This document discusses the optimization of a dual expression vector design in the CHOZN® GS-/- expression system to enhance recombinant protein production by minimizing sequence redundancy and improving selection stringency. Various promoter designs and poly A signals were tested, leading to the identification of vector #77 as the top performer with expression levels comparable to the control vector. The modifications aimed to facilitate cloning, propagation, and analysis while maintaining high expression levels in stable cell lines.

1. Improved vector design eases cell
line development workflow in the
CHOZN®
GS-/-
Expression System
Amber Petersen and Trissa Borgschulte, MilliporeSigma, St. Louis, MO
hCMV Promoter HC#77
mCMV
Promoter,
Exon, Intron
LC
GS
Selection
Cassette
hCMV Promoter LC
hCMV
Poly A
#37
mCMV
Promoter,
Exon, Intron
HC
GH-Bt
Poly A
GS
Selection
Cassette
hCMV
Poly A
GH-Bt
Poly A
hCMV Promoter LC#79
mCMV
Promoter, EF1a
Intron
HC
GS
Selection
Cassette
hCMV Promoter HC#78
mCMV
Promoter, EF1a
Intron
LC
GS
Selection
Cassette
hCMV Promoter LC#39
mCMV
Promoter, EF1a
Intron
HC
GS
Selection
Cassette
hCMV
Poly A
hCMV
Poly A
hCMV
Poly A
GH-Bt
Poly A
GH-Bt
Poly A
GH-Bt
Poly A
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Introduction
Expression vectors are critical determinants of the
outcome of a cell line development process, influencing titer
and stability of recombinant protein expression. Vector
elements of critical importance include the promoters,
enhancers and introns that regulate transcription, poly A
signals that impact translation and the selection cassette
which influences the stringency of selection, impacting the
resulting titers in surviving pools of cells. Each of these vec-
tor components can be optimized for maximum production
from the stable cell line.
In addition to recombinant protein production, vector
design can also impact the ease of cloning and stability
for propagation in E. coli and the mammalian host cell
line. In dual expression vectors, both expression
cassettes may contain the same promoter or poly A
sequences. Sequence redundancy in the two expression
cassettes can lead to recombination during propagation
in E. coli, resulting in loss of vector elements that alters
oreliminatesrecombinantproteinproduction.Sequence
redundancy also complicates sequencing of the vector,
identification of insertion sites in the host cell genome
and copy number analysis.
For these reasons, we have sought to eliminate sequence
redundancy with modifications of the poly A signals and
promoter sequences of a dual expression vector. Here,
we have tested the effects of various promoter designs
and poly A signals on recombinant protein production
in stable pools and single-cell clones.
Figure 1. Vector Modifications
The control vector and modified vectors are shown above. Each vector was constructed with
either the LC and HC of an IgG or GFP (in the LC expression cassette) and RFP (in the HC
cassette).
Table 1. Selected Promoters Influence Expression Levels
The percentage of GFP and RFP double-positive cells was determined for each vector.
Mean channel fluorescence (MCF) was also measured and the GFP to RFP ratio is shown
as an indication of the relative expression strengths of the promoters driving LC and HC.
Methods
Gene synthesis and vector construction of IgG1 and fluoro-
phore-expressing vectors was done by Atum. Vectors were
transfected into CHOZN®
GS-/-
cells via electroporation. For analysis
of GFP and RFP expression, cells were collected 48 hours post-
transfection and then sorted using a MACSQuant instrument.
Selection and generation of stable pools from transfections with
IgG-encoding vectors was performed as described in the CHOZN®
technical bulletin. Briefly, minipools were plated in glutamine-
lacking media at 5,000 cells per well 24 hours post-transfection.
After recovery from selection, minipools were gradually scaled
up to shake flask. Titer analysis was performed in static (96 well
plate), in a 7 day TPP assay and in a 14 day fed batch assay using
a QK ForteBio. Single-cell clones were generated by limiting-
dilution cloning.
Vector % GFP(LC)/RFP(HC)+
GFP(LC): RFP(HC)
#37 62.7 0.15
#39 50.4 0.30
#41 41.6 0.37
#43 43.3 1.73
#45 44.9 0.91
Figure 2. Vector #39 Out-Performs Other Promoter Designs
IgG titers of select minipools during the scale-up process.
Figure 3. Vector Modifications in Response to Initial Screen
The mean channel fluorescence ratio of vector #37 indicated high expression from the HC-expressing cassette, providing a potential explanation for the poor results seen in the initial screen.
Therefore, an additional vector was created in which the relative positions of the LC and HC were switched (A). Vector #39, identified as the top performer in the initial screen, was similarly modified
(B). In addition, the orientation of the GS cassette was reversed in vector #39 with the intent to improve titers through increased selection stringency (B).
Figure 4. Top Performing Vector Design Created by Switching the Relative Positions of the Light and Heavy Chain of Vector #39
IgG titers of select minipools during the scale-up process.
Figure 5. Stable Minipools and Single-Cell Clones Generated from Vector #77 Produce Levels of IgG Comparable to the Control Vector
IgG titers from minipools and single-cell clones throughout the scale-up process.
0
5
10
15
20
25
30
35
40
45
50
#37 #39 #41 #43 #45 Control
Titer[mg/LIgG]
Vector #
0
10
20
30
40
50
60
#37 #77 #39 #78 #79 Control
Titer[mg/LIgG]
Vector #
0
10
20
30
40
50
60
70
80
90
Titer[mg/LIgG]
#77 Control
0
50
100
150
200
250
300
#39 #41 #43 #45 Control
Titer[mg/LIdddgG]
Vector #
0
200
400
600
800
1000
1200
1400
#39 #41 #45 Control
MaximumTiter[mg/LIgG]
Vector #
0
50
100
150
200
250
300
#37 #77 #39 #78 #79 Control
Titer(mg/LIgG)
Vector #
0
500
1000
1500
2000
2500
3000
3500
4000
4500
#77 Control
MaximumTiter[mg/LIgG]
Vector #
0,0
500,0
1000,0
1500,0
2000,0
2500,0
3000,0
#77 Control
Vector #
MaximumTiter[mg/LIgG]
0
50
100
150
200
250
300
350
Titer[mg/LIgG]
Control #77
0
500
1000
1500
2000
2500
3000
3500
4000
4500
#77 Control
Vector
MaximumTiter[mg/LIgG]
A. 7 Day Static Assay (80 minipools)
A. 7 Day Static Assay (24 minipools)
A. 7 Day Static Assay
(640 minipools)
A.
B. 7 Day Batch TPP Assay (Top 10 minipools)
B. 7 Day TPP Assay (10 minipools)
B. 14 Day Fed Batch Assay
(Top 25 minipools)
B.
C. 14 Day Fed Batch Assay (Top 10 minipools)
C. 14 Day Fed Batch Assay (10 minipools)
C. 7 Day Static Assay
(300 Single-Cell Clones)
D. 14 Day Fed Batch Assay
(Top 25 Single-Cell Clones)
Results
Modifications to our dual expression vector were made in order to minimize sequence similarity,
easing cloning and analysis of sequence integration into the host genome (Figure 1). Generation and characterization of stable
minipools expressing a benchmark humanized IgG1 revealed a substantial influence of these modifications on titer. IgG expression
was negatively impacted by several of the promoter modifications, including #37, #41 and #43 (Figure 2). The lead vector
identified in the initial screen was vector #39.
In addition to lower titers, vector #37 also resulted in relatively few minipools expressing detectable levels of IgG in contrast to all
other vectors, from which nearly all minipools were expressing detectable levels of IgG. Analysis of GFP and RFP expression from
the modified vectors indicated relatively high expression from the RFP/HC expression cassette of vector #37 (Table 1). A stronger
promoter resulting in overabundance of HC, known to be toxic to cells, provides a possible explanation for the poor results with
this vector. Interestingly, swapping the positions of the LC and HC in #37 resulted in a vector, #77, that out-preformed the initially
identified lead vector (Figure 4). This same change was made to vector #39 without any resulting improvement in titers
(vector #78, Figure 3). Interestingly, vector #39 had a smaller difference in relative promoter strengths, based on mean channel
fluorescence ratio of GFP to RFP (Table 1), suggesting that overabundance of HC was not an impediment to IgG expression from
#39. Poor titers were also seen with a modified version of vector #39 (vector #79, Figure 4) in which the glutamine synthethase
selection cassette was in the reverse orientation. This second screen identified vector #77 as the lead vector design (Figure 4).
A full comparative study of vector #77 and the control vector was performed, cumulating in the generation and comparison of
single-cell clones from each. These studies have demonstrated the equivalence of these vectors in terms of IgG titer (Figure 5).
Further analysis will include stability studies on the clones generated as well as comparison of expression of other, more
difficult-to-express antibodies. In summary, this work has resulted in the identification and characterization of a dual expression
vector with minimized similarity between the two cassettes to ease cloning, propagation and analysis of vector integration in
stable cell lines while maintaining the high expression of the original vector design.
hCMV Promoter LC / GFP
hCMV
Poly A
#37
mCMV Promoter,
Exon, Intron
HC / RFP
GH-Bt
Poly A
hCMV Promoter LC / GFP
hCMV
Poly A
#39
mCMV Promoter,
EF1a Intron
GH-Bt
Poly AHC / RFP
hCMV Promoter LC / GFP
SV40
Poly A
#41 hCMV Promoter
GH-Bt
Poly AHC / RFP
hCMV Promoter LC / GFP
HSV
TK
Poly A
#45
Insulators, hEf1a
Promoter, Intron
GH-BT
PolyAHC / RFP
hCMV Promoter LC
SV40
Poly A
Control
Vector
hCMV Promoter HC
SV40
Poly A
hCMV Promoter LC / GFP
HSV
TK
Poly A
#43
GH-Bt
Poly AHC / RFP
Insulators,
hGAPDH Promoter,
hIE1 Intron
Control
Vector
hCMV Promoter LC
SV40
Poly A
hCMV Promoter HC
SV40
Poly A
GSSelectio
n
Cassette LC Express
ion
Cassette
HCExpression
C
assetteKan.
SelectionCassette