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APPLICATION NOTE
The Expi293™ Expression System
The Expi293™ Expression System
Optimizing protein yield
The Expi293™ Expression System is
a mammalian serum-free transient
transfection system designed to produce
high levels of recombinant protein and to
scale easily from sub-milliliter to multiliter formats while maintaining consistent
volumetric protein yields.
The system includes Expi293F™ Cells,
Expi293™ Expression Medium, and the
ExpiFectamine™ 293 Transfection Kit,
which includes a transfection reagent
as well as proprietary transfection
enhancers. The Expi293™ Expression
Medium is capable of supporting
extremely high cell densities and enables
transient transfection at increased cell
densities, thus enhancing the protein
production capacity per milliliter of
culture media. The increased cell density,
in combination with a high-efficiency
transfection reagent and transfection
enhancers, leads to significant increases
in overall volumetric protein yields. The
high-density Expi293™ Expression System
can produce up to ten times more protein
than the FreeStyle™ 293 system.
The standard protocol provided in the Expi293™ Expression System Users
Manual is a robust method that results in high-level expression of a wide
variety of proteins. However, as proteins and equipment differ from lab to lab,
additional optimization may further increase individual protein yields.
Here we describe details about critical protocol steps, equipment, and
performance characteristics that may impact protein yield.
Materials
• Expi293™ Expression System (Cat. No.A14524)
• Opti-MEM® Reduced Serum Medium (Cat. No. 31985)
• Reporter gene: Human IgG cloned into pcDNA™ 3.4 vector (Cat. No. A14697)
• Antibody-Expressing Positive Control Vector (Rabbit IgG) (Cat. No. A14662)
• Erlenmeyer shake flasks with vented caps
Equipment setup
Equipment needed
• Shaker platform with 0.75–1 inch throw, capable of speeds 50–200 rpm
• Flask clamps or shaker platform sticky tape/sticky mat
• CO2 incubator set to 37oC, 8% CO2
Procedure
1.Place the shaker platform (such as the New Brunswick, Innova 2100) inside
the CO2 incubator, making sure there is sufficient clearance for movement.
Alternatively, a shaker incubator system such as an Infors Multitron or
Kuhner incubator shaker can be used.
2.Line the shaker platform with sticky tape or use flask clamps to keep culture
flasks secure while in rotation.
• The day before transfection,
Expi293F™ Cells were seeded
at 2.0 x 106 cells/mL in fresh
Expi293™ Expression Medium.
• The day of transfection, cells were
diluted to 2.9 x 106 cells/mL in fresh
Expi293™ Expression Medium.
For each 1 mL of culture to be
transfected, 1 µg of vector DNA was
diluted in Opti-MEM® medium to a
total of 50 µL. In a separate tube, 2.7
µL of ExpiFectamine™ 293 Reagent
was diluted in Opti-MEM® medium
to a total of 50 µL. Both tubes were
incubated at room temperature for
5 minutes.
• The DNA solution was added to
the ExpiFectamine™ 293 solution,
mixed well, and incubated at room
temperature for 20 min to allow
DNA complexes to form.
• The DNA complexes were then
added to the cell culture, and
Transfection Enhancers 1 and 2
were added 16–24 hours posttransfection.
Results
In developing the new Expi293™
Expression System, we determined
that the transfection conditions used
in the FreeStyle™ 293 Expression
System were inefficient at the
high cell densities used in the new
Expi293™ system. We examined key
protocol and equipment parameters
that impact transient protein yields
Optimization of ExpiFectamine™ 293
Transfection Reagent dosage
The new ExpiFectamine™ 293
Transfection Reagent allows highefficiency transfection of high-density
cultures of Expi293™ Cells, critical to
obtaining increased protein yields. The
volumetric yield of human IgG (protein
produced per culture volume) was
found to increase significantly with
increasing dosage of ExpiFectamine™
293 reagent; maximal performance
occurring at a dosage of 2.7 μL of the
transfection reagent per milliliter
of cell suspension (Figure 1). While
this result likely holds true for
other suspension 293F cell lines,
optimization of ExpiFectamine™ 293
reagent dosage is recommended if
the Expi293™ system is used with an
alternative 293 cell line.
Lipid dose
700
600
500
400
300
200
100
0
3.0
2.7
2.4
2.1
ExpiFectamine™ 293 dose (µL/mL culture)
Figure 1. ExpiFectamine™ 293 Transfection
Reagent dosage vs. yield of human IgG protein.
Yield of IgG increases with increasing dosage of
ExpiFectamine™ 293 reagent, with optimal yield
occurring at a dose of 2.7 μL of transfection
reagent per mL of cell suspension.
Optimization of DNA
We also examined the impact of
DNA dose on protein expression.
Interestingly, the Expi293™ Expression
System tolerated wide variation
in DNA dosage while maintaining
consistently high protein yields
(Figure 2). In this experiment, maximal
yield was obtained at 0.9–1.0 µg DNA
per milliliter of transfected culture.
DNA concentrations from 0.6 µg/mL
to 1.1 µg/mL also provided very high
levels of hIgG expression.
DNA dose
700
600
hIgG (mg/L)
Methods
Transfections were performed as
directed in the Expi293™ Expression
System Users Manual except as
described here:
and optimized the Expi293™ system
formulations and protocol to maximize
recombinant protein production.
However, depending on your
particular protein or laboratory setup,
additional optimization of transfection
cell density, DNA concentration,
ExpiFectamine™ 293 dose, and
Transfection Enhancer timing may
further increase recombinant protein
yields.
hIgG (mg/L)
A setting of 8% CO2 was determined
to be optimal for suspension
culture of Expi293F™ Cells in
Expi293™ Expression Medium.
Other CO2 settings (5%) can be
used if necessary but may result
in decreased cell health or protein
production.
500
400
300
200
100
0
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
DNA dose (µL/mL)
Figure 2. DNA dosage vs. yield of human IgG
protein.
Note: When expressing antibody
molecules with the heavy and light
chains encoded on two separate
plasmids, we also recommend
optimizing the ratio of heavy chain
to light chain for each individual
antibody, as the polypeptide transcription, folding, and degradation
rates can vary for different plasmids
and amino acid sequences. We
recommend initial testing of heavy
chain: light chain ratios between 2:1
and 1:4.
Enhancer time of addition
700
hIgG (mg/L)
600
500
Day 3
Day 6
Day 7
400
300
200
100
0
3 hr
24 hr
Time of enhancer addition
Figure 3. Time of post-transfection addition
of Transfection Enhancers 1 and 2, vs. yield of
human IgG protein.
Unfortunately, different counting
methods and instruments can give
widely varying results, confounding
efforts to recommend a cell density
for maximal protein expression.
In Figure 4, two automated cell
counting devices were used to
measure the density of an Expi293™
system cell suspension prior to
transfection. Transfections set up
using information from Cell Counter 1
generated increasing levels of protein
as the seeding density increased,
approaching the recommended
range. In contrast, transfections set
up using data from Cell Counter 2,
which underestimated cell density,
generated decreasing levels of protein
as the seeding density increased.
Due to the underestimated cell
count, the actual cell density in the
transfection experiments exceeded
the recommended level and led to
decreased protein yields. These
differences between different counting
devices will not prevent researchers
from obtaining high protein yields
from the Expi293™ Expression
System. However, to ensure optimal
protein yield, we recommend that
researchers determine the optimal
transfection cell density using their
own cell counting equipment.
Seeding density vs. protein yield
800
Cell Counter 1
700
Cell Counter 2
600
hIgG (mg/L)
Optimization of cell density at time of
transfection
Expi293™ Expression Medium
supports untransfected cell densities
up to 14 x 106 cells/mL or greater.
However, the cell density at the time
of transfection greatly impacts final
protein yield; low densities result in
a smaller protein production engine,
while densities which are too high
can result in lowered transfection
efficiency and rapid exhaustion of
medium nutrients. An optimal balance
of cell density and yield was achieved
with essentially a plateau of efficiency
between 2.5 and 3.0 x 106 cells/mL at
time of transfection (Figure 4).
500
400
300
200
100
0
2
2.5
2.75
3
Seeding density (x106 cells/mL )
Figure 4. Cell seeding density vs. yield of
human IgG protein.
Antibody-Expressing Positive Control
Vector
To assist end users in testing and
optimizing the Expi293™ Expression
System, we have provided a rabbit IgG
expression control, which optimally
generates 250–300 mg/L rabbit IgG in
the Expi293™ Expression System
(Figure 5). This control can also be
used to validate system performance
when scaling beyond conditions
validated by the manufacturer or in
alternative culture formats.
Control Rabbit IgG express scaleability
350
300
Rabbit IgG (mg/L)
Time of addition of Transfection
Enhancer
The ExpiFectamine™ 293 Transfection
Enhancers 1 and 2 are designed to
work with the ExpiFectamine™ 293
Transfection Reagent and Expi293™
Expression Medium to help achieve
high transfection efficiencies and
further increase recombinant protein
yields. We have determined that the
enhancers can be added 12–24 hours
post-transfection, with the optimal
time of addition typically at 16–18
hours post-transfection. We do not
recommend adding the enhancers
at too early a time point, as this will
result in reduced expression
(Figure 3). Adding enhancers after
24–30 hours post-transfection can
also result in decreased expression;
however, this is preferred over
not using the enhancers at all,
which has an even more significant
negative impact on protein yield. For
convenience, Transfection Enhancers
1 and 2 may be mixed together in a
cocktail that can be added directly to
the transfected culture in one shot.
This solution is stable at 4°C for at
least a week.
250
Day 5
Day 7
200
150
100
50
0
1 mL
30 mL
200 mL
Culture volume
Figure 5. Cell culture volume vs. pervolume yield of rabbit IgG protein from the
Antibody-Expressing Positive Control Vector
(Rabbit IgG).
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