Optimization of heterologous protein production in Chinese hamster ovary cells under overexpression of spliced form of human X-box binding protein
© Gulis et al.; licensee BioMed Central Ltd. 2014
Received: 23 December 2013
Accepted: 8 April 2014
Published: 11 April 2014
The optimization of protein production is a complex and challenging problem in biotechnology. Different techniques for transcription, translation engineering and the optimization of cell culture conditions have been used to improve protein secretion, but there remain many open problems involving post-translational modifications of the secreted protein and cell line stability.
In this work, we focus on the regulation of secreted protein specific productivity (using a recombinant human immunoglobulin G (IgG)) by controlling the expression of the spliced form of human X-box binding protein (XBP-(s)) in Chinese hamster ovary cells (CHO-K1) under doxycycline (DOX) induction at different temperatures. We observed a four-fold increase in specific IgG productivity by CHO cells under elevated concentrations of DOX at 30°C compared to 37°C, without detectable differences in binding activity in vitro or changes in the structural integrity of IgG. In addition, we found a correlation between the overexpression of human XBP-1(s) (and, as a consequence, endoplasmic reticulum (ER) size expansion) and the specific IgG productivity under DOX induction.
Our data suggest the T-REx system overexpressing human XBP-1(s) can be successfully used in CHO-K1 cells for human immunoglobulin production.
KeywordsCHO cells Heterologous protein production X-box binding protein T-REx™ system Doxycycline
The optimization of the production of secreted proteins, such as therapeutic monoclonal antibodies (mAbs), is still a challenging problem in pharmaceutical biotechnology. Although biopharmaceutical products can be produced by many host cell systems, eukaryotic cells are preferred due to their ability to correctly process and modify human proteins. The primary goal is to establish the ideal combination of a rapid accumulation of productive biomass and the maintenance of cell viability for as long as possible. Many different strategies have been considered for improving both cell viability and the productivity of recombinant proteins, including mAbs. These strategies include physiological optimization and genetic and metabolic engineering [1, 2].
The most common problem during the optimization of protein production is an error in protein folding in the endoplasmic reticulum (ER). The inhibition of protein folding activates the unfolded protein response (UPR), which is a signal transduction network. Overcoming UPR is one of the many strategies for optimizing protein productivity. For instance, protein production has been tested under the expression of survival proteins that play important roles in UPR, including B-cell lymphoma protein 2 (bcl-2), B-cell lymphoma-extra-large protein (bcl-XL) [3–5], caspase inhibitors  and molecular chaperones/heat shock proteins (HSP70) . The role of the spliced form of X-box binding protein (XBP-1(s)) (which plays an important role in regulation processes, such as physical expansion of the ER, increasing the mitochondrial mass and function, increasing the cell size and enhancing total protein synthesis) in optimizing protein production has also been studied . This approach to increasing the secretion capacity of mammalian cells by overexpressing the transcription factor XBP-1(s) was successful in CHO cells; the production of the secreted proteins alkaline phosphatase (SEAP) and alpha-amylase (SAMY) was enhanced upon XBP-1(s) overexpression , as was the production of antibody . However, these studies have shown that using overexpression systems without regulation leads to cell apoptosis due to the accumulation of the produced proteins .
To overcome accumulation-induced apoptosis, other strategies have been applied to regulate the protein production, such as the use of induction systems. For instance, tetracycline has been used to optimize the overexpression of glycosyltransferases under the control of the Tet on/off system in CHO cells, but unfortunately, a high expression of glycosyltransferases still led to growth inhibition . Furthermore, interesting work using the same expression system has been conducted to control the overexpression of human transferrin (hTf) in human embryonic kidney (HEK-293) cells. That study found favorable concentrations of tetracycline at which the overexpression of hTf was optimal, but again, the high levels of expression limited the cell viability. Such impairment might have been a consequence of the overexpression of the protein of interest, which might have altered the quality of this cell product or even been toxic to the cells . Some studies have attempted to investigate the effect of the expression of an ER-resident molecular chaperone, protein disulfide isomerase (PDI), on the specific production levels of thrombopoietin (TPO) and antibody (Ab) in Chinese hamster ovary cells. Mohan and colleagues used the Tet-off system (in the absence of tetracycline) to regulate PDI, TPO and Ab expression in CHO cells under doxycycline (DOX; a chemical analogue of tetracycline) induction. However, only a small increase in antibody production was observed, and the production of TPO was not affected by PDI expression .
Moreover, the optimization of protein production in CHO cells cultured at different temperatures has been addressed. For instance, lowering the temperature from 37°C to 33°C increased the production of erythropoietin (EPO) by approximately four-fold, but at the same time, a low cultivation temperature suppressed cell growth . In addition, a temperature reduction from 37°C to 33°C in the culture of a CHO cell line producing recombinant human granulocyte/macrophage colony-stimulating factor (CHO-K1-hGM-CSF) led to a reduced growth rate, increased cell viability, improved cellular protein production and decreased cell metabolism . One study on the optimization of protein production at 32°C also demonstrated that the specific growth rate of CHO cells producing human mAb decreased by 30–63% at 32°C compared to 37°C. However, the specific antibody productivity of these cells was significantly enhanced at 32°C . Lowering the cultivation temperature even more, from 37°C to 30°C, caused growth arrest associated with a 1.7-fold increase in the specific production of secreted alkaline phosphatase (SEAP) in CHO cells .
Cell lines and media
The CHO-K1 (ATCC®CCL-61™) and Raji (ATCC®CCL-86™) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). CHO-K1 cells were grown and maintained at 37°C or 30°C with 70% humidity and 5% CO2 in HAM F12 media (Gibco, Big Cabin, OK, USA) supplemented with 2% fetal bovine serum (FBS, Gibco, Big Cabin, OK, USA) and were used in experiments on protein production. Raji cells were grown and maintained at 37°C with70% humidity and 5% CO2 in RAMP media (Gibco, Big Cabin, OK, USA) supplemented with 10% FBS and were used in FACS direct ligation experiments.
Plasmids and cloning
pCOMIRES HIL anti-CD20 is a tricistronic vector that encodes both the heavy and the light chains of an anti-CD20 antibody along with a neomycin resistance gene under the control of a synthetic CMV promoter. This vector was transfected into CHO-K1 cells to obtain IgG (anti-CD20)-producing cells. The human xbp-1(s) coding sequence was chemically synthesized by GeneScript (Piscataway, NJ, USA). The restriction enzymes Hind III and BamH I (Fermentas, Ontario, Canada) were used to obtain the xbp-1(s) insert and then clone it into the inducible expression plasmid pcDNA™4/TO/myc-His A from the Invitrogen T-REx™ system (Invitrogen, Carlsbad, CA, USA). This plasmid was used to co-transfect IgG-producing stable clones of CHO cells along with the regulatory plasmid pcDNA6/TR (Invitrogen, Carlsbad, CA, USA). To confirm xbp-1(s) cloning, XL1-blue bacterial cells (Stratagene, La Jolla, CA, USA) were transformed with ligated DNA. Ampicillin (Sigma, Ronkonkoma, NY, USA)-selected colonies were isolated and processed for DNA extraction and purification, which was performed using a QIAprep Miniprep Kit (Qiagen, Valencia, CA, USA). Restriction analysis and sequencing (using CMV forward primer 5′-CGCAAATGGGCGGTAGGCGTG-3′ and BGH reverse primer 5′-TAGAAGGCACAGTCGAGG-3′) confirmed the cloning of the xbp-1(s) insert.
Transfection with pCOMIRES anti-CD20 DNA (IgG-encoding plasmid) into CHO cells and generation of stable IgG-producing cells
The transfection of pCOMIRES HIL anti-CD20 plasmid (encoding an anti-CD 20 (IgG) antibody, a secretable protein with molecular weight 150 kDa (two light chains, each with molecular weight 25 kDa, and two heavy chains, each with molecular weight 50 kDa)) into CHO cells was performed using a PolyPlus (JetPrime, New York, NY, USA) kit in six-well test plates (TPP, San Diego, CA, USA) according to the manufacturer’s instructions. The clones harboring the pCOMIRES HIL anti-CD20 transgene were selected from a mixed population by the single-cell dilution method. Geneticin (Roche, Gaillard, France) was used for selection at 800 μg/mL.
Transfection with the T-REx™ -XBP-1(s) system into stable IgG-producing clones of CHO cells and generation of stable double clones (IgG-T-REx-XBP-1(s) cells)
The co-transfection of T-REx-xbp-1(s) plasmid (encoding a spliced form of human apoptotic XBP-1 protein with predicted molecular weight 40 kDa) along with regulatory plasmid pcDNA6/TR into one of the stable IgG-producing clones was performed using a PolyPlus (JetPrime, New York, NY, USA) kit according to the manufacturer’s instructions in six-well test plates (TPP, San Diego, CA, USA). Blasticidin (Sigma, Ronkonkoma, NY, USA) and Zeocin (Sigma, Ronkonkoma, NY, USA) were added to a final concentration of 0.5 μg/mL and 50 μg/mL, respectively. The selective markers encoded by regulatory plasmid pcDNA6/TR and expression plasmid pcDNA™4/TO/myc-His A are against blasticidin and Zeocin, respectively.
Selected IgG-T-REx-XBP-1(s) cells (after the first transfection, IgG clones; after the second, co-transfection, T-REx-XBP-1(s) clones) were induced by DOX at different concentrations: 0 μg/mL for control, 0.1 μg/mL, 0.5 μg/mL and 1 μg/mL. We used these concentrations because we found out that 5 μg/ml and 7.5 μg/ml of doxycycline completely inhibits cells growth for clones and wild type CHO-K1 cells. DOX induction was performed 24 hr after IgG-T-REx-XBP-1(s) cells seeding at a uniform cell density (0.5 × 105 cells/mL) in tissue culture flasks (75 cm2, TPP, San Diego, CA, USA) and then incubated for seven days at 37°C or 30°C. All cultures reached at least 80% under these conditions. Samples were collected for viable cell density, Ab detection by ELISA, nuclear extract isolation and ER staining. Half of the cells in each group continued to grow for seven more days in DOX-free medium after DOX wash-out. Independently, IgG-T-REx-XBP-1(s) cells were incubated for 42 days at 30°C (150 cm2 flasks, TPP, San Diego, CA, USA) with or without 1 μg/mL DOX. In all DOX induction experiments, DOX was added (at an appropriate concentration) every three days to the cell culture. Induction experiments were performed twice in duplicate (four independent culture samples per group).
The viable cell density of the IgG-T-REx-XBP-1(s) cells were tested under different DOX concentrations (0 μg/mL, 0.1 μg/mL, 0.5 μg/mL or 1 μg/mL) every day during seven days of cell growth at 37°C and 30°C. Seeding was performed at a uniform cell density (0.06 × 105 cells/mL) in six-well tissue culture plates (TPP, San Diego, CA, USA). At the seventh day all cultures reached at least 80% under these conditions. In addition, the viable cell density of IgG-T-REx-XBP-1(s) cells was tested on the seventh day of growth with DOX and on the seventh day after wash-out in DOX-free medium. In addition, IgG-T-REx-XBP-1(s) cells were tested every seventh day during 42 days of cell growth under 1 μg/mL DOX (or 0 μg/mL as control) at 30°C. The viable cell density was measured using the trypan blue (Sigma, Ronkonkoma, NY, USA) exclusion method with a hemocytometer and light microscope for manual cell counting. Every viable cell density experiment was performed twice in duplicate (single determination from each of two independent culture samples per group in two independent experiments).
The supernatants of IgG-T-REx-XBP-1(s) cells in the presence or absence of DOX were collected every seventh day of 37°C or 30°C growth for two weeks or every seventh day for six weeks and processed for analysis by enzyme-linked immunosorbent assay (ELISA) (duplicate determination from each of two independent culture samples per group in two independent experiments). The Lunc/Maxisorp Immunoplate (Thermo Scientific, Waltham, MA, USA) was incubated with primary antibody (goat anti-human IgG (H + L), 1:3000 dilution; Thermo Scientific, Waltham, MA, USA) and blocked with 3% fat-free dehydrated milk solution. After blocking and washing the plate, the supernatants were applied to the plate and incubated for 2 hr. The plate was washed again, and secondary antibody (anti-human IgG Fc-specific, alkaline phosphatase-conjugated, produced in goat, 1:3000 dilution; Sigma, Ronkonkoma, NY, USA) was applied for 1 hr. The plate was washed again, and at the end of the procedure, the signal of absorbance was read at 405 nm by a microplate reader (ELx800 96-well Microplate Reader, MTX Lab Systems, Inc., Vienna, VA, USA) after 4-Nitrophenyl phosphate disodium salt solution (pNPP) (Invitrogen, Carlsbad, CA, USA) addition. In addition, human IgG (whole molecule; Thermo Scientific, Waltham, MA, USA) was used in different concentrations as a control on the same plate.
Isolation and purification of produced proteins
The IgG produced under different temperature conditions by IgG-T-REx-XBP-1(s) cells was purified on the HiTrap™ Protein A HP 1 mL (GE Life Sciences, Pittsburgh, PA, USA) column. The column was first equilibrated with 10 mL Protein A IgG Binding Buffer (Thermo Scientific, Waltham, MA, USA) at a rate of 1 mL/min. Then, the supernatant from IgG-T-REx-XBP-1(s) cells was applied to the equilibrated column. The column was washed with 30 mL Protein A IgG Binding Buffer (Thermo Scientific, Waltham, MA, USA). Then, the protein was eluted with 50 mL IgG Elution Buffer (Thermo Scientific, Waltham, MA, USA), and 2 mL per fraction was collected. Fractions were neutralized with Tris–HCl pH 9.0. The Ab present in the fractions was immunodetected in a dot blot assay. Five microliters of each fraction was directly pipetted onto a nitrocellulose Hybond-C Extra membrane (Amersham® Bioscience, Piscataway, NJ, USA). The membrane was blocked with 3% fat-free milk solution and incubated with anti-human IgG (Fc-specific, alkaline phosphatase-conjugated, produced in goat, 1:3000 dilution) (Sigma, Ronkonkoma, NY, USA), and the proteins were revealed using a BCIP/NBT substrate Kit (Invitrogen, Carlsbad, CA, USA). The Ab-containing fractions were selected for dialysis, which was performed using a Centricon YM-50 (Amicon Bioseparations, Billerica, MA, USA) in PBS buffer (10 mM NaH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).
Anti-CD20 antibody was also detected by western blotting. Five hundred nanograms of IgG sample was loaded in each well of a Bis-Tris gel (NuPAGE® Novex 4-12% Bis-Tris Gel, Invitrogen, Carlsbad, CA, USA) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to the manufacturer’s instructions. The proteins were transferred to the Hybond-C Extra nitrocellulose membrane (Amersham® Bioscience, Piscataway, NJ, USA) and blocked in 3% fat-free milk PBS solution. The immunodetection was performed using anti-human IgG (Fc-specific, alkaline phosphatase-conjugated, produced in goat (1:3000 dilution) (Sigma, Ronkonkoma, NY, USA) with a BCIP/NBT substrate Kit™ (Invitrogen, Carlsbad, CA, USA).
XBP-1(s) was also probed by western blotting. The nuclear extracts from the IgG-T-REx-XBP-1(s) cells were prepared as described by Becker and colleagues . Briefly, the nuclear extracts were prepared from 5×106 cells/per sample and equal volumes of nuclear extracts were loaded into a Bis-Tris gel (NuPAGE® Novex 4-12% Bis-Tris Gel, Invitrogen, Carlsbad, CA, USA), and SDS-PAGE was performed according to the manufacturer’s instructions. Samples were transferred to the Hybond-C Extra nitrocellulose membrane (Amersham® Bioscience, Piscataway, NJ, USA), and after blocking with 3% fat-free milk PBS solution, rabbit anti-human-XBP-1(s) (1:1000 dilution; Sigma, Ronkonkoma, NY, USA) was added, followed by alkaline phosphatase-conjugated anti-rabbit IgG incubation (1:1000 dilution; Sigma, Ronkonkoma, NY, USA). The proteins were revealed using a BCIP/NBT substrate Kit™ (Invitrogen, Carlsbad, CA, USA).
Fluorescence-activated cell sorting (FACS)-ER staining
The IgG-T-REx-XBP-1(s) cells that were grown for seven days under DOX induction and those that were grown for one more week after wash-out were collected at 3×105 cells/per staining and washed with HBSS buffer (140 mM NaCl, 4.7 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4). After washing with HBSS buffer, the cells were labeled with 250 nM of ER-Tracker™ Green Dye (ER-Tracker™ Green Dye for Live-Cell Endoplasmic Reticulum, Molecular Probes, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. The samples were washed again with HBSS buffer and analyzed using a BD FACS Verse flow cytometer (BD Bioscience, San Jose, CA, USA). Ten thousand events were collected per sample using no gate for acquisition. The dead cells were not excluded in the analysis. We used BD FACSuite to data acquisition. The experiment was performed twice in duplicate.
FACS direct ligation assay
Raji cells were grown for five passages as described above, collected and resuspended in 1 part RAMP media with 10% FBS and 1 part FACS buffer (PBS supplemented with 2% FBS) at 3×106 cells/well in a 96-well plate (TPP, San Diego, CA, USA). After centrifugation, the cells were blocked with FcR blocking reagent (MACS, Biotec, Bergisch Gladbach, Germany) on ice for 30 minutes according to the manufacturer’s instructions. Purified and dialyzed IgG samples, which were produced by IgG-T-REx-XBP-1(s) cells at 37°C and 30°C, and commercial IgG (rituximab, MabThera, Genetech Inc., South San Francisco, CA, USA) as a positive control were added at 100 ng per well. Samples were incubated on ice for 1 hr and centrifuged after the addition of FACS buffer. The cells were washed twice with FACS buffer and incubated with mouse FITC anti-human IgG (BD Pharmingen™, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s manual. The cells were incubated on ice for 30 minutes in the dark, washed again twice with FACS buffer and processed for fluorescence intensity measurements using a BD FACS Verse flow cytometer (BD Bioscience, San Jose, CA, USA). Each experiment was performed twice in duplicate.
Results and discussion
Viability and IgG production under induction with DOX in IgG-T-REx-XBP-1(s) cells cultivated at 37°C and 30°C
The supernatants from all cells were collected after seven days of induction and tested by ELISA to determine their IgG yields. The specific IgG productivity depended on the concentration of DOX: under 0.5 μg/mL and 1 μg/mL DOX, the increase of specific IgG productivity was 40% and 66%, respectively, compared to the basal level of specific IgG productivity (0 or 0.1 μg/mL DOX) at 37°C (Figure 2C). These data demonstrate that IgG-T-REx-XBP-1(s) cells produced three-fold more IgG compared to untreated cells, even at low viable cell density. At 0.1 μg/mL DOX, there was no improvement in specific IgG productivity at 37°C. Moreover, the data from ELISA indicate that protein production in the cells incubated at 30°C increased four-fold and three-fold under 1 μg/mL and 0.5 μg/mL DOX, respectively. Once more, induction at a low concentration of DOX (0.1 μg/mL) did not increase specific IgG productivity at 30°C, as above at 37°C. In contrast, the specific IgG productivity by IgG-T-REx-XBP-1(s) cells at 30°C increased by 31.5% and 43.5% compared to induction at 37°C under 1 μg/mL and 0.5 μg/mL DOX concentrations, respectively (Figure 2C). However, we did not detect any effect of low temperature on specific IgG productivity per se (without the induction of DOX). Tigges and Fussenegger  reported the same lack of effect in CHO cells expressing SEAP, whereas other authors reported an increase in the production of different proteins at low temperature and with no inductor [15–18]. These deviations in experimental results may be due to differences in the proteins and cell lines used in these studies. In conclusion, our data demonstrate a successful improvement of specific IgG productivity using 1 μg/mL DOX in IgG-T-REx-XBP-1(s) cells at 30°C.
Effect of XBP-1(s) expression and ER size expansion on protein production in IgG-T-REx-XBP-1(s) cells
Binding activity of the recombinant proteins produced at different temperatures
Establishing a stable protein-producing cell line
Many studies have been published on improving recombinant protein production. In general, the published data suggest that the optimization of the production of specific target proteins requires specifically adjusted growth conditions and a carefully chosen cell line. In the present study, we optimized the conditions for IgG (human anti-CD20) specific productivity in CHO-K1 cells. We showed that the combination of low temperature (30°C) and XBP-1(s) overexpression regulated by DOX induction significantly improved anti-CD20 specific productivity: under 1 μg/mL DOX treatment, specific IgG productivity was increased by 32% compared to the cells grown under the same concentration at 37°C and 74% compared to the cells grown without DOX induction at 37°C or 30°C. Moreover, the results of our study indicate the direct dependence of specific IgG productivity on the concentration of DOX (under 0.5 μg/mL, the increase was 2.7-fold, and under 1 μg/mL, the increase was 3.9-fold), which allows for the precise regulation of specific IgG productivity in CHO-K1 cells. In addition to the concentration dependence, we demonstrated the possibility of returning the specific IgG over productivity to the basal level of specific productivity by removing DOX. This step also restored the viable cell density, which permitted the cells to overcome the problem of accumulation of the target protein. In the production of proteins, it may be possible to use the T-Rex-XBP-1(s) system to turn up and down the production of protein, repeating this cycle several times to accumulate higher amounts of target protein without a loss of cell viability. We also observed a DOX concentration-dependent relationship involving XBP-1(s) overexpression (western analysis), ER size expansion (FACS measurements) and specific IgG productivity (ELISA). Finally, our data demonstrate that it is possible, under DOX induction at low temperature, to produce a target protein for an extended period of time. Taken together, our data suggest the T-REx-XBP-1(s) system can be used in CHO-K1 cells for human immunoglobulin production.
B-cell lymphoma protein 2
B-cell lymphoma-extra-large protein
Chinese hamster ovary cells
CHO cell line producing recombinant human granulocyte/macrophage colony-stimulating factor
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Human embryonic kidney cells 293
Heat shock proteins 70
Human immunoglobulin G
Median fluorescence intensity
Protein disulfide isomerase
4-Nitrophenyl phosphate disodium salt solution
Secreted alkaline phosphatase proteins
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
Unfolded protein response
Spliced form of human X-box binding protein.
The work was funded by grant from BNDES, Brazil. GG is a fellow of PNPD postdoctoral program from CAPES. KCRS is a fellow of the CAPES graduate program.
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