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\begin{document}

\title{Genomic Features of Transposase and Randomly Derived Recombinant CHO
Clones}



\author[1]{Steven Huhn}%
\author[1]{Meiping Chang}%
\author[1]{B. Jiang}%
\author[1]{Xiaoyan Tang}%
\author[2]{Michael Betenbaugh}%
\author[1]{Zhimei Du}%
\affil[1]{Merck \& Co Inc}%
\affil[2]{Johns Hopkins University Department of Chemical and Biomolecular Engineering}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
The use of transposase in cell line development (CLD) programs has
experienced increased popularity over the past decade. However, few
studies have described the mechanism of action and the genomic and
phenotypic characteristics of clones derived from transposase.
Additionally, how these traits impact long-term bioproduction is
unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to
characterize the unique fingerprints associated with transposase-derived
clones. Transposase reduces the cellular pool of transient vector as
early as three days post transfection following transfection and
expedites stable pool establishment by up to two weeks. Furthermore, ,
recombinant DNA expression is significantly improved up to
\textasciitilde{}3 fold along with a greater balance of antibody heavy
and light chain transcripts, resulting in higher titers in transposase
generated pools. Transposase derived pools contained an often
innumerable number of integration sites, representing a vast increase in
integration site diversity over randomly generated pools, which were
bottlenecked at 1-3 integration sites per pool. These transposase
mediated integrations typically occurred in clean singlets, free of
genomic scars such as deletions, inversions, and other modifications
associated with legacy transfection methods which exhibited higher copy
numbers per integration site. Also we observed a relative decline in
gene expression with copy number increase in the randomly generated, but
not the transposase derived clones. Furthermore, transposase-derived
clones were more likely to exhibit enhanced a long term stability
profile, including product quality attributes such as Mannose-5. This
improved stability may result from circumventing mechanisms associated
with the silencing of tandem repeats. Thus, transposase-mediated
approaches can provide multifaceted molecular and phenotypic advantages
in cell line development when compared to legacy random-integration
methods.%
\end{abstract}%



\sloppy


\textbf{Introduction}

The transfection of exogenous DNA into a host organism is a fundamental
technique in molecular biology and the first step in CHO bioproduction
(Wurtele, Little, \& Chartrand, 2003). A typical transfection scheme
applies chemical or physical methods to permeabilize the cell membrane
to nucleic acid, resulting in nuclear uptake and expression by the
native transcription machinery. This process results in transient
episomal expression which typically peaks around 48-72 hours post
transfection and then declines thereafter with active cell divisions
(Cervera, Gutierrez-Granados, Berrow, Segura, \& Godia, 2015; Wurtele et
al., 2003). Regulatory agencies mandate that biotherapeutic material
must be derived from a single cell clone that exhibits stable expression
and consistent product quality attributes (Frye et al., 2016). These
features are obtained when exogenous DNA is permanently incorporated
into the host genome. The stable integration of nucleic acids by
traditional methods is both a rare (\textasciitilde{}0.01\% in CHO) and
poorly understood event (Abraham, Tyagi, \& Gottesman, 1982). Thus, the
process of stable cell line generation represents a critical pain-point
during cell line development (Wurtele et al., 2003).

Conventional stable CHO cell line establishment is reliant on random DNA
integration via illegitimate repair (Wurtele et al., 2003). This process
is dependent on active DNA double strand breaks and subsequent
recognition of foreign DNA by host DNA repair factors (Kostyrko et al.,
2017; Wurtele et al., 2003). Foreign DNA is first detected by endogenous
nucleases and ligases, leading to the concatemerization of many
exogenous molecules (Kostyrko et al., 2017; Sargent, Brenneman, \&
Wilson, 1997; Wurm \& Petropoulos, 1994; Wurtele et al., 2003). This
process is inherently mutagenic (Wurtele et al., 2003). The ends of the
concatemer are resectioned and ligated to an active DNA double strand
break via the non-homologous end joining pathway (NHEJ), resulting in
stable transgenic integration (Kostyrko et al., 2017; Sargent et al.,
1997; Wurm \& Petropoulos, 1994; Wurtele et al., 2003). In most
randomized integrations these events happen once to an acceptor locus
and are concurrent with chromosomal reorganization (Aeschlimann et al.,
2019; Smith, 2001). The damaged acceptor locus undergoes a dynamic
post-repair process that can introduce genetic changes to the integrated
transgene (Sargent et al., 1997; Singer, Mesner, Friedman, Trask, \&
Hamlin, 2000; Sishc \& Davis, 2017; Wurtele et al., 2003). Gene
amplification by agents such as methotrexate accelerate such changes,
and these transgenic arrays are prone to homologous recombination and
epigenetic silencing (Sargent et al., 1997; Singer et al., 2000; Wurtele
et al., 2003). Therefore, processes that are intrinsic to illegitimate
repair by NHEJ contribute to both genetic and epigenetic instability
(Chang et al., 2022; Chusainow et al., 2009; S. Huhn et al., 2022; S. C.
Huhn et al., 2021).

Transposons are DNA segments that can freely change positions within the
genome (Ding et al., 2005; Hickman \& Dyda, 2016). Transposon systems
have been widely exploited in molecular biology as an efficient tool to
improve integration frequency during transfection (Ding et al., 2005).
Transposon mediated integration differs from illegitimate repair in that
it requires the donor sequence to be flanked with specialized motifs
known as inverted terminal repeats (ITRs) (Ding et al., 2005; Mitra,
Fain-Thornton, \& Craig, 2008). Supplementation with an independent
transposase enzyme catalyzes excision of the donor sequence from the ITR
region, exposing staggered TTAA motifs with free hydroxyl groups (Mitra
et al., 2008). The exposed hydroxyls then perform a nucleophilic attack
on the DNA backbone of a TTAA rich acceptor locus, resulting in
integration of the donor DNA onto the acceptor site (Mitra et al.,
2008). Transposase mediated integration has the added benefit of
preferential insertion into active chromatin (Hickman \& Dyda, 2016;
Tsukiyama et al., 2011). These features make transposase driven DNA
integration an attractive option in cell line development pipelines.
While transpose utilization has been described in terms of antibody
yield and productivity, little work is available on how transposase
derived clones differ from those derived from non-homologous
recombination (Ahmadi et al., 2017; Matasci et al.).

Therefore, in this work we characterize the intrinsic features of CHO
clones derived from transposase-based integration and compare these to
those derived from randomized methods. We observe that the use of
transposase dramatically shortens CLD timelines, balances expression of
antibody chains, enhances recombinant DNA transcription and pool titer,
and improves product quality during cell line development. Furthermore,
we show that transposase lead to a marked increase in the diversity of
cell integration patterns which can have benefits in terms of clone
consistency. Lastly, we illustrate fundamental differences in the
genomic landscape of NHEJ repaired versus transposase derived clones:
while transposase mediated integrations occurred in singlets, free of
genomic scars associated with legacy transfection methods, randomly
generated clones were associated with changes to the host genome and
transgenetic cassette. These features offer a potentially protective
tool to limit the genetic silencing often observed in randomly derived
clones. Taken as a whole, we demonstrate from a mechanistic perspective
how the use of transposes improves cell line development platforms in a
multifaceted manner.

\textbf{Materials and Methods}

\textbf{Generation and propagation of recombinant cells}

CHO hosts were grown in CD-CHO medium (Gibco, Cat: 10743029) containing
1\selectlanguage{ngerman}×HT Supplement (Gibco, Cat: 11067030) and L-Glutamine (Gibco, Cat:
25030081). CHO cells were continuously cultured in shaking incubator
(Kuhner) at 37°C, 5\% CO\textsuperscript{2} with or without L-glutamine.

CHO cells were transfected by electroporation with GS (glutamine
synthetase) expression vector plus or minus transposase mRNA (in
titrated amounts) according to manufacturer's protocol (Neon, Thermo
Fisher Scientific). The transfected cells were then selected for the
expression of GS with CD-CHO media without glutamine, supplemented with
0-12.5 \selectlanguage{greek}µ\selectlanguage{english}M methionine sulfoximine (MSX). After continuous subculture,
stable pools were cryopreserved and evaluated by fed-batch production
assay. The vector system used in this manuscript utilizes a LC-GS (light
chain linked to GS which also contains the IRES element) expression
system, with the HC (heavy chain) being linked to a neomyicin resistance
gene.

For cloning, the bulk pools were cloned into 96-well plates through
limiting dilution or single cell sorting by using FACS Fusion sorter
(BD). After approximately 10 to14 days, each colony was scaled to
appropriately sized vessel plate.

\textbf{Transposase generation}

mRNAs were prepared from super transposase containing plasmid developed
internally. The plasmid was first linearized, and then subjected
to\emph{In Vitro} transcription using the HiScribe T7 ARCA mRNA Kit
(NEB, cat: E2060S). The mRNAs were purified using the MegaClear Kit
(Thermo, Cat: AM1908).

\textbf{Determination of genomic DNA copy numbers and RNA expression
levels}

Genomic DNAs were extracted from CHO cultures using the DNeasy Blood and
Tissue Kit (QIAGEN, Germany).

For gene expression, total RNAs from CHO cells were extracted using the
RNeasy Plus Mini Kit from QIAGEN (QIAGEN, Germany). cDNAs were prepared
from the RNA samples by reverse transcription using the SuperScript IV
VILO Master Mix (Thermo Fisher Scientific, MA).

The QX200 Droplet Digital PCR (ddPCR) System (Bio-Rad, Hercules, CA) was
used to quantify the DNA copy number or the RNA transcript levels of the
respective antibody chains.. Fluorescent-labeled oligo nucleotide probes
for the ddPCR reactions were designed using the Primer Express Software
(Applied Biosystems, Thermo Fisher Scientific, MA), and synthesized by
Invitrogen (Thermo Fisher Scientific, MA).

\textbf{Cytogenetics}

Suspension CHO cells were treated with 0.2 \selectlanguage{greek}µ\selectlanguage{english}g/mL Colcemid for 4-5 hrs at
37C. Cells were then adjusted with Pre-Hypotonic Swelling Solution
(Genial Helix, Cat: GGS-JL-007) and pelleted at 200g for 10 minutes. All
but 0.5 mL of the remaining culture media was aspirated, and cells were
resuspended in the residual media. The remaining cell volume was
adjusted dropwise to 1 mL of 0.075M KCL and then to 13 mL 0.075M KCL
with a serological pipette. Cultures were then incubated 20 minutes,
pelleted as above, resuspended in 0.5 mL KCL, and fixed with dropwise
addition of 3:1 methanol acetic acid. The cultures were then washed
three times in 3:1 methanol acetic acid and aliquots of cells were
dropped onto slides using a Hanabi-PVI cell-spreader. Slides were baked
overnight at 50\textsuperscript{o}C before any analysis. For
karyotyping, only cells with non-overlapping chromosomes were considered
for quantitation.

\textbf{FISH}

Cytogenetic spreads were first transferred to prewarmed PBS for 5-10
minutes and then transferred to 0.2 M HCL at 37C for 30 minutes.
Following several washes, the spreads were digested with RNAse A (Sigma,
Cat: R6148), Pepsin (Abcam, Cat: ab64201), and fixed in 1\%
paraformaldehyde for 10 minutes. The slides were then serially
dehydrated in 80, 90 and 100\% ethanol and allowed to air dry.

Slides were then denatured in water bath at 72\textsuperscript{o}C for
30 minutes in 2X SSC (Lonza, Cat: 51205), allowed to cool to room
temperature for 20 minutes, and immediately transferred to coplin jars
containing: 0.1X SSC, 0.07 N NaOH, 0.1X SSC at 4C and 2X SSC at 4C for 1
min each. The slides were then serially dehydrated as above, warmed to
37C and denatured probe containing 1-2.5 ng/\selectlanguage{greek}µ\selectlanguage{english}L probe DNA was applied to
the slide. The slide was covered with a coverslip, sealed with rubber
cement, and incubated at 37\textsuperscript{o}C for at least 18 hrs.
Following hybridization, the slides were submerged in 2X SSC + 0.1\%
tween, the rubber cement was removed, and coverslips were floated off.
Slides were washed at 72\textsuperscript{o}C in 0.4X SSC for 2 minutes,
and then washed in 2X SSC + 0.1\% tween for 5 minutes. Slides were then
equilibrated in PBS, blocked in 0.5\% Blocking Reagent (Perkin Elmer,
Cat: FP1012), and incubated with anti-DIG POD (Roche, Cat: 11207733910
at 1:500) overnight. Slides were then developed using the TSA Plus
Cyanine 3.5 (Perkin Elmer, Cat: NEL763001KT) exactly to manufacturer's
specifications. Slides were mounted with Vectashield + DAPI
(Vectashield, Cat: H-1200) before viewing on a Motorized Carl Zeiss
Microscope AxioImager Z2 using ISIS software (Metasystems, Inc).

\textbf{Probe Generation}

1-5 \selectlanguage{greek}µ\selectlanguage{english}g of plasmid was digested using the Roche DIG Nick translation kit
for 2-5 hours, until an approximate probe size of \textasciitilde{}500
bp was visualized on a 1.5\% agarose gel. The probe was purified using
the QIAquick Gel Extraction Kit (Qiagen, Cat: 28704 and 28706) and
eluted in purified water. Prior to use, the probe was combined with 12.5
\selectlanguage{greek}µ\selectlanguage{english}g of salmon sperm DNA (Invitrogen, Cat: AM9680), adjusted to 0.5M NaCl
and 70\% ice-cold ethanol, and incubated at -20C for 2 hrs. The probe
was precipitated by centrifugation at 16,000g for 1hr at
4\textsuperscript{o}C, washed twice in 70\% ethanol, and dissolved by
incubating in 5-10 \selectlanguage{greek}µ\selectlanguage{english}l of 100\% formamide using a thermomixer. The probe
was then adjusted to 95\% FISH Hybridization Buffer (Abnova, Cat:
U0029), mixed, and denatured at 75\textsuperscript{o}C for 10 minutes.

\textbf{Chromosome Painting}

Slides were prepared identically to those utilized in generic FISH, but
probe was substituted for 12X Chinese Hamster Probe (Metasystems, Cat:
D-1526-060-DI). Chromosome paints were visualized using ISIS software
(Metasystems, Inc).

\textbf{Targeted Locus Amplification}

5-10 million cells were processed for Targeted Locus Amplification (TLA)
according to the manufacturer's instructions (Cergentis, The
Netherlands). The amount of unique integration events identified by TLA
was tallied on a scaffold-by-scaffold basis per clone and is represented
in heatmaps. Transpoase specific integrations were identified as
sequences containing vector data extending past the ITR.

\textbf{Fed Batch Production}

Cells were seeded in in-house production media, glucose and lactate
levels were measured daily using the RANDOX RX imola chemistry analyzer
(Crumlin, UK). Cell density and viability were measured using a Beckman
Coulter ViCELL cell counter (Beckman Coulter, Indianapolis, IN). mAb
production levels were determined by Protein-A UPLC. In the mock
perfusion assay, cells were seeded at a density of 10 million per ml.
Viable cell density, mAb titer measurement, and complete media exchange
was performed daily for four days.

\textbf{ProA Purification}

Cell-free supernatants were adjusted to 500ul in PBS and purified using
PhyTips (PhyNexus, San Jose, CA; Cat: PTM 95-40-07). Briefly, tips were
equilibrated in PBS, and washed in 10 mM Sodium Phosphate, 500 mM NaCl,
pH 6.5 for three cycles. The captured mAb was eluted in 20 mM Sodium
Acetate, pH 3.5. Protein Concentration was assessed with UV spectroscopy
(Spectramax M5e, Molecular Devices, San Jose, CA) using at 260/280.

\textbf{N-Glycan}

Two micrograms of ProA purified material was subjected to HILIC and
N-Glycan analysis using the AdvanceBio Glycan Map, Rapid Resolution
(Agilent, Cat: 859700-913) column according to the manufacturer's
instructions. Peak identity was resolved using Empower 3.0 software.

\textbf{Statistical Analysis}

Statistics (ANOVA or student's T-test) was calculated using Graphpad
Prism.

\textbf{Results}

\textbf{Transposase accelerates CLD timelines and improves pool
productivity}

Transposase mediated transgenic integration is a ubiquitous tool in
molecular biology but is not well described in the CHO cell line
development landscape. In order to better understand the differences
between transposase-mediated versus random integration clones, host cell
lines were transfected with circularized DNA with or without titrated
amounts of transposase mRNA \textbf{(Figure 1).} At three days post
transfection, cells were cultured in media to select for the expression
of transgene and underwent regular passaging with complete media
exchange (see Materials and Methods). We observed that the +transposase
derived pools suffered little loss in viability (see red viability curve
representing the +Transposase clones in \textbf{Fig. 1A} ), and by
passage six, the pool reached maturity with a doubling time of
\textasciitilde{}28 hours \textbf{(red bars in Fig. 1B)} . Diluting the
transposase concentration ten-fold (green) and one-hundred-fold (purple)
resulted in a dose-dependent delayed recovery throughout selection
(\textbf{Figure 1A,B} purple, and green curves/histograms). In these
pools, doubling times did not reach maturity (\textasciitilde{}28 hours)
until passage 8 for 1:10 dilution and 10 for the 1:100 dilution. The
absence of transposase (blue) lowered post-selection viabilities over
all but 2 passages with stable pool establishment at 9 passages, 3
passages later than that with transposase at maximum concentration
(\textbf{Figure 1B} , red bars).

Next, the pools were grown in fed batch cultures and evaluated for
growth and antibody production. For a 14 day fed-batch production
process, a transpose dependent increase in the production of mAb was
observed \textbf{(Figure 1C,} right two graphs\textbf{).} The
+transposase and 1:10 derived pools were observed to produce 11 g/L and
7 g/L of mAb, respectively \textbf{(Figure 1C,} left hand plot) while a
1:100 dilution and complete omission of transposase resulted in a
striking and significant decrease in titer. To further characterize the
impact of transposase implementation during stable pool development,
aliquots of stable pools were collected, lysed, and extracted for total
RNA and DNA every 2-3 days for up to 45 days post transfection. As shown
in \textbf{Figure 1D,} the levels of cellular vector heavy chain DNA
decreased dramatically over time during the transient phase of
transfection (\textasciitilde{}3 weeks), but this rate of decline was
dependent on the amount of transposase. This result suggests that the
transposase enzyme begins exhausting the pool of cellular plasmid
immediately and perhaps prior to the first measurement, ultimately
generating stable clones as early as day 3 after transfection (910 \&
730 copies of heavy chain in the +transposase group {[}red line{]} vs
1680 \& 1350 copies for -transposase group {[}blue line{]} on D3 and D7,
respectively). A transposase dose-dependent increase of recombinant
vector mRNA expression was also observed \textbf{(Figure 1E)} ,
especially for the antibody chains, consistent with titer results.
Therefore, we surmised that transpose is most important for vector
elements which are not as subject to selective pressure, given that the
heavy chain was exposed to much less stringent selection in our
expression platform (see Materials and Methods). Considering the
expression patterns of mAb heavy and light chains at highest transposase
level (\textbf{Figure 1E} ), these data suggest that an added benefit of
transposase incorporation is to facilitate a more balanced ratio of
antibody chain expression, when compared to the random integration
alternative.

\textbf{Cytogenetic characterization of integration sites}

We then sought to better characterize some of the cellular properties
that transposase conferred to established stable pools. First, analysis
by digital PCR demonstrated a progressive increase in copy number at
increasing transposase concentrations during the transfection process
which suggests a transposase-specific effect in driving transgene copy
number (see solid bars in \textbf{Figure 2A} ). We also evaluated the
levels of the integrated bacterial fragment (see Bacterial Backbone
{[}BB{]} in schematic of \textbf{Figure 2A} and checkered bars in the
histogram), a marker for whole-vector integration through non-homologous
recombination. These levels indicate the existence of random integration
events in the +transposase pools, which was on average one-third of the
total integrations (\textbf{Figure 2A,} compare solid and checkered
histogram). Conversely, the quantity of bacterial backbone almost
exactly matched the amount of heavy chain copies in the -transposase and
1:100 pools, demonstrating complete random integration of the plasmid
for these cases. Next, in order to evaluate the means of integration,
the pools were treated for cytogenetic analysis and probed with a
cocktail of biotin labeled Bacterial backbone (a marker for random
integration) and DIG labeled ITR (a marker for the transposase mediated
integration) fragments \textbf{(Figure 2B\&C)} . Titration of
transposase resulted in a dose dependent progressive increase in total
integration sites, confirming our ddPCR results \textbf{(Figure 2 A-C).}
Furthermore the +transposase pools had multiple integration foci
scattered across several chromosomes \textbf{(Figure 2B)} . Both
findings were in stark contrast to the 1:100 or -transposase pools,
which tended to exhibit a single integration event with a high
transgenic probe intensity. In these pools, the bacterial backbone
always colocalized with the oft singular integration event
\textbf{(Figure 2B)} . Conversely, in the +transposase or 1:10 pools,
the Bacterial BB probe appeared to colocalize with only the brightest
ITR foci, suggesting that these random integration sites contained a
relatively higher number of copies than those resulting from transposase
(\textbf{Figure 2B} , top panel, compare red arrowheads, indicating
random integrants, to white arrows indicating +transposase sites).
Together, these data suggest that transposase favors enhancement of
transgenic insertions albeit with fewer copies per site.

The interaction of the transgene with the host chromosome plays a
critical role in gene expression, and the transfection modality can play
a critical role in the transgenic landscape (Wurm \& Petropoulos, 1994).
In order to better understand the transgene architecture with and
without transposase, we utilized FISH and whole chromosome painting
(mFISH) to characterize chromosomal integration sites in the
transposase-derived and randomly integrated pools. We examined a 24
clones in the +transposase pool and observed that each clone
demonstrated a unique configuration of integration sites among
chromosomes \textbf{(Figure 3A,B)} , with up to 15 foci on one
chromosome while others had only one or two (compare the zoomed images
comparing Chr1 and ChrX amongst clones 1 \& 2 in the lower panel of
\textbf{Figure 3B} ). In stark contrast, individual clones from the
-transposase pools demonstrated only a narrow distribution comprised of
only a few unique integrations sites throughout the population (largely
consisting of just Chr5 and Chr8) for the analyzed cells \textbf{(Figure
3A,B)} . Interestingly, while integration site diversity in the
-transposase pools was clearly bottlenecked, we did observed a drastic
distribution of copy number at these integration sites, which could be
visualized by the probe intensity at the integrated foci (compare the
zoomed images comparing Chr5 amongst clones 3 \& 4 in the lower panel
of\textbf{Figure 3B} ).

\textbf{Characterization of integration sites using deep sequencing}

We next sought to further elaborate on our findings using DNA
sequencing. Targeted deep sequencing when married to approaches such as
targeted locus amplification (TLA) allows for enhanced resolution across
transgenic arrays (Aeschlimann et al., 2019; Hottentot, van Min,
Splinter, \& White, 2017). We were disappointed to see that conventional
whole genome sequencing was not able to generate a clear and
quantitative picture of pool diversity in the +transposase pools, though
for the -transposase pool one major integration site could be visualized
(data not shown). Therefore, to circumvent this issue, we screened and
identified 19 of top producing clones from the pools (with and without
transposase) identified in \textbf{Figure 3} and sequenced them using
TLA (See Materials and Methods). These sequencing results closely
mirrored those visualized by FISH, in which all --transposase pools
demonstrated near homogeneity across a few integration sites
(\textbf{Figure 4A} ). In contrast, the clones derived by +transposase
exhibited many different integrations sites across multiple
loci\textbf{(Figure 4A).} Interestingly, the number and intensity of the
heavy chain was higher than for light chain for these +transposase
clones, which may be indicative of selection stringency of each chain.
Additional examination of the TLA-derived genomic landscape revealed
several the features associated with NHEJ in -transposase pools such as
deletions (yellow arrows), inversions (blue arrows) and complex (green
arrows) rearrangements (\textbf{Figure 4B,} see representative coverage
plots from 3 clones, top panel). In contrast, the integration sites
identified as transposase demonstrated little or no change to the
surrounding chromosome and exhibited clean peaks with decreasing
coverage with distance from the primer pair (\textbf{Figure 4B,} see
representative coverage plots from 3 clones, bottom panel). When the
copy number of these clones was analyzed by ddPCR \textbf{(Figure
5A),}the +transposase clones demonstrated a relatively higher number of
copies on average. The copy number of the +transposase clones closely
mirrored that of the total number of identified integration sites,
suggesting that transposase tends to integrate in singlets rather than
in tandem (compare histograms of the +transposase clones
across\textbf{Figures 5A,B} )\textbf{.} Additionally, we detected
randomly integrated (see y-axis in \textbf{Figure 5B} labeled unknown or
random) DNA in +transposase clones that did not have a 1:1 relationship
between copy number and the number of integration sites. This suggests
that these randomly integrated sites likely contained multiple copies,
in agreement for what is observed in clones derived without
transposase\textbf{.} In agreement with \textbf{Figures 2 and 3} ,
-transposase clones exhibited diversity in copy number
only\textbf{(Figure 5A)} . Furthermore, a clear trend emerged for the
subset of -transposase clones with identical integration sites; as copy
number rose, gene expression tended to decline (negative correlation
R=-0.75)\textbf{(Figure 5C)} . This observation is consistent with the
observation that expansion of tandem arrays leads to genetic silencing
(Chusainow et al., 2009; Kostyrko et al., 2017). This same trend was not
evident for the +transposase clones, although these clones contained
different configurations of integration sites\textbf{(Figure 5B and D)}
. Taken together, these data are in agreement with the proposition that
the presence of a transposase results in an increase of transgene copy
number scattered across many integration sites rather than one. This
spreading of integration sites may be inherently more protective against
the silencing gene expression of tandem repeats, which occurs during
random integration.

\textbf{Assessment of cell and product quality stability in transposase
and randomly derived clones}

Establishment of cell lines with stable expression and consistent
product quality attributes over time plays a vital role in regulatory
clearance (Chusainow et al., 2009; Frye et al., 2016). In order to
demonstrate stable productivity and product quality of the +transposase
clones, we aged the nineteen clones in \textbf{Figure 5} to PDL75 (75
population doublings) and assessed cell productivity and percentages of
Mannose-5 structures at day fourteen during fed batch culture. The
specific productivity (top panel, \textbf{Figure 6} ) and percentage of
Mannose-5 (bottom panel, \textbf{Figure 6} ) in the +transposase clones
were comparable between the PDL0 and PDL75 clones for the +transposase
clones (bottom panel, \textbf{Figure 6} ). However, the standard
deviation (Std of 8.3 for transposase clones vs 17.8 for random clones)
of cell productivity was higher in the randomly derived vs the
+transposase clones, demonstrating enhanced consistency in the yield of
producers in the + transposase group. Additionally, amongst the
-transposase clones, a greater variability in Mannose-5 was observed as
the cell aged and the Mannose-5 levels were on average higher in these
clones when compared to their counterparts. Thus, transposase-mediated
integration does not affect stability and may provide positive benefits
in terms of product quality consistency.

\textbf{Discussion}

In this work we contrasted the fundamental features associated with
recombinant CHO clones and pools generated with and without use of
transposase. We demonstrated that transposase resulted in considerably
more diverse pools that are established in a shorter time frame and with
an impact on cell titer and qP consistent with previous results (Kowarz,
L\selectlanguage{ngerman}öscher, \& Marschalek, 2015) (Ahmadi et al., 2017; Matasci et al.).
However, little has been done to molecularly characterize and compare
clones derived from both processes. To address this question we applied
molecular genetics tools for an in depth characterization of pools and
clones obtained from both methodologies. Our work demonstrates that
transposase derived clones contained many permutations of unique
integration events which preferred the euchromatic regions of the CHO
genome (S. Huhn et al., 2022). We also observed that these integrations
tended to occur as clean, scattered singlets free of genomic scarring,
rather than rearranged tandem repeats which can also be accompanied by
deletions and inversions or other genomic events present for random
integrations. Expression levels, especially of heavy chains (which
exhibited weak selection), were also observed to decline with days
post-transfection for those without transposase, suggesting gene
silencing for random integration. Furthermore, transposase derived
clones did not appear to have liability issues in terms of long-term
expression, and instead offered enhancements in long-term product
quality, and enhancements in clonal expression consistency.

It is well understood that transposase prefers open chromatin
architecture during the integration process (Raurell-Vila,
Ramos-Rodriguez, \& Pasquali, 2018). Interestingly, the integration
patterns for clones derived from transposase differ greatly than those
observed without it. We cannot rule out that components of our vector
drive the preferences observed in \textbf{Figure 3} , and furthermore,
the lack of genetic diversity makes it difficult to derive commonalities
in regions prone to integration by illegitimate repair in CHO.
Nevertheless, it has been observed that fragile sites, AT rich regions
associated with increased risk of DNA double strand breaks, are
associated with increased integration by viruses and foreign DNA in
human cells (Ma et al., 2012; Rassool et al., 1991). It is possible that
many of loci associated with illegitimate DNA integration in CHO are
associated with similar ``fragile'' features. Despite this, to our
knowledge, no comprehensive evaluation of these random integration
patterns have been demonstrated in CHO.

Several key questions and technical challenges must be resolved in order
to better utilize transposase in the context of bioproduction. Chiefly,
we observed the substantial persistence of random integrations in
transposase derived pools. These random sites integrated in tandem and
were subject to the same liabilities as any site derived from
non-homologous repair. Thus, preventing these events represent an area
for improvement. In addition, we observed a poor correlation between
copy number and gene expression in the transposase derived clones. These
data suggest that, not all transposase sites contribute equally to gene
expression. Thus future refinements are still needed in order to truly
harness the capabilities of transposase for cell line development.

\textbf{Acknowledgements}

We would like to thank Dr. Luke Nelson for his review of this
manuscript.

\textbf{Declaration of interests}

S. Huhn, B. Jiang, M. Chang, X. Tang, Z. Du are employees of Merck Sharp
\& Dohme Corp., a subsidiary of Merck \& Co., Inc., Kenilworth, NJ, USA
and potentially own stock and/or hold stock options in Merck \& Co.,
Inc., Kenilworth, NJ, USA. Merck \& Co., Inc provided support in the
form of salaries for all authors but did not have any additional role in
the study design, data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of these authors are
articulated in the ``author contributions'' section.

Figure Legends

\textbf{Figure 1: Establishment of Recombinant CHO Pools with and
without transposase}

Host CHO cells were transfected with recombinant DNA in addition to
transposase mRNA, with transposase mRNA diluted serially to 1:10 or
1:100, or transposase entirely omitted (-transposase). The recombinant
pools were subjected to selection and passaged ten times. The
viabilities are plotted in \textbf{(A)} while associated and doubling
times are plotted in \textbf{(B).}The dashed black line in \textbf{(B)}
represents pool maturity (about a 28-hour doubling time). Mature pools
were then subjected a 14-day fed batch procedure \textbf{(C).} Titer
(left panel) and qP (right panel) were measured on the indicated time
points (in Days) following Protein A column purification. A red asterisk
represents statistical significance (p\textless{}0.05) of D14
+transposase titer vs. D14 data points in other groups. \textbf{(D)}
Cells were transfected with recombinant DNA with titrated transposase
mRNA (blue curve without transposase, purple curve 1:100 transposase
dilution, green curve 1:10 transposase dilution, and red curve undiluted
transposase). Cell aliquots were then harvested, lysed, and extracted
for gDNA at the indicated daily timepoints post transfection (x-axis).
The DNA was then processed by ddPCR and the copy number of the antibody
heavy chain was determined (y-axis). \textbf{(E)}Cell aliquots were then
harvested, lysed, and extracted for mRNA at the indicated daily
timepoints post transfection (bottom x-axis). The mRNA was then
processed and the gene expression of the antibody recombinant chains was
quantified (red curve heavy chain, blue curve light chain, green curve
glutamine synthetase). The top x-axis demonstrates the category of
transposase titration.

\textbf{Figure 2: Copy Number and Integration Site Mediated Changes to
Transposase Derived Clones} \textbf{and Pools}

Cells were transfected with recombinant DNA with (including 1:10 or
1:100 dilutions) or without transposase mRNA. \textbf{(A)} The
recombinant pools were subjected to selection, passaged, and then DNA
extracted for copy number analysis with ddPCR. The average number of
heavy chain copies is shown by solid bars. The average number of
bacterial backbone copies (indicative of total vector incorporation) is
shown by dashed bars. The number of transposase mediated copies can be
estimated by the difference between the bacterial backbone and heavy
chain (HC). A red asterisk represents statistical significance
(p\textless{}0.05) of the HC copy number +transposase data point vs HC
copy number in other groups. A schematic of the plasmid integration
vector is shown at right. \textbf{(B)} Recombinant pools were fixed in
3:1 methanol acetic acid, spread onto slides, and probed with FISH
probes specific for the bacterial backbone (green, whole-vector
integration) or the transgene (red, transposase mediated integration).
White arrows indicate pure transposase mediated integrations (note the
absence of co-localization with green probe), while red arrowheads mark
randomized integrations where both probes colocalize. Note the intensity
of random integrations (red foci intensity) compared to transposase
mediated integrations, indicating higher copy number. The number of red
foci per metaphase spread in +transposase, 1:10 dilution, and
-transposase pools were quantified in \textbf{(C)} . Each dot represents
an individual cell within the bulk pool. A red asterisk represents
statistical significance (p\textless{}0.05) of +transposase foci number
vs. all other groups, while a double red asterisk represents
significance vs. 1:100 or -transposase only.

\textbf{Figure 3: Integration site diversity.} Recombinant CHO cells
were transfected with recombinant DNA containing transposase mRNA
(+Transposase) or buffer control (-Transposase). Following selection and
stable pool establishment, the pools were fixed in 3:1 methanol acetic
acid, dropped onto slides, and co-stained for recombinant DNA and
chromosome painting. \textbf{(A)} The number of observed FISH foci in
metaphase chromosome territories was quantified in heatmap form among 24
+Transposase and 36 -Transposase metaphase spreads (clones). Rows
represent individual clones (in the pool) and columns represent
metaphase chromosomes. As the number of observed integrations increases,
heatmap coloring shifts from green (low integration number) to red (high
integration number). \textbf{(B)} Representative images used in Figure
3A of a +Transposase cell (left panel) and a -Transposase clones (right
panel). Chromosomes that are boxed in orange are zoomed in the bottom
panel. The zoomed images demonstrate dramatically different integration
patterns in the +transposase clone chromosomes 1 and X. Note the
dramatic difference in fluorescence (copy number) in the -transposase
clones, but not integration sites.

\textbf{Figure 4: Integration Site Characteristics and Integrity in
+Transposase and -Transposase derived Clones.} Stable pools from
transposase derived and random transfection were cloned, scaled, and
subjected to fixation and transgene locus amplification
(TLA).\textbf{(A)} The number of integration sites per clone was
quantified and bucketed into individual genomic scaffolds; the large
genomic assemblies that make up the CHO chromosomes. Columns represent
individual clones and rows represent the number of integrations
occurring at the indicated scaffold (genomic coordinate). As the number
of observed integrations increases, heatmap coloring shifts from (low
integration number) to red (high integration number). Note the
completely unique composition of sites found in transposase derived
clones and the scaffold/site frequently used in random
integration.\textbf{(B)} The sequence read coverage profiles of loci
flanking the integration sites within three +Transposase and three
-Transposase clones were characterized by TLA following read mapping and
bioinformatics analysis. The presence of a clean singular peak
represents a locus with no obvious higher-level structural changes (see
representative sites from transposase clones, boxed blue bottom panels).
In the case of the clones with random integration (top red boxed
panels), the sequence read coverage profiles and positions of the
transgene -- genome breakpoints indicate DNA structure changes including
deletion (yellow arrows), inversion (blue arrows) and complex
rearrangements (green arrows).

\textbf{Figure 5: Relationship between Copy Number and Integration Sites
in +Transposase and -Transposase derived Clones. (A)} Stable pools from
transposase derived and random transfection were cloned, scaled, and
subjected DNA extraction and copy number analysis via ddPCR.\textbf{(A)}
Histogram of DNA copy numbers (y-axis) of the heavy chain (HC) and light
chain (LC) of the antibody (noted in the x-axis). Each color represents
a unique clone, and the method each clone was derived is denoted under
the bars on the x-axis. \textbf{(B)} Histogram of the total number of
integration sites (quantified in y-axis) in each clone was bucketed into
transposase mediated heavy chain specific, transposase mediated light
chain specific, random, or unknown using TLA (quantified in x-axis).
Each color represents a unique clone and matches\textbf{(A)} . The
dashed line is drawn through both graphs at the 10 copy or 10
integration mark, respectively, and used to compare copies to
integration sites. \textbf{(C)} ddPCR was used to measure the RNA
expression of the heavy and light chain (barplot, left-y axis) among the
random integration clones with an identical integration site and
contrasted to transgene copy number (black dotted line, right y-axis).
The clone ID is depicted in the x-axis. \textbf{(D)} This process was
also used to measure the expression of the heavy and light antibody
chains among the transposase derived clones. The correlation coefficient
R between the RNA levels of the heavy chain and copy number is indicated
in the top right hand corner of the graph. (HC=Antibody heavy chain,
LC=Antibody heavy chain).

\textbf{Figure 6: Stability of Productivity and Antibody Characteristics
in Transposase Derived and Random Integration Clones.} Stable pools from
transposase derived and random transfection at 0 PDL (population
doubling level) or 75 PDL were grown in fed batch culture for 14 days,
then cell free supernatants were collected and subjected to ProA
purification and N-Glycan analysis. The top and bottom panels show the
cell specific productivity, and the percentage of Mannose-5 identified
in each mAb, respectively. The dotted line separates the transposase
derived clones (left) and the random integration clones (right). Clones
are represented in different colors (as in Figure 5) with solid colors
representing PDL0 and checkered color representing PDL75. The PDL level
of clones is also shown in the x-axis. Statistical significance (p
\textless{} 0.05) between a clone's PDL0 and PDL75 trait marked by a red
asterisk.

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