Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc
Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry Takeshi Okumura,1, 2, * Kenji Masuda,1 Kazuhiko Watanabe,1 Kenji Miyadai,1 Koichi Nonaka,1 Masayuki Yabuta,1 and Takeshi Omasa2 R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan1 and Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan2 Received 14 October 2014; accepted 7 January 2015 Available online xxx
To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2e1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4e4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Monoclonal antibody; Chinese hamster ovary cell; Cell line development; Flow cytometry; Side scatter; Forward scatter]
The Chinese hamster ovary (CHO) cell is widely used for the production of therapeutic proteins such as monoclonal antibody (mAb) (1e6). Many expression systems in CHO cells have been developed to improve the productivity of target proteins for reducing the cost of goods. In the current available expression systems, glutamine synthetase (GS) (7), dihydrofolate reductase (DHFR) (8,9) and some antibiotic resistance genes (10,11) are used as a selective marker to isolate a transfectant which harbors an expression vector. Also, promoters such as SV40 (12,13), CMV (14,15), and elongation factor-1 (EF-1) (16,17), and functional DNA elements that enable improvement of productivity and genetic stability such as ubiquitous chromatin opening elements (UCOE) (18,19) and matrix attachment regions (MAR) (20,21) are also used to construct an expression vector. However, the expression level and genetic stability of genes introduced in the isolated transfectant are unpredictable due to a position effect by a random integration in many cases (22,23). Screening of a desired cell that is genetically stable and shows higher productivity with preferable quality is generally laborious and time-consuming. On the other hand, site-specific recombination techniques might provide a predictable expression level and genetic stability of genes of interest (GOI) in a transfectant obtained from minimum screening for the
* Corresponding author at: R&D Division, Daiichi Sankyo Co., Ltd., Gunma, 3700503, Japan. Tel./fax: þ81 276 86 7357. E-mail address: [email protected] (T. Okumura).
isolation of a higher producing cell (24e28). However, the cells constructed by the site-specific recombination technique have not provided satisfactory productivity because productivity from a single copy of GOI is generally lower than that from amplified multiple copies of GOI (29). So far, a random integration technique has been widely applied to isolate high-producing cells with laborious screening although it is not possible to evaluate productivity in all obtained transfectants. Therefore, high throughput screening/evaluation systems are required to isolate a desired high producing cell. Flow cytometry (FCM) is able to perform a high throughput screening by continuous cell sorting based on intra- or extracellular fluorescein detection. In fact, some FCM-based screening strategies for the isolation of high-producing cells have been reported by some research groups (30e34). Meng et al. (30) and DeMaria et al. (31) suggested a method for selection of highproducing cell using coexpressed green fluorescent protein and cell surface protein CD20, respectively, as a reporter. It is an unfavorable method to express untargeted protein as impurity in biopharmaceutical manufacturing. Yoshikawara et al. (32) used fluorescein methotrexate, which is a chemical compound that is easy to remove and binds DHFR, as a reporter to screen high-producing cells. However, this method has efficacy only for the expression system that uses DHFR as a selective marker. Moreover, these three methods are an indirect method by using reporter. On the other hand, Brezinsky et al. (33) demonstrated that the secreted antibody as a target is transiently present on the cell surface and it can be
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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stained with a fluorescence anti-IgG antibody. It is also useful for screening high-producing cells, however, cell size is not regarded in this method. Shi et al. (34) focused on cell size and intracellular density, and set a gate based on forward scatter (FSC) versus side scatter (SSC) for screening, however, this was only for removing dead cells. We predicted that fluorescence intensity might have depended on the cell size because larger cells could present a significant amount of expressing mAb on a cell surface and we could improve the screening strategy to enrich high-producing cells by regarding cell size and intracellular density. In this study, we report a new effective screening strategy for the isolation of higher mAb producing cells using FCM by gating based on FSC and SSC. MATERIALS AND METHODS Host cell line CHOeO1, which is derived from CHOeK1 ATCC CCL61 (American Type Culture Collection, Manassas, VA, USA) and has been adapted to serum-free and suspension culture conditions, was used as a host cell. Cells used for transfection were maintained in CD-CHO (Life Technologies Corporation, Grand Island, NY, USA) with 4 mM alanyl-glutamine (Sigma, St. Louis, MO, USA), 0.2% anti-clumping agent (Life Technologies Corporation) at 37 C in a 5% CO2 atmosphere. Expression vector The double gene vector carrying light chain and heavy chain genes of an mAb, pDSLH4.1, was designed and constructed as an expression vector for a model mAb and used for transfection. An ampicillin resistance marker was used for plasmid propagation in Escherichia coli. A neomycin resistance gene was used for transfectant selection in CHO cells. Vector configuration is provided in supportive data (Fig. S1). Transfection and stable pool preparation The expression vector pDSLH4.1 was transfected to CHOeO1 using Neon Transfection System (Invitrogen, Carlsbad, CA, USA). The transfected cells were cultured in C/E medium, composed of CD-CHO (Life Technologies Corporation) with 40% (v/v) EX-CELL 325 PF (SAFC, St. Louis, MO, USA), 2% (v/v) CHO Feed Bioreactor Supplement (SAFC), 4 mM alanyl-glutamine (Sigma), 10 mg/L HT supplement (Life Technologies Corporation), and 0.2% anticlumping agent (Life Technologies Corporation) in a T-25 tissue culture flask at 37 C in a 5% CO2 atmosphere. Twenty-four hours after transfection, geneticin (Life Technologies Corporation) was added to the culture for transfectant selection. Six to eight days after transfection, the cells were transferred to a 125mL Erlenmeyer flask and cultured at 37 C in a 5% CO2 atmosphere. Fourteen days after transfection, a stable pool which showed a resistance to Geneticin was obtained. Flow cytometry Cell sorting was performed using BD FACSAria Fusion sorter (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). 2 108 cells were pelleted by centrifugation for 3 min at 200 g 4 C and suspended in cold wash buffer, phosphate-buffered saline (PBS) w/o calcium and magnesium (Life Technologies Corporation) with 2% (w/v) of bovine serum albumin (BSA) (Bovogen Biologicals, Essendon, Victoria, Australia). After recentrifugation, the resulting cell pellet was resuspended in cold wash buffer. The recovered cells were stained with 1328 mg fluorescein isothiocyanate (FITC)-conjugated Goat F(ab0 )2 Fragment Anti-Human IgG (H þ L) (Beckman Coulter Company, Marseille, France) or phycoerythrin (PE)-conjugated Goat F(ab0 )2 Fragment Anti-Human IgG (H þ L) (Beckman Coulter Company) for 30 min at 4 C. The stained cells were washed and resuspended with cold wash buffer at a density of 5 106 cells/mL. The resulting cells were sorted on BD FACSAria Fusion sorter using a laser diode emitting at 488 nm and detecting FITC emission with a 530/30 bandpass filter.
FIG. 1. Simple gate sorting of a stable pool by FCM. (A) A histogram of fluorescence intensity obtained from cells stained with a PE-conjugated anti-IgG antibody in the stable pool (SP-1). P1 means a fraction showing the top 10% fluorescence intensity. (B) Evaluation of final product concentration (closed bars) and viable cell density (open circles) in a fed-batch culture of unsorted (stable pool) and sorted cells (P1 gate fraction) in three stable pools (SP-1, -2, -3). stained with Guava Cell Cycle Reagent. The cell cycle in the stained cells was analyzed by Guava easyCyte HT (EMD Millipore Corporation).
RESULTS
Fed batch culture Sorted cells from a stable pool were evaluated by a fedbatch culture in a 125 mL Erlenmeyer flask. Cell count and viability analyses were conducted using Guava PCA (EMD Millipore Corporation, Hayward, CA, USA). Initial cell density was adjusted at 3 105 viable cells/mL in production culture of which the medium was custom-made basal media DA1 (Life Technologies Corporation) with 20 mM HEPES (Life Technologies Corporation) and 4 mM Lglutamine (Life Technologies Corporation). A production culture was performed at 37 C and 120 rpm in a 5% CO2 atmosphere for 14 days. Custom-made feed media DAFM3 (Life Technologies Corporation) was added to the production culture at 10% of the working volume on days 4, 6, 8, and 10. Cell density analyzed by Guava easyCyte HT (EMD Millipore Corporation), viability, and antibody concentrations analyzed by ProteinA-HPLC were monitored during cultivation.
Cell sorting by simple gating After pDSLH4.1 was transfected to CHOeO1, a stable pool that has a huge variety of GOI expression levels was obtained under selection pressure by Geneticin and sorted by FCM. Transfection was conducted three times, and three stable pools named SP-1, -2, -3 were obtained and sorted. The fraction (P1 gate) which showed the top 10% fluorescence intensity in the cells stained with PE-conjugated anti-IgG antibody was collected and independently expanded (Fig. 1A). The mAb final product concentration of the expanded P1 gate fraction was compared to that of the unsorted cells in a fed-batch culture. The final product concentration of the sorted cells (P1) was 1.2e1.3-times higher than that of unsorted cells (Fig. 1B).
Cell cycle analysis Cell cycle analysis was performed by a flow cytometer with Guava Cell Cycle Reagent (EMD Millipore Corporation) according to the manufacturer’s instructions. Tested cells were pelleted by centrifugation and suspended in PBS. The recovered cell pellet was resuspended in residual PBS and fixed in icecold 70% ethanol for an hour at 4 C. The fixed cells were washed by PBS and
Cell sorting by gating based on FSC and SSC Fig. 1, the P1 gating fraction, showing the top 10% intensity, gave us the possibility to isolate the producing cells. However, fluorescence intensity
As shown in fluorescence higher mAb might have
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
VOL. xx, 2015 depended on the cell size because bigger cells could present a significant amount of mAb expression on a cell surface. If our assumption was correct, a different parameter set would be useful for screening mAb high-producing cells. To develop a screening process by FCM, it was hypothesized that fluorescence intensity obtained from a cell surface had a correlation with not only mAb productivity but also with the FSC and SSC of a cell. The cells stained with FITC-conjugated anti-IgG antibody were equally divided by the SSC value on the basis of SSC-area vs. FSCarea dot plots (Fig. 2). Then, each fraction was divided into quarters for further FSC-area gating. Gates P3, P4, P5, and P6 were arranged in order of FSC-area increments. Gates P7, P8, P9, and P10 were completed in order, in the same manner. The top 5% fluorescence intensity in gate P3 was fractionated to gate P11. The same gating manner was adapted to isolate gates P12, P13, P14, P15, P16, P17, and P18 from the P4, P5, P6, P7, P8, P9, and P10, respectively (Fig. 2A, B). From each gates, 15e25 104 cells were recovered. The means of the FSC-area, SSC-area and FITC-area against each value of unsorted cells in SP-1 are shown in Table 1. The mean of the FSCarea increased from P11 to P14 and from P15 to P18 as gated. The mean of the SSC-area from P11 to P14 also increased, however the SSC-area from P15 to P18 was almost constant. The mean of the FITC-area of P14 was the highest in all sorted cells. The FITC-area
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TABLE 1. Relative value of areas sorted by FSC, SSC and FITC in SP-1. Group
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FITC
100 60 93 133 174 25 75 98 138
100 223 269 294 344 46 72 72 73
100 1461 1194 1349 1551 269 85 83 96
tended to increase with the FSC-area increment in P11eP14 and P14 showed the highest FITC-area in all sorted cells. On the other hand, the trend in the P15eP18 was not investigated and the FITCarea of P15 was the highest in the group. The final product concentration of unsorted cells and eight sorted cells (named P11, P12, P13, P14, P15, P16, P17 and P18) were evaluated by fed-batch culture for three stable pools (Fig. 2C). All sorted cells indicated higher final product concentration than unsorted cells. Among them, P15, which showed the smallest value of
FIG. 2. Gating strategy of a stable pool sorting by FCM. (A) Dot plots of cells gated based on the SSC-area and FSC-area and a fractionated area to isolate gates P3, P4, P5, P6, P7, P8, P9, and P10 in the stable pool (SP-1). (B) Gating strategy of P11 included in the top 5% fluorescence intensity of P3 in SP-1. The same gating manner was adapted to isolate gates P12, P13, P14, P15, P16, P17, and P18 from the P4, P5, P6, P7, P8, P9, and P10, respectively. (C) Final product concentration (closed bars) and viable cell density (open circles) in a fed-batch culture of unsorted cells and eight sorted fractions in three stable pools (SP-1, -2, -3).
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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TABLE 2. Cell-cycle phase ratio of G0/G1, S, and G2/M phases in SP-1. Group
Unsorted P3 P4 P5 P6 P7 P8 P9 P10
Percentage of each cell cycle (%) G0/G1 phase
S phase
G2/M phase
49 35 41 51 45 68 73 67 70
21 22 21 18 20 21 17 19 19
30 43 38 32 35 11 11 14 11
FSC-area and SSC-area in the P11eP18, indicated the highest final product concentration among them. Especially, the P15 showed 3.4e4.7-fold higher final product concentration than unsorted cells. Cell cycle analysis of cell fractions gated based on FSC and SSC The cell cycle of unsorted cells and sorted cells, gates P3, P4, P5, P6, P7, P8, P9 and P10, were investigated to analyze the cell state at the moment of FSC-/SSC-area gating. The ratio of cells in G0/G1, S, and G2/M in SP-1 is shown in Table 2. The cell-cycle phase ratios of G0/G1, S, and G2/M in unsorted cells showed 49, 21 and 30%, respectively. Higher SSC fractions, P3eP6, included fewer cells in the G0/G1 phase and many more cells in the G2/M phase. On the other hand, lower SSC-area fractions, P7eP10, included a larger amount of cells in the G0/G1 phase and fewer cells in the G2/M phase. However, there was no correlation between the cellcycle phase and value of the FSC-area. Enrichment of mAb-high producing cells by multiplerounds of FCM-based sorting The FCM-sorted fraction, P15, showed the highest final product concentration among all other fractions. It was speculated that mAb-high producing cells were included in the FCM-sorted fraction, P15. To isolate a higher fraction by multiple-rounds of sorting, we performed two more rounds of sorting in the same manner by fractionating P15 in SP1. The 2- and 3-round sorting fractions were named P15eP15 and P15eP15eP15, respectively. The final product concentration after multiple-rounds of sorting was evaluated in a fed-batch culture as shown in Fig. 3. The final product concentration was enhanced by multiple-rounds of sorting and the P15eP15eP15 represented 8.5-fold final product concentration in comparison to unsorted cells. To compare detailed characteristics of sorted cells, the SSC, FSC and FITC of each cell were analyzed by a flow cytometer. The distribution of unsorted and sorted cells are dot-plotted in Fig. 4A based on the SSC-area vs. FSC-area. Although the sorted cells were collected as the smallest size of the FSC-area and the lowest density of the SSC-area, cells sorted in the SSC-FSC manner showed almost the same distribution as unsorted cells after cell expansion. The FITC-area distribution of unsorted and sorted cells is shown in Fig. 4B, in a histogram format. The FITC-area was upper-shifted after multiple-rounds of sorting. DISCUSSION In this study, our goal is to demonstrate that our new method using high-throughput equipment, FCM, is useful to enrich highproducing cells and to reduce efforts for screening. However, the highest mAb-producing cell could not be isolated by a simple gating procedure based on higher fluorescence intensity (Fig. 1). On the other hand, it was found that a grouping/gating strategy, which had never been conducted before, on the basis of FSC and SSC worked well to enrich higher mAb producers (Fig. 2). The
FIG. 3. The final product concentration of multiple-round sorted cells in SP-1. Final product concentration (closed bars) and viable cell density (open circles) in a fed-batch culture of multiple-round sorted cells (P15, P15eP15 and P15eP15eP15) was graded as a value relative to that of unsorted cells.
FCM-based P15 fraction, which showed the smallest size of the FSC-area and the lowest density of the SSC-area, indicated the highest final product concentration in a fed-batch culture. The FITC-area of the P15 fraction was surprisingly lower than those of other fractions. When enriching higher producing cells, not only the FITC-area of the cells but also the FSC-area and SSC-area of the cells should be considered. As shown in Table 2, higher SSC-area fractions, P3eP6, included fewer cells in the G0/G1 phase and many more cells in the G2/M phase. On the other hand, lower SSCarea fractions, P7eP10, included larger amount of cells in the G0/ G1 phase and fewer cells in the G2/M phase. This means that the cell cycle is synchronized at the moment of FSC-/SSC-area gating. In addition, multiple rounds of cell sorting in this manner were adapted for more efficient enrichment of the highest mAb producing cells as shown in Fig. 3. About 8.5-fold final product concentration was shown in a fed-batch culture after 3 rounds of FCM-based enrichment from SP-1. We also tried multiple rounds of cell sorting from SP-2, SP-3 and an additional round of cell sorting from P15eP15eP15, but higher producing cells were not enriched any more (data not shown). This phenomenon would be explained as follows: higher producer enrichment from above experiments reached a saturation point at around 1500 mg/L under current conditions such as expression vector, host and production media and an enrichment efficiency was not a uniform state as expected. The distribution of the FSC-area and SSC-area of cells represents a variety and complexity of cellular morphology including the cell cycle as shown in Table 2, and currently, cellular morphology of sorted cells only reflects a partial cell state of all cell states. This might be a reason why cells sorted in the SSC-FSC manner showed almost the same distribution as unsorted cells after cell expansion (Fig. 4), although the sorted cells were collected at the smallest size of the FSC-area and the lowest density of the SSC-area. The peak of the FITC-area was upper-shifted after multiple-rounds of sorting. This result allows us to expect that the specific production rate of sorted cells might have been improved after multiple-rounds of sorting and also the final product concentration has been improved by multiple-rounds of sorting.
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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FIG. 4. SSC-area vs. FSC-area dot plots and an FITC-area histogram of unsorted and multiple-round sorted cells in SP-1. (A) SSC-area vs. FSC-area dot plots of unsorted cells (top), P15 (upper-middle), P15eP15 (lower-middle), and P15eP15eP15 (bottom) were plotted. (B) A distribution of means of the FITC-area of unsorted cells (top), P15 (upper-middle), P15eP15 (lower-middle), and P15eP15eP15 (bottom) in a histogram were plotted.
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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Our results for the P15 fraction, which showed the smallest size of the FSC-area and the lowest density of the SSC-area, indicated the highest final product concentration and proved, contrary to our expectations, that the final product concentration of all fractions would be almost at the same level. Another two results, in which different CHO cell lines with the same expression vector pDSLH4.1 and different expression vector carrying a different antibody gene were used, showed that the P15 fraction in a fed-batch culture indicated the highest product concentration among all fractions (data not shown). This phenomenon, as shown in supportive data (Fig. S2), suggested a negative correlation between the FSC-/SSCarea and mAb production. These results suggested that it might have been general manner and our proposed new screening procedure might have been applicable to another CHO expression systems. In general, the cell growth rate of high-producing cells is slow, because its energy is used for production more than growth. The cell volume and cell weight change along with changes in the cell culture conditions and specific growth rate (35,36). It is reported that there is a close relationship among the chromosomal number, cell size, and specific growth rate (37). Because SSC indicates intracellular density, it is thought that there are some differences in the organelle relating protein secretion such as an endoplasmic reticulum and Golgi body among each sorted cell from a stable pool. Therefore, we need further investigations to clarify the causal connections between productivity and cell size, productivity and intracellular density by evaluation of cell proliferation, chromosomal karyotype confirmed by multicolor fluorescence in situ hybridization, and observation of organelles by a transmission electron microscope (TEM). The result that there was a difference in cell cycles between higher SSC-area fractions and lower SSC-area fractions might be an important clue for further investigation of this phenomenon. We concluded that high-producing cells would be enriched in the fraction having the smallest value of the FSC-area and SSC-area, and our new screening procedure based on FSC and SSC gates by using FCM could be a powerful tool for the isolation of higher-producing cells. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2015.01.007. ACKNOWLEDGMENTS This work is partially supported by project focused on developing key technology of discovering and manufacturing drug for next-generation treatment and diagnosis. The authors are grateful to Ms. Keiko Okado in the Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. for served as scientific advisors and Mr. Naoto Ishikawa in the Biologics Technology Research Laboratories, Daiichi Sankyo Co., Ltd. for collected data. References 1. Werner, R. G., Noé, W., Kopp, K., and Schlüter, M.: Appropriate mammalian expression systems for biopharmaceuticals, Arzneimittelforschung, 48, 870e880 (1998). 2. Chu, L. and Robinson, D. K.: Industrial choices for protein production by largescale cell culture, Curr. Opin. Biotechnol., 12, 180e187 (2001). 3. Andersen, D. C. and Krummen, L.: Recombinant protein expression for therapeutic applications, Curr. Opin. Biotechnol., 13, 117e123 (2002). 4. Andersen, D. C. and Reilly, D. E.: Production technologies for monoclonal antibodies and their fragments, Curr. Opin. Biotechnol., 15, 456e462 (2004). 5. Yoon, S. K., Hong, J. K., Choo, S. H., Song, J. Y., Park, H. W., and Lee, G. M.: Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production, J. Biotechnol., 122, 463e472 (2006). 6. Rita Costa, A., Elisa Rodrigues, M., Henriques, M., Azeredo, J., and Oliveira, R.: Guidelines to cell engineering for monoclonal antibody production, Eur. J. Pharm. Biopharm., 74, 127e138 (2010).
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USA, 93, 9067e9072 (1996). 26. Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A., Kerr, W. G., and Soriano, P.: Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells, Proc. Natl. Acad. Sci. USA, 94, 3789e3794 (1997). 27. Irion, S., Luche, H., Gadue, P., Fehling, H. J., Kennedy, M., and Keller, G.: Identification and targeting of the ROSA26 locus in human embryonic stem cells, Nat. Biotechnol., 25, 1477e1482 (2007). 28. Wirth, D., Gama-Norton, L., Riemer, P., Sandhu, U., Schucht, R., and Hauser, H.: Road to precision: recombinase-based targeting technologies for genome engineering, Curr. Opin. Biotechnol., 18, 411e419 (2007). 29. Obayashi, H., Kawabe, Y., Makitsubo, H., Watanabe, R., Kameyama, Y., Huang, S., Takenouchi, Y., Ito, A., and Kamihira, M.: Accumulative gene integration into a pre-determined site using Cre/loxP, J. Biosci. Bioeng., 113, 381e388 (2012). 30. Meng, Y. G., Liang, J., Wong, W. L., and Chisholm, V.: Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells, Gene, 242, 201e207 (2000). 31. DeMaria, C. T., Cairns, V., Schwarz, C., Zhang, J., Guerin, M., Zuena, E., Estes, S., and Karey, K. P.: Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen, Biotechnol. Prog., 23, 465e472 (2007). 32. Yoshikawa, T., Nakanishi, F., Ogura, Y., Oi, D., Omasa, T., Katakura, Y., Kishimoto, M., and Sugaet, K.: Flow cytometry: an improved method for the
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VOL. xx, 2015 selection of highly productive gene-amplified CHO cells using flow cytometry, Biotechnol. Bioeng., 74, 435e442 (2001). 33. Brezinsky, S. C., Chiang, G., Szilvasi, A., Mohan, S., Shapiro, R., MacLean, A., Sisk, W., and Thillet, G.: A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity, J. Immunol. Methods, 277, 141e155 (2003). 34. Shi, S., Condon, R. G., Deng, L., Saunders, J., Hung, F., Tsao, Y. S., and Liu, Z.: A high-throughput automated platform for the development of manufacturing cell lines for protein therapeutics, J. Vis. Exp., 55, 3e7 (2011). 35. Frame, K. K. and Hu, W. S.: Cell volume measurement as an estimation of mammalian cell biomass, Biotechnol. Bioeng., 36, 191e197 (1990).
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36. Omasa, T., Furuichi, K., Iemura, T., Katakura, Y., Kishimoto, M., and Suga, K.: Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture, Bioprocess Biosyst. Eng., 33, 117e125 (2010). 37. Takahashi, M., Kimura, S., Haghparast, S. M., Cao, Y., Itoi, T., Shirai, A., Onitsuka, M., and Omasa, T.: Relationship between chromosomal instability and cell characterization in Chinese hamster ovary cell lines, p. 108, in: Abstracts of the 25th Japanese Association for Animal Cell Technology (JAACT) Meeting, Nagoya. Japanese Association for Animal Cell Technology, Tokyo (2012).
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Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry Takeshi Okumura,1, 2, * Kenji Masuda,1 Kazuhiko Watanabe,1 Kenji Miyadai,1 Koichi Nonaka,1 Masayuki Yabuta,1 and Takeshi Omasa2 R&D Division, Daiichi Sankyo Co., Ltd., Gunma 370-0503, Japan1 and Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan2 Received 14 October 2014; accepted 7 January 2015 Available online xxx
To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2e1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4e4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Monoclonal antibody; Chinese hamster ovary cell; Cell line development; Flow cytometry; Side scatter; Forward scatter]
The Chinese hamster ovary (CHO) cell is widely used for the production of therapeutic proteins such as monoclonal antibody (mAb) (1e6). Many expression systems in CHO cells have been developed to improve the productivity of target proteins for reducing the cost of goods. In the current available expression systems, glutamine synthetase (GS) (7), dihydrofolate reductase (DHFR) (8,9) and some antibiotic resistance genes (10,11) are used as a selective marker to isolate a transfectant which harbors an expression vector. Also, promoters such as SV40 (12,13), CMV (14,15), and elongation factor-1 (EF-1) (16,17), and functional DNA elements that enable improvement of productivity and genetic stability such as ubiquitous chromatin opening elements (UCOE) (18,19) and matrix attachment regions (MAR) (20,21) are also used to construct an expression vector. However, the expression level and genetic stability of genes introduced in the isolated transfectant are unpredictable due to a position effect by a random integration in many cases (22,23). Screening of a desired cell that is genetically stable and shows higher productivity with preferable quality is generally laborious and time-consuming. On the other hand, site-specific recombination techniques might provide a predictable expression level and genetic stability of genes of interest (GOI) in a transfectant obtained from minimum screening for the
* Corresponding author at: R&D Division, Daiichi Sankyo Co., Ltd., Gunma, 3700503, Japan. Tel./fax: þ81 276 86 7357. E-mail address: [email protected] (T. Okumura).
isolation of a higher producing cell (24e28). However, the cells constructed by the site-specific recombination technique have not provided satisfactory productivity because productivity from a single copy of GOI is generally lower than that from amplified multiple copies of GOI (29). So far, a random integration technique has been widely applied to isolate high-producing cells with laborious screening although it is not possible to evaluate productivity in all obtained transfectants. Therefore, high throughput screening/evaluation systems are required to isolate a desired high producing cell. Flow cytometry (FCM) is able to perform a high throughput screening by continuous cell sorting based on intra- or extracellular fluorescein detection. In fact, some FCM-based screening strategies for the isolation of high-producing cells have been reported by some research groups (30e34). Meng et al. (30) and DeMaria et al. (31) suggested a method for selection of highproducing cell using coexpressed green fluorescent protein and cell surface protein CD20, respectively, as a reporter. It is an unfavorable method to express untargeted protein as impurity in biopharmaceutical manufacturing. Yoshikawara et al. (32) used fluorescein methotrexate, which is a chemical compound that is easy to remove and binds DHFR, as a reporter to screen high-producing cells. However, this method has efficacy only for the expression system that uses DHFR as a selective marker. Moreover, these three methods are an indirect method by using reporter. On the other hand, Brezinsky et al. (33) demonstrated that the secreted antibody as a target is transiently present on the cell surface and it can be
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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stained with a fluorescence anti-IgG antibody. It is also useful for screening high-producing cells, however, cell size is not regarded in this method. Shi et al. (34) focused on cell size and intracellular density, and set a gate based on forward scatter (FSC) versus side scatter (SSC) for screening, however, this was only for removing dead cells. We predicted that fluorescence intensity might have depended on the cell size because larger cells could present a significant amount of expressing mAb on a cell surface and we could improve the screening strategy to enrich high-producing cells by regarding cell size and intracellular density. In this study, we report a new effective screening strategy for the isolation of higher mAb producing cells using FCM by gating based on FSC and SSC. MATERIALS AND METHODS Host cell line CHOeO1, which is derived from CHOeK1 ATCC CCL61 (American Type Culture Collection, Manassas, VA, USA) and has been adapted to serum-free and suspension culture conditions, was used as a host cell. Cells used for transfection were maintained in CD-CHO (Life Technologies Corporation, Grand Island, NY, USA) with 4 mM alanyl-glutamine (Sigma, St. Louis, MO, USA), 0.2% anti-clumping agent (Life Technologies Corporation) at 37 C in a 5% CO2 atmosphere. Expression vector The double gene vector carrying light chain and heavy chain genes of an mAb, pDSLH4.1, was designed and constructed as an expression vector for a model mAb and used for transfection. An ampicillin resistance marker was used for plasmid propagation in Escherichia coli. A neomycin resistance gene was used for transfectant selection in CHO cells. Vector configuration is provided in supportive data (Fig. S1). Transfection and stable pool preparation The expression vector pDSLH4.1 was transfected to CHOeO1 using Neon Transfection System (Invitrogen, Carlsbad, CA, USA). The transfected cells were cultured in C/E medium, composed of CD-CHO (Life Technologies Corporation) with 40% (v/v) EX-CELL 325 PF (SAFC, St. Louis, MO, USA), 2% (v/v) CHO Feed Bioreactor Supplement (SAFC), 4 mM alanyl-glutamine (Sigma), 10 mg/L HT supplement (Life Technologies Corporation), and 0.2% anticlumping agent (Life Technologies Corporation) in a T-25 tissue culture flask at 37 C in a 5% CO2 atmosphere. Twenty-four hours after transfection, geneticin (Life Technologies Corporation) was added to the culture for transfectant selection. Six to eight days after transfection, the cells were transferred to a 125mL Erlenmeyer flask and cultured at 37 C in a 5% CO2 atmosphere. Fourteen days after transfection, a stable pool which showed a resistance to Geneticin was obtained. Flow cytometry Cell sorting was performed using BD FACSAria Fusion sorter (Becton, Dickinson and Company, BD Biosciences, San Jose, CA, USA). 2 108 cells were pelleted by centrifugation for 3 min at 200 g 4 C and suspended in cold wash buffer, phosphate-buffered saline (PBS) w/o calcium and magnesium (Life Technologies Corporation) with 2% (w/v) of bovine serum albumin (BSA) (Bovogen Biologicals, Essendon, Victoria, Australia). After recentrifugation, the resulting cell pellet was resuspended in cold wash buffer. The recovered cells were stained with 1328 mg fluorescein isothiocyanate (FITC)-conjugated Goat F(ab0 )2 Fragment Anti-Human IgG (H þ L) (Beckman Coulter Company, Marseille, France) or phycoerythrin (PE)-conjugated Goat F(ab0 )2 Fragment Anti-Human IgG (H þ L) (Beckman Coulter Company) for 30 min at 4 C. The stained cells were washed and resuspended with cold wash buffer at a density of 5 106 cells/mL. The resulting cells were sorted on BD FACSAria Fusion sorter using a laser diode emitting at 488 nm and detecting FITC emission with a 530/30 bandpass filter.
FIG. 1. Simple gate sorting of a stable pool by FCM. (A) A histogram of fluorescence intensity obtained from cells stained with a PE-conjugated anti-IgG antibody in the stable pool (SP-1). P1 means a fraction showing the top 10% fluorescence intensity. (B) Evaluation of final product concentration (closed bars) and viable cell density (open circles) in a fed-batch culture of unsorted (stable pool) and sorted cells (P1 gate fraction) in three stable pools (SP-1, -2, -3). stained with Guava Cell Cycle Reagent. The cell cycle in the stained cells was analyzed by Guava easyCyte HT (EMD Millipore Corporation).
RESULTS
Fed batch culture Sorted cells from a stable pool were evaluated by a fedbatch culture in a 125 mL Erlenmeyer flask. Cell count and viability analyses were conducted using Guava PCA (EMD Millipore Corporation, Hayward, CA, USA). Initial cell density was adjusted at 3 105 viable cells/mL in production culture of which the medium was custom-made basal media DA1 (Life Technologies Corporation) with 20 mM HEPES (Life Technologies Corporation) and 4 mM Lglutamine (Life Technologies Corporation). A production culture was performed at 37 C and 120 rpm in a 5% CO2 atmosphere for 14 days. Custom-made feed media DAFM3 (Life Technologies Corporation) was added to the production culture at 10% of the working volume on days 4, 6, 8, and 10. Cell density analyzed by Guava easyCyte HT (EMD Millipore Corporation), viability, and antibody concentrations analyzed by ProteinA-HPLC were monitored during cultivation.
Cell sorting by simple gating After pDSLH4.1 was transfected to CHOeO1, a stable pool that has a huge variety of GOI expression levels was obtained under selection pressure by Geneticin and sorted by FCM. Transfection was conducted three times, and three stable pools named SP-1, -2, -3 were obtained and sorted. The fraction (P1 gate) which showed the top 10% fluorescence intensity in the cells stained with PE-conjugated anti-IgG antibody was collected and independently expanded (Fig. 1A). The mAb final product concentration of the expanded P1 gate fraction was compared to that of the unsorted cells in a fed-batch culture. The final product concentration of the sorted cells (P1) was 1.2e1.3-times higher than that of unsorted cells (Fig. 1B).
Cell cycle analysis Cell cycle analysis was performed by a flow cytometer with Guava Cell Cycle Reagent (EMD Millipore Corporation) according to the manufacturer’s instructions. Tested cells were pelleted by centrifugation and suspended in PBS. The recovered cell pellet was resuspended in residual PBS and fixed in icecold 70% ethanol for an hour at 4 C. The fixed cells were washed by PBS and
Cell sorting by gating based on FSC and SSC Fig. 1, the P1 gating fraction, showing the top 10% intensity, gave us the possibility to isolate the producing cells. However, fluorescence intensity
As shown in fluorescence higher mAb might have
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
VOL. xx, 2015 depended on the cell size because bigger cells could present a significant amount of mAb expression on a cell surface. If our assumption was correct, a different parameter set would be useful for screening mAb high-producing cells. To develop a screening process by FCM, it was hypothesized that fluorescence intensity obtained from a cell surface had a correlation with not only mAb productivity but also with the FSC and SSC of a cell. The cells stained with FITC-conjugated anti-IgG antibody were equally divided by the SSC value on the basis of SSC-area vs. FSCarea dot plots (Fig. 2). Then, each fraction was divided into quarters for further FSC-area gating. Gates P3, P4, P5, and P6 were arranged in order of FSC-area increments. Gates P7, P8, P9, and P10 were completed in order, in the same manner. The top 5% fluorescence intensity in gate P3 was fractionated to gate P11. The same gating manner was adapted to isolate gates P12, P13, P14, P15, P16, P17, and P18 from the P4, P5, P6, P7, P8, P9, and P10, respectively (Fig. 2A, B). From each gates, 15e25 104 cells were recovered. The means of the FSC-area, SSC-area and FITC-area against each value of unsorted cells in SP-1 are shown in Table 1. The mean of the FSCarea increased from P11 to P14 and from P15 to P18 as gated. The mean of the SSC-area from P11 to P14 also increased, however the SSC-area from P15 to P18 was almost constant. The mean of the FITC-area of P14 was the highest in all sorted cells. The FITC-area
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TABLE 1. Relative value of areas sorted by FSC, SSC and FITC in SP-1. Group
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Relative rate (%, means of area) FSC
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FITC
100 60 93 133 174 25 75 98 138
100 223 269 294 344 46 72 72 73
100 1461 1194 1349 1551 269 85 83 96
tended to increase with the FSC-area increment in P11eP14 and P14 showed the highest FITC-area in all sorted cells. On the other hand, the trend in the P15eP18 was not investigated and the FITCarea of P15 was the highest in the group. The final product concentration of unsorted cells and eight sorted cells (named P11, P12, P13, P14, P15, P16, P17 and P18) were evaluated by fed-batch culture for three stable pools (Fig. 2C). All sorted cells indicated higher final product concentration than unsorted cells. Among them, P15, which showed the smallest value of
FIG. 2. Gating strategy of a stable pool sorting by FCM. (A) Dot plots of cells gated based on the SSC-area and FSC-area and a fractionated area to isolate gates P3, P4, P5, P6, P7, P8, P9, and P10 in the stable pool (SP-1). (B) Gating strategy of P11 included in the top 5% fluorescence intensity of P3 in SP-1. The same gating manner was adapted to isolate gates P12, P13, P14, P15, P16, P17, and P18 from the P4, P5, P6, P7, P8, P9, and P10, respectively. (C) Final product concentration (closed bars) and viable cell density (open circles) in a fed-batch culture of unsorted cells and eight sorted fractions in three stable pools (SP-1, -2, -3).
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TABLE 2. Cell-cycle phase ratio of G0/G1, S, and G2/M phases in SP-1. Group
Unsorted P3 P4 P5 P6 P7 P8 P9 P10
Percentage of each cell cycle (%) G0/G1 phase
S phase
G2/M phase
49 35 41 51 45 68 73 67 70
21 22 21 18 20 21 17 19 19
30 43 38 32 35 11 11 14 11
FSC-area and SSC-area in the P11eP18, indicated the highest final product concentration among them. Especially, the P15 showed 3.4e4.7-fold higher final product concentration than unsorted cells. Cell cycle analysis of cell fractions gated based on FSC and SSC The cell cycle of unsorted cells and sorted cells, gates P3, P4, P5, P6, P7, P8, P9 and P10, were investigated to analyze the cell state at the moment of FSC-/SSC-area gating. The ratio of cells in G0/G1, S, and G2/M in SP-1 is shown in Table 2. The cell-cycle phase ratios of G0/G1, S, and G2/M in unsorted cells showed 49, 21 and 30%, respectively. Higher SSC fractions, P3eP6, included fewer cells in the G0/G1 phase and many more cells in the G2/M phase. On the other hand, lower SSC-area fractions, P7eP10, included a larger amount of cells in the G0/G1 phase and fewer cells in the G2/M phase. However, there was no correlation between the cellcycle phase and value of the FSC-area. Enrichment of mAb-high producing cells by multiplerounds of FCM-based sorting The FCM-sorted fraction, P15, showed the highest final product concentration among all other fractions. It was speculated that mAb-high producing cells were included in the FCM-sorted fraction, P15. To isolate a higher fraction by multiple-rounds of sorting, we performed two more rounds of sorting in the same manner by fractionating P15 in SP1. The 2- and 3-round sorting fractions were named P15eP15 and P15eP15eP15, respectively. The final product concentration after multiple-rounds of sorting was evaluated in a fed-batch culture as shown in Fig. 3. The final product concentration was enhanced by multiple-rounds of sorting and the P15eP15eP15 represented 8.5-fold final product concentration in comparison to unsorted cells. To compare detailed characteristics of sorted cells, the SSC, FSC and FITC of each cell were analyzed by a flow cytometer. The distribution of unsorted and sorted cells are dot-plotted in Fig. 4A based on the SSC-area vs. FSC-area. Although the sorted cells were collected as the smallest size of the FSC-area and the lowest density of the SSC-area, cells sorted in the SSC-FSC manner showed almost the same distribution as unsorted cells after cell expansion. The FITC-area distribution of unsorted and sorted cells is shown in Fig. 4B, in a histogram format. The FITC-area was upper-shifted after multiple-rounds of sorting. DISCUSSION In this study, our goal is to demonstrate that our new method using high-throughput equipment, FCM, is useful to enrich highproducing cells and to reduce efforts for screening. However, the highest mAb-producing cell could not be isolated by a simple gating procedure based on higher fluorescence intensity (Fig. 1). On the other hand, it was found that a grouping/gating strategy, which had never been conducted before, on the basis of FSC and SSC worked well to enrich higher mAb producers (Fig. 2). The
FIG. 3. The final product concentration of multiple-round sorted cells in SP-1. Final product concentration (closed bars) and viable cell density (open circles) in a fed-batch culture of multiple-round sorted cells (P15, P15eP15 and P15eP15eP15) was graded as a value relative to that of unsorted cells.
FCM-based P15 fraction, which showed the smallest size of the FSC-area and the lowest density of the SSC-area, indicated the highest final product concentration in a fed-batch culture. The FITC-area of the P15 fraction was surprisingly lower than those of other fractions. When enriching higher producing cells, not only the FITC-area of the cells but also the FSC-area and SSC-area of the cells should be considered. As shown in Table 2, higher SSC-area fractions, P3eP6, included fewer cells in the G0/G1 phase and many more cells in the G2/M phase. On the other hand, lower SSCarea fractions, P7eP10, included larger amount of cells in the G0/ G1 phase and fewer cells in the G2/M phase. This means that the cell cycle is synchronized at the moment of FSC-/SSC-area gating. In addition, multiple rounds of cell sorting in this manner were adapted for more efficient enrichment of the highest mAb producing cells as shown in Fig. 3. About 8.5-fold final product concentration was shown in a fed-batch culture after 3 rounds of FCM-based enrichment from SP-1. We also tried multiple rounds of cell sorting from SP-2, SP-3 and an additional round of cell sorting from P15eP15eP15, but higher producing cells were not enriched any more (data not shown). This phenomenon would be explained as follows: higher producer enrichment from above experiments reached a saturation point at around 1500 mg/L under current conditions such as expression vector, host and production media and an enrichment efficiency was not a uniform state as expected. The distribution of the FSC-area and SSC-area of cells represents a variety and complexity of cellular morphology including the cell cycle as shown in Table 2, and currently, cellular morphology of sorted cells only reflects a partial cell state of all cell states. This might be a reason why cells sorted in the SSC-FSC manner showed almost the same distribution as unsorted cells after cell expansion (Fig. 4), although the sorted cells were collected at the smallest size of the FSC-area and the lowest density of the SSC-area. The peak of the FITC-area was upper-shifted after multiple-rounds of sorting. This result allows us to expect that the specific production rate of sorted cells might have been improved after multiple-rounds of sorting and also the final product concentration has been improved by multiple-rounds of sorting.
Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
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Our results for the P15 fraction, which showed the smallest size of the FSC-area and the lowest density of the SSC-area, indicated the highest final product concentration and proved, contrary to our expectations, that the final product concentration of all fractions would be almost at the same level. Another two results, in which different CHO cell lines with the same expression vector pDSLH4.1 and different expression vector carrying a different antibody gene were used, showed that the P15 fraction in a fed-batch culture indicated the highest product concentration among all fractions (data not shown). This phenomenon, as shown in supportive data (Fig. S2), suggested a negative correlation between the FSC-/SSCarea and mAb production. These results suggested that it might have been general manner and our proposed new screening procedure might have been applicable to another CHO expression systems. In general, the cell growth rate of high-producing cells is slow, because its energy is used for production more than growth. The cell volume and cell weight change along with changes in the cell culture conditions and specific growth rate (35,36). It is reported that there is a close relationship among the chromosomal number, cell size, and specific growth rate (37). Because SSC indicates intracellular density, it is thought that there are some differences in the organelle relating protein secretion such as an endoplasmic reticulum and Golgi body among each sorted cell from a stable pool. Therefore, we need further investigations to clarify the causal connections between productivity and cell size, productivity and intracellular density by evaluation of cell proliferation, chromosomal karyotype confirmed by multicolor fluorescence in situ hybridization, and observation of organelles by a transmission electron microscope (TEM). The result that there was a difference in cell cycles between higher SSC-area fractions and lower SSC-area fractions might be an important clue for further investigation of this phenomenon. We concluded that high-producing cells would be enriched in the fraction having the smallest value of the FSC-area and SSC-area, and our new screening procedure based on FSC and SSC gates by using FCM could be a powerful tool for the isolation of higher-producing cells. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2015.01.007. ACKNOWLEDGMENTS This work is partially supported by project focused on developing key technology of discovering and manufacturing drug for next-generation treatment and diagnosis. The authors are grateful to Ms. Keiko Okado in the Discovery Science and Technology Department, Daiichi Sankyo RD Novare Co., Ltd. for served as scientific advisors and Mr. Naoto Ishikawa in the Biologics Technology Research Laboratories, Daiichi Sankyo Co., Ltd. for collected data. References 1. Werner, R. G., Noé, W., Kopp, K., and Schlüter, M.: Appropriate mammalian expression systems for biopharmaceuticals, Arzneimittelforschung, 48, 870e880 (1998). 2. Chu, L. and Robinson, D. K.: Industrial choices for protein production by largescale cell culture, Curr. Opin. Biotechnol., 12, 180e187 (2001). 3. Andersen, D. C. and Krummen, L.: Recombinant protein expression for therapeutic applications, Curr. Opin. Biotechnol., 13, 117e123 (2002). 4. Andersen, D. C. and Reilly, D. E.: Production technologies for monoclonal antibodies and their fragments, Curr. Opin. Biotechnol., 15, 456e462 (2004). 5. Yoon, S. K., Hong, J. K., Choo, S. H., Song, J. Y., Park, H. W., and Lee, G. M.: Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production, J. Biotechnol., 122, 463e472 (2006). 6. Rita Costa, A., Elisa Rodrigues, M., Henriques, M., Azeredo, J., and Oliveira, R.: Guidelines to cell engineering for monoclonal antibody production, Eur. J. Pharm. Biopharm., 74, 127e138 (2010).
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Please cite this article in press as: Okumura, T., et al., Efficient enrichment of high-producing recombinant Chinese hamster ovary cells for monoclonal antibody by flow cytometry, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.01.007
