Materials and Methods
Plasmid design and construction. All EpoFc-expressing clones were made from the parent plasmid, pCMV-EpoFc-pA (Lattenmayer et al., 2007). EpoFc-siRNA1 (ES) plasmid (Supplementary Figure 1a) was constructed by cloning the siRNA1/BART10 cassette between theSacI and XbaI sites upstream of the EpoFc protein.D HFR- E poFc-N EO (EDN) plasmid (Supplementary Figure 1b) expressing EpoFc along with DHFR and NeoR (aminoglycoside 3’-phosphotransferase) genes was constructed by inserting a cassette containing the DHFR and NeoR genes (under the control of SV40 promoters along with their respective poly A sequences) into the EcoRIsite. EGFP-siRNA1 plasmid (Figure 2a) was constructed by cloning EGFP and the siRNA1/BART10-intron cassette between the XbaI andEcoRV sites, replacing EpoFc. Plasmid containing EpoFc along with siRNA1 and Puromycin resistance (ESP, Supplementary Figure 1c) was generated by inserting PurR ( puromycin N-acetyl-transferase) into the EcoRI site and cloning the siRNA1/BART10 cassette into the XbaI site. Finally, HC1 (heavy chain along with siRNA1/BART10) -LC2 (light chain along with siRNA2)-GS plasmid (Figure 4a) expressing adalimumab and glutamine synthetase was cloned by inserting siRNA1-HC at XbaI , LC at HindIII , siRNA2 cassette at KpnI , and GS gene at EcoRV sites of pCMV-EpoFc-pA. All cloning was performed by Genscript (Piscataway, NJ) and confirmed by sequencing. Individual clones were delivered as an industrial grade plasmid DNA with a minimum seven stringent QC parameters analyzed (https://www.genscript.com/industrial-grade-plasmid.html).
Host cell lines and culture maintenance. CHO-K1 cells adapted to grow in suspension were used as host cell lines for both EpoFc and mAb expression and have been described previously (Bort et al., 2010). Cells were cultured in CD CHO medium (Thermo Fisher Scientific), supplemented with 8mM Glutamax and anti-clumping agent (Thermo Fisher Scientific). Cultures were routinely passaged twice a week at 1.5 x 105 cells/mL in 125 mL culture flasks (Thomson Instrument Company, Oceanside, CA) and incubated in a humidified 5% CO2/air mixture at 37 °C, on a shaker at 120 rpm. Viable cells were distinguished from dead cells using trypan blue dye exclusion, and the viable cell concentration (VCC) was quantified using a Countess II® FL automated cell counter (Thermo Fisher Scientific).
Functional mRNA synthesis. All mRNAs containing siRNAs and all control mRNAs were generated by in vitro transcription. Plasmids expressing appropriate inserts were linearized using a unique restriction enzyme. The DNA was purified using QiaQuick PCR Purification kit (Qiagen, Germantown, MD), as per manufacturer’s instructions. From the linearized DNA, RNA was transcribed using MEGAscript T7 transcription kit (Thermo Fisher Scientific) and then purified using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific) as per manufacturer’s instructions. The capping (ScriptCap m7G Capping System) and tailing (Tailing – A-Plus Poly(A) Polymerase Tailing Kit) was then performed sequentially using kits from CELLSCRIPT (Madison, WI). The RNA was then purified again using the MEGAclear Transcription Clean-Up Kit.
Stable cell line development by PTSelect™. CHO-K1 (80 x 106cells) were electroporated with plasmid DNA using a Maxcyte-ATX (MaxCyte, Inc., Gaithersburg, MD), according to the manufacturer’s protocol. After transfection, cells were seeded into pre-warmed CD-CHO media at 200,000 cells/mL and grown under normal growth conditions. After 48 hours, at least 80 x 106cells were transfected with 100-1000 ng of CD4/siRNA mRNA per million cells and incubated in pre-warmed CD-CHO media under normal growth conditions. After 4 to 6 hours, cells expressing CD4 on their cell surface (without GOI) were isolated using EasySep™ Release Human CD4 Positive Selection kit by STEMCELL Technologies (Vancouver, Canada), as per manufacturer’s instructions with a key modification of keeping the supernatant (cells with GOI and not expressing CD4 on their cell surface). The cells in supernatant were incubated in CD-CHO media under normal growth conditions. This depletion process was typically performed on days 3, 5 and 10 after transfection with GOI or when the cells had reached 80 x 106 in number.
Stable cell line development by conventional methods. CHO-K1 cells were electroporated with plasmid DNA using a Maxcyte-ATX (MaxCyte, Inc., Gaithersburg, MD), according to the manufacturer’s protocol. After transfection, cells were seeded at 1.5 x 105 cells/ml in 30 mL of CD-CHO medium in 125 mL shaker flasks. Selection was performed by adding either G418 (400 µg/mL) or Puromycin (10 µg/mL). For generating mAb-producing cell lines, selection was performed in a mixture of 20% PowerCHO-2 CD (Lonza Bioscience, Walkersville, MD) and 80% ExCell CHO cloning medium (Sigma-Aldrich, St. Louis, MO) with GS expression medium supplement (GSEM, Sigma-Aldrich), and MSX (25 µM) (Sigma-Aldrich, St. Louis, MO). Cell counts and viability were monitored twice a week, and a stable pool population was obtained when the viability reached greater than 90 percent. To obtain single cell-derived clones, cell pools were subjected to limiting dilution using growth media in 96-well plates (Supplementary Figure 5). After three weeks, media from established clones were harvested and ELISA (EpoFc, R&D Systems, Minneapolis, MN and Human IgG, STEMCELL Technologies, Vancouver, Canada) was performed, as per manufacturer’s protocol. The top forty clones were expanded and frozen.
Short-term productivity assay and titer measurements. Four-day batch cultures were performed to determine specific productivity of both Epo-Fc and IgG (adalimumab). Cells were seeded in duplicate, at 1.0 x 105 cells/mL in 10 mL of CD CHO medium supplemented with 8 mM Glutamax and anti-clumping agent in 50 mL bio-reaction tubes on a shaker set at 195rpm. VCC and viability were measured daily as described above. On day 4, media were harvested from duplicate tubes, combined, centrifuged, and stored in aliquots at -20 °C. The EpoFc fusion protein and human IgG expression levels in CHO cell culture supernatant were measured as described above. Qp(pg/cell/day) was calculated by dividing titer obtained from ELISA by integrated viable cell density (IVCD). IVCD was calculated as, IVCD1+(VCD1+VCD2)/2*(t2-t1)/24, wherein VCD (cells/day/mL) is viable cell density and t1 and t2 are two different time points in hours.
Analysis of clone stability. The six highest producing EpoFc-clones generated by PTSelect™ and Neo/DHFR amplification were chosen. Cells were seeded at 1.5 x 105 cells/mL in 30 mL of CD CHO medium supplemented with 8mM Glutamax and anti-clumping agent in 125 mL shaker flask. Cells were passaged every 3-4 days and cultured for three months/12 weeks. Cells from weeks 0, 4, 8 and 12, as counted from the start of the long-term cultivation were analyzed for protein expression levels by ELISA as described above and for clonal variation. To test for clonal drift, cells from each time point were transfected with a plasmid expressing dTomato cloned with sequences that can be regulated by the siRNA in the EpoFc-plasmid and GFP-mRNA (transfection control). Parental CHO-K1 cells were also transfected with either dTomato/siRNA1 or GFP/siRNA2 to serve as control for compensation of the fluorochromes. Flow cytometry analysis of the transfected cells was performed using FACSAria II (BD Biosciences) at the Neural Stem Cell Institute (Rensselaer, NY). Five hours after transfection, cells were analyzed by flow cytometry; GFP signals were collected in the FITC channel and dTomato signals in the PE channel. Experiments were done in duplicate. A total of 50,000 events were collected in the analysis. Data analysis was performed using FlowJo 10 (FlowJo LLC).