Plasmid vector construction
As luciferase reporter and control vectors, pGL4.10 and pGL4.74 (Promega Biosciences Inc., San Luis Obispo, CA, USA) were used, respectively. pGL4.10 contains the firefly luciferase reporter gene luke2, and each candidate promoter was inserted into a multiple cloning site upstream of luke2. pGL4.74 contains the HSV-TK promoter and the Renilla luciferase gene hRluc.
For mAb expression, pEF1/myc-His B (ThermoFisher Scientific, Waltham, MA, USA) was used as a backbone vector, and mAb expression genes (heavy chain and light chain) were inserted. (Supplementary Fig. S8).
Culture of CHO cells for transcriptome analysis
Two strains of CHO-K1 (CCL-61; ATCC, Manassas, VA, USA)37 mAbs expressing (IgG1) were cultured in custom medium G13 (Fujifilm Wako Pure Chemical, Osaka, Japan) and CD DA1 (ThermoFisher Scientific) in a 1 L animal cell culture vessel (ABLE Corporation, Tokyo, Japan). Cultures were performed for 14 days and 1 × 106 viable cells were acquired on culture days 4, 7, 9, 11 and 14, washed (300 rpm, 5 min) with PBS and stored at -80°C after freezing with liquid nitrogen.
RNA extraction and transcriptome analysis
Total RNA was extracted from thawed cells using RNAiso Plus (Takara Bio Inc., Shiga, Japan). After RNA quality analysis using Nanodrop (ThermoFisher Scientific) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), a sequence library was prepared using the TruSeq v2 RNA sample prep kit (Illumina, San Diego, CA, USA) and an automated device (Agilent Technologies). PolyA+ RNAs were isolated, fragmented and cDNA was synthesized. After blunting and phosphorylation of both ends of the synthesized cDNA, 3′-dA protrusion treatment was performed and the adapters were ligated. DNA was amplified by PCR using double-stranded cDNA with an adapter as template. Then, the PCR product obtained by the magnetic bead method using AMPure XP (Beckman Coulter, Marseille, France) was purified to generate a sequence library. High-throughput sequencing analysis was performed with the HiSeq system (Illumina) using a sequence library. A cluster that served as the sequence template was generated and the base sequence (fastq format) of the template DNA was acquired. The main sequences obtained by sequence analysis were mapped onto the genome sequences. The gene expression level (RPKM) normalized based on the position information was calculated.
Transient expression of luciferase in CHO cells
A culture containing 5 × 105 host CHO cells were centrifuged (240 g, 24°C for 3 min), after which the medium was removed. Then, the cell pellet was suspended in 2 mL of Opti-MEM medium (ThermoFisher Scientific). After removal of the supernatant by centrifugation, the remainder was resuspended in 2 ml of Opti-MEM medium and 1 ml of the culture was distributed in two wells of a 24-well culture plate. Mixtures of 3.2 μg of expression vectors and 68 μL of Opti-Pro SFM medium (ThermoFisher Scientific) were mixed with a mixture of 8 μL of gene transfer reagents and 68 μL of Opti-Pro SFM medium and reacted at room temperature for 20 min. Half of the reagents were added to each of the two dispensed wells and statically incubated in a CO2 incubator under 5% CO conditions2 and 37°C.
After transfection of the vector, the cells were cultured overnight and then the entire culture was centrifuged (9000 g, 5 min) to obtain cell pellets. Cells were washed with PBS and 100 µL of 1× lysis buffer was added, followed by incubation at room temperature for 5 min. The Dual-Luciferase® Reporter Assay System (Promega Biosciences Inc) was used for the luciferase assay. The prepared cell lysate was diluted 100 times with distilled water and 20 µL of the diluted solution and 100 µL of Luciferase II assay reagent were mixed. Using this sample, firefly luciferase activity (reporter) was measured with a luminometer (OT245-01; Berthold Technologies, Bad Wildbad, Germany) for 10 s. Then, 100 µL of Stop & Glo® reagent was added and Renilla luciferase activity (internal control) was measured for 10 s with a luminometer. The result of the firefly luciferase measurement was divided by that of Renilla luciferase measurement to calculate luciferase activity.
Construction of stable expression pools for luciferase and mAbs
After transfection and culture for 24 h, two wells of culture medium were collected in a single tube. The culture medium was centrifuged (240 g, 24°C for 3 min) to remove the supernatant, after which the cell pellet was suspended in 4 mL of transfection medium containing 800 µg/mL of geneticin. Subsequently, this suspension was transferred to a well of a six-well culture plate, followed by incubation in a CO2 incubator (37°C, 5% CO2) to initiate selection with Geneticin. Media was regularly replaced with transfection medium containing geneticin (final concentration of 800 μg/mL) and expanded in T-25 culture flasks using selective expansion medium with geneticin (final concentration of 800 µg/mL) in accordance with cell growth. After incubation for 3-4 days, the culture was expanded to a 125 ml Erlenmeyer flask and used as a stable pool.
Acquisition of monoclonal cell lines
To 100 mL of semi-solid medium was added cell culture medium diluted 100 times with C/E medium38 at a viable cell density of 48 cells/mL. The mixture was mixed gently so that bubbles did not form, and the stable pool was seeded at approximately 2 ml/well onto a six-well plate for semi-solid medium. Cells were centrifuged at 240g for 5 min at 4°C and then cultured for 14 days in CO2 incubator at 37°C and 5% CO2. Colonies were picked with an automated animal cell colony picker (Molecular Devices, LLC, Sunnyvale, CA, USA). Each colony picked was seeded into a 96-well plate for cell culture, in which 150 µL of C/E medium was dispensed into each well and cultured in CO2 incubator at 37°C and 5% CO2. After passage, the monoclonal cells were evaluated in fed-batch culture.
Stable pools and monoclonal strains expressing luciferase and mAb were evaluated by batch culture in a 125 ml Erlenmeyer flask or a small 15 ml culture reactor (Sartorius, Göttingen, Germany). Production cultures in Erlenmeyer flasks were grown for 14 days in 5% CO2 at 37°C and 120 rpm. Cultures in the small 15 ml culture reactors were conducted for 14 days at 37°C and 850 rpm, with an air saturation of 50% dissolved oxygen at pH 7.00 ± 0.05 (controlled by co2 and Na2CO3). Custom G13 medium and custom F13 feed medium were used. Cell concentration and viability were measured using Vi-CELL (Beckman Coulter). Metabolites were analyzed using Bio Profile FLEX2 (Nova Biomedical, Waltham, MA, USA). Antibody concentrations were analyzed by HPLC (Agilent Technologies) with a Protein A affinity column (PA ID Sensor Cartridge Φ2.1 mm × 30 mm; ThermoFisher Scientific). mAb aggregation was detected by HPLC with a size exclusion chromatography column (ACQUITY UPLC BEH200, SEC, 1.7 μm, φ4.6 mm × 300 mm; Waters, Milford, MA, USA) as high molecular weight species. For transcriptional analysis, cell pellets containing 1 × 106 cells were acquired during the culture period and cryopreserved at -80°C.
RNA extraction and reverse transcription
RNeasy Micro Kit (Qiagen, Hilden, Germany) was used for RNA extraction from cultured cells (1 × 106 cells) and the PrimeScript™ RT-PCR kit (Takara Bio) was used for reverse transcription. One microliter of dNTP mix (10 mM each dNTP), 1 µL of random 6-mers (20 µM), 1 µL of template RNA, and 7 µL of RNase-free dH2O were mixed and annealed using a thermocycler at 65°C for 5 min. Ten microliters of the reaction solution was mixed with 4 µL of PrimeScript 5× buffer, 0.5 µL of RNase inhibitor, 0.5 µL of PrimeScript RTase, and 5 µL of RNase-free dH.2O, and the reverse transcription reaction was performed at 42°C for 30 min, followed by 95°C for 5 min, and the resulting cDNA solution was collected.
ddPCR™ EvaGreen Supermix (Bio-Rad, Berkeley, CA, USA) was used for quantitative PCR. A total of 2.2 µL of cDNA diluted in distilled water, 11 µL of 2 × ddPCR Evagreen Supermix, 1.1 µL of forward primer (5 µL), 1.1 µL of reverse primer (5 µL) and 6.6 µL of water were mixed, and droplets were made from this mixture using an automated droplet generator (Bio-Rad). Subsequently, the PCR reaction was carried out under the conditions of 95°C for 5 min, 40 cycles of 95°C for 30 s and 62°C for 1 min, 4°C for 5 min and 90°C for 5 min. . The number of cDNA copies amplified using Droplet Reader was quantified. household genes gapdh and actβ were used as internal controls.
In silico promoter analysis
The sequences of hspa5 upstream regions and transcriptional origin information (cDNA sequences) were extracted from the NCBI database. GENETYX-SV/RC Ver 13.1.1 software (GENETYX, Tokyo, Japan) was used for sequence analysis. The cutoff values for the polymerase II promoter assay were: TATA box -8.16, Cap signal -3.75, CCAAT box -4.54, and GC box -4.9. The CpG island analysis conditions were defined as: window size 100, average range 10, minimum CpG island length 200, minimum GC content 50% and minimum Obs/Exp CpG 0.6. BLAST analysis was used to analyze the homology of upstream gene regions.
Added ER stress inducers
The CHO-K1 cell clone expressing IgG1 under Hspa5p was cultured. Then the following were added as ER stress inducers on day 7 of fed-batch culture and mAb and hspa5 gene transcription levels were analyzed: DTT (final concentrations of 1, 2, 5 and 10 mM), tunicamycin (final concentrations of 0.01, 0.05, 0.1, 0.2 and 0.5 µg/ mL) and thapsigargin (final concentrations of 0.002, 0.005 and 0.01 µg/mL). Cells for transcriptional analysis were obtained on day 10 of culture, and IgG and endogenous transcript levels hspa5 have been analyzed.