Results
Creation of the Parental Dock Cell Line
Docks were placed in transcriptionally active sites throughout the
genome of CHOZN® GS-/- cells with sequential rounds of transduction
using GPEx® retrovector technology (Figure 1). The Dock cell pool
contained approximately 75 Dock copies per cell on average. To test the
integration efficiency and expression of the pool, we co-transfected
with Phi-C31 Recombinase and Boat construct expressing an Fc fusion
protein (Figure 2). After transfection, pools were allowed to recover
for 2-4 days prior to selection by removal of glutamine from the media.
The system utilizes multiple methods to reduce GS expression resulting
in survival of only high copy number clones regardless of the initial
copy number of the pool (see methods). For example, a pool containing 31
inserted copies per cell on average prior to selection via glutamine
withdrawal resulted in 63 copies after selection while another pool that
contained 64 copies per cell average before selection reached 94 copies
after selection. Even though the second pool already contained 64 copies
per cell on average, selection still resulted in a substantial increase
in copy number.
After pools recovered from selection, QPCR using the attR primers, which
only detect Boat that has integrated into the Dock site, revealed that
these pools contained approximately 30 copies of recombined Boat per
cell. In generic fed-batch production, these pools produce up to 5 g/L
indicating the system was working as designed.
To isolate a high Dock copy number clone that had good growth
characteristics, the Dock pool was subjected to single cell cloning
using the Berkeley Lights Beacon® instrument. Multiple clones with the
highest number of Dock copies were co-transfected with plasmid encoding
the Phi-C31 recombinase and the same test Boat construct expressing an
Fc fusion protein. After recovering from selection, the cell pool
generated from the top clone contained about 40 integrated Boat copies
on average. This clone was found to have suitable reproducibility of
insertion, averaging 40 to 130 copies per pool. Robust growth
characteristics and high protein expression were observed; therefore,
this clone was used as the parental Dock cell line for all further
process optimization studies and projects.
Creation of Cell Pools Expressing Proteins of
Interest
To test the insertion efficiency, expression, and consistency of the
technology, we also utilized a Boat expression construct that contains
two cloning sites for the independent expression of both heavy chain and
light chain sequences in the same Boat as opposed to the single chain
construct described above. The GPEx® Lightning Parental Dock line was
co-transfected with Phi-C31 recombinase plasmid and each Boat plasmid
construct in triplicate. To select high expressing cells, glutamine was
removed from the media and viability was monitored during selection
(Figure 3A). Each triplicate transfection showed very consistent
recovery from selection, and selection dynamics were very similar
between molecules. Pools consistently achieved about 50 integrated Boat
copies per cell on average (Figure 3B).
Fed Batch Production from Cell
Pools
Pools were subjected to a fed-batch production using a generic,
unoptimized feeding strategy (GFB1) for each of the three replicate
pools. All three replicate pools of mAb1 reached approximately 20 x
106 cells per mL, maintained high viability for the
full 20 days of production, and produced very consistent titers of about
6.5 g/L (Figure 4A). The same feed strategy was used for production of
the Fc fusion protein (test molecule) and three other mAb molecules.
Average viable cell densities for the three replicates of each molecule
reached 18-26 million cells per mL and viability remained high
throughout production (Figure 4B). Average final titers ranged from 3.5
to 5.5 g/L across these molecules. These data indicate that the
technology consistently produces robust and high titer producing pools.
Upstream process development was performed on mAb1 pool population #3.
Media/feed scouting experiments identified the GFB2 strategy as superior
to GFB1 (Figure 5). The Ambr® 250 bioreactor system was used for further
control of the GFB2 process. Using the optimized strategy, the pool
reached 28 million cells per mL viable cell density, maintained high
viability throughout production, and attained a final titer of
approximately 12 g/L.
Stability of Cell Pools
To determine the stability of pools expressing the Fc fusion protein,
three unique pools made at three different generations from the GPEx®
Lightning Dock Pool (not clone) were subjected to stability analysis.
For this study, two different media and feed strategies were employed.
GFB1 typically provides robust titer and culture longevity for most
products. GFB2 is more variable but often results in higher cell density
and significantly higher titers than GFB1. Final titers and average
number of integrated transgenes for all generations in each of the two
medias remained stable for more than 40 generations (Figure 6A).
The genetic and production stability of pools expressing mAb1 and mAb2
made using the Parental Dock clone were also assessed using GFB1 (Figure
6B). These pools also showed high production and genetic stability
across more than 40 generations. Together, these data indicate robust
stability of transgene copy number and protein expression. Stability and
consistency of expression are critical requirements for the potential
use of these stable cell pools in drug substance manufacturing.
Expression and Stability of Clones
To determine the expression and stability of clones, the highest titer
pool expressing the Fc Fusion molecules (pool population 4, Figure 6)
was cloned using the Berkeley Lights
Beacon® instrument. Clones were
ranked and exported based on relative expression using the
SpotlightTM Assay. 26 clones were expanded and their
attR copy numbers ranged from 37 to approximately 200. At high copy
numbers such as this, intrinsic variability in QPCR assays make it
difficult to accurately measure precise copy number (D’haene, 2010).
Since QPCR is better able to quantify lower copy numbers, we used
primers that amplify attP, which is only present in empty dock sites.
Highlighting the efficiency of the process, the attP amplicon could not
be detected in one of the clones indicating that this clone had all dock
sites filled.
Next, we performed a small-scale 16-day production run on these 26
clones using the GFB1 strategy. Titers ranged from 2.5 to 5 g/L in this
production run. We found that Boat copy number correlated well with
final titer (Figure 7A). To determine the stability of the three highest
titer clones, a study was performed, this time for over 60 generations.
As with cell pools, the clones showed the same robust stability of
transgene copy number (not shown) and product expression (Figure 7B).
Protein Quality Characterization of Pools and
Clones
To examine the consistency of the protein product quality, a
generational stability study was conducted using two pools made at
different times, but expressing the same Fc-fusion protein, which
contains five potential N-glycan attachment sites (Figure 8A and Table
1). While there are minor differences between the quantity of each
particular N-glycan between the two pools, different generations within
each pool are essentially identical, demonstrating consistent quality
over the generations studied.
Comparisons of product quality between pools and their clones were
performed for 3 different antibody products. To create sample diversity
for the evaluation, we chose clones that produced significantly more
product than the pool (mAb05), less product than the pool (mAb06), and
about the same amount of product as the pool (mAb07). No matter how the
clones produced relative to the pool, no significant differences in
glycosylation pattern or levels were observed (Figure 8B and Table 2).
In each case the pools and their corresponding clone(s) are
superimposable. Table 2 shows more detail and concordance for each
species between the pool and the clone(s). Of particular interest is the
similarity in high mannose and total fucosylated species.