APPLICATION NOTE
Improving expression and purification of mAb using
FectoCHO® Expression System and MabXpure™.
Jonathan Havard1, Fabien Rousset2, Mathieu Porte1 & Alengo Nyamay’Antu1.

INTRODUCTION
Transient gene expression (TGE) is commonly used for
medium scale production of recombinant proteins andantibodies in suspension CHO cell lines. This approach allows
production of sufficient protein amounts for “proof ofconcept” studies or tools validation, prior to moving on totime-consuming, but cost-effective production of stable celllines at large scale. Speed and flexibility of TGE has allowed
this technique to be widely adopted in early phase discovery.
There are two main bottlenecks in protein production thatneed to be addressed to make TGE profitable: increasingprotein production yield and improving protein recoveryyield.
To achieve high transient protein yields, TGE is dependent on
an efficient transfection method. Polyplus-transfection® has
therefore developed a protein production system, named
FectoCHO® Expression System for optimal protein
production in several commercially available suspension CHO
cell lines. FectoCHO® Expression System consists of theassociation of FectoPRO® transfection reagent with a novel
chemically-defined CHO medium: FectoCHO® CD Expression
Medium. The latter is developed to improve protein
production in various strains of CHO cells such as CHO-K1,
CHO-S and ExpiCHO-S™. FectoPRO®-mediated transienttransfection of CHO cell lines in this chemically defined
medium leads to highest production yields of protein andantibody; with up to 4-fold increase in protein yields
compared to competitors, while using low DNA amount (< 1µg/mL of cell culture).
To recover the target protein, we chose flowthrough (FT)
purification technologies, as they improve the throughput by
reducing time and facilitating the operation. With
MabXpure™, a novel truly single-use chromatography media,
we increased recovery yield by combining size exclusion
chromatography (SEC) and anion exchange (AEX). With SEC,
we avoid the penetration of large proteins (e.g. mAbs) to thebeads and their collection in the FT fraction. In the same time,
AEX allows to capture the bioburden, such as host cell DNA,
host cell proteins and other small molecules (Figure 1). The
multimodal depletion of MabXpure™ also binds other classes

of impurities that are usually depleted by hydrophobicinteraction (HIC), cation exchange (CEX), mixed-mode (MM),dipole-dipole interactions, etc., which confers on this FT
purification step an exceptional performance.
Synergistically, we show here that combining FectoCHO®
Expression System (Polyplus-transfection®) with MabXpure™
(DAICEL Bioseparations) provides high expression andrecovery of antibodies.

Figure 1: Multimodal
MabXpure™.

depletionmechanismsofDAISEPMATERIAL & METHODS
Mammalian cell culture
Commercially available ExpiCHO-S™ cells (Life Technologies,Carlsbad, CA) were grown in suspension culture inFectoCHO® CD Expression Medium supplemented with 100
U/mL of penicillin and 100 µg/mL of streptomycin, 1 % of
Pluronic™-F68, and 8 mM L-glutamine. Cell cultures weremaintained between 0.25 and 10 x 106 cells/mL in 125 mL
Erlenmeyer flasks with vented cap, shaken at 130 rpm in an
incubator at 37 °C with 8 % CO2.

Transfection
ExpiCHO-S™ cells, cultured in FectoCHO® CD Expression
Medium, were transfected with FectoPRO® transfection
reagent, by using vectors coding for the heavy (HC) and the1 Polyplus-transfection®, 850 Bld Sébastien Brant, 67400 Illkirch-Graffenstaden, France, support@polyplus-transfection.com

2 Daicel Bioseparations, 160 Bld Gonthier d’Andernach, 67400 Illkirch-Graffenstaden, France, bioseparations@cte.daicel.com

APPLICATION NOTE
light (LC) chain of a recombinant chimeric IgG1 (Claudiximab).

IgG quantification

The day of transfection, cells were counted and viability wasmeasured before seeding 6 x 106 cells/mL in 90 mL ofcomplete fresh FectoCHO® CD Expression Medium.

IgG1 protein quantification was performed using the BLItz®
System (ForteBIO®) according to the manufacturer’s
instruction for protein A Biosensors (Dip and Read™ ProteinA (ProA)). The quantity of IgG1 in each sample was measured
in duplicates with the integrated BLItz Pro® software, using apreviously established standard curve for Claudiximab. IgG1
quantification was performed at each purification step.

For the transfection with FectoPRO®, 0.8 µg of DNA was used
per mL of cell culture, with a 1:2 (µg to µL) DNA to FectoPRO®
ratio. DNA was pre-diluted in 10 mL of non-supplementedfresh FectoCHO® CD Expression Medium (corresponding to10 % of the final culture volume), briefly vortexed and added
to pure FectoPRO®. After 10 minutes of complexation atroom temperature, the DNA:FectoPRO® complexation mix
was added to the ExpiCHO-S™ cell culture. The cell culture
was placed in an incubator at 37 °C; 8 % CO2, under agitation(130 rpm). Protein yields were analyzed on Day 7 posttransfection.

Clarification
On Day 7 post-transfection, the whole cell culture wascentrifuged (5 min, 210 g) and the supernatant was passed
through a 0.22 µm filter. Clarified samples were then purified
using MabXpure™ resin or protein A resin column.

MabXpure™
Clarified samples were purified with MabXpure™ using
SpinXL device. The MabXpure™ resin was first placed in theSpinXL device and centrifuged (5 min, 1500 g) to remove theethanol preservation buffer. Two successive washes with 15mL of NaCl 150 mM were performed by centrifugation (5 min,1500 g). Clarified samples were then added to theMabXpure™ resin and placed under gentle agitation for 30
min. Two v/v ratios were chosen: 10 mL of sample for 2 mLof MabXpure™ (ratio 1:5) and 10 mL of sample for 4 mL of
MabXpure™ (ratio 1:2.5). Purified samples were collected bycentrifugation (10 min, 1500 g).

Protein A
Clarified samples were purified using a protein A agarosebased affinity resin column (Praesto® Jetted A50 (1 mL) HTColumn from Purolite®), following the provider’srecommendations. The protein A affinity resin was firstwashed with 10 mL of binding buffer (NaHPO4 20 mM, NaCl
150 mM pH 7-7.4) at a rate of 1 mL per minute. Clarifiedsamples were passed on the column at a similar rate for atotal volume of 5 mL. After washing the resin with 10 mL ofbinding buffer at a rate of 1 mL per minute, IgG1 was eluted
using 5 mL of acetic acid 0.1 M at a similar rate of 1 mL per
minute and collected in a tube containing 500 µL of Tris-HCl
1 M buffer (100 µL/mL of sample).

Qualitative analysis of IgG production – SDS-PAGE
Clarified and purified samples were qualitatively analyzed byusing BioRad Criterion™ TGX™ system, including BioRadCriterion™ Cell and Criterion™ TGX™ 4-15 % 12+1 well gels.
Tris/Glycine running buffer and loading buffer wererespectively supplemented with 0.1 % and 2 % SDS. Biorad
Bio-Safe™ Coomassie staining was used for detection of total
protein in samples.

Host Cell Proteins quantification
Samples were analyzed for Host Cell Proteins (HCP).
Quantification of HCPs was performed using a CHO HCP ELISA
kit (Cygnus™ Technologies) at 450 nm using Bio-Tek EL800™
plate reader. Samples were first 10-fold serial diluted down
to 10-6. Serial dilutions of 10-2 to 10-6 were analyzed at eachstep of the purification, except for samples after protein Apurification, which were also analyzed at 100 and 10-1 serial
dilutions. All samples were measured in duplicates.

DNA quantification
DNA extraction of the samples was performed using Cygnus
DNA Extraction kit in tubes (Ref. D100T). Then, DNA
quantification was performed by using Quanti-iT PicoGreen
dsDNA Reagent kit from Invitrogen (ultra-sensitive
fluorescent nucleic acid stain for double-stranded DNA insolution). The samples’ fluorescence was measured using aVictor X microplate reader (Perkin Elmer) at standard
fluorescein wavelengths (excitation ~480 nm, emission ~520nm).

APPLICATION NOTERESULTS & CONCLUSIONRESULTS & CONCLUSION

IgG1 yields / recovery

SDS-PAGE

Figure 2: IgG1 yields/recoveries are significantly higher when using
MabXpure™ resin compared to Protein A resin, with all recoveries
higher than 90 %. IgG1 quantification was performed at eachpurification step using the BLItz® system (ForteBIO®) with protein A
biosensors (Dip and Read™ Protein A (ProA)).

Host Cell Proteins (HCP)

Figure 3: MabXpure™ resin has proven to be very effective indepleting Host Cell Proteins in clarified samples. HCP quantification
was performed using a CHO HCP ELISA kit (Cygnus™ Technologies) at
450 nm using Bio-Tek EL800™ plate reader.

DNA quantification
DNA quantification was performed using Cygnus DNA
extraction kit and Invitrogen Quanti-iT PicoGreen dsDNA kit.
In all analyzed samples, DNA concentration was found below
the limit of quantification (1 ng/mL) with starting materials
containing 50 to 100 ng/mL, representing up to 2 Log
Reduction Value (LRV) for DNA.

Figure 4: Qualitative analysis on SDS-PAGE reveals that clarified
samples (A) contain a high quantity of impurities, including HCP.
MabXpure™ resin used at ratio 1:5 (B) or 1:2.5 (C) allows asignificant depletion of these impurities, without visible loss ofIgG1. Protein A resin (D) purification shows a total depletion ofimpurities and a significant loss of IgG1. SDS-PAGE was performed
using BioRad Criterion™ TGX™ system including Criterion™ Cell andCriterion™ TGX™ 4-15 % gels.

Transient transfection using FectoCHO® Expression Systemfrom Polyplus-Transfection® has successfully enabled
expression with high titers of mAbs after 7 days oftransfected cells culture. Thus, in order to preserve thebenefits of a fast production system, the use of singlestep/single-use flowthrough purification techniques wasfavored to "bind and elute" technologies that are greedy forhandling multi-steps samples. DAISEP MabXPure™ has
achieved high depletion rates, while ensuring good antibody
recovery yields, which was the ultimate goal (mAb yield >90%, HCP depletion >85 %, DNA depletion >99 %).
In the field of medium-scale recombinant proteins andantibodies production in suspension CHO cells, transienttransfection using FectoCHO® Expression System can quickly
and efficiently provide high mAbs titers. When combinedwith MabXpure™ purification technologies, it allows thecompensation of expensive affinity resin use after acentrifugation step.

APPLICATION NOTERESOURCESPOLYPLUS-TRANSFECTION

Phone: +33 (0)3 90 40 61 87
Email: support@polyplus-transfection.com
Web site: www.polyplus-transfection
ProductRef N°

Expression Medium

Transfection Reagent

Expression Booster

FectoCHO® Expression System

716-01LKIT

1L

1 mL

1 mL

116-001

-

1 mL

1 mL

116-010-10 mL10 mL

116-040-

4 x 10 mL

4 x 10 mL

716-01L

1L--FectoPRO® Transfection Reagent
FectoCHO® CD Medium

DAICEL BIOSEPARATIONS

Phone: +33 (0)3 88 79 52 00
Email: bioseparations@cte.daicel.com
Web site: www.daicelbioseparations.com

ProductType

DAISEP™ Spin tubes

Filter tubes

MabXpureTM Bulk

Slurry bottle

MabXpureTM FT

Prepacked columns

Ref N°

VolumeUnitsDSPXS0040

0.6 mL40

DSPXL0008

22 mL

8

DMXBK0050

50 mL

1DMXBK0500

500 mL

1DMXBK0001

1 mL

5DMXBK0005

5 mL5