-xL-off-DG. Cultures were performed in the presence of doxycycline (䊉) and the absence of doxycycline (䊊). The error bars represent the standard deviations calculated from three independent experiments.
Materials and Methods Cell line and culture maintenance The rCHO cell lines producing Fc-fusion protein (DUKX-Fc and DG44-Fc) were obtained from ISU ABXIS (Seoul, Korea). The stable rCHO cell lines, DUKX-Fc and DG44-Fc, were derived from DUKX-B11 (ATCC CRL-9096) and DG44, respectively. The Fc-fusion protein is a homodimer having three glycosylation sites in the head protein and one in the Fc region of human IgG4. The double-stable Tet-off cell lines overexpressing hamster Bcl-xL (Bcl-xL-off-DX and Bcl-xL-off-DG) were established as described previously.9 They were adapted to grow in suspension culture with a serum-free medium (SFM) (HyQ SFM4CHO; HyClone, Logan, UT) supplemented with 4 mM of glutamine (HyClone).
Corning, NY) containing 50 mL of SFM. The shake flasks (Corning, Corning, NY) were rotated at 110 rpm in a shaking CO2 incubator (Adolf Kuhner AG, Birsfelden, Switzerland) set at 50 mm shaking amplitude, 5% CO2, 85% humidity, and 378C. For a control culture without Bcl-xL overexpression, doxycycline was added to the medium at 1 lg/mL every 3 days. The samples were taken at indicated times. Culture supernatants, after centrifugation, were aliquoted and kept frozen at 2708C for further analyses. Evaluation of specific protein productivity The specific protein productivity (qp) was evaluated from a plot of the Fc-fusion protein concentration against the time integral values of the viable cell growth curve.10
Batch culture
Analytical methods
Exponentially growing cells were inoculated at a concentration of 3 3 105 cells/mL into 125-mL shake flasks (Corning,
Cell concentration was estimated using a hemocytometer. Viable cells were distinguished from dead cells using the
Biotechnol. Prog., 2015, Vol. 31, No. 4
1135
Figure 2. IEF gel analysis for purified Fc-fusion protein produced in (A) Bcl-xL-off-DX and (B) Bcl-xL-off-DG. The “1” and “2” denote the presence and absence of doxycycline, respectively. Relative sialic acid content of purified Fc-fusion protein produced in (C) Bcl-xL-off-DX and (D) Bcl-xL-off-DG in the presence (W) and absence of doxycycline (w). Culture supernatants were harvested at indicated times of the cultures shown in Figure 1. The error bars represent the standard deviations calculated from technical triplicate samples. *P 0.05.
trypan blue dye exclusion method. Secreted Fc-fusion protein concentration was quantified by an enzyme-linked immunosorbent assay (ELISA) as described previously.11 Western blot analysis Western blot analysis was performed as described previously.9 Antibodies used for Western blot analysis were anti-Bcl-xL antibody (54H6) (1:1,000, rabbit monoclonal, Cell Signaling Technology #2764) and anti-b-actin antibody (Clone AC-74) (1:5,000, mouse monoclonal, Sigma-Aldrich #A2228). Isoelectrofocusing gel analysis of purified Fc-fusion protein Culture supernatants were harvested at indicated times, and then centrifuged and filtrated to remove the cell debris. The Fc-fusion protein was purified byTMprotein A affinity chromatography using MabSelect SuRe (GE Healthcare) € and a chromatography system (AKTA explorer 100Air; GE Healthcare). Equal amounts of purified protein were analyzed on NovexV pH 3–7 isoelectrofocusing (IEF) Gels TM (Invitrogen) and the gel was stained with InstantBlue 11 (Expedeon) as described previously. R
Sialic acid content analysis The sialic acid content ofTMpurified Fc-fusion protein was determined using EnzyChrom sialic acid assay kit (BioAssay Systems, Hayward, CA) as described previously.12
Lactate dehydrogenase and sialidase activity assays Lactate dehydrogenase (LDH) activity in the culture supernatant was measured using a metabolite analyzer (Cedex Bio, Roche). The enzyme activity of extracellular sialidase was measured using a neuraminidase assay kit (ab138888, Abcam, Cambridge, MA) according to the manufacturer’s protocols.
Results and Discussion To investigate the effect of Bcl-xL overexpression on the sialylation of recombinant protein in rCHO cells, we established two Fc-fusion protein-producing rCHO cell lines with regulated expression of Bcl-xL (Bcl-xL-off-DX and Bcl-xL-off-DG) using the Tet-off system. Two cell lines, derived from DUKX-B11 and DG44, respectively, were employed to generalize the effect of Bcl-xL overexpression. In addition, a controlled expression system was used to exclude the possibility of clonal variability usually encountered in constitutive overexpression experiments. Western blot analysis revealed that the Bcl-xL of Bcl-xL-off-DX and Bcl-xL-off-DG was completely repressed at 1 lg/mL doxycycline (data not shown). To evaluate the effect of Bcl-xL overexpression on cell growth and Fc-fusion protein production, Bcl-xL-off-DX and Bcl-xL-off-DG cells were cultivated in the absence and the presence of 1 lg/mL doxycycline. For both cell lines, inducibility of Bcl-xL overexpression was sustained during batch culture (Figures 1A,B). Bcl-xL was overexpressed in the absence of doxycycline, and repressed in the presence of
1136
Biotechnol. Prog., 2015, Vol. 31, No. 4
Figure 3. Time-course changes in extracellular LDH activity in the culture medium of (A) Bcl-xL-off-DX and (B) Bcl-xL-off-DG in the presence of doxycycline (䊉) and the absence of doxycycline (䊊). Extracellular sialidase activity in the culture medium of (C) Bcl-xL-off-DX and (D) Bcl-xL-off-DG in the presence (W) and absence of doxycycline (w). Culture supernatants were harvested at indicated times of the cultures shown in Figure 1. The error bars represent the standard deviations calculated from three independent experiments. *P 0.05.
doxycycline. Consistent with a previous report,13 Bcl-xL overexpression in both cell lines did not significantly affect specific growth rate, maximum viable cell concentration, or qp, as shown by statistical analysis using the t-test (P > 0.05). In addition, the specific metabolites uptake or production rates of glucose (qGlc), glutamine (qGln), lactate (qLac), and ammonia (qAmm) were not significantly affected by Bcl-xL overexpression in either cell line (Supporting Information Table S1). However, Bcl-xL overexpression extended culture longevity in batch culture, and hence increased maximum glycoprotein production by 39% for Bcl-xL-off-DX and 10% for Bcl-xLoff-DG, respectively (Figures 1C,D). Cultivation with other Bcl-xL-overexpressing cell lines (Bcl-xL-off-DX#2 and Bcl-xL-off-DG#2) confirmed that the effect of Bcl-xL overexpression on cell growth and Fc-fusion protein production was not a feature unique to the specific clones used for this study (Supporting Information Figure S1). To determine the effect of Bcl-xL overexpression on the isoform distribution and sialic acid content of glycoprotein, Fc-fusion protein was purified from culture supernatants sampled at indicated times from the batch cultures shown in Figure 1 by protein A chromatography. The isoform distribution and the sialic acid content of purified Fc-fusion protein were determined by a Coomassie blueTM stained IEF gel analysis and an EnzyChrom sialic acid kit, respectively. As shown in Figures 2A,B, Bcl-xL overexpression did not affect the isoform distribution of Fc-fusion protein significantly until day 5 for Bcl-xL-off-DX and day 6 for Bcl-xLoff-DG. However, toward the end of batch culture, the proportion of acidic isoforms in Fc-fusion protein produced with Bcl-xL overexpression was higher than that without Bcl-xL overexpression for both cell lines. Similarly, the sialic
acid content in Fc-fusion protein on day 5 for Bcl-xL-off-DX and on day 6 for Bcl-xL-off-DG was not significantly affected by Bcl-xL overexpression (P > 0.05) (Figures 2C,D). The relative sialic acid content in Fc-fusion protein during batch cultures of Bcl-xL-off-DX and Bcl-xL-off-DG was obtained from the normalization with the sample on day 5 for Bcl-xL-off-DX with Bcl-xL overexpression and on day 6 for Bcl-xL-off-DG with Bcl-xL overexpression, respectively. In contrast, the sialic acid content of Fc-fusion protein with Bcl-xL overexpression was significantly higher than that without Bcl-xL overexpression toward the end of cultures for both cell lines (P 0.05). Thus, it was found that Bcl-xL overexpression could reduce the decrease in the proportion of acidic isoforms and sialic acid content of Fc-fusion protein produced in rCHO cells in batch cultures. To quantify the degree of cell lysis, the activities of LDH and sialidase released from plasma membrane-damaged cells in the culture supernatants were sampled at indicated times in batch cultures as shown in Figure 1 and measured. Sialidase is responsible for removing sialic acid on N-glycan residue of glycoprotein. Consistent with the changes in cell viability during batch cultures (Figures 1C,D), the LDH activity, regardless of Bcl-xL overexpression, remained low until day 5, and, thereafter, started to increase rapidly for both cell lines (Figures 3A,B). However, the LDH activity increased much more slowly in Bcl-xL-overexpressing cells, compared with Bcl-xL-nonoverexpressing cells. Unlike LDH activity, sialidase activity increased during the exponential phase of growth, suggesting that sialidase was also released from viable cells (Figures 3C,D). Bcl-xL overexpression did not affect sialidase activity significantly until day 6 for BclxL-off-DX and until day 5 for Bcl-xL-off-DG (P > 0.05). Afterward, the sialidase activity, like LDH activity, increased much
Biotechnol. Prog., 2015, Vol. 31, No. 4
more slowly in Bcl-xL-overexpressing cells, compared with Bcl-xL-nonoverexpressing cells. Thus, Bcl-xL overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures, and hence exerted a favorable influence on the sialylation of Fc-fusion protein. Besides, Bcl-xL overexpression may also positively influence the sialylation of Fc-fusion protein by affecting sialidase synthesis in the decline phase of growth. As shown in Figure 3, extracellular accumulation of sialidase, one of the glycosidases, in the culture supernatant during rCHO cell cultures was owing to the secretion from viable cells and/or release from dead cells upon cell lysis. There are four types of mammalian sialidases, Neu1 (lysosomes), Neu2 (cytosol), Neu3 (plasma membrane), and Neu4 (lysosomes and mitochondria).14 When gene expression of Neu2 in rCHO cells was reduced by RNAi, sialic acid content of recombinant human interferon gamma (rhIFN-c) was improved only when cells were in the death phase.15 In contrast, after knocking down Neu3 expression by siRNA, rhIFN- c sialylation was enhanced when cells were at both the live and the death phases, suggesting that Neu3 was secreted from viable cells.16 Accordingly, extracellular degradation of glycoprotein oligosaccharides in rCHO cells cannot be prevented completely by overexpressing Bcl-xL alone. Active secretion of glycosidases including sialidase from viable cells may also be minimized to further reduce the impairment of sialylation of glycoprotein. To do so, genetic engineering of N-glycosylation-related genes in CHO cells can be considered. Alternatively, culture condition can also be optimized to reduce the expression level of glycosidases including sialidase. Recently, it was reported that sodium butyrate concentration and ammonium concentration significantly affected the expression level of sialidase.11,17
Conclusions In conclusion, Bcl-xL overexpression in rCHO cell culture not only increased Fc-fusion protein production, but also reduced the impairment of sialylation of Fc-fusion protein by maintaining high viability during the culture.
Acknowledgements This research was supported in part by a grant from the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (MEST) (2011-0031962), the Fundamental R&D Program for Technology of World Premier Materials funded by the Ministry of Knowledge Economy (MKE) (10037842), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the MEST (2012R1A1A2007666).
1137
Literature Cited 1. Elliott S, Lorenzini T, Asher S, Aoki K, Brankow D, Buck L, Busse L, Chang D, Fuller J, Grant J, Hernday N, Hokum M, Hu S, Knudten A, Levin N, Komorowski R, Martin F, Navarro R, Osslund T, Rogers G, Rogers N, Trail G, Egrie J. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol 2003;21:414–421. 2. Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009;19: 936–949. 3. Weiss P, Ashwell G. The asialoglycoprotein receptor: properties and modulation by ligand. Prog Clin Biol Res 1989;300:169–184. 4. Gramer MJ, Goochee CF. Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant. Biotechnol Prog 1993;9:366–373. 5. Gu X, Harmon BJ, Wang DI. Site-and branch-specific sialylation of recombinant human interferon-c in Chinese hamster ovary cell culture. Biotechnol Bioeng 1997;55:390–398. 6. Arden N, Betenbaugh MJ. Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 2004;22:174–180. 7. Kim JY, Kim YG, Lee GM. CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 2012;93:917–930. 8. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 1992;89:5547–5551. 9. Kim YG, Kim JY, Mohan C, Lee GM. Effect of Bcl-xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient-deprived condition. Biotechnol Bioeng 2009;103:757–766. 10. Renard JM, Spagnoli R, Mazier C, Salles MF, Mandine E. Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotechnol Lett 1988;10:91–96. 11. Lee SM, Kim YG, Lee EG, Lee GM. Digital mRNA profiling of N-glycosylation gene expression in recombinant Chinese hamster ovary cells treated with sodium butyrate. J Biotechnol 2014;171:56–60. 12. Ha TK, Kim YG, Lee GM. Effect of lithium chloride on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture. Appl Microbiol Biotechnol 2014;98:9239–9248. 13. Kim YG, Lee GM. Bcl-xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 338C and 378C. Biotechnol Prog 2009;25:252–256. 14. Miyagi T, Yamaguchi K. Mammalian sialidases: physiological and pathological roles in cellular functions. Glycobiology 2012; 22:880–896. 15. Ngantung FA, Miller PG, Brushett FR, Tang GL, Wang DI. RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 2006;95:106–119. 16. Zhang M, Koskie K, Ross JS, Kayser KJ, Caple MV. Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells. Biotechnol Bioeng 2010;105:1094–1105. 17. Ha TK, Kim YG, Lee GM. Understanding of altered Nglycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting. Biotechnol Bioeng 2015. doi: 10.1002/bit.25568. [Epub ahead of print] Manuscript received Jan. 10, 2015, and revision received Mar. 8, 2015.
Yeon-Gu Kim Biotechnology Process Engineering Center, KRIBB, Ochang 363-883, Republic of Korea
Gyun Min Lee Dept. of Biological Sciences, KAIST, Daejeon 305-701, Republic of Korea DOI 10.1002/btpr.2115 Published online June 26, 2015 in Wiley Online Library (wileyonlinelibrary.com)
The sialic acid of glycoproteins secreted by recombinant Chinese hamster ovary (rCHO) cells can be impaired by sialidase under culture conditions which promote the extracellular accumulation of this enzyme. To investigate the effect of Bcl-xL overexpression on the sialylation of glycoproteins produced in rCHO cell culture, two rCHO cell lines producing the same Fc-fusion protein, which were derived from DUKX-B11 and DG44, respectively, were engineered to have regulated Bcl-xL overexpression using the Tet-off system. For both cell lines, Bcl-xL overexpression improved cell viability and extended culture longevity in batch cultures. As a result, a maximum Fc-fusion protein titer increased by Bcl-xL overexpression though the extent of titer enhancement differed between the two cell lines. With Bcl-xL overexpression, the sialylation of Fc-fusion protein, which was assessed by isoelectric focusing gel and sialic acid content analyses, decreased more slowly toward the end of batch cultures. This was because Bcl-xL overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures. Taken together, Bcl-xL overexpression in rCHO cell culture increased Fc-fusion protein production and also reduced the impairment of sialylation of Fc-fusion protein by maintaining high viability durC 2015 American Institute of Chemical Engineers Biotechnol. Prog., ing batch cultures. V 31:1133–1137, 2015 Keywords: CHO cells, Fc-fusion protein, sialylation, apoptosis, Bcl-xL, inducible expression
Introduction Chinese hamster ovary (CHO) cells are one of the most popular hosts for therapeutic protein production. In the production of a therapeutic glycoprotein using recombinant CHO (rCHO) cells, sialylation, the attachment of sialic acid residues to a protein, is a critical factor in terms of biological functions such as solubility, thermal stability, biological activity, immunogenicity, and pharmacokinetics.1,2 Sialic acid, the terminal carbohydrate for N-linked complex glycan structures, generally increases the in vivo circulatory half-life of a glycoprotein because of its ability to prevent the degradation of glycoprotein by asialoglycoprotein receptors.3 Previously, it was suggested that the oligosaccharides of glycoproteins secreted by rCHO cells can potentially be impaired by sialidase released upon cell lysis during cell culture.4 In addition, a continuous loss of terminal sialic acids of interferon-c secreted by rCHO cells was observed coincident with the loss of
Additional Supporting Information may be found in the online version of this article. Correspondence concerning this article should be addressed to G. M. Lee at [email protected]. (or) Y.-G. Kim at [email protected]. C 2015 American Institute of Chemical Engineers V
cell viability in a batch culture, indicating that the decrease of sialylation after the loss of cell viability was owing to the extracellular accumulation of sialidase resulting from cell lysis.5 Therefore, to avoid the reduction in sialylation of glycoproteins by sialidase, it is important to minimize cell lysis by delaying the onset of cell death in rCHO cell cultures. During rCHO cell cultures, cell death is triggered by a variety of stresses including nutrient depletion, accumulated toxic byproducts, and shear stress.6 Bcl-xL, a member of the bcl-2 family, is a well-known antiapoptotic protein that inhibits the release of proapoptotic molecules from mitochondria. In rCHO cell culture, numerous reports have shown that Bcl-xL overexpression extended culture longevity by suppressing apoptotic cell death and resulted in improved glycoprotein production.7 However, the potential of Bcl-xL overexpression to prevent the reduction in the sialylation of glycoprotein, resulting from cell lysis in rCHO cell culture, has not been examined yet. In this study, we investigated the effect of Bcl-xL overexpression on the sialylation of glycoprotein in two rCHO cell lines producing the same Fc-fusion protein. The two cell lines were derived from DUKX-B11 and DG44, respectively. For Bcl-xL overexpression, we used the Tet-off system, a doxycycline-controlled expression system.8 1133
1134
Biotechnol. Prog., 2015, Vol. 31, No. 4
Figure 1. Batch cultures of Bcl-xL-off-DX and Bcl-xL-off-DG in the absence and the presence of doxycycline. (A) Inducible overexpression of Bcl-xL regulated by doxycycline during cultures of Bcl-xL-off-DX and (B) Bcl-xL-off-DG. (C) Culture profiles of cell growth, viability, and Fc-fusion protein production of Bcl-xL-off-DX and (D) Bcl-xL-off-DG. Cultures were performed in the presence of doxycycline (䊉) and the absence of doxycycline (䊊). The error bars represent the standard deviations calculated from three independent experiments.
Materials and Methods Cell line and culture maintenance The rCHO cell lines producing Fc-fusion protein (DUKX-Fc and DG44-Fc) were obtained from ISU ABXIS (Seoul, Korea). The stable rCHO cell lines, DUKX-Fc and DG44-Fc, were derived from DUKX-B11 (ATCC CRL-9096) and DG44, respectively. The Fc-fusion protein is a homodimer having three glycosylation sites in the head protein and one in the Fc region of human IgG4. The double-stable Tet-off cell lines overexpressing hamster Bcl-xL (Bcl-xL-off-DX and Bcl-xL-off-DG) were established as described previously.9 They were adapted to grow in suspension culture with a serum-free medium (SFM) (HyQ SFM4CHO; HyClone, Logan, UT) supplemented with 4 mM of glutamine (HyClone).
Corning, NY) containing 50 mL of SFM. The shake flasks (Corning, Corning, NY) were rotated at 110 rpm in a shaking CO2 incubator (Adolf Kuhner AG, Birsfelden, Switzerland) set at 50 mm shaking amplitude, 5% CO2, 85% humidity, and 378C. For a control culture without Bcl-xL overexpression, doxycycline was added to the medium at 1 lg/mL every 3 days. The samples were taken at indicated times. Culture supernatants, after centrifugation, were aliquoted and kept frozen at 2708C for further analyses. Evaluation of specific protein productivity The specific protein productivity (qp) was evaluated from a plot of the Fc-fusion protein concentration against the time integral values of the viable cell growth curve.10
Batch culture
Analytical methods
Exponentially growing cells were inoculated at a concentration of 3 3 105 cells/mL into 125-mL shake flasks (Corning,
Cell concentration was estimated using a hemocytometer. Viable cells were distinguished from dead cells using the
Biotechnol. Prog., 2015, Vol. 31, No. 4
1135
Figure 2. IEF gel analysis for purified Fc-fusion protein produced in (A) Bcl-xL-off-DX and (B) Bcl-xL-off-DG. The “1” and “2” denote the presence and absence of doxycycline, respectively. Relative sialic acid content of purified Fc-fusion protein produced in (C) Bcl-xL-off-DX and (D) Bcl-xL-off-DG in the presence (W) and absence of doxycycline (w). Culture supernatants were harvested at indicated times of the cultures shown in Figure 1. The error bars represent the standard deviations calculated from technical triplicate samples. *P 0.05.
trypan blue dye exclusion method. Secreted Fc-fusion protein concentration was quantified by an enzyme-linked immunosorbent assay (ELISA) as described previously.11 Western blot analysis Western blot analysis was performed as described previously.9 Antibodies used for Western blot analysis were anti-Bcl-xL antibody (54H6) (1:1,000, rabbit monoclonal, Cell Signaling Technology #2764) and anti-b-actin antibody (Clone AC-74) (1:5,000, mouse monoclonal, Sigma-Aldrich #A2228). Isoelectrofocusing gel analysis of purified Fc-fusion protein Culture supernatants were harvested at indicated times, and then centrifuged and filtrated to remove the cell debris. The Fc-fusion protein was purified byTMprotein A affinity chromatography using MabSelect SuRe (GE Healthcare) € and a chromatography system (AKTA explorer 100Air; GE Healthcare). Equal amounts of purified protein were analyzed on NovexV pH 3–7 isoelectrofocusing (IEF) Gels TM (Invitrogen) and the gel was stained with InstantBlue 11 (Expedeon) as described previously. R
Sialic acid content analysis The sialic acid content ofTMpurified Fc-fusion protein was determined using EnzyChrom sialic acid assay kit (BioAssay Systems, Hayward, CA) as described previously.12
Lactate dehydrogenase and sialidase activity assays Lactate dehydrogenase (LDH) activity in the culture supernatant was measured using a metabolite analyzer (Cedex Bio, Roche). The enzyme activity of extracellular sialidase was measured using a neuraminidase assay kit (ab138888, Abcam, Cambridge, MA) according to the manufacturer’s protocols.
Results and Discussion To investigate the effect of Bcl-xL overexpression on the sialylation of recombinant protein in rCHO cells, we established two Fc-fusion protein-producing rCHO cell lines with regulated expression of Bcl-xL (Bcl-xL-off-DX and Bcl-xL-off-DG) using the Tet-off system. Two cell lines, derived from DUKX-B11 and DG44, respectively, were employed to generalize the effect of Bcl-xL overexpression. In addition, a controlled expression system was used to exclude the possibility of clonal variability usually encountered in constitutive overexpression experiments. Western blot analysis revealed that the Bcl-xL of Bcl-xL-off-DX and Bcl-xL-off-DG was completely repressed at 1 lg/mL doxycycline (data not shown). To evaluate the effect of Bcl-xL overexpression on cell growth and Fc-fusion protein production, Bcl-xL-off-DX and Bcl-xL-off-DG cells were cultivated in the absence and the presence of 1 lg/mL doxycycline. For both cell lines, inducibility of Bcl-xL overexpression was sustained during batch culture (Figures 1A,B). Bcl-xL was overexpressed in the absence of doxycycline, and repressed in the presence of
1136
Biotechnol. Prog., 2015, Vol. 31, No. 4
Figure 3. Time-course changes in extracellular LDH activity in the culture medium of (A) Bcl-xL-off-DX and (B) Bcl-xL-off-DG in the presence of doxycycline (䊉) and the absence of doxycycline (䊊). Extracellular sialidase activity in the culture medium of (C) Bcl-xL-off-DX and (D) Bcl-xL-off-DG in the presence (W) and absence of doxycycline (w). Culture supernatants were harvested at indicated times of the cultures shown in Figure 1. The error bars represent the standard deviations calculated from three independent experiments. *P 0.05.
doxycycline. Consistent with a previous report,13 Bcl-xL overexpression in both cell lines did not significantly affect specific growth rate, maximum viable cell concentration, or qp, as shown by statistical analysis using the t-test (P > 0.05). In addition, the specific metabolites uptake or production rates of glucose (qGlc), glutamine (qGln), lactate (qLac), and ammonia (qAmm) were not significantly affected by Bcl-xL overexpression in either cell line (Supporting Information Table S1). However, Bcl-xL overexpression extended culture longevity in batch culture, and hence increased maximum glycoprotein production by 39% for Bcl-xL-off-DX and 10% for Bcl-xLoff-DG, respectively (Figures 1C,D). Cultivation with other Bcl-xL-overexpressing cell lines (Bcl-xL-off-DX#2 and Bcl-xL-off-DG#2) confirmed that the effect of Bcl-xL overexpression on cell growth and Fc-fusion protein production was not a feature unique to the specific clones used for this study (Supporting Information Figure S1). To determine the effect of Bcl-xL overexpression on the isoform distribution and sialic acid content of glycoprotein, Fc-fusion protein was purified from culture supernatants sampled at indicated times from the batch cultures shown in Figure 1 by protein A chromatography. The isoform distribution and the sialic acid content of purified Fc-fusion protein were determined by a Coomassie blueTM stained IEF gel analysis and an EnzyChrom sialic acid kit, respectively. As shown in Figures 2A,B, Bcl-xL overexpression did not affect the isoform distribution of Fc-fusion protein significantly until day 5 for Bcl-xL-off-DX and day 6 for Bcl-xLoff-DG. However, toward the end of batch culture, the proportion of acidic isoforms in Fc-fusion protein produced with Bcl-xL overexpression was higher than that without Bcl-xL overexpression for both cell lines. Similarly, the sialic
acid content in Fc-fusion protein on day 5 for Bcl-xL-off-DX and on day 6 for Bcl-xL-off-DG was not significantly affected by Bcl-xL overexpression (P > 0.05) (Figures 2C,D). The relative sialic acid content in Fc-fusion protein during batch cultures of Bcl-xL-off-DX and Bcl-xL-off-DG was obtained from the normalization with the sample on day 5 for Bcl-xL-off-DX with Bcl-xL overexpression and on day 6 for Bcl-xL-off-DG with Bcl-xL overexpression, respectively. In contrast, the sialic acid content of Fc-fusion protein with Bcl-xL overexpression was significantly higher than that without Bcl-xL overexpression toward the end of cultures for both cell lines (P 0.05). Thus, it was found that Bcl-xL overexpression could reduce the decrease in the proportion of acidic isoforms and sialic acid content of Fc-fusion protein produced in rCHO cells in batch cultures. To quantify the degree of cell lysis, the activities of LDH and sialidase released from plasma membrane-damaged cells in the culture supernatants were sampled at indicated times in batch cultures as shown in Figure 1 and measured. Sialidase is responsible for removing sialic acid on N-glycan residue of glycoprotein. Consistent with the changes in cell viability during batch cultures (Figures 1C,D), the LDH activity, regardless of Bcl-xL overexpression, remained low until day 5, and, thereafter, started to increase rapidly for both cell lines (Figures 3A,B). However, the LDH activity increased much more slowly in Bcl-xL-overexpressing cells, compared with Bcl-xL-nonoverexpressing cells. Unlike LDH activity, sialidase activity increased during the exponential phase of growth, suggesting that sialidase was also released from viable cells (Figures 3C,D). Bcl-xL overexpression did not affect sialidase activity significantly until day 6 for BclxL-off-DX and until day 5 for Bcl-xL-off-DG (P > 0.05). Afterward, the sialidase activity, like LDH activity, increased much
Biotechnol. Prog., 2015, Vol. 31, No. 4
more slowly in Bcl-xL-overexpressing cells, compared with Bcl-xL-nonoverexpressing cells. Thus, Bcl-xL overexpression delayed the extracellular accumulation of sialidase activity by reducing cell lysis during batch cultures, and hence exerted a favorable influence on the sialylation of Fc-fusion protein. Besides, Bcl-xL overexpression may also positively influence the sialylation of Fc-fusion protein by affecting sialidase synthesis in the decline phase of growth. As shown in Figure 3, extracellular accumulation of sialidase, one of the glycosidases, in the culture supernatant during rCHO cell cultures was owing to the secretion from viable cells and/or release from dead cells upon cell lysis. There are four types of mammalian sialidases, Neu1 (lysosomes), Neu2 (cytosol), Neu3 (plasma membrane), and Neu4 (lysosomes and mitochondria).14 When gene expression of Neu2 in rCHO cells was reduced by RNAi, sialic acid content of recombinant human interferon gamma (rhIFN-c) was improved only when cells were in the death phase.15 In contrast, after knocking down Neu3 expression by siRNA, rhIFN- c sialylation was enhanced when cells were at both the live and the death phases, suggesting that Neu3 was secreted from viable cells.16 Accordingly, extracellular degradation of glycoprotein oligosaccharides in rCHO cells cannot be prevented completely by overexpressing Bcl-xL alone. Active secretion of glycosidases including sialidase from viable cells may also be minimized to further reduce the impairment of sialylation of glycoprotein. To do so, genetic engineering of N-glycosylation-related genes in CHO cells can be considered. Alternatively, culture condition can also be optimized to reduce the expression level of glycosidases including sialidase. Recently, it was reported that sodium butyrate concentration and ammonium concentration significantly affected the expression level of sialidase.11,17
Conclusions In conclusion, Bcl-xL overexpression in rCHO cell culture not only increased Fc-fusion protein production, but also reduced the impairment of sialylation of Fc-fusion protein by maintaining high viability during the culture.
Acknowledgements This research was supported in part by a grant from the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (MEST) (2011-0031962), the Fundamental R&D Program for Technology of World Premier Materials funded by the Ministry of Knowledge Economy (MKE) (10037842), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the MEST (2012R1A1A2007666).
1137
Literature Cited 1. Elliott S, Lorenzini T, Asher S, Aoki K, Brankow D, Buck L, Busse L, Chang D, Fuller J, Grant J, Hernday N, Hokum M, Hu S, Knudten A, Levin N, Komorowski R, Martin F, Navarro R, Osslund T, Rogers G, Rogers N, Trail G, Egrie J. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol 2003;21:414–421. 2. Hossler P, Khattak SF, Li ZJ. Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 2009;19: 936–949. 3. Weiss P, Ashwell G. The asialoglycoprotein receptor: properties and modulation by ligand. Prog Clin Biol Res 1989;300:169–184. 4. Gramer MJ, Goochee CF. Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant. Biotechnol Prog 1993;9:366–373. 5. Gu X, Harmon BJ, Wang DI. Site-and branch-specific sialylation of recombinant human interferon-c in Chinese hamster ovary cell culture. Biotechnol Bioeng 1997;55:390–398. 6. Arden N, Betenbaugh MJ. Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 2004;22:174–180. 7. Kim JY, Kim YG, Lee GM. CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol 2012;93:917–930. 8. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 1992;89:5547–5551. 9. Kim YG, Kim JY, Mohan C, Lee GM. Effect of Bcl-xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient-deprived condition. Biotechnol Bioeng 2009;103:757–766. 10. Renard JM, Spagnoli R, Mazier C, Salles MF, Mandine E. Evidence that monoclonal antibody production kinetics is related to the integral of the viable cells curve in batch systems. Biotechnol Lett 1988;10:91–96. 11. Lee SM, Kim YG, Lee EG, Lee GM. Digital mRNA profiling of N-glycosylation gene expression in recombinant Chinese hamster ovary cells treated with sodium butyrate. J Biotechnol 2014;171:56–60. 12. Ha TK, Kim YG, Lee GM. Effect of lithium chloride on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture. Appl Microbiol Biotechnol 2014;98:9239–9248. 13. Kim YG, Lee GM. Bcl-xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 338C and 378C. Biotechnol Prog 2009;25:252–256. 14. Miyagi T, Yamaguchi K. Mammalian sialidases: physiological and pathological roles in cellular functions. Glycobiology 2012; 22:880–896. 15. Ngantung FA, Miller PG, Brushett FR, Tang GL, Wang DI. RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 2006;95:106–119. 16. Zhang M, Koskie K, Ross JS, Kayser KJ, Caple MV. Enhancing glycoprotein sialylation by targeted gene silencing in mammalian cells. Biotechnol Bioeng 2010;105:1094–1105. 17. Ha TK, Kim YG, Lee GM. Understanding of altered Nglycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting. Biotechnol Bioeng 2015. doi: 10.1002/bit.25568. [Epub ahead of print] Manuscript received Jan. 10, 2015, and revision received Mar. 8, 2015.
