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).