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Transient Production of scFv-Fc Fusion Proteins in Mammalian Cells
Chapter · January 2010
DOI: 10.1007/978-3-642-01147-4_30
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Vol. II Chapter 20
Transient production of scFv-Fc fusion proteins in mammalian cells
Thomas Schirrmann*, Konrad Büssow
Technische Universität Braunschweig
Institut für Biochemie und Biotechnologie
Department of Biotechnology
Spielmannstr. 7
38106 Braunschweig
th.schirrmann@tu-bs.de
Konrad Büssow
Helmholtz Centre for Infection Research
Department of Structural Biology
Inhoffenstr. 7
38124 Braunschweig
konrad.buessow@helmholtz-hzi.de
*
corresponding author
Abstract
Single chain Fv (scFv) antibody fragments fused to the human immunoglobulin G (IgG) Fc moiety obtain
IgG-like properties but nevertheless they are still encoded by a single gene. Since transient production of
scFv-Fc proteins in mammalian can easily achieve milligram amounts, this antibody format is particularly
suitable for many research applications. Here, we describe two protocols for the transient secretory
production of scFv-Fc antibodies in transformed adherent human embryonic kidney cells HEK293T and
suspension HEK293-6E cells. More than 10 µg/mL yield of scFv-Fc protein can be obtained every day for
one to two weeks by transient production in HEK293T cells, whereas soluble HEK293-6E cells produce
more than 100 µg/mL scFv-Fc protein within 5 days under serum-free conditions.
1. Introduction
Today 60-70% of recombinant protein pharmaceutics and all currently approved therapeutic antibodies are
produced in mammalian cells although cultivation requirements are expensive and handling them is
somewhat difficult. In contrast to alternative production systems like bacteria, yeast, insect cells or
transgenic plants (Schirrmann et al. 2008), the advanced folding, secretion and post-translational apparatus
of mammalian cells is best suited to produce antibodies which are indistinguishable from those produced
in the human body with least concerns for immunogenic modifications. In addition the still continuing
progress of the mammalian cell culture technology has already reached IgG production levels of more
than 5 g/L in the industry (Wurm 2004). Major parameters responsible for this development are the
improved generation of high producer cell lines, optimized serum-free media and prolonged production at
very high cell densities. For most laboratory applications, however, transient and semi-stable production
of recombinant antibodies is more suitable because it does not require the generation of stable producer
cell lines which is laborious and time consuming. By combination of transient transfection with batch or
fed-batch processes in bioreactors up to 80 mg/L IgG in scales of 3 – 150 litre have been reported (Baldi
et al. 2007). Recently, more than 1 g/L antibody titers were described after co-transfection of antibody
gene expression vectors together with vectors encoding cell cycle regulators (p18, p21, acidic fibroblast
growth factor), transfection at high cell densities (2x107 cell/mL), exposure of cells with valproic acid and
cultivation at high cell densities (4x106 cells/mL) for up to two weeks (Backliwal et al. 2008). Thus,
transient antibody production is not only suitable for small scale applications like antibody screening
(Jostock et al. 2004) but also for the production of milligrams, even grams of antibody. Progresses of
transient protein expression in mammalian cells are recently reviewed (Geisse and Henke 2005).
1.1 Mammalian cell lines for transient antibody production
The production of vaccines and therapeutic proteins is performed exclusively in diploid mammalian cell
lines to prevent contaminations by unknown factors with infectious, pathogenic or oncogenic potential
that might be released by human tumor cell lines. Therefore, only neoplastic diploid cell lines are the
standard for antibody production. Chinese hamster ovarian (CHO) cells represent the most common used
cell line for stable antibody production. Other mammalian cell lines but less frequently used for
recombinant antibodies production are Baby hamster kidney (BHK) cells or murine myeloma cell lines
like NS0 or SP/0. Mammalian cell lines produce glycosylation patterns very similar to that in humans, but
even small differences can influence pharmacokinetics and effector functions of antibodies (Baker et al.
2001, Gramer et al. 1995, Lifely et al. 1995, Umana et al. 1999). Against that, human cell lines are thought
to generate human glycosylation patterns with the lowest immunogenicity (Jones et al. 2003).
The human embryonic kidney cell line HEK293 and the human retinal cell line Per.C6 (Crucell,
NL) are two cell lines that were generated from human embryonal tissue by transformation with
Adenovirus 5 (Ad5) DNA. Both “designer cell substrates” have the advantage that the transformation
event is well understood and both gained regulatory approval for recombinant protein production for
therapy. Derivatives of the human embryonic kidney (HEK) cell line 293 transformed either with the
simian virus 40 (SV40) large T antigen, termed HEK293T, or the Epstein Barr virus (EBV) nuclear
antigen 1 (EBNA1), termed HEK293E, combine high transfection efficiency with the semi-stable
episomal propagation of expression plasmids containing a origin of replication (ori) of SV40 or EBV (ori
P), respectively. Here, production can be maintained for several weeks. There are also suspension adapted
HEK293S cells that can be grown in high cell densities in shake flasks, roller or spinner flasks or
bioreactors. Some suspension HEK293 cell derivatives are adapted to the growth in defined serum-free
medium (e.g., Invitrogen’s FreeStyle™ 293 Expression system) facilitating downstream processing by
reducing unspecific product contamination.
In the following protocol we focus on the transient production of recombinant antibodies in
adherent HEK293T cells. Their adherent growth in serum containing standard medium allows relatively
simple handling and cultivation. HEK293T cells can be efficiently transfected with plasmid vectors using
lipid or polymer based transfection reagents. The second protocol employs suspension HEK293-6E cells
that are cultured in serum-free medium in higher cell densities obtaining higher antibody yields.
1.2 Transient transfection of HEK293 cells with polyethylenimine
Transient transfection of HEK293T cells with plasmid DNA can be efficiently performed using cationic
liposomes or polyethylenimine (PEI) (Thomas and Klibanov 2003), even with calcium phosphate
transfection (Meissner et al. 2001). Commercially available transfection reagents based on cationic
liposomes and polymers (e. g. Lipofectamine®, HEKfectin®, Nanofectin® etc.) are very efficient for
transfection of HEK293 cells, but most of them are expensive. Against that, calcium phosphate
transfection is very inexpensive, but the formation of small DNA::CaPi coprecipitate complexes is timesensitive and can be technically challenging. In contrast, the cationic polymer, polyethyleneimine (PEI),
exhibits several properties which are extremely important for high efficient gene delivery (Boussif et al.
1995, Boussif et al. 1996). PEI has been successfully used for the transfection of a broad range of cell
lines including HEK293 cells, shows a relatively low toxicity and simple handling. It has been used in
serum containing as well as serum free media. Moreover, PEI is very inexpensive and therefore useful for
scale up experiments (Durocher et al. 2002, Durocher et al. 2007, Schlaeger and Christensen 1999, Wurm
and Bernard 1999). Transfection of HEK293E cells using PEI was found to depend on the presence of
serum (Durocher, Perret and Kamen 2002), but it is more likely that additives of serum-free media that
promote growth in single cell suspension like heparin or dextran-sulfate inhibit the cellular uptake of
PEI::DNA complexes. HEK293 cell derivatives have been developed that can be transfected with PEI in
the absence of serum, e.g. the HEK293E derived clone HEK293-6E (Loignon et al. 2008, Zhang et al.
2009). In our group we have also successfully transfected FreeStyle™ 293-F cells in the protein-free
FreeStyle™ medium (Invitrogen) using PEI. PEI is available in both, linear and branched isoforms of
different molecular weight and polydispersities (Godbey et al. 1999b) but mostly the linear 25 kDa form is
used for transfection. Its cationic charge results from the large number of protonable amino groups which
is thought to participate in DNA complexation (Boussif, Lezoualc'h, Zanta, Mergny, Scherman, Demeneix
and Behr 1995, Boussif, Zanta and Behr 1996). Moreover, these protonable amino groups may protect
DNA from degradation in cytoplasmic endosomes due to their high pH buffering capacity and mediate a
proton sponge effect which is postulated to cause an early escape of DNA::PEI complexes from lysosomes
(Godbey et al. 1999a).
1.2 Mammalian expression vector pCMV-hIgG1-Fc-XP
The mammalian expression vector pCMV-hIgG1-Fc-XP (Figure 1) used in the following experiments is
designed for one step cloning of scFv gene fragments from antibody gene libraries generated in phagemids
like pHAL14 (e.g. HAL4/7), pIT2 (e.g. Tomlinson) or pSEX81. The pCMV-hIgG1-Fc-XP vector drives
scFv-Fc gene expression by the human immediate early Cytomegalovirus (CMV) promoter. The scFv is
introduced into the restriction sites NcoI and NotI downstream from the mouse IgG heavy chain signal
peptide and upstream to the hinge CH2 and CH3 domains of the human IgG1. The signal peptide is
responsible for secretory production and contains an intron that stabilizes transgene expression. The
human IgG1 Fc moiety leads to an efficient dimerization and enhanced secretion of the scFv-Fc protein.
The short bovine growth hormone (BGH) poly A signal increases mRNA stability for improved gene
expression. In addition, the pCMV-hIgG1-Fc-XP vector contains a neomycin phosphotransferase (Neo)
expression cassette for antibiotic selection (i. e. with G418) of transfected mammalian cells which is not
required for transient productions. Note, in contrast to the parental HEK293 cell line, HEK293T and
HEK293E cells already express the Neo selection marker. Therefore, stable transfection with the pCMVhIgG1-Fc-XP vector is not possible with this cell lines. Its SV40 promoter comprises also the SV40 ori
for episomal replication in cell lines expressing the SV40 large T antigen like HEK293T cells.
2. Material
2.1 Cell lines
-
Human embryonic kidney cell line HEK293T/17 from American type culture collection (ATCC),
LGC Standards GmbH, Wesel, Germany: ATCC-No. CRL-11268 (http://www.lgcstandards-atcc.org/)
-
HEK293-6E, subline of HEK293E (NRC-BRI)
2.3 Material for transient production in adherent HEK293T cells
-
PEI (polyethylenimine), linear, 25 kDa (Polysciences, Warrington, PA, USA,
http://www.Polysciences.com): Prepare 1 mg/ml PEI in deionized water, neutralize with HCl and
filter sterilize (CA membrane, 0.2 µm). Store aliquots at -20°C.
-
Phosphate buffered saline (PBS): 10x stock solution contains 1.37 M NaCl, 26 mM KCl, 80 mM
Na2HPO4, 15 mM KH2PO4 (1x PBS should have pH 7.4), sterilise by autoclaving.
-
Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4.5 g/L) and 2 mM L-glutamine
(PAA, Parching, Germany).
-
100xPS (PAA): penicillin (10.000 U/mL) /streptomycin (10 mg/mL) concentrate in 0.9% (w/v) NaCl;
store at -20°C (shelf time: 2 years), stability < 5 d at 37°C.
-
200 mM L-glutamine (PAA)
-
Fetal calf serum (FCS) (PAA); for complement inactivation incubate 500 mL FCS flask for 1 h at
56°C. Before heat activation shake the FCS flask several times until solution become homogenous.
-
Ultra-low IgG FCS or IgG stripped FCS (PAA); complement inactivation as described above.
-
Trypsin (5.0 mg/mL) / EDTA (Titriplex III, 2.2 mg/mL) (PAA), store at -20°C (shelf time: 2 years).
-
Medium 1: DMEM / 4-8% (w/v) fetal calf serum (FCS) / 1x PS.
-
Medium 2: DMEM / 4% (w/v) ultra-low FCS / 1x PS
-
High quality Plasmid-DNA preparation; it is recommended to prepare plasmid DNA with purification
kits based on anion exchange chromatography (e. g. from Machery Nagel, Qiagen, etc.) to obtain high
quality DNA with an optimal concentration of about 1 µg/µL.
-
Tissue culture plates for adherent cells (e. g. Cellstar® from Greiner, Bio-One, Frickenhausen)
-
10 cm tissue culture dishes for adherent cells (e.g. Sarstedt, Nürnbrecht, Germany)
-
0.01% (75 - 150 kDa) poly-L-lysine, sterile, cell-culture grade (Sigma)
2.3 Additional material for transient production in suspension HEK293 cells
-
Linear shaker Incutec K15-500 in a CO2 incubator with humidified atmosphere.
-
Flow cytometer, Guava EasyCyte mini (Guava Technologies, Hayward, CA, USA)
-
96-well cell culture plates (Brand)
-
24-well cell culture plates (Falcon Multiwell 353047, tissue-culture treated, polystyrene, flat-bottom
with lid, Becton Dickenson)
-
12-well cell culture plates (Falcon Multiwell 353043, tissue-culture treated, polystyrene, flat-bottom
with lid, Becton Dickenson)
-
96-, 24- or 6-well polystyrol cell culture plates (e. g. Cellstar® Greiner Bio-One)
-
G418 solution, 50 mg/ml (PAA, Pasching, Austria)
-
TN1: Tryptone N1 (Organotechnie S.A.S., La Courneuve, France). Prepare 20% w/v stock solution in
Medium 3 (Pham et al. 2005).
-
F17 medium, Freestyle™ version of CD17, Formula No. 05-0092DK (Invitrogen) contains 0.1 g/L
pluronic
-
Medium 3: F17 supplemented with 1 g/L pluronic, 25 mg/L G418, 7.5 mM glutamine
-
Pluronic F-68 (Sigma) Sigma P-1300, prepare a 10% (w/v) solution in water, filter sterilize and
refrigerate.
2.4 Plasmid vectors
-
pCMV-scFv-hIgG1-Fc-4E3 encodes a CD30-specific scFv-Fc antibody fragment
-
pEF-FS-EGFP (control for transfection efficiency)
3. Protocols
3.1. Transient transfection protocol of adherent HEK293T cells
1. Grow HEK293T cells in Medium 1.
2. According to table 1 seed HEK293T cells in culture Medium 1 (DMEM / 4-8% (v/v) FCS / 1% (v/v)
PS) into a flat bottom tissue culture plates or dishes for adherent cells (see also troubleshooting 5.1
note 2) and incubate overnight at 37°C, 7% CO2 and 95% humidity.
3. On the next day cells should have grown to 75-80% confluence (see also troubleshooting 5.1 note 3).
4. Prepare the vector DNA::PEI transfection mix with the amounts and volumes listed in table 1 and as
briefly described:
4.1. Dilute PEI in appropriate volume DMEM in a polystyrol plate or tube (Do not use polypropylene
tubes!).
4.2. Dilute Plasmid-DNA (see also troubleshooting 5.1 note 4) in appropriate volume DMEM and
mix with the PEI suspension.
4.3. Incubate at RT for 15-30 min to allow formation of PEI::DNA complexes.
4.4. Disperse PEI::DNA suspension evenly over the cells.
5. Change medium about 24 h after transfection. If plates are not coated with poly-L-lysine be very
careful not to detach cells from the bottom of the plate. Switch to Medium 2 if IgGs or Fc fusion
proteins shall be purified by protein A/G affinity chromatography to minimize co-purification of
bovine immunoglobulins.
6. Harvest culture supernatant every day and change medium. Depending on the cell growth production
can be maintained for 1 – 2 weeks (see also troubleshooting 5.1 note 5).
7. Test yield of human IgG or Fc fusion protein using an IgG/Fc capture ELISA.
3.2 Transient transfection of scFv-Fc protein in suspension HEK293-6E cells
1. Grow HEK293-6E cell in Medium 3.
2. Seed 1 mL of 5x105 cell/mL HEK293-6E cells in Medium 3 per well of a 12-well plate.
3. Shake the plate at 150 rounds per minute (rpm) on a linear shaker at 5% CO2 and 95% humidity for
two days.
4. Prepare the DNA::PEI transfection solution as follows:
4.1. Dilute the 5 µg of PEI in 50 µL F17-medium for each transfection using a polystyrol plate or
polystyrol tube (Do not use polypropylene tubes!).
4.2. For each transfection, prepare 1 µg plasmid DNA in 50 µl F17-Medium in a 96-well plate. If
required, add 50-100 ng DNA of the pEF-FS-EGFP reporter plasmid for the measurement of
transfection efficiency by flow cytometry.
4.3. Mix 50 µL diluted PEI and 50 µl diluted DNA per transfection sample in a 96-well polystyrol
plate by pipetting up and down several times.
4.4. Incubate at RT for 15-30 min to complex PEI::DNA.
5. Add 100 µL transfection mix per well to the cells. Mix immediately by swirling the plate.
6. Shake the plate for two days at 150 rpm.
7. Take a small sample to check the transfection efficiency by flow cytometry and count the proportion
of (E)GFP+ cells in a flow cytometer.
8. Feed the cells by adding TN1 to 0.5% and continue to shake the plate.
9. Harvest the medium supernatant five days after the transfection by centrifugation at 850xg for 4 min.
10. Test yield of human IgG or Fc fusion protein using an IgG/Fc capture ELISA.
4. Results
4.1 Transient semistable production of scFv-Fc protein in adherent HEK293T cells
HEK293T cells were transfected in a 24 well plate according to protocol 3.1 with different amounts of
plasmid DNA and PEI (Figure 2). The plasmid pCMV-scFv-hIgG1-Fc-4E3 encoded the CD30 specific
scFv-Fc antibody (Menzel et al. 2008). Results shown in Figure 2 are from samples taken 40 h after
transfection. The concentration of the scFv-Fc antibody was tested by a human IgG/Fc capture ELISA as
described elsewhere. Samples harvested 40 h after transfection contained up to 8.5 µg/mL scFv-Fc
protein. The production yield depended from the transfection with an optimal specific ratio of plasmid
DNA to PEI of 1:4 – 1:8. In 24 well plates and 600 µL culture volume 0.5-1 µg plasmid DNA and 4 µg
PEI were optimal, respectively. Up-scaling to 6 well plates or 10 cm plates resulted in even higher yields
of up to 20 µg/mL (data not shown). The productivity can be estimated with 5 – 20 pg/cell/day one to days
after transfection. If the medium is exchanged every day the production yield is stable for several days
until cells grow too dense and begin to detach or become apoptotic. The production time is usually 1 – 2
weeks. It should be noted that the production depends strongly on the individual antibody clone. We
observed yields of less than 1 µg/mL for some scFv-Fc antibodies whereas other clones achieved yields of
more than 20 µg/mL.
4.2 Transient production of scFv-Fc protein in suspension HEK293-6E cells
HEK293-6E cells were cultured and transfected with 1 µg plasmid pCMV-scFv-hIgG1-Fc-4E3 encoding a
CD30 specific scFv-Fc antibody fragment according to protocol 3.2. A total of 50 ng pEF-FS-EGFP was
co-transfected as reporter plasmid. In this experiment different amounts of PEI (2, 3, 5 and 7 µg) were
tested. After two days transfection efficiencies were analyzed by flow cytometry (figure 3). After 5 days
production the culture supernatant was harvested and analyzed by a human IgG/Fc capture ELISA.
HEK293-6E cells transfected with 1 µg pCMV-scFv-hIgG1-Fc-4E3 and 3-5 µg PEI obtained the highest
transfection efficiencies of 40 – 45% (e.g. EGFP+ cells) and the highest antibody yields of up to 140
µg/mL.
5. Troubleshooting
5.1 Transient semistable antibody production in adherent HEK293T cells
1. Incubate adherent HEK293T cells with about 1 mL Trypsin/EDTA per 10 cm dish for a few minutes
at 37°C. Keep incubation as short as possible but also avoid large cell aggregates. Trypsin will be
inactivated by washing cells once with medium.
2. We observed strong differences between the various suppliers of tissue culture plates/dishes. For
improved adherence of HEK293T cells coat plates with poly-L-lysine or use commercially available
plates. For coating 10 cm dishes with poly-L-lysine incubate 5 mL poly-L-lysine for at least 30 min at
37°C and wash dishes twice with sterile PBS.
3. Certain sublines of HEK293T cells show different growth properties. Therefore, the initial cell
number should be adapted to obtain 75-80% confluence on the day of transfection.
4. For transfection of mammalian cells it is recommended to use high quality DNA preparations. Anion
exchange column based plasmid DNA preparation kits usually obtained best results. An optimal
plasmid DNA concentration is about 1 µg/µL.
5. For up-scaling to 10 cm dishes it must be considered that the typical culture volume is about 12 - 15
mL and the cell density is about two-fold higher. Increase culture volume during transfection to 25
mL or reduce transfection mix by half.
5.2 Additional notes for transient antibody production in supension HEK293-6E cells
1. For small scale production in multi-well plates linear shakers are recommended over orbital shakers to
prevent enhanced accumulation and aggregation of the cells in the middle of the wells.
2. Mammalian expression vectors containing an EBV ori P (e.g. pCEP vectors of Invitrogen) allow
(semi-)stable production in HEK293-6E cells.
6. References
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design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1
g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36:e96
Baker KN, Rendall MH, Hills AE, Hoare M, Freedman RB and James DC (2001) Metabolic control of
recombinant protein N-glycan processing in NS0 and CHO cells. Biotechnol Bioeng 73:188-202
Baldi L, Hacker DL, Adam M and Wurm FM (2007) Recombinant protein production by large-scale
transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol Lett
29:677-84
Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B and Behr JP (1995) A
versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.
Proc Natl Acad Sci U S A 92:7297-301
Boussif O, Zanta MA and Behr JP (1996) Optimized galenics improve in vitro gene transfer with cationic
molecules up to 1000-fold. Gene Ther 3:1074-80
Durocher Y, Perret S and Kamen A (2002) High-level and high-throughput recombinant protein
production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res
30:E9
Durocher Y, Pham PL, St-Laurent G, Jacob D, Cass B, Chahal P, Lau CJ, Nalbantoglu J and Kamen A
(2007) Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of
293 suspension cells. J Virol Methods 144:32-40
Geisse S and Henke M (2005) Large-scale transient transfection of mammalian cells: a newly emerging
attractive option for recombinant protein production. J Struct Funct Genomics 6:165-70
Godbey WT, Wu KK, Hirasaki GJ and Mikos AG (1999a) Improved packing of poly(ethylenimine)/DNA
complexes increases transfection efficiency. Gene Ther 6:1380-8
Godbey WT, Wu KK and Mikos AG (1999b) Size matters: molecular weight affects the efficiency of
poly(ethylenimine) as a gene delivery vehicle. J Biomed Mater Res 45:268-75
Gramer MJ, Goochee CF, Chock VY, Brousseau DT and Sliwkowski MB (1995) Removal of sialic acid
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human IgG antibodies derived from Fab-on-phage display libraries. J Immunol Methods 289:65-80
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CAMPATH-1H expressed in different cell lines and grown under different culture conditions.
Glycobiology 5:813-22
Loignon M, Perret S, Kelly J, Boulais D, Cass B, Bisson L, Afkhamizarreh F and Durocher Y (2008)
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protein targeting CD30+ lymphomas. Blood 111:3830-7
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protein synthesis. Biotechnol Bioeng 90:332-44
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Microbiol Biotechnol 62:27-34
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antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol
17:176-80
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Tables
Table 1 – Cultivation and transfection conditions for adherent HEK293T cells
24-well
12-well
6-well
∅100 mm*
Culture area (factor)
1.9 cm2 (1x)
3.5 cm2 (1.8x)
9.6 cm2 (5x)
78.5 cm2 (41x)
HEK293T preculture**
1.5x105
3x105
7.5x105
4 - 6x106
Suggested culture volume
0.6 mL
1,2 mL
3 mL
12.5 mL (25 mL)
PEI (1 mg/mL)
4 µL
8 µL
20 µL
80 µL (160 µL)
DMEM for dilution of PEI
30 µL
60 µL
125 µL
600 µL (1.2 mL)
0.5 - 1 µg
1 µg
1.25-2.5 µg
10 µg (20 µg)
30 µL
60 µL
150 µL
600 µL (1.2 mL)
Tissue culture plate/dish
Transfection mix
DNA (see Note 2)
DMEM for dilution of DNA
*
**
See 5.1 troubleshooting note 5
Next day cells should be 75-80% confluent.
Figure legends
Figure 1 – Mammalian expression vector pCMV-hIgG1-Fc-XP
Mammalian expression vector pCMV-hIgG1-Fc-XP contains an NcoI – NotI cloning cassette that allows
one step subcloning of scFv genes from common antibody gene libraries into the dimeric scFv-Fc
antibody format. The scFv-Fc antibody fragments form homodimers with IgG like properties and are
efficiently secreted by mammalian cell lines. The Fc moiety allows tag-less purification (protein A/G) and
detection (standard polyclonal secondary antibody conjugates) and mediates enhanced protein stability,
prolonged serum half-life and IgG effector functions.
Figure 2 – Chessboard transfection of HEK293T cell in a 24 well plate
HEK293T cells were transfected in 24-well scale according to the protocol 3.1 but with different amounts
of the plasmid pCMV-scFv-hIgG1Fc-4E3 and different volumes of 1 mg/mL PEI. Samples taken 40 h
after transfection were analyzed using a human IgG capture ELISA.
Figure 3 – Transient production of scFv-Fc in suspension HEK293-6E cells in 12-well plate
HEK293-6E cells were transfected with 1 µg of the plasmid pCMV-scFv-hIgG1Fc-4E3 and 50 ng of an
EGFP reporter plasmid (pEF-FS-EGFP) according to protocol 3.2. The propotion of GFP positive cells
was recorded two days after transfection by flow cytometry to determine the transfection efficiency
(crosses). Production was continued for another three days before supernatant was harvested. Titers of
scFv-Fc antibody were analyzed by a human IgG/Fc capture ELISA (bars). Controls are only transfected
with the reporter plasmid.
Figures
Figure 1 - pCMV-hIgG1-XP-scFv-Fc.pdf
scFv gene fragment
VH
Linker
VL
scFv-Fc
Figure 2 - Adherent HEK293T cells.pdf
Figure 3 - Suspension HEK293 cells.pdf
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