large-scale transient transfection of serum-free suspension-growing hek293 ebna1 cells: peptone...

Large-Scale Transient Transfection ofSerum-Free Suspension-Growing HEK293EBNA1 Cells: Peptone Additives ImproveCell Growth and Transfection Efficiency

Phuong Lan Pham, Sylvie Perret, Huyen Chau Doan, Brian Cass,Gilles St-Laurent, Amine Kamen, Yves Durocher

Animal Cell Technology Group, Bioprocess Platform, BiotechnologyResearch Institute, National Research Council Canada, 6100 RoyalmountAvenue, Montreal, Canada H4P 2R2; Telephone: 514-496-6192;fax: 514-496-6785; e-mail: [email protected] 28 January 2003; accepted 5 June 2003

Published online 21 August 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10774

Abstract: Large-scale transient transfection of mamma-lian cells is a recent and powerful technology for the fastproduction of milligram amounts of recombinant pro-teins (r-proteins). As many r-proteins used for therapeu-tic and structural studies are naturally secreted or engi-neered to be secreted, a cost-effective serum-free culturemedium that allows their efficient expression and purifi-cation is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates oncell proliferation, transfection efficiency, and volumetricproductivity was evaluated using green fluorescent pro-tein (GFP) and human placental secreted alkaline phos-phate (SEAP) as reporter genes. The suspension grow-ing, serum-free adapted HEK293SF-3F6 cell line was sta-bly transfected with an EBNA1-expression vector toincrease protein expression when using EBV oriP bear-ing plasmids. Compared to our standard serum-free me-dium, concomitant addition of the gelatin peptone N3and removal of BSA slightly enhanced transfection effi-ciency and significantly increased volumetric productiv-ity fourfold. Using the optimized medium formulation,transfection efficiencies between 40–60% were routinelyobtained and SEAP production reached 18 mg/L−1. Todate, we have successfully produced and purified overfifteen r-proteins from 1–14-L bioreactors using this se-rum-free system. As examples, we describe the scale-upof two secreted his-tagged r-proteins Tie-2 and Neuropi-lin-1 extracellular domains (ED) in bioreactors. Each pro-tein was successfully purified to >95% purity following asingle immobilized metal affinity chromatography(IMAC) step. In contrast, purification of Tie-2 and Neuro-pilin-1 produced in serum-containing medium was muchless efficient. Thus, the use of our new serum-freeEBNA1 cell line with peptone-enriched serum-free me-dium significantly improves protein expression com-pared to peptone-less medium, and significantly in-creases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive andexpensive chromatographic steps, and allows for thesimple, reliable, and extremely fast production of milli-gram amounts of r-proteins within 5 days posttransfec-tion. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 332–342, 2003.

Keywords: polyethyleneimine; secreted proteins; biore-actor; human embryonic kidney cells; immobilized metalaffinity chromatography

INTRODUCTION

The great advances made in genomics and proteomics dur-ing the past decade have positioned recombinant proteins(r-proteins) as major therapeutic biomolecules and drug tar-gets (Chu and Robinson, 2001; Kelley, 2001). The increas-ing demand for milligrams of r-proteins to be used in pre-clinical, biochemical, and biophysical studies justifies theneed of a rapid and scaleable expression system. Whilebacterial expression systems often satisfy these needs, ex-pressed proteins frequently suffer from poor solubility orlack of proper processing. In this regard, mammalian ex-pression systems are more appropriate for providing solubleand fully processed r-proteins. Although the usual means ofproducing r-proteins in mammalian cells is to establishclones stably expressing the gene of interest, this technologyis not readily amenable to a high-throughput mode. Therecent large-scale transient transfection technology is nowgenerating great interest because of its demonstrated abilityto produce large amounts of r-proteins within a few days(Durocher et al., 2002; Girard et al., 2002; Jordan et al.,1998; Meissner et al., 2001; Schlaeger and Christensen,1999). The use of nonviral gene-transfer systems such ascationic liposomes and polymers, or calcium-phosphate pre-cipitates has recently been shown to be highly effective intransfecting cells grown in suspension, a prerequisite forscale-up. The cationic polymers polyethyleneimines (PEIs)further offer the advantages of being cost-effective, sim-ple to use, non-cytotoxic, and stable (Boussif et al., 1995).Polyethyleneimines polymers are available in linear andbranched forms with various molecular weights and poly-dispersities. The most important characteristic of PEIs con-sists of their cationic charge density due to the presence ofa potentially protonable amino nitrogen at every third atomwhich may participate in DNA condensation. These pro-Correspondence to: Yves Durocher

© 2003 Wiley Periodicals, Inc.

tonable amino groups may also play a protective role inpreventing DNA degradation in cytoplasmic endosomes.The so-called proton sponge effect was postulated to causethe early escape of DNA/PEI complexes from lysosome,thus avoiding lysosomal degradation (Boussif et al., 1995).This hypothesis however, has been recently challenged be-cause of the apparent lack of lysosomal involvement incellular DNA/PEI trafficking (Godbey et al., 2000). Thehuman embryonic kidney 293 cell line (293) is suitable fortransient transfection technology as it can be efficientlytransfected in suspension using cost-effective vehicles suchas polyethyleneimine (Durocher et al., 2002; Schlaeger etal., 1999) or calcium-phosphate (Girard et al., 2002; Jordanet al., 1998; Meissner et al., 2001). Moreover, a 293 geneticvariant stably expressing the EBV EBNA1 protein (293E)has been shown to provide significantly higher protein ex-pression when EBV’s oriP is present in the vector backbone(Durocher et al., 2002; Parham et al., 2001; Schlaeger et al.,1999). The increased expression obtained by the use oforiP/EBNA1 systems appears to be independent of episomalreplication when performing transient transfection. This issupported by the fact that removal of the DS domain oforiP, which is responsible for initiation of DNA replicationin EBNA1 positive cells (Wysokenski and Yates, 1989)does not reduce transgene expression, while removal of FRbut not DS strongly reduces expression (data not shown;Langle-Rouault et al., 1998). The increased expression isthus likely due to the combined effect of the EBNA1-dependent enhancer activity of oriP (Reisman and Sugden,1986) and to the increased nuclear import of plasmids ow-ing to the presence of a nuclear localization signal inEBNA1 (Ambinder et al., 1991).

We have previously shown that serum significantly in-creases r-protein expression following PEI-mediated trans-fection of 293E cells (Durocher et al., 2002). However,when one needs to produce secreted proteins, serum greatlyinterferes with their subsequent purification. As we were notsuccessful in adapting 293E cells to our standard serum-freemedium, our goal was to establish a new stable EBNA1-expressing cell line based on our serum-free adapted 293SFclone (Cote et al., 1998). We also modified our serum-freeformulation by using peptone additives to increase volumet-ric r-protein productivity. As peptones are low-molecular-weight peptides issued from enzymatic digestion of animalor vegetal proteins, they are more easily eliminated duringthe first chromatographic steps of purification, hence,greatly improving the purification of r-proteins. These com-ponents are also known to stimulate DNA synthesis andmitosis (Jan et al., 1994). Moreover, the presence of theseprotein hydrolysates may reduce proteolytic and/or intrinsicdegradation of the secreted r-proteins (Heidemann et al.,2000).

In this study, we show that the use of our new 293SF-EBNA1 (293SFE) cell line significantly improves transientr-protein expression compared to the original 293SF clonewhen both are grown and transfected in the original serum-free medium. Furthermore, peptone addition and BSA re-

moval from the medium formulation greatly enhance volu-metric productivity. This significantly improves purificationof the target r-proteins using immobilized metal affinitychromatography (IMAC) compared to production in serum-containing medium. Scaleability and robustness of the pro-cess were demonstrated by producing and purifying twosecreted r-proteins in bioreactors at the 14-L scale.

MATERIALS AND METHODS

Cell Lines

The human embryonic kidney 293 cell line stably express-ing Epstein Barr virus Nuclear Antigen-1 (293E) wasadapted to suspension culture in low-calcium-SFM (LC-SFM, Invitrogen, Grand Island, NY) supplemented with0.1% Pluronic F-68, 1% bovine calf serum (BCS), 50 �g/mL Geneticin and 10 mM Hepes (Durocher et al., 2002).The suspension-growing 293SF-3F6 clone (Cote et al.,1998) was maintained in LC-SFM supplemented with 0.1%lipid mixture (1000×; Sigma, St. Louis, MO) and 0.1% BSA(from a Bovuminar Microbiological 30% solution, IntergenNorcross, GA). This medium is termed LC-SFM-LB. In thisstudy, we used a derivative of 293SF cell stably expressingthe EBNA1 protein (293SFE). The 293 SFE cell line wasobtained by stable transfection of 293 SF with an expressionplasmid encoding the EBNA1 protein and conferring ge-neticin resistance.

The 293SFE cells were maintained at the exponentialphase in suspension in culture flasks containing LC-SFM-LB, 10 �g/mL of Geneticin and 10 mM Hepes.

Peptones

All peptones were obtained from OrganoTechnie S.A. (LaCourneuve, France). Stock solution (5%, w/v) were pre-pared in LC-SFM, sterilized by filtration through 0.2 �mfilters and stored at 4°C until use.

Construction and Purification of Plasmids

The pTT/GFPq and pTT/SEAP vectors (Durocher et al.,2002) were employed in this study to optimize the transfec-tion and expression parameters. The other cDNA expressedin this study was tagged with a H8GGQ epitope at theirC-terminus. Plasmids were amplified using the E. coliDH5� strain grown in CircleGrow broth (Qbiogene, Carls-bad, CA), supplemented with ampicillin (100 �g/mL) andpurified using Maxi/Giga plasmid purification kits (Qiagen,Valencia, CA). DNA concentration was measured by UVabsorbance at 260 nm in 50 mM Tris-HCl pH 8.0. Onlyplasmids with A260/280 ratio 1.80–1.95 were used.

Transfection Reagent

Stock solution (1 mg/mL) of 25-kDa linear PEI (Poly-sciences, Warrington, CA) was prepared in water, acidified

PHAM ET AL.: LARGE-SCALE TRANSIENT TRANSFECTION OF MAMMALIAN CELLS 333

with HCl to pH 2 until dissolved, then neutralized withNaOH, sterilized by filtration (0.2 �m), aliquoted, andstored at −20°C.

PEI/DNA Complex Formation

PEI/DNA complexes were prepared by adding PEI to DNAdiluted in LC-SFM/10 mM Hepes followed by vigorousvortexing as previously reported (Durocher et al., 2002). AllPEI/DNA complexes were incubated at room temperaturefor 15 min before adding to cell culture.

Static Cultures

To study the effect of various peptones on cell growth, cellswere plated in 24-well plates at an initial cell density of50,000 cells per well in 0.5 mL of LC-SFM-LB mediumsupplemented with 1% (w/v) of peptone. For transfection in12-well plates, cells were seeded in 0.9 mL of fresh medium3 h before transfection at a concentration of 0.5 × 106 cells/mL. One hundred microliters of DNA/PEI complexes wereadded to each well and cells were analyzed 96–120 h post-transfection (hpt) by flow cytometry for GFP expressionand SEAP measurement (cells were cotransfected with 10%pTT/GFPq and 90% pTT/SEAP).

Large-Scale Transfection Conditions

Large-scale production of r-proteins using the 293SFE cellline was performed in bioreactors of 1 L (Biostat Q, B.Braun, Germany), 10 L (BioFlo 110, New Brunswick, NJ)or 14 L (Chemap, Switzerland). The working volumes wereadjusted to about 65–90% of total volume. The fermentorswere equipped with 45° pitched-blade impellers and thestirring speeds were 100 rpm for 1-L and 70 rpm for 10-Land 14-L bioreactors. Surface aeration was applied with agas mix of nitrogen, carbon dioxide and oxygen at gas-flowrate of 100 sccm (1-L bioreactor) and 300 sccm (10 and14-L bioreactor). The dissolved oxygen tension (pO2) wasmonitored by a polarographic oxygen electrode (Mettler-Toledo, Urdorf, Switzerland) and controlled at 40% air satu-ration by surface aeration during the run. The temperaturewas maintained at 37°C by heated water circulating througha jacket (Biostat Q and Chemap) or by using a heatingblanket (BioFlo 110). The pH was measured with a gel-filled electrode (Mettler-Toledo) and controlled at 7.15 withCO2 at the beginning of the run and with NaHCO3 7.5%(w/v) during the cell growth phase. The medium utilizedwas LC-SFM supplemented with 0.1% (v/v) lipid mixtureand 0.5% (w/v) of GPN3 (Gelatin Peptone N3 termed LC-SFM-LG. Bioreactors were seeded at 0.25 × 106 viablecells/mL and transfected with the DNA/PEI complexes 24 hpost-inoculation when viable cell density was typicallyaround 0.5 × 106 cells/mL. Samples for cell counts andmetabolites analyses were taken at regular intervals andtreated as described below.

Purification of Secreted r-Proteins

Culture media harvested from bioreactors were centrifugedto eliminate the cells. The supernatant was passed through a0.45 �m filter then concentrated approximately 20 times bytangential flow filtration using 10 kDa cut-off membranes(Pall Filtron, Northborough, MA). The concentrates wereloaded onto a TALONTM cobalt-based IMAC column (10-mL bed volume) (Clontech, Palo Alto, CA). After extensivewashing (wash buffer: 50 mM NaH2PO4 pH 7.0, 300 mMNaCl and 5 mM imidazole) to eliminate the nonadsorbedmaterial, bound proteins were eluted with 150 mM imidaz-ole in wash buffer. To assess the purity level, proteins wereanalyzed by SDS-PAGE (4–12% NuPAGE Bis-Tris gradi-ent gel) followed by Coomassie staining.

Cell Counts

Cell density and viability were determined using a haema-cytometer (Hausser Scientific, Horshaw, PA) after Erythro-sine B staining. For the study on the effect of peptones oncell growth, cell count was estimated by staining the cellswith crystal violet and treated with citric acid/Triton × 100.Briefly, wells with 0.5 mL of medium containing differentpeptones at 1% (w/v) were seeded with 50,000 cells andincubated at 37°C for one week. The spent medium wasdiscarded and 0.5 mL of crystal violet solution (0.05%, w/v)that contained 2.1% of monohydrate citric acid (w/v) and0.1% Triton × 100 (v/v) was added to each well. The cellswere harvested in eppendorf tubes, vigorously vortexed forone minute and passed for nuclei counting in a haemacy-tometer (Lin et al., 1991).

Flow Cytometric Assay

GFP-positive cells were estimated by flow cytometry usingan Epics Profile II (Coulter, Hialeah, FL) equipped with a15-mW argon-ion laser. For each assay, cells were analyzedusing appropriate gating to exclude dead cells, debris, andaggregates.

SEAP Measurement

Determination of SEAP activity (�A410/min) was done aspreviously described (Durocher et al., 2002). Briefly, super-natants were diluted with water to obtain para-nitrophenylphosphate (pNPP) hydrolysis rates lower than 0.2 absor-bance units at 410 nm per min. The enzymatic reaction wasinitiated when 50 �L of SEAP buffer (1M diethanolamine,pH 9.8, 1 mM MgCl2) containing 20 mM pNPP was addedto 50 �L of diluted supernatants in a 96-well plate. All dataare representative of at least two independent experiments.Error bars shown in figures represent standard deviationcalculated from one experiment done in duplicate.

334 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 3, NOVEMBER 5, 2003

Glucose, Lactate, Ammonia, and AminoAcid Analysis

Glucose, lactate, and ammonia assays were performed usinga Biolyzer Analyzer (Kodak, New Haven, CT). Amino acidconcentrations in fresh media and supernatants were quan-tified by HPLC (Waters Alliance System, Waters Corp.,Milford, MA) using a modification of the Waters AccQ.Tagmethod as described by Cohen (2000).

SDS-PAGE and Western Blot Analyses

Recombinant proteins produced from different transfectionruns were diluted (3:1, v/v) in NuPAGE 4× sample buffer(Invitrogen, Carlsbad, CA) containing 50 mM DTT and thenheated at 70°C for 10 min. SDS-PAGE was carried out on4–12% Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad,CA) using MOPS-SDS running buffer for 1 h at 200 V. Thegels were rinsed with water and the protein bands werevisualized by staining with Coomassie Blue R250. Molecu-lar weights were determined by using Mark 12 wide-rangeprotein standard (Invitrogen). Western blots were per-formed by transferring to nitrocellulose membrane usingTris-glycine buffer for 1 h at 300 mA, then blotting with theappropriate antibody.

RESULTS

Stable Expression of EBNA1 in 293SF Cells

EBNA1 expression in emerging clones was verified byWestern blotting (Fig. 1A) using the monoclonal 1H4 anti-EBNA1 antibody (Grundhoff et al., 1999). EBNA1-positiveclones were tested for expression following transient trans-fection with the pTT/SEAP vector in the presence of 1%BCS (Fig. 1B). Based on the SEAP expression level reachedat 96 hpt, the clone 41 was selected (subsequently referredto as the 293SFE cell line) for further experiments. Thesuspension-growing 293SFE cell line was re-adapted to se-rum-free medium by stepwise reduction of BCS from 1% to0% in LC-SFM-LB containing 10 �g/mL G418. The cellswere thereafter routinely maintained by regular dilution in125-mL shake flasks in a humidified incubator under 5%CO2. While the LC-SFM-LB medium proved to be suitablefor growth of 293SF cells (Cote et al., 1998), transient trans-fection of SFE cells in this medium showed poor perfor-mance in terms of SEAP expression (Fig. 2). Only one thirdto one half of SEAP activity was achieved when growingand transfecting 293SFE in LC-SFM-LB compared to theuse of LC-SFM media containing 1% BCS. To increasetransfection efficiency and gene expression in the absenceof serum, the effect of medium supplementation with vari-ous peptones was evaluated.

Effect of Peptones on Cell Growth

The potency of peptones to sustain the growth of 293SFEcells in serum-free medium was monitored after an incuba-

tion period of one week in a 24-well plate by countingnuclei using the crystal violet method (Fig. 3). A total ofnine peptones were tested by addition to LC-SFM at a con-centration of 1.0% (w/v). The negative control was LC-SFM-LB and the positive controls were LC-SFM-LB/1%

Figure 2. Protein expression in various LC-SFM formulations. 293SFEcells (clone 41) were grown and transfected in LC-SFM/1% BCS, LC-SFM-LB/1% BCS or LC-SFM-LB, while 293E cells were grown andtransfected in LC-SFM/1% BCS. SEAP activity was measured at 120 hpt.

Figure 1. EBNA1 expression in 293 clones. The 293SF cells were trans-fected with an EBNA1 expression vector using lipofectamine. Two daysfollowing transfection, 100 �g/mL of geneticin was added and the selec-tion was maintained for 2 weeks. Cells were then cloned by limitingdilution by plating at 0.5 cell/well in 96-well plates in 1% BCS-sup-plemented LC-SFM. Panel A: cells lysates of emerging clones were ana-lyzed by Western blot using a monoclonal anti-EBNA1 antibody. Panel B:293 clones were transfected with pTT/SEAP and SEAP activity was mea-sured 96 hpt as described in Materials and Methods.


BCS and LC-SFM/1% BCS. Most peptones (GPN3, CPN1,CPE1, GPN2, CPN3, MPS1, MPN1, MPN2) were found tobe effective in promoting cell growth while the meat pep-tone MPS2 showed some negative effect. The eight pep-tones that promoted the cell growth were further studied inthe experiment reported below.

Effect of Peptones on Transfection andGene Expression

As one of our objectives was to reach a high productivity ofr-proteins, it was essential to evaluate the effect of peptoneson transfection. This experiment was performed with CPE1,CPN1, CPN3, GPN2, GPN3, MPN1, MPN2, and MPS1 in12-well plates. All of the tested peptones were incorporatedin LC-SFM-LB medium at 1% (w/v). The 293SFE cellswere cotransfected with pTT/GFP and pTT/SEAP vectors.Transfection efficiency was evaluated at 96 hpt by flowcytometry (Fig. 4, panel A) as indicated in Materials andMethods. SEAP expression was also monitored as shown inFigure 4, panel B. Microscopic observation (data notshown) showed that meat peptones (MPN1, MPN2, andMPS1) strongly induced cell aggregation. When present at1%, CPE1 and especially CPN3 inhibited the transfectionefficiency (Fig. 4, panel A) while other peptones (CPN1,MPN1) showed a slight positive effect compared to thenegative control. The two peptones GPN3 and MPS1 gavethe best results in transfection efficiency but as GPN3 al-lowed a better protein expression (Fig. 4, panel B), it wasselected for the next series of experiments.

Effect of GPN3 Concentration on TransfectionEfficiency and Protein Expression

Four concentrations of GPN3 (0.25; 0.5; 1.0; and 1.5%,w/v) were tested in this experiment. As shown in Figure 5,increasing peptone concentrations (1.0 and 1.5%) led to asignificant decrease in percentage of transfected cells whilethe fluorescence intensity (X-mean) slightly increased untilreaching a maximum at 1.0% then decreased slightly at1.5%. Highest SEAP activity (panel B) was obtained when0.5% of GPN3 was added to LC-SFM-LB medium. Thus,this quantity of GPN3 appeared to be optimal in maintaininga reasonable cell growth, an effective transfection, and ahigh expression level.

Impact of BSA on Transfection Efficiency andProtein Expression

We wished to eliminate BSA from our formulation in ourefforts to design a low-protein medium and because BSAappears to inhibit protein expression (Fig. 2). We evaluated

Figure 3. Effect of peptone addition on cell growth. 293SFE cells wereseeded in a 24-well plate (50,000 cells/well) with a final volume of 0.5 mLLC-SFM-LB. Peptones were added into each well at 1% (w/v) concentra-tion. Cell growth was estimated 7 days later by performing nuclei countingas described in Materials and Methods. CPE1: casein peptone E1; CPN1:casein peptone N1; CPN3: casein peptone N3; GPN2: gelatine peptone N2;GPN3: gelatin peptone N3; MPN1: meat peptone N1; MPN2: meat peptoneN2; MPS1: meat peptone S1; MPS2: meat peptone S2; LB-BCS: LC-SFM-LB/1% BCS; BCS: LC-SFM/1% BCS; CTRL: LC-SFM-LB.

Figure 4. Effect of peptones on transfection efficiency and gene expres-sion. 293SFE cells were plated in a 12-well plate and transfected withpTT/GFP and pTT/SEAP vectors (1:9). At 96 hpt, the transfected cells andsupernatants were harvested and analyzed for GFP and SEAP expression,respectively. Panel A � gray bar: % GFP-positive cells; black bar: fluo-rescence intensity (X-mean); open diamond: total GFP. Total GFP expres-sion was calculated by multiplying the percentage of GFP-positive cells(GFP+) by the mean fluorescent intensity (X-mean). Panel B � SEAPactivity.


the effect of BSA in LC-SFM-lipids supplemented with0.5% GPN3 on transfection and protein expression. BSAhad an inhibitory effect on protein expression (Fig. 6, panelB) as they were reduced by almost 50%. A relatively lowpercentage of GFP-transfected cells for both conditions wasfound as these values were measured at 120 hpt to maximizeSEAP expression (Fig. 6, panel A). Our previous study(Durocher et al., 2002) showed that the percentage of GFP-positive cells peaks at 72 hpt and then gradually decreases.As cell growth was not affected in the absence of BSA (datanot shown), BSA-free LC-SFM/lipids/GPN3 (LC-SFM-LG)was used in the following experiments.

Effect of Lipids Concentration on Transfectionand Protein Expression

Transfections were performed in 12-well plates using293SFE cell line in LC-SFM-LG employing four concen-trations of lipid mixture: 0, 0.1, 0.2, and 0.5% (v/v). Allwells were seeded with 0.5 × 106 cells in one mL of mediumand cotransfected with pTT/GFP and pTT/SEAP plasmids(ratio 1:9). Increasing lipids concentration up to 0.5% al-

lowed higher percentage of transfected cells, but resulted ina gradual reduction of fluorescence intensity value (X-mean) as depicted in Figure 7A. Thus, 0.5% lipids caused adecrease in the overall GFP expression probably due totoxicity. The profile of GFP (Fig. 7, panel A) and SEAPexpression (Fig. 7, panel B) showed that a lipid concentra-tion of 0.1% was sufficient to achieve good cell growth(Fig. 7, panel C), effective transfection efficiency, and pro-tein expression.

GPN3—A Peptone That Improves Transfection inSerum-Free Medium

To confirm the positive effect of GPN3 on transient trans-fection in serum-free medium, we compared SEAP expres-sion using 293E cells grown and transfected in LC-SFMsupplemented with 1% BCS to 293SFE cells grown andtransfected in LC-SFM-LB or in LC-SFM-LG (Fig. 8). Weobserved almost a fourfold increase in SEAP activity whenusing SFE cells in LC-SFM-LG compared to LC-SFM-LB.The level of SEAP expression represented about 88% of theactivity obtained with 293E cells in 1% serum-containingmedium. Thus, the incorporation of the peptone GPN3 andremoval of BSA allowed the transfection to be performed in

Figure 5. Effect of GPN3 concentrations on transfection efficiency andprotein expression. 293SFE cells were plated in LC-SFM-LB containing0.25%, 0.5%, 1.0%, and 1.5% (w/v) of GPN3 and transfected with pTT/GFP and pTT/SEAP vectors (1:9). Cells were harvested and analyzed at120 hpt. Panel A � gray bar: GFP positive cells in percentage; black bar:fluorescent intensity (X-mean); open diamond: total GFP. Total GFP ex-pression was calculated by multiplying the percentage of GFP-positivecells (GFP+) by the mean fluorescent intensity (X-mean). Panel B �

SEAP activity.

Figure 6. Effect of BSA on transfection and SEAP expression. 293SFEcells were plated in a 6-well plate in LC-SFM-LG with or without supple-mentation with 0.1% BSA. Cells were then co-transfected with pTT/GFPand pTT/SEAP vectors (1:9) and analyzed at 120 hpt later for GFP (panelA) and SEAP expression (panel B) as described in Materials and Methods.Panel A � gray bar: % GFP-positive cells; black bar: fluorescent intensity(X-mean); open diamond: total GFP. Panel B � SEAP activity.


serum-free medium with an expression level close to thatobtained with 1% serum-supplemented medium.

Large-Scale Transient Transfection: Production ofTie-2 and Neuropilin-1 ED

The scaleability and robustness of serum-free transienttransfection using our SFE cells adapted in LC-SFM-LGwas evaluated using extracellular domains of Tie-2 andNeuropilin-1. Tie-2 is a receptor tyrosine kinase for thenovel family of angiopoietin growth factors. Its ED wastransiently expressed in a 1-L bioreactor (Biostat Q) andscaled-up in a 14-L bioreactor (Chemap). The kinetics ofcell growth and metabolites consumption/production duringthe transfection in a 1-L bioreactor are shown in Figure 9A.The bioreactor was seeded at 0.28 × 106 cells/mL and thecell density reached 0.56 × 106 cells/mL 24 h later. At thistime, the cells were transfected with the pTT vector encod-ing myc-H8GGQ-tagged Tie-2 ED. The growth was almostarrested and cells entered lag phase after addition of PEI-plasmid complexes. This lag phase was most likely due to a

stress induced by the transfection. The cells resumed theirgrowth 72 hpt and grew steadily up to 1.2 × 106 viablecells/mL at 144 hpt. This maximum cell concentration islower than the maximum viable cell density attainable inuntransfected cultures (about 4 × 106 cells/mL, data notshown).

The glucose concentration profile showed a 50% reduc-tion at 24 hpt. Although cells entered the lag phase between24 and 72 hpt, glucose continued to decrease rapidly,and was depleted at 96 hpt. The glutamine concentrationdropped from 5.4 mM to 1.2 mM at the end of the culture.Maximal lactate concentration was observed at 96 hptreaching values of 43 mM, which corresponded to the glu-cose depletion point, while the ammonia level reached 5.2mM at the end of fermentation. Hence, 22 mM of glucosewas transformed into 43 mM of lactate, resulting in an over-all yield of 1.9 mol lactate/mol glucose. These concentra-tions of lactate and ammonia might represent the criticalinhibition concentrations for 293 cells (Nadeau et al., 1996).Amino acid analysis showed a rapid depletion of asparticacid at 48 hpt. Serine was also completely consumed after120 hpt (data not shown). Most of the other amino acidswere still present in the medium at the end of the culture.The non-essential amino acids such as glycine, alanine, pro-line, and glutamate were all produced by the cells in thebatch culture. A Western blot of the supernatants drawn atdifferent times (Fig. 9B) showed the accumulation profile ofTie-2 secreted in the medium. Product expression was de-tected after only one day posttransfection and continued toincrease steadily up to 96 hpt. Although cell growth stillincreased after 96 hpt (Fig. 9A), extracellular product ac-cumulation seemed to reach its maximal level at this point.Moreover, no product degradation was observed eventhough the harvest time was 144 hpt, suggesting low pro-tease level in the medium. The production of Tie-2 has alsobeen done in a 14-L Chemap bioreactor (data not shown). In

Figure 7. Effect of lipids concentrations on transfection and expression.293SFE cells were inoculated at 0.5 × 106 cells/mL in LC-SFM-LG me-dium supplemented with various concentrations of lipids. The cells werethen co-transfected with pTT/GFP and pTT/SEAP (1:9). GFP (panel A)and SEAP (panel B) were analyzed 96 hpt. Panel A � gray bar: %GFP-positive cells; black bar: fluorescent intensity (X-mean); open dia-mond: total GFP. Panel B � SEAP activity. Panel C � gray bar: viablecell density; black bar: total cell density.

Figure 8. Performance of 293SFE cells in LC-SFM supplemented with0.5% (w/v) of GPN3. Three cell line/medium combinations were tested: (1)293E cells grown and transfected in LC-SFM supplemented with 1% BCS;(2) 293SFE cells grown and transfected in LC-SFM-LB; (3) 293SFE cellsgrown and transfected in LC-SFM-LG. The cells were seeded in a 6-wellplate and co-transfected with pTT vectors expressing GFP and SEAP (1:9).SEAP activity was analyzed 120 hpt.


this case, the total cell density reached a peak value of 2.1× 106/mL at 120 hpt. Oxygen demand increased up to 65–70% of gas flow (300 mL/min) at 72 hpt and this value wasmaintained during most of the cultivation time but slowlydecreased to 60% at 120 hpt.

Neuropilin-1 is a receptor for the collapsing/semaphoringfamily of proteins that mediates neuronal cell guidance.This protein is expressed by endothelial and tumor cells asan isoform-specific receptor for vascular endothelial growthfactor VEGF (Soker et al., 1998). Neuropilin-1 ED wasproduced transiently in a 14-L bioreactor (BioFlo 110) witha working volume of 10 L. The bioreactor was seeded at0.20 × 106 cells/mL and transfected 24 h later with the pTT

plasmids encoding the protein with a H8GGQ C-terminaltag. The growth curve following transfection was similar tothe 14-L Tie-2 run and showed a maximal viable cell den-sity of 1.60 × 106 cells/mL at 120 hpt. IMAC purification ofthe two proteins expressed by transient transfection in bio-reactors is depicted in Figure 10. Panel A represents thepurification of Tie-2 ED produced by 293SFE cells in LC-SFM-LG and harvested at 120 hpt (14-L bioreactor), whilepanel B shows the same protein expressed by 293E cells in1% serum-supplemented LC-SFM (3-L bioreactor). Fromthe 3-L bioreactor, 9 mg of partially purified Tie-2 wasobtained, while 33 mg of >95% pure Tie-2 was harvestedfrom the 14-L serum-free production. The purified Neuro-

Figure 9. Production of Tie-2 ED in 1-L bioreactor: growth kinetic and metabolite profiles. Panel A: 293SFE were seeded in a 1-L bioreactor at 0.28× 106 cells/mL with a final volume of 610 mL, and transfected with pTT vector encoding myc-H8GGQ-tagged Tie-2 ED 24 h later. The bioreactor washarvested 144 hpt. Closed circles: viable cell density; open circles: viability; closed squares: glucose; closed up triangle: lactate; closed down triangle:ammonium; open squares: aspartic acid; open up triangle: glycine; open down triangle: glutamine. Panel B � Tie2 expression kinetic following transfectionin 1-L bioreactor. Lane 1 as detected using an anti-HisG antibody (Invitrogen, Carlsbad, CA).


pilin-1 ED expressed by 293SFE grown in LC-SFM-LG andharvested at 120 hpt is shown in panel C. From this 10-Ltransient transfection, 35 mg of >95% pure Neuropilin1 wasobtained.

DISCUSSION

The HEK 293 cell line was derived from primary humanembryonic kidney cells transformed with sheared humanAd5 DNA. Adaptation of the suspension adapted 293S cellsto serum-free medium was carried out in a LC-SFM-LBfollowed by two cloning steps (Cote et al., 1998). The293SF-3F6 clone was isolated and selected for its ability togrow at the highest specific cell growth rate and cell density,while forming the least and the smallest cell aggregates inshake-flask suspension culture. The 293SF-3F6 cell linewas then stably transfected with a plasmid driving the ex-pression of EBNA1 to create a 293SF cell line constitutivelyexpressing the EBNA1 protein (293SFE cells). The pres-ence of EBNA1 enhances protein expression and allowsepisomal maintenance when the plasmid DNA contains theEBV oriP. We have previously shown that the combinationof oriP-bearing vector with the EBNA1-expressing cell line

293E provided very high gene expression following tran-sient transfection (Durocher et al., 2002). Here we providefurther evidence that this effect is mainly due to EBNA1(when comparing 293SF to 293SFE clones) and also to cellline characteristics (e.g., transfectability) as we observedsignificant differences in SEAP expression when using vari-ous 293SFE clones. By using the oriP-containing pTT ex-pression plasmid with the 293E cell line, the expressionlevel of intracellular proteins reached as high as 20% oftotal cellular protein (Durocher et al., 2002). In this study,transfection of 293SFE cells led to a fivefold and eightfoldincrease in total GFP and SEAP activity, respectively, com-pared to transfection of 293SF cells (Fig. 1, panel B). Thesedata confirm that the combination of oriP and EBNA1 con-tributes to a significant increase in gene expression. Thisimprovement might be due, in part, to an enhancement ofnuclear import of oriP bearing plasmids, but also to thecis-acting FR-EBNA1 transcriptional enhancer activity(Langle-Rouault et al., 1998; Reisman and Sugden, 1986).However, there was no correlation between EBNA1 levelsand transgene expression.

The recent increase in the use of mammalian cells togenerate large amounts of r-proteins emphasizes the need

Figure 10. Purification of Tie-2 and Neuropilin-1 ED. Tie-2 and Neuropilin-1 ED were purified on a 10 mL TALON column as described in Materialsand Methods. Panel A: Tie-2 ED produced by 293SFE in serum-free medium at the 14-L scale. Lane 1: concentrated culture medium; Lane 2: TALONflow-through; Lane 4: 150 mM imidazole eluate. Panel B: Purified Tie-2 ED produced by 293E in 1% serum-containing medium at the 3-L scale. PanelC: Neuropilin-1 produced by 293SFE in serum-free medium at the 10-L scale. Lane 1: concentrated culture medium; Lane 2: IMAC flow-through; Lane3: wash fraction; Lane 4: 150 mM imidazole eluate. The positions of proteins of interest are indicated by arrows.


for ways other than stable clones to generate them. Thelarge-scale transient transfection approach has recentlydemonstrated its suitability for such a need, and offers theadvantage of being extremely rapid (Durocher et al., 2002;Girard et al., 2002; Jordan et al., 1998; Meissner et al.,2001; Schlaeger and Christensen, 1999). While the produc-tion of r-proteins in serum-free medium offers significantadvantages, we observed a significant decrease in the ex-pression level when using our original LC-SFM-LB formu-lation (Fig. 2). Many other commercially available serum-free media have been developed for 293 cells; however,many of them do not support good transfection when usingPEI (data not shown) and their cost-effectiveness is ratherquestionable. This is most likely due to the presence ofpolyanions (heparin, dextran sulphate) that are added toprevent cell aggregation (Schlaeger and Christensen, 1999,and unpublished data). Among substances used for reduc-tion or substitution of serum, many efforts have concen-trated on low-cost commercial protein hydrolysates. In thisstudy, only animal-derived peptones were selected as mostplant peptones strongly induced cell aggregation and/or hadsignificant inhibitory effect on cell growth (data not shown).From a mass-balancing viewpoint, amino acids and peptidesrepresent by far the most important compounds that hydro-lysates may supply. The composition of free amino acidsand peptides in each peptone preparation should largelycontribute to promoting or inhibiting cell growth. Peptonesused in our screening experiment (Fig. 3) were obtainedfrom enzymatic hydrolysis of animal sources such as meat(MPN1, MPN2, MPS1, MPS2), gelatin (GPN2, GPN3) andcasein (CPN1, CPE1, CPN3). Some researchers attributedthe growth-promoting effect to the presence of low molecu-lar peptides (Taylor, 1981) or ethanolamine and phospho-ethanolamine (Murakami et al., 1982). However, the natureof compounds that promote biosynthetic and mitotic eventsremains unclear. Through metabolic flux analysis, it wasshown that the growth of hybridoma cells is more efficientwhen medium was enriched with meat peptone (PrimatoneRL) rather than with free amino acids (Nyberg et al., 1999).In addition, 50% less lactate was produced and twice asmuch substrate was used in the tricarboxylic acid (TCA)cycle in comparison with the equivalent free amino acidmixture. One possible reason is that the amino acid balanceof the peptone-enriched medium allowed more efficient up-take by the cell line. Furthermore, the authors believed thatthe peptide transport into the cell is more energy efficientthan that for free amino acids. Other studies showed thatcultured animal cells can metabolize dipeptides such asglycyl-L-glutamine and L-alanyl-L-glutamine (Minamoto etal., 1991) and so one would expect that other peptides couldbe utilized as well. The uptake of peptones is linked tospecialized transport systems, which are different fromamino acid transporters. Inside the cells, the peptones arefirst clipped by proteases and the resulting free amino acidscan then be used as nutrients via the TCA cycle or as pre-cursors for other amino acids, nucleic acids, or incorporatedinto proteins. It was found that the active transport of pep-

tides in kidney is energized by an H+ gradient rather than aNa+ gradient commonly seen in amino acid and glucosetransport systems (Hopfer, 1997). Unfortunately, detailedinformation concerning peptide uptake in cultured cells isunavailable so far.

The experiment carried out on effect of hydrolysates ontransfection efficiency (Fig. 4) shows that while some pep-tones favor cell growth, they can be inhibitory to transfec-tion. While most meat peptones support good transfectionefficiency, it is not the case for casein peptones (CPE1,CPN3). At concentration of 1% (w/v), CPN3 strongly in-hibited transfection (Fig. 4) even though it stimulated cellgrowth (Fig. 3). Supplementation with gelatin peptone(GPN3) resulted in the highest level of SEAP activity com-pared to other peptones. In addition, GPN3 does not inducecell aggregation in contrast to meat peptones (data notshown). The positive effect of GPN3 may be due not onlyto nutritional aspects, but also to the presence of undefinedcomponents that might improve transfection efficiency.This beneficial behavior of gelatin peptone makes it a po-tential candidate to replace serum. To date, few data areavailable on the effect of peptones on transfection proce-dure. For example, Primatone RL (a tryptic meat digest)was reported to enhance the biological activity of the trans-fection complexes (Schlaeger and Christensen, 1999). Thisanimal tissue digest consists of 37% free amino acids and41% peptides with a majority of them ranging from 200–500 Da (di- to tetrapeptide). The absence of Primatone RLin the DNA/PEI complex formation medium decreases thetransfection efficiency by about 30–40% compared to thecontrol sample, which contains 0.3% hydrolysates.

While BSA is usually added to serum-free medium for-mulations to transport lipids, minerals, and hormones, andto protect cells, it appears to be dispensable when GPN3 isused. The removal of BSA from the medium routinely usedfor 293SF allowed a significant enhancement in terms oftransfection and protein expression (Fig. 6). Furthermore,BSA removal also contributes to lowering the protein con-tent of the basal medium.

Our results demonstrated the positive effect of GPN3 onprotein expression using the 293SFE cell line growing inserum-free medium. While other peptones were also active,they strongly induced cell aggregation. Up to a fourfoldincrease in SEAP activity was detected when using 293SFEgrown in presence of 0.5% GPN3 compared to the conven-tional LC-SFM-LB medium (Fig. 8). This value also rep-resents 88% of that obtained when 293E cells were grownand transfected in 1% serum supplemented LC-SFM.Hence, the SFE constitutes a new and robust cell line thatmight be especially useful when production in serum-freemedium is needed. Peptones do not appear to interfere withdownstream processes. All of the peptones tested have mo-lecular weights of less than 3000 Da that greatly facilitatestheir separation from r-proteins. In the present work, wedescribed the robustness of our new serum-free system byproducing two r-proteins at significant expression levels. Todate, we have successfully produced and purified over fif-


teen r-proteins from 1–14-L bioreactors using this serum-free system. As an example we compared the productionand purification of Tie-2 ED in 293E cell line in LC-SFM/1% BCS to 293SFE in LC-SFM-LG. Both cell lines allowedharvesting and purification of over 30 mg of proteins within5 days. When purifying r-proteins by IMAC from 1% BCScontaining medium, two major contaminant proteins (55and 57 kDa, Fig. 10B) were systematically present, creatinga need for a second purification step. Using the new serum-free formulation and the 293SFE cell line, Tie-2 ED waspurified to >95% using a single IMAC step. The perfor-mance of this fast and reliable serum-free transfection tech-nology was also demonstrated with the production of Neu-ropilin-1 ED. Again, one step purification by IMAC al-lowed us to get a >95% pure protein, as determined bySDS-PAGE.

In conclusion, we have established a new HEK293/EBNA1 cell line (293SFE) capable of growing in a cost-ef-fective serum-free medium. We demonstrated the positiveeffect of using GPN3 as a potential serum replacement thatsustains both cell growth and PEI-mediated transfection.The use of serum-free medium greatly facilitates r-proteinpurification, eliminating the need for extra chromatographicsteps, thus lowering the overall cost of operations, and in-creasing the production throughput. An improved and inte-grated system that includes aspects of medium formulation,feeding strategies, and expression vector to improve cellculture duration and volumetric protein yield is ongoing.

We thank Rosa Tran for her useful help in ultrafiltration opera-tions, Louis Bisson for HPLC amino acid analysis and ChunlinXin for his technical assistance in cDNA cloning and plasmidpurification.

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