Rapid, high-yield protein production from transiently ... - Invitrogen

Transfection
Rapid, high-yield protein production from
transiently transfected suspension cultures
FreeStyle MAX System
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An optimized expression system for CHO and 293 suspension cultures
Functional proteins with mammalian-type posttranslational modifications
Easy scale-up and purification with serum- and animal origin–free components
The FreeStyle MAX System is a breakthrough protein
production technology for large-scale, rapid generation of
posttranslationally modified, functional proteins. The excep-
tional productivity and speed with this technology is achieved
starting with adapted CHO and 293 cells grown in suspension
culture, and then transiently transfecting these cultures with the
new high-efficiency FreeStyle MAX Reagent. This system reli-
ably produces milligram quantities of human IgG (Table 1), and
provides an advanced alternative to conventional stable cell line
generation or transient transfection of adherent cultures in mul-
tiple roller bottles (Figure 1).
The FreeStyle MAX System offers a completely novel
cationic lipid–based transfection reagent, optimized expres-
sion kits, and protein production services. The FreeStyle MAX
Expression System (kit) includes FreeStyle MAX Reagent, GIBCO®
FreeStyle Expression Media, FreeStyle CHO or 293 cells , and
OptiPRO SFM. You can also purchase the system components
separately. All components of the FreeStyle MAX System are
completely animal origin free and are optimized for supporting
the growth and transfection of CHO and 293 cells in suspension
conditions.
Table 1—High protein yield in FreeStyle MAX CHO suspension cells. Human
IgG, Factor IX, human growth hormone (HGH), and erythropoietin (EPO)
were produced in FreeStyle CHO-S cells. Cells were cultured to a density
of 1 x 10 6 cells/ml in 30 ml of FreeStyle CHO Expression Medium in a
125 ml shaker flask. The transfection complexes were formed using 1.25 µg
of plasmid DNA and 1.25 µl of FreeStyle MAX Reagent per ml of culture
volume to be transfected, in OptiPRO SFM. At 24 hours post-transfection,
transfection efficiency was 60% as measured by GFP expression. Protein
production was measured by ELISA, in mg/L.
Protein Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Human IgG 6 18 28 36 42 47
Factor IX 0.72 1.6 2.24 2 2.32 2.16
HGH 50 150 270 330 350 400
EPO 1 2 3 6 6 7
Transfect
Select and subclone
Adapt to
serum-free
suspension
Bank and expand
Protein
Stable
2–6
months
2–7
days
Transfect
Protein
Transient
Harvest
CHO or 293
suspension
cells
Figure 1 —Transient transfection in suspension culture compared to stable transfection.
The FreeStyle MAX System offers exceptional productivity and
speed for generating milligram quantities of proteins and antibodies by
using suspension-adapted mammalian CHO or 293 cells in shake or spinner
cultures and the new high-efficiency FreeStyle MAX Reagent.

Convenience and speed
Large-scale, transient transfection of mammalian cells in suspen-
sion culture is a reliable and rapid method to generate milligram
quantities of mammalian recombinant proteins for functional,
therapeutic, and structural studies. With the FreeStyle MAX
System, you only need a sterile work bench, a CO 2 incubator,
and a platform shaker with shake flasks or a spinner culture.
Stable cell line development requires labor-intensive screening
and adaptation procedures to identify highly expressing clones.
The overall process requires months from the initial selection
of a stably transfected cell line to production of final product.
In comparison, the FreeStyle MAX System achieves large-scale
protein production in days, and is easily scalable from 30 ml to
10 L cultures (Figure 2). Detailed protocols for scale-up are avail-
able at www.invitrogen.com/transfection.
Protein production services
FreeStyle MAX Services are also available to produce your
proteins under optimized conditions in suspension CHO or
Ordering Information
The FreeStyle MAX System includes everything you need to get large quantities of functional protein from your mammalian expression
vector. The FreeStyle MAX CHO Expression System includes FreeStyle MAX Reagent, GIBCO® FreeStyle CHO Expression Medium, Free-
style CHO-S Cells, and OptiPRO SFM. The FreeStyle MAX 293 Expression System includes FreeStyle MAX Reagent, GIBCO® FreeStyle
293 Expression Medium, Freestyle 293-F Cells, and OptiPRO SFM. Visit www.invitrogen.com/transfection to learn how get the highest
transfection efficiencies and to order the FreeStyle MAX System.
293 systems based on the scale you request. We offer time-
course studies, and protein production in 1–50 L cultures. We
can provide you with the cell pellet, or even better, the purified
protein, as the final deliverable. Talk to your Invitrogen Account
Manager or call Tech Support at 800 955 6288, ext 2, to discuss
your needs and to initiate a project.
5
Days after transfection
Figure 2—A highly scalable, robust solution for rapid protein production. IgG
yields are shown for 30 ml and 1 L FreeStyle CHO-S cell cultures transfected
with the FreeStyle MAX Reagent. Transfection conditions for both scales
were 1.25 μg DNA and 1.25 μl FreeStyle MAX Reagent per ml of culture
volume to be transfected; 1 x 106 cells/ml; FreeStyle CHO-S cell passage
number 15, and a DNA/lipid incubation time of 10 min.
Product Quantity Cat. no.
FreeStyle MAX Reagent 1 ml 16447-100
FreeStyle MAX CHO Expression System 1 kit K9000-20
GIBCO® FreeStyle CHO Expression Medium 1 L 12651-014
GIBCO® FreeStyle CHO Expression Medium 6 × 1 L 12651-022
FreeStyle CHO-S Cells 1 × 10 7 cells R800-07
FreeStyle MAX 293 Expression System 1 kit K9000-10
GIBCO® FreeStyle 293 Expression Medium 1 L 12338-018
GIBCO® FreeStyle 293 Expression Medium 6 × 1 L 12338-026
FreeStyle 293-F Cells 1 × 10 7 cells R790-07
OptiPRO SFM (1X) 100 ml 12309-050
Human IgG yields (µg/ml)
70
60
50
40
30
20
10
0
30 ml culture
1 L culture
27.4 28.7
©2006 Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or www.invitrogen.com). By use of these products you accept the
terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. T-066147-r1 US 0606
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