Figure Legends
Figure 1. GPEx® Technology
Overview
A. The GPEx® cell line development process utilizes retrovector
technology to insert the product expressing gene of interest (GOI) into
transcriptionally active sites in the host genome and can be used for
most mammalian cell lines. B. GPEx® Lightning combines the benefits of
GPEx® with GS selection and targeted recombination technology. GPEx®
technology was leveraged to place >200 Dock sites, which
contain recombinase attachment sequences (att), into transcriptionally
active sites throughout the genome, creating a parental GPEx Lightning
Dock cell line. Transfection of this parental line is performed with a
Boat construct containing att, GS, and the GOI, plus a separate
recombinase construct (not shown). After transfection, selection results
in the rapid and efficient insertion of up to approximately 200 copies
of the GOI into transcriptionally active sites in the genome.
Figure 2. GPEx® Lightning Boat Insertion
Process
Dock sites containing the attP site flanked by modified retroviral long
terminal repeats (mLTR) sequences were placed in transcriptionally
active sites throughout the CHO genome using GPEx® technology. See
Figure 1 and main text for detailed description of Dock site placement
and GPEx® Lightning Parental Dock cell line development. The parental
Dock cell line is co-transfected with a Boat construct (containing attB,
GS, and the GOI) and a construct expressing recombinase. Recombinase
protein mediates the insertion of the entire Boat construct into the
attP site in the Dock; this process occurs simultaneously across many
dock sites. Following integration, the upstream att site, now called
attR, is comprised of the upstream portion of attP (light blue) and the
downstream portion of attB (light purple). Conversely, the downstream
att site, attL, is comprised of the upstream portion of attB (light
purple) and the downstream portion of attP (light blue). The location of
forward (green) and reverse (red) primers used in the attR QPCR assay to
quantify integration are indicated.
Figure 3. Pool Selection
A. Percent cell viability over time for pools expressing four different
monoclonal antibodies (mAb) or an Fc fusion protein. B. Approximate
number of integrated Boat copies per cell in each pool. Error bars
represent standard deviation of triplicate transfections.
Figure 4. Pool Generic Fed-Batch
Production
Results of fed-batch production from pools using the GFB1 strategy.
Viable cell density, viability, and titer for A) three replicate pools
expressing mAb1, B) average of replicates for pools expressing mAb2
(n=2), mAb3 (n=3), mAb4 (n=3), and an Fc Fusion protein (n=1). Average
standard deviation for each molecule across the time course for each of
the 4 mAbs were less than 10%, 3%, and 13% for VCD, viability, and
titer, respectively (not displayed).
Figure 5. Pool Ambr® 250 Bioreactor
Production
Viable cell density, viability, and titer for mAb1 pp3 production using
GFB2 and the optimal GFB2-based production strategy identified in an
Ambr® 250 process optimization campaign. Results from the GFB1 strategy
(from Figure 4) are shown for comparison.
Figure 6. Pool Stability
A. Final titer and integrated Boat copy number from a stability study of
40+ generations of 3 independent pools expressing an Fc fusion protein.
Each pool stability study was performed in 2 different media. Error bars
intentionally omitted for clarity. B. Final titer and integrated Boat
copy number from a 40+ generation stability study on 2 pools expressing
mAb 1 and mAb2. Production was performed using the GFB1 strategy in
duplicate. Error bars indicate standard deviation.
Figure 7. Clone Generic Fed-Batch Production and
Stability
A. Linear regression of final titer from a 16-day productivity using
GFB1 plotted against percent Dock fill for 26 clones isolated from Fc
Fusion pp2. B. Final titer from a 40+ generation stability study on the
top 3 clones from Fc Fusion pp2. Productivity was performed using the
GFB1 strategy. Error bars indicate standard deviation.
Figure 8. Released N-glycan Profile Stability and Pool to
Clone
comparison
A. Overlay of UPLC traces of fluorescently labeled and released
N-glycans from two different Fc-fusion pools. Terminal samples from
productivity of the indicated generations (Gen) were analyzed. B.
Overlay of UPLC traces of fluorescently labeled and released N-glycans
from three different monoclonal antibodies. Terminal samples from
productivity of a pool and a clone derived from that pool were analyzed.
Table 1. Released N-glycan Profile Stability
A comparison of individual N-glycan species released from Figure 9.
Numbers shown are percentages.
Table 2. N-glycan Species from Pools and
Clones.
A comparison of individual N-glycan species released from Figure 10.
Numbers shown are percentages. ND = not detected or below 0.4% area
reporting threshold.