Emu

AKa

b

c

a

ARR1AA

KIPPABAAN

1

peMapso(pAp

fn

0d

Journal of Chromatography A, 1216 (2009) 7851–7864

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

ngineering of a two-step purification strategy for a panel ofonoclonal immunoglobulin M directed against

ndifferentiated human embryonic stem cells

nne Tscheliessniga,∗, Danny Onga, Jeremy Leea, Siqi Pana, Gernalia Satianegaraa,ornelia Schriebla, Andre Chooa,b, Alois Jungbauerc

Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, 138668 SingaporeDivision of Bioengineering, Faculty of Engineering, National University of Singapore, SingaporeDepartment of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria

r t i c l e i n f o

rticle history:eceived 10 August 2009eceived in revised form7 September 2009ccepted 23 September 2009vailable online 26 September 2009

eywords:

a b s t r a c t

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5–7.7)produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pHand mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD,a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer,the binding capacity was still higher compared to a convection driven resin (monolith). This purificationstrategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could

gMEGrecipitationnion-exchange chromatographyenzonasedsorption isotherm

be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%).The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement ofrecovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using anendonuclease, benzonase. So far this strategy has been applied for the purification of up to 2 l hybridomasupernatants.

© 2009 Elsevier B.V. All rights reserved.

dsorption kineticsumber of binding sites

. Introduction

Various purification strategies have been developed for theurification of immunoglobulin M (IgM) antibodies from differ-nt sources, such as serum, ascites or cell culture supernatants.ost of those strategies comprise techniques based on size sep-

ration, such as size-exclusion chromatography (SEC) [1–11] orolyethylene glycol (PEG) precipitation [3,12]. Additionally, highlyelective techniques such as affinity chromatography using a rangef different ligands [1,13–23], anion-exchange chromatographyAEC) [1–3,5,7,11,24–27] as well as hydroxyapatite chromatogra-hy [4,28–31] have been applied. In particular, the combination ofEC with a subsequent SEC step [1,2,5,7,11] was successful for the

urification of IgM from ascites and cell culture supernatants.

PEG precipitation is advantageous as an initial enrichment stepor large proteins such as IgM (960 kDa). The predominant mecha-ism in PEG precipitation is the exclusion of the proteins from the

∗ Corresponding author. Tel.: +65 6478 8909; fax: +65 6478 9561.E-mail address: anne.tscheliessnig@gmx.at (A. Tscheliessnig).

021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.09.059

suspension volume which is taken up by dissolved PEG (“excludedvolume”) [32,33]. Differently sized proteins can be easily and selec-tively separated depending on the PEG size and concentration[34–36]. While excluded volume is the major effect, it has beenshown that other parameters, such as temperature, pH or ionicstrength can affect the solubility significantly [35]. In particular,conditions that favor the self-association of proteins are advanta-geous, e.g. a pH close to the isoelectric point [37]. Therefore, designof an efficient PEG precipitation step not only includes selection ofPEG size and concentration but also the screening for other param-eters such as pH or salt concentration.

Due to their size, IgM antibodies also carry many chargedgroups, which enable adsorption to ion-exchange chromatogra-phy resins even at moderate pH, thereby reducing the potentialfor degradation during purification [30,38]. In particular, anion-exchange chromatography has proven to be efficient in the

purification of IgM from cell culture supernatant [7,30]. Its majordisadvantage, however, is the adsorption of most other host cellproteins leading to a reduced capacity for IgM. In contrast, cation-exchange chromatography, while having the advantage of minimalhost cell protein adsorption, is less generic for adsorption of IgM.

7 matog

Ot

itdswmoutoeIpkdhomFtmctta(o

bhioitfieocmttftscstcu

2

2

(atABuIn

852 A. Tscheliessnig et al. / J. Chro

ften the pH of the supernatant has to be adjusted to allow adsorp-ion [30].

Besides the selection of the chromatographic mode, it is alsomportant to consider the pore geometry of the resins [39]. Conven-ional porous resins provide a large surface area. However, the poreiameter might not be sufficiently wide to allow large molecules,uch as IgM, to diffuse to the binding site. In contrast, not onlyill the transport be slow due to hindered diffusion but adsorbedolecules could potentially block the pore preventing diffusion

f further molecules into the pore. It has been suggested that fornhindered diffusion, the pore diameter should be at least 10 timeshe molecule diameter [39]. For IgM with a hydrodynamic radiusf 20 nm [7] this would require a pore of 200 nm diameter. How-ver, the average pore diameter for porous resins is 50–100 nm [39].nterestingly we found that for one resin, Fractogel EMD, the ratio ofrotein to pore size is not a constraining factor [40]. The adsorptioninetics of proteins in their unfolded state (∼20 nm, hydrodynamiciameter) were only 1/3 to 2/3 that of the native proteins (6–10 nm,ydrodynamic diameter) when using the Fractogel EMD [40]. Forther resins, e.g. Toyopearl, the uptake was severely impeded. Theechanism of the mass transfer of the unfolded proteins in the

ractogel is not understood but we would expect a similar fast massransfer for the IgM. Alternatively, convection driven resins, such as

embranes and monoliths have been recommended for the purifi-ation of IgM [7,30]. While the binding capacity is reduced dueo a lower surface area, their advantage is the fast mass transferhat allows processing of large volumes in short times. Gagnon etl. [30] reported flow rates between 2.5 and 12.0 column volumesCVs)/min for a separation of an IgM from cell culture supernatantn a cation-exchange monolith.

At BTI we have raised a panel of hybridoma producing IgM anti-odies selectively targeting surface markers on undifferentiateduman embryonic stem cells (hESC) [41]. One of the antibod-

es, mAb 84, not only binds but also kills hESC within 30 minf incubation [41]. Initially all hybridoma clones were culturedn media containing 10% fetal bovine serum (FBS). A purifica-ion strategy, developed for mAb 84, comprising tangential flowltration (TFF), anion-exchange chromatography (AEC) and size-xclusion chromatography (SEC) was only minimally successful inbtaining purified mAb 84 [7]. After adaptation of the hybridomalone to serum-free media we not only obtained highly pureAb 84 but could also simplify the purification process by omit-

ing the initial TFF without affecting the final purity. However,he developed purification strategy had only a low concentrationactor (∼2.7) [7]. Therefore we decided to modify the purifica-ion strategy by replacing the final SEC step that resulted in highample dilution, with an initial enrichment step using PEG pre-ipitation. For AEC we compared different chromatographic resins,uch as Fractogel EMD, Toyopearl and Capto Q for their adsorp-ion equilibrium and kinetics. With DNA being one of the majorontaminants, a DNA removal step using benzonase was also eval-ated.

. Material and methods

.1. Chemicals

Chemicals of analytical grade were purchased from MerckDarmstadt, Germany) or BDH Prolabo (Poole, UK). Bovine serumlbumin (BSA) and o-phenylenediamine dihydrochloride (OPD)ablets were purchased from Sigma–Aldrich (St. Louis, USA).

ll buffers were prepared using ultra-pure water (18.2 M� cm).uffers used for the chromatography experiments were filteredsing 0.2 �m nitrocellulose membranes (Millipore, Carrigtwohill,

reland). Sodium hydroxide solutions were filtered using 0.2 �mylon membranes (Sartorius, Goettingen, Germany). Gel elec-

r. A 1216 (2009) 7851–7864

trophoresis buffers were purchased from Invitrogen (Carlsbad, CA,USA).

2.2. Cultivation conditions

Hybridoma were cultured in a continuously stirred 5 l or 3 lbioreactor (Sartorius Stedim Biotech, Melsungen, Germany). Allhybridoma clones had been adapted step-wise to a protein-free,chemically defined media (proprietary to BTI). Temperature waskept at 37 ◦C and a silicon membrane tubing basket (B. Braun, Mel-sungen, Germany) for bubble-less aeration was used. The dissolvedoxygen concentration (DO) was maintained at 30% of air saturationusing an Air/N2 mix (early phase) or O2/Air mix (late phase) set at1 l/min. pH in the culture was adjusted to 7.10 using intermittentCO2 addition to the gas mix or 7.5% (w/v) NaHCO3 (Sigma–Aldrich)solution. The protein-free, chemically defined feed was based ona 10× DMEM/F12 (Sigma). For fed-batch cultures (5 l) feeding wasset to maintain glutamine above a preset glutamine set-point in theculture. The culture broth was harvested when the viability reached60% and clarified by centrifugation at 4000 × g (Sigma 8k, Sarto-rius Stedim Biotech, Goettingen, Germany) for 10 min followed byfiltration using a 0.2-�m depth filter (Sartorius).

2.3. PEG precipitation

For the solubility experiments, 4 ml of a 5–35% (w/v) PEG 6000(BDH Prolabo) was added to an equi-volume of hybridoma 84supernatant and the solution was incubated at 4 ◦C for up to 4 h.The suspension was then centrifuged (∼5900 × g) and the col-lected precipitate dissolved in 2 ml AEC equilibration buffer (30 mMsodium phosphate, 50 mM NaCl, pH 7.5). mAb 84 present in thedissolved PEG precipitate and clarified supernatant was quantifiedusing ELISA.

For measurement of the precipitation kinetics a similar exper-imental set-up was used: 100 ml of a 20% (w/v) PEG 6000 (BDHProlabo) was added to an equi-volume of hybridoma mAb 84 super-natant. In one set-up the solution was mixed to obtain homogeneityand then incubated at 4 ◦C without further mixing. Alternatively,to evaluate the influence of mixing during precipitation, the samemixture was continuously mixed at 4 ◦C using a magnetic stirrer.At indicated times, samples (4 ml) were drawn, centrifuged andthe precipitate obtained dissolved in 2 ml AEC equilibration buffer.The concentration of mAb 84 in the dissolved PEG precipitate wasmeasured by ELISA.

The small-scale and large-scale PEG precipitation was per-formed similar to the kinetics experiments: An equi-volume of 20%(w/v) PEG 6000 (BDH Prolabo) was mixed with hybridoma super-natant and the solution stirred using a magnetic stirrer at 4 ◦C for aminimum of 2 h. The precipitate was then harvested by centrifuga-tion (5900 × g, Sigma 8k) and dissolved in 25% of the suspensionsvolume using AEC equilibration buffer. The dissolved PEG precip-itate was filtered using a 0.2-�m depth filter (Sartorius StedimBiotech) to remove particulate matter.

2.4. Anion-exchange chromatography

The anion-exchange chromatography was performed on theÄkta Explorer 100 (GE Healthcare, Uppsala, Sweden) using Frac-togel EMD DEAE (M), a weak anion-exchange media with a particlediameter of 40–90 �m and pore diameter of approximately 80 nm(Merck).

For linear gradient elution a Tricorn 5/50 (GE Healthcare) waspacked with approximately 1 ml of Fractogel EMD DEAE (M). Theflow rate was 1 ml/min if not specified otherwise. The column wasfirst equilibrated with AEC equilibration buffer followed by loadingof 100–200 ml of dissolved PEG precipitate at 1 ml/min. Unbound

matog

pspA7tbwI

cSEfoaIAwNlc

2

nts1bcozcTdchaS

mttcat

2

gJcMc9mm9la

q

A. Tscheliessnig et al. / J. Chro

roteins were washed out with AEC equilibration buffer until a con-tant UV (280 nm) and conductivity baseline were observed. Theroteins were then eluted using a linear gradient from 0% to 100%EC elution buffer (30 mM sodium phosphate, 1000 mM NaCl, pH.5) over 20 column volumes (CVs) and the eluate collected in frac-ions of 1 ml. The column was regenerated with 10 CVs AEC elutionuffer, followed by 0.5 M NaOH until a steady UV (280 nm) baselineas observed for at least 10 CVs. The fractions were analyzed for

gM and total protein concentration.For the step gradient elution either a Tricorn 5/50 (GE Health-

are, Uppsala, Sweden) or a Tricorn 10/100 (GE Healthcare, Uppsala,weden) were packed with approximately 1 ml or 8 ml FractogelMD DEAE (M), respectively. The flow rate was either 1 ml/minor the 1 ml column or 4 ml/min for the 8 ml column. 100–1000 mlf clarified dissolved PEG precipitate was loaded onto the columnnd unbound proteins washed out using AEC equilibration buffer.gM was, depending on the respective mAb, eluted with 23–27%EC elution buffer and fractions of 1 ml were collected. The columnas regenerated using 100% AEC elution buffer, followed by 0.5 MaOH until a constant UV (280 nm) baseline was observed for at

east 10 CVs. The fractions were analyzed for IgM and total proteinoncentration.

.5. Benzonase treatment

For measurement of the kinetics of the benzonase, 1 ml of super-atant was mixed with benzonase (purity grade II, >90%, Merck)o give a final concentration of 10 U/ml, 50 U/ml or 100 U/ml. Theamples were mixed at 4 ◦C, 37 ◦C and room temperature and after5, 30, 45, 60 and 120 min the benzonase activity was inhibitedy addition of 1 ml of 400 mM EDTA. For all samples, the DNAoncentration was analyzed as given below. For the comparisonf the purification with and without benzonase treatment, ben-onase was added to 200 ml of hybridoma 84 supernatant to a finaloncentration of 50 U/ml and mixed for 1 h at room temperature.he benzonase-treated supernatant was filtered through a 0.2-�mepth filter (Sartorius Stedim Biotech) and subjected to PEG pre-ipitation and AEC as described previously. For the control, theybridoma 84 supernatant was also stirred for 1 h at room temper-ture and thereafter filtered using a 0.2-�m depth filter (Sartoriustedim Biotech) subject to PEG precipitation and AEC.

For large scale purifications, benzonase treatment was slightlyodified: To the hybridoma 84 supernatant, benzonase was added

o a final concentration of 25 U/ml. After mixing for 30 min at roomemperature, additional benzonase was added to give a final con-entration of 50 U/ml and the supernatant mixed for another 30 mint room temperature. The suspension was then filtered and subjecto PEG precipitation and AEC as described previously.

.6. Adsorption isotherms

The particulate resins, Fractogel EMD DEAE (M) (Merck), Fracto-el EMD DEAE (S) (Merck), Toyopearl DEAE-650 M (Tosoh, Tokyo,apan), Toyopearl DEAE-650S (Tosoh) and Capto Q (GE Health-are) were pre-equilibrated in AEC equilibration buffer. Using theediaScout ResiQuot (Atoll GmbH, Weingarten, Germany) with the

orresponding perforated plate, 7.8 �l of resin were aliquoted into a6-well micro plate. Different concentrations of 0.1–0.5 ml purifiedAb 84 were added and the suspension incubated for approxi-ately 6 h on a rotator (SB 3, Barloworld Scientific, Stone, UK). The

6-well micro plate was then centrifuged and the supernatant col-

ected and analyzed. From the mass balance, the amount of mAb 84dsorbed onto the media could be calculated:

= c0V0 − cV

Vg(1)

r. A 1216 (2009) 7851–7864 7853

with c0 and V0 as the mAb 84 concentration and volume in thebeginning and c and V as the final mAb 84 concentration and thetotal volume of the suspension. Vg is the settled media volume inthe slurry.

For the EDA CIM disk (monolith) a different set-up was used:EDA CIM MiniDisks (gift from BIA Separations, Ljubljana, Slovenia)with 34 �l column volume were placed in the designated housing(gift from BIA Separations) and connected to the injection valve ofthe Äkta explorer 100. Using the sample pump P960, 2.0–2.9 ml ofmAb 84 at different concentration were circulated over the columnat 0.2 ml/min for 5 h. After washing the disk until a constant UVbaseline (280 nm) was obtained, bound mAb 84 bound was elutedusing AEC elution buffer. The mAb 84 content in the collected frac-tion was quantified using ELISA.

The obtained data points, c and q, were then fitted to the Lang-muir isotherm using TableCurve 2D software to give the apparentmaximum adsorption capacity, qm and the apparent adsorptionequilibrium parameter, KA:

q = qmKAc

1 + KAc(2)

2.7. Adsorption kinetics

The adsorption kinetics were measured using the method ofshallow bed [42]. In short, approximately 20 �l of a 1:2 slurryof pre-equilibrated resin was placed into a Tricorn 5/20 column(GE Healthcare) and mixed with 200 �l of Sephadex G25 to emu-late conditions of a fixed bed. The column was connected to anÄkta Purifier 10 (GE Healthcare) and equilibrated with AEC loadingbuffer at 4 ml/min. Using the sample pump P960, 90–450 ml of puri-fied mAb 84 (∼80 �g/ml) as well as dissolved PEG precipitate frombenzonase treated and untreated hybridoma 84 supernatant (each∼80 �g/ml mAb 84) was circulated over the column at 4.0 ml/min.At specified times, loading was stopped, the column washed untila constant UV (280 nm) baseline was obtained before bound pro-teins are eluted using AEC elution buffer. The collected fraction wasanalyzed for DNA, IgM and total protein concentration.

To evaluate the adsorption kinetics of the EDA (monolith), a 34-�l CIM EDA MiniDisk (gift from BIA Separations) was used. Theflow rate had to be reduced to 1–2 ml/min to accommodate for thepressure increase during loading.

2.8. Number of binding sites

Measurement of the number of binding sites was performedaccording to Yamamoto and Ishihara [43]. A Tricorn 5/100 (GEHealthcare) packed with the respective resin or a CIM Disk housing(BIA Separations) loaded with 4 EDA CIM Disks (BIA Separations)were connected to an Äkta Explorer 100. The flow rate throughoutthe experiment was 1 ml/min. The column was first equilibratedwith 2.5 CV of AEC loading buffer followed by loading 1 mg of mAb84 (1 mg/ml). After washing of the resin with AEC loading buffer(2 CVs), the bound mAb 84 was eluted using a linear gradient of AECelution buffer with slopes of different steepness (30–90 CVs). Theelution of mAb 84 was observed by measurement of the absorptionat 280 nm. Then, correlating the normalized gradient slope, �, to theNaCl concentration of the peak maximum of the elution peaks, thenumber of binding sites (z) can be calculated according to

CR(z+1)

M

� =A(z + 1)

(3)

with

� = ˇ(Vt − Vv) (4)

7 matog

ˇCA

A

wi

2

2

saNIH3mK7(i1tiI(attaStaOsuwco(

2

clpMaasc

2

UNTtI

2

Bt

854 A. Tscheliessnig et al. / J. Chro

is the gradient slope and v the interstitial mobile phase velocity.RM is the modifier concentration at the peak maxima and parameteris defined as

= Ke�B (5)

ith Ke as the equilibrium association constant and � as the totalon-exchange capacity.

.9. Analytical methods

.9.1. Enzyme-linked immunosorbent assay (ELISA)The IgM content was quantified using a high-sensitivity

andwich enzyme-linked immunosorbent assay (ELISA). First,microtiter plate (Immuno 96 MicroWell Plates, MaxiSorb,

UNC, Roskilde, Denmark) was coated with rabbit anti-mousegM (�-chain specific) antibody (0.005 mg/ml, Open Biosystems,untsville, USA) in 0.05 M sodium carbonate, pH 9.6 for 2 h at7 ◦C. The plates were then blocked with 3% bovine serum albu-in (BSA, Sigma–Aldrich) in PBS/Tween (137.0 mM NaCl, 2.7 mM

Cl, 10.0 mM Na2HPO4, 1.8 mM KH2PO4, 0.1% Tween 20, pH.4) for 1 h at 37 ◦C. For each plate, a duplicate standard curve0.500–0.003 �g/ml) was prepared by serial dilution (1:2) using ann-house standard (protein A—purified mAb 84, 0.5 �g/ml in PBS) in% BSA in PBS/Tween. The samples were diluted likewise and afterransferring samples and standards to the microtiter plate, it wasncubated for 1 h at 37 ◦C. After incubation with a goat anti-mousegM (�-chain specific) antibody horseradish peroxidase conjugateHRP) (1:5000, Sigma–Aldrich) in 1% BSA in PBS/Tween for 1 ht 37 ◦C, O-phenylenediamine dihydrochloride (Sigma FastTM OPDablets, Sigma–Aldrich) was added for staining. The enzymatic reac-ion was stopped by adding 3 M HCl to each well. The absorbancet 492 nm was read using a Tecan Sunrise plate reader (Tecan,alzburg, Austria) using a reference wavelength of 620 nm. Usinghe decimal logarithm of the IgM concentration of the standardsnd their measured absorbance a linear regression was performed.nly data points which gave a fit with an R2 larger 0.99 were con-

idered. The IgM concentration of the samples was determinedsing the obtained calibration curve. For each sample duplicatesere performed where a minimum of 3 dilutions had to have a

oncentration deviating less than ±10% from the average. A limitf quantitation (LOQ) was 0.019 �g/ml and the limit of detectionLOD) was 0.009 �g/ml.

.9.2. Analytical size-exclusion chromatographyA G4000SWxl size exclusion column (Tosoh, Tokyo, Japan) was

onnected to the HPLC system (Shimadzu, Kyoto, Japan) and equi-ibrated with SEC running buffer (0.2 M sodium phosphate, 0.1 Motassium sulfate, pH 6.0) at 0.6 ml/min. 100 �l of filtered (0.2 �m,illipore, Carrigtwohill, Co. Cork, Ireland) sample were injected

nd the protein detected by measuring the absorbance at 280 nmt the column outlet. mAb 84 present in supernatant and purifiedamples could be quantified in the range of 20–627 �g/ml using aalibration curve of protein A-purified mAb 84.

.9.3. Total protein quantificationAbsorbance was measured at 280 nm using either the

V–visible photometer UV-1601 (Shimadzu, Kyoto, Japan) or theanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA).he concentration of total protein was calculated using an extinc-ion coefficient of 1.2 (AU ml/cm mg), which has been suggested forgM [30].

.9.4. Reduced SDS-PAGE electrophoresis and immunoblottingReduced SDS-PAGE was performed using 4–12% NuPAGE

is–Tris gels (Invitrogen) according to the manufacturer’s instruc-ions. The samples were diluted with NuPAGE LDS Sample Buffer

r. A 1216 (2009) 7851–7864

(Invitrogen), NuPAGE Reducing Agent (Invitrogen) and RO-waterto give a maximum load 0.25–0.30 �g IgM for Coomassie stain-ing and 0.83–0.99 �g IgM for Western blot. After incubation at95 ◦C for 10 min, the samples were loaded onto precast 4–12%NuPAGE Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) and separatedfor 35 min using NuPAGE MES buffer (Invitrogen) and a Novex XCellSureLock Mini-Cell PAGE system (Invitrogen) with the voltage setto 200 V.

For Coomassie staining, the gels were incubated in ∼100 mlCoomassie Blue staining solution (0.1% (w/v) Coomassie brilliantblue R-250, 30% (v/v) absolute ethanol, 10% (v/v) glacial aceticacid) for approximately 30 min. The gel was then incubated in thedestaining solution (10% (v/v) absolute ethanol and 10% (v/v) glacialacetic acid) until a clear background was observed.

For immunoblotting, the proteins were transferred from the gelonto a 0.2-�m polyvinylidene difluoride (PVDF) membrane (Mil-lipore) using a Mini Trans-Blot tank blotter (80 mA, 1 h, Bio-Rad)containing NuPAGE transfer buffer (Invitrogen) with 20% methanol(BDH, Poole, UK). After transfer, the membrane was blocked with 3%non-fat milk powder in washing buffer (0.1% Tween 20-PBS) for atleast 1 h at room temperature. IgM was detected using a goat anti-mouse IgM alkaline phosphatase conjugated secondary antibody(1:5000, Calbiochem, La Jolla, CA, USA) in 1% non-fat milk pow-der in washing buffer. After 2 washing steps with washing bufferfor 10 min each, the IgM was visualized using NBT/BCIP substrate(Bio-Rad) as directed by the manufacturer’s instructions.

2.9.5. DNA quantificationDNA was quantified using the Quant-iT PicoGreen dsDNA kit

(Invitrogen) in microplate format. All buffers and reagents wereprepared according to the manufacturer’s instructions. Using a96-well microplate the samples and standards (diluted to either200 or 1000 �g/ml Lambda DNA standard) were 1:2 serial dilutedwith TE-buffer. 100 �l of sample were transferred into a black96-well Polystyrene Microplate (Greiner-Bio, Frickenhausen, Ger-many) 100 �l of the Quant-iT PicoGreen dsDNA reagent added andthe solutions gently mixed. The fluorescence intensity was mea-sured using the Tecan Infinite M200 (excitation: 480 nm with 9 nmbandwidth, emission: 520 nm with 20 nm bandwidth, automaticgain, 10 reads with 20 �s integration time). A linear calibrationcurve was fitted to the standard dilutions and used for calculationof DNA concentration of the standard.

2.9.6. Isoelectric focusingAll samples were first buffer-exchanged into 0.05 M NH4HCO3

using PD-10 Desalting Columns (GE Healthcare) according to themanufacturers instructions and then concentrated using Vivaspin500 (MWCO 100 kDa, Sartorius Stedim Biotech) to a concentra-tion of approximately 5 mg/ml. IsoGel® Agarose IEF Plates, pH 3–10(Lonza, Rockland, USA) were set up in the Multiphor (GE Health-care) according to the manufacturer’s instructions. 10 �g of sample(2 �l of 5 mg/ml), 1 �l of the Broad Range marker (pI 4.45–9.60, Bio-Rad) and 5 �l of the High Range marker (pI 5.0–10.5, GE Healthcare)were loaded and the gel run for 60–90 min with the initial settingsof 25 W, 1000 V. The gel was fixed and Coomassie stained accordingto the manufacturer’s instructions.

2.9.7. Flow cytometryFor all clones, supernatants and purified IgM were brought

to a concentration of 25 �g/ml, either by concentrating using aVivaspin 500 (Sartorius Stedim Biotech) with a MWCO of 10 kDa

or by dilution with PBS for supernatant or 1% BSA/PBS for puri-fied IgM. The assay was performed as described elsewhere [41]. Inshort, 2 × 105 HES-3 cells, harvested as single cell suspension usingtrypsin, were resuspended in 10 �l of 1% BSA/PBS and 200 �l ofthe purified IgM or supernatant added. After incubation for 30 min

A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864 7855

mpar

ofrDp(su

2

fiPfhiartpu

3

mh5Ifp(IatwSci

3

Ims

ment the pH adjustment in the purification strategy even thoughthe results were favorable as there is product loss (pH 6.0). In addi-tion an additional clearance step is required to remove impuritiesprecipitated by the pH shift.

Fig. 1. IEF gel for the panel of clones. The pI of each clone can be estimated from co

n ice, the cells were washed with cold 1% BSA/PBS and incubatedor another 15 min (4 ◦C) with a goat anti-mouse antibody fluo-escein isothiocyanate conjugate (FITC) (1:500, DAKO, Glostrup,enmark). After washing with 1% BSA/PBS, the cells were resus-ended in 200 �l 1% BSA/PBS and 1.25 mg/ml propidium iodidepI, Sigma–Aldrich) and analyzed using FACScan (Becton, Dickin-on and Company, Franklin Lakes, USA). An anti SSEA-1 (IgM) wassed as negative control.

.9.8. Cytotoxicity assay for mAb 84 and mAb 85Hybridoma 84 and hybridoma 85 supernatant as well as puri-

ed mAb 84 and mAb 85 were brought to 25 �g/ml by dilution withBS (supernatant) or 1% BSA/PBS (purified IgM). The assay was per-ormed as described elsewhere [41]. In short, 2 × 105 HES-3 cells,arvested as single cell suspension using trypsin, were resuspended

n 10 �l of 1% BSA/PBS and 150 �l of the supernatant or purified IgMdded. After incubation for 45 min on ice the cells were washed andesuspended in 1% BSA/PBS and 1.25 mg/ml pI using FACScan (Bec-on, Dickinson and Company). To validate the results obtained usingI exclusion assays, viability for each sample was also determinedsing tryphan blue exclusion.

. Results and discussion

Our aim was to develop a purification strategy for a panel ofonoclonal IgM antibodies from hybridoma. The IgM in the panel

ave different isoelectric points (pI), ranging from approximately.5 for the most acidic IgM (mAb 5) to around 7.7 for the more basic

gM (mAb 14) (Fig. 1). The majority of the IgM have a range of dif-erent isoforms with isoelectric points spreading over around oneH unit, only mAb 5 and mAb 14 have a narrow isoform spectrumFig. 1). Recently we have developed a purification strategy for angM from this panel using anion-exchange chromatography (AEC)nd size-exclusion chromatography (SEC) [7]. While we were ableo obtain a high purity, the concentration factor of this purificationas low; therefore we decided to modify this strategy: the final

EC step was replaced by an initial enrichment step using PEG pre-ipitation and for the AEC step, a panel of resins were screened todentify one with high binding capacity.

.1. PEG precipitation

We used PEG 6000 which is a common precipitant for IgG andgM [3,12]. First we determined the PEG concentration required for

aximum precipitation by mixing equal volumes of hybridoma 84upernatant and PEG 6000 to obtain a final concentration of 17.5%

ison to the Broad Range marker (BR marker) and High Range marker (HR marker).

in steps of 2.5% (Fig. 2). The highest yield was measured for 10%PEG 6000. At higher PEG 6000 concentrations, the yield decreased.This is maybe due to the increased viscosity of the solution thatprevents smaller precipitates to settle during centrifugation. Theformation of small precipitates might have been due to the lack ofmixing during precipitation. Thus in later experiments all sampleswere mixed, if not mentioned otherwise.

Adjustment of the pH closer to the isoelectric point has beensuggested to improve the precipitation yield [37]. For mAb 84 (pI6.5–7.0) adjusting the pH to pH 5.0 and pH 6.0 resulted in a higheryield at lower PEG concentrations (Fig. 3). Only as little as 7.5% PEGwere sufficient for high yield of mAb 84 for both either pH 6.0 or pH5.0. From the pI it would be expected that mAb 84 has a positive netcharge at pH 5.0 and pH 6.0 as a negative net charge at pH 7.4. There-fore, one would expect a similar precipitation yield at a positive andnegative charge. However, from the results, we could assume thatthe surface charge distribution between the positively and neg-atively charged IgM is sufficiently different to give this differentprecipitation behavior. Either there is more attraction between themAb 84 molecules at pH 5.0 and 6.0 or less repulsion leading toprecipitation at lower PEG 6000 concentrations. We did not imple-

Fig. 2. Precipitation of mAb 84 from serum-free hybridoma supernatant using dif-ferent final concentrations of PEG 6000.

7856 A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864

Fsb

tkpfitastksaetttfdpwsbm

Fciq

ig. 3. Comparison of the precipitation yield of mAb 84 from serum-free hybridomaupernatant which has been adjusted to pH 5.0 (black), pH 6.0 (light grey) or haseen used without prior adjustment, pH 7.5 (dark grey).

Considering our reduced yield observed for unmixed precipi-ated samples, we studied the influence of mixing on precipitationinetics at a larger scale. We incubated sets of precipitated sam-les with and without mixing. As can be seen from Fig. 4, for therst 15 min there is little influence from mixing on the precipi-ation kinetics. This is likely so because the precipitate particlesre still small enough to be unaffected by mixing. For particlesmaller than 1 �m diameter, it has been suggested that precipi-ation is mainly governed by diffusion, meaning the precipitationinetics are influenced by the viscosity of the solution, particleize and diffusivity. This is usually referred to as the perikineticggregation [44]. When particles become larger than 1 �m in diam-ter, fluid motion will cause particles to collide and aggregate. Athis stage, mixing becomes pivotal to obtaining a high recovery;his is referred to as orthokinetic precipitation [44]. We observedhat for mAb 84, precipitation particles around 1 �m diameter areormed after 15–30 min [45] which is in good agreement with theata presented here: between 15 and 60 min, the yield of the sus-ension which was mixed while incubation increases significantly

hereas the suspension which was not mixed during incubation

howed only little increase in yield over time. This can most likelye explained by the formation of larger precipitate particles uponixing which results in a higher yield during centrifugation.

ig. 4. Precipitation kinetics for mAb 84 from hybridoma supernatant at a finaloncentration of 10% (w/v) PEG 6000. The suspension was incubated with mix-ng (inverse triangle) and without mixing (full circle). Samples were drawn foruantification at specified time steps.

Fig. 5. AEC chromatogram for linear gradient elution of mAb 84. The fractions com-prising mAb 84 and the impurities are indicated.

We selected the following conditions for the PEG precipita-tion as the initial enrichment step for all hybridoma supernatants:continuous mixing of the hybridoma supernatant at a final PEG con-centration of 10% (w/v) with a magnetic stirrer for a minimum of2 h at 4 ◦C.

3.2. Anion-exchange chromatography (AEC)

The AEC step was developed based on a previous purificationstrategy for mAb 84 [7]. From measurement of the breakthroughcurves of the dissolved PEG precipitate for different AEC resins (datanot shown), we selected Fractogel EMD DEAE (M) instead of thepreviously used EDA disk (monolith) due to the higher capacityobserved.

First, we performed a linear gradient elution (Fig. 5) to obtain theconditions needed for the step gradient elution. mAb 84 eluted inthe first peak was already at a high purity (data not shown) becausethe impurities were mostly found in a later eluting peak. For thefirst batch of hybridoma 84 supernatant, the amount of impuritieswas low, as can be seen from Fig. 5. For the following batches ofmAb 84 as well as all other clones the amount of impurities wassignificantly higher than the amount of mAb 84 present; it is notclear why the level of impurities was so much lower for the firstfed-batch run. For the step elution of mAb 84, 24% elution buffercorresponding to approximately 280 mM NaCl (Fig. 6A) was used.Only minor amounts of mAb 84 were found in the flowthrough andregenerate (data not shown) and as can be seen from the analyticalSEC (Fig. 6B). mAb 84 of high purity can be obtained using thistwo-step purification strategy.

The same process, linear gradient followed by step gradientelution, was performed for the other IgMs. For some IgMs, the con-centration of the elution buffer in the AEC step had to be adjusted(Table 1). Table 1 gives the mass balance showing the concentra-tion of IgM and total protein in the supernatant as well as the IgMconcentration, purity and yield for the PEG precipitation and AECstep elution.

For mAb 95, mAb 375 and mAb 432, the low yield in the PEGprecipitation can be explained by the solubility of the IgM at 10%PEG 6000. Assuming a similar solubility for all IgMs, we can usethe solubility of mAb 84 (1–2 �g/ml) for some simple calculations:When mixing an equi-volume of supernatant with a 20% PEG, themAb concentration is diluted 1:2, e.g. for mAb 95, that would bring

it down from ∼6.9 �g/ml to ∼3.45 �g/ml. Assuming an average sol-ubility of ∼1.5 �g/ml, a theoretical yield of only ∼50% is possible formAb 95. This is in agreement with the data observed. The same istrue for mAb 375 and mAb 432. For these IgMs, PEG precipitationwould be more efficient if an initial concentration step is introduced

A. Tscheliessnig et al. / J. Chromatog

Fig. 6. (A) AEC chromatogram of purification of mAb 84 with step gradient. Thefractions comprising mAb 84 and the impurities are indicated. (B) The analyticalS(op

oaiftmsos

t

TTT

EC chromatogram of the hybridoma 84 supernatant (solid line), PEG precipitateblack dashed line) and AEC eluate (grey dashed-dotted line) shows that the majorityf impurities can be removed during PEG precipitation while AEC leads to furtherurification and concentration of mAb 84.

r if the PEG is at a higher initial concentration, e.g. 50%. For mAb 5nd mAb 14, which also have yields <80%, the initial concentrations sufficiently high so we would assume that their significantly dif-erent isoelectric points as compared to the other IgM resulted inhe lower precipitation efficiency. At pH 7.5, both IgMs (mAb 5 and

Ab 14) would be strongly charged hence intra-molecule repul-

ion results in hindered precipitation. For such cases, adjustmentf the pH closer to its isoelectric point might increase the yieldignificantly.

The recovery for the AEC step is around 79–93% with the excep-ion of mAb 375 and mAb 529 which have a recovery of 55–61%.

able 1he mass balance is given for the small-scale purification including hybridoma supernatahe recovery is always given as step recovery/overall recovery. For AEC, the NaCl concentra

mAb 5 mAb 14 mAb 63 mAb 84

SNVolume [ml] 190 200 200 100IgM [�g/ml] 57.7 60.4 50.7 93.0Total protein [mg/ml] 2.90 1.80 4.35 0.62

PEGIgM [�g/ml] 205.2 213.0 91.0 229.2Purity [%] 30.0 63.1 35.0 35.0Recovery [%] 71.1/71.1 79.3/79.3 89.7/89.7 91.4/91.4

AECNaCl for elution [mM] 288 288 278 278IgM [�g/ml] 1122.4 1563.5 2605.0 1233.1Purity [%] 85.0 >99.0 93.0 >99.0Recovery [%] 79.0/56.2 82.0/65.0 79.3/71.1 87.0/79.5

r. A 1216 (2009) 7851–7864 7857

This is due to the strong tailing of the elution peak for mAb 375 andmAb 529 which was not collected to avoid excessive dilution of thepurified mAb. We feel that such losses will be less pronounced ata larger scale because due of the observed higher concentrationsof IgM in the eluate a less conservative peak collection could beapplied. From Table 1 we can also see that most mAbs have a puritygreater than 90%, with the exception of mAb 5 and mAb 375 whichhave a purity of 85.0% and 46.6%, respectively. The analytical SECfor mAb 5 does not show significant presence of other proteins, adiscrete symmetrical peak at the elution time of an IgM is observed(data not shown). It is possible that the presence of DNA affectedthe final purity, as DNA will contribute to UV absorbance at 280 nm.

For mAb 375, the analytical SEC showed the presence of otherproteins besides mAb 375. An additional purification step will beneeded to obtain the required purity. We found that hydroxyap-atite gave good results in this case: mAb 375 is first diluted to aphosphate concentration of 10 mM and then eluted with 300 mMphosphate in the presence of 10% (w/v) PEG 600 (data not shown).This method was suggested by Gagnon for the removal of antibodyfragments and aggregates [29]. The difference in the purificationefficiency between mAb 375 and other antibodies with a similar pI,e.g. mAb 529 or mAb 95, is surprising but could be explained bydifferences in the surface charge distribution. Comparing the con-centration of NaCl needed for elution, it can be seen that mAb 375actually requires the highest NaCl concentration (∼300 mM NaCl)for elution, suggesting that the binding of mAb 375 to the AEC resinis stronger than for the other mAbs. As we noticed, the impuritiesusually have a stronger binding to the AEC resin, we assumed thatthey would be co-eluting with mAb 375 at the selected NaCl con-centration. Besides adding an additional step, the purification couldbe improved by changes of pH or by switching to different resinchemistries, e.g. cation-exchange chromatography.

For all IgMs, the supernatant and final purified IgM afterAEC were loaded onto SDS-PAGE (4–12%) and visualized usingCoomassie staining (Fig. 7) or immunoblotting (Fig. 8). The pro-tein transfer during immunoblot was not efficient, in particularfor small-molecular weight proteins, which can be seen from themissing light chain (Fig. 8). Additional bands below 50 kDa can beobserved for some of the AEC purified IgM in the Coomassie stain.These are most likely fragments visualized in the immunoblot. FormAb 375, the bands below 50 kDa on the Coomassie stain have nocorresponding bands in the immunoblot. This corresponds to theobservations of the mass balance and analytical SEC that suggested

that impurities are present in the AEC eluate.

The antibody activity was evaluated for all antibodies exceptmAb 84 by comparing the binding to hESC before and after purifica-tion using flow cytometry. As can be seen from Fig. 9, no significantdifference was found between the mAbs present in the supernatant

nts (SN) using PEG precipitation (PEG) and anion-exchange chromatography (AEC).tion needed for elution is given for each mAb.

mAb 85 mAb 95 mAb 375 mAb 432 mAb 529

100 155 200 200 200139.5 6.9 13.8 23.0 78.95.55 1.78 3.23 2.84 2.26

356.4 23.4 28 55.3 282.752.8 17.6 13.5 36.4 57.589.9/89.9 50.5/50.5 73.2/73.2 78.8/78.8 85.3/85.3

278 278 307 297 2781080.3 62.4 955.6 1008.1 2531.3>99.0 >99.0 46.6 95.5 >99.093.1/83.7 55.7/28.1 61.3/44.9 84.2/66.3 67.1/57.3

7858 A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864

) and A

awItmnssf(

3

hwlpotDas

Fig. 7. Coomassie stain of SDS-PAGE (4–12%) of supernatant (SN

nd corresponding purified mAb solutions. As mAb 84 kills hESCithin 30 min, no signal can be obtained by flow cytometry [41].

nstead we compared the killing efficiency of the purified mAb 84o that of the supernatant. mAb 85 binds to the same epitope as

Ab 84 but has no effect on cell viability and is therefore used as aegative control (isotype control). We found that our purificationtrategy did not affect the activity of either mAb: mAb 84 had theame cytotoxic effect when used in supernatant or in its purifiedorm while neither fraction of mAb 85 exhibited cytotoxic activitydata not shown).

.3. Benzonase treatment of hybridoma supernatant

A challenge of this purification process was the presence ofigh concentrations of DNA in the hybridoma supernatant. Thisas particularly obvious in an mAb 84 purification performed at

arger scale where DNA precipitation was observed during PEGrecipitation (data not shown). During this step, a large amount

f mAb 84 (30%) was lost, which we assume was due to adsorp-ion to the precipitated DNA. Additionally we found that part of theNA forms a soluble precipitate that is co-purified with proteinsnd contaminates the dissolved precipitate. In the following AECtep, the DNA binds to the resin, resulting in a lower binding capac-

Fig. 8. Immunoblot of SDS-PAGE (4–12%) of supernatant (SN) and AE

EC eluate (AEC) of all clones (M1, See Blue Plus2; M2, Mark 12).

ity as well as a lower purity of the eluate. We therefore proposedtreating of the hybridoma supernatant with DNAase prior to furtherpurification.

Benzonase is a genetically engineered endonuclease originatingfrom Sewatia murcescens that can hydrolyze single- or double-stranded, linear, circular or supercoiled DNA as well as RNAresulting in the formation of oligonucleotides of 3–5 bases length[46]. The enzyme is active between pH 6.0–10.0 and from 0 ◦C to40 ◦C but its activity can be affected by the presence of phosphate(>100 mM) or monovalent cations (>150 mM). Low concentrationsof Mg2+ is crucial for its activity. The enzyme can be inactivatedby addition of EDTA (>5 mM). It is necessary to test the optimumcleavage conditions, time and temperature, for the respective solu-tion.

Preliminary studies showed that addition of 50 U benzonaseper ml of supernatant reduced the DNA level from ∼15 �g/ml to∼2 �g/ml within 1 h when incubated at room temperature (data notshown). At 4 ◦C the final level was only reduced to ∼7 �g/ml. Lower

concentrations (10 U/ml) were not as efficient whereas higher con-centrations (100 U/ml) showed no significant improvement (datanot shown). The conditions for this step were therefore set to 50 Ubenzonase per ml of hybridoma supernatant and incubation for 1 hat room temperature with stirring.

C eluate (AEC) of all clones (M1, See Blue Plus2; M2, Mark 12).

A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864 7859

F IgM oc tly st

8pwwtTdan

TCa(r

ig. 9. Flow cytometry assay for evaluating the binding efficiency of the differentontrol). The signal for the purified mAb (solid line) is in all cases similar if not sligh

We compared the purification of antibodies from hybridoma4 supernatant which has been treated with benzonase to theurification of one without benzonase (Table 2). During incubationith benzonase, the supernatant turned turbid; this precipitateas not removed and we assume that it formed the majority of

he non-dissolvable precipitate observed after PEG precipitation.he dissolved PEG precipitate was filtered prior to AEC. However,ue to the large amount of non-dissolvable precipitates presentfter benzonase treatment, a filter with a larger surface area waseeded. The loss of 30 ml of dissolved PEG precipitate obtained from

able 2omparison of the mass balance of a mAb 84 purification from benzonase-treatednd untreated hybridoma supernatant (SN). The recovery during PEG precipitationPEG) and anion-exchange chromatography (AEC) is given as step recovery/overallecovery.

mAb 84

No benzonase With benzonase

SNVolume [ml] 400IgM [�g/ml] 62.6Total protein [mg/ml] 5.14DNA [ng/ml] 20980.5

PEGVolume [ml] 200 175IgM [�g/ml] 121.2 124.8Total protein [mg/ml] 0.71 0.28DNA [ng/ml] 18222.6 1924.9

Purity [%] 17.1 45.1Recovery [%] 96.4/96.4 88.2/88.2

AECVolume [ml] 7.00 8.00IgM [�g/ml] 1192.0 1982.0Total protein [mg/ml] 1.8 2.0DNA [ng/ml] 15796.9 312.9Purity [%] 67.3 99.1Recovery [%] 40.0/38.5 70.5/62.2

Column capacity [mg/ml] 1.1 1.7

n hESC. The shaded peak gives the auto-fluorescence signal of the hESC (negativeronger than for mAb present in the supernatant (dotted line).

the benzonase-treated supernatant can be ascribed to the use ofa larger filter area (Table 2). Comparison of the filtered dissolvedPEG precipitate with and without benzonase treatment (Fig. 10A)using analytical size-exclusion chromatography showed a signif-icant difference: after benzonase treatment, all high-molecularweight impurities were removed. This suggests that these impu-rities are actually proteins bound to co-precipitating DNA resultingin apparent high-molecular weight proteins.

For the AEC, the same volume of dissolved PEG precipitate wasloaded, in both cases the column was overloaded and mAb 84 wasfound in the flowthrough resulting in a low step recovery (Table 2).Fig. 10B shows the analytical size-exclusion chromatography ofboth AEC steps. Though the AEC eluate from the benzonase-treatedsupernatant showed presence of fragments and/or small-molecularweight impurities, it is most likely that these are also present inthe AEC eluate in the untreated supernatant although they are lessobvious because of the lower signal. Due to the adsorption behaviorof IgM and the impurities present in the dissolved precipitate, over-loading of the column leads to the replacement of IgM by impuritiesresulting in a lower purity of the eluate. This is the main reasonfor the lower purity of the purification without benzonase treat-ment (67.3%). Due to the lower amount of impurities present inthe dissolved PEG precipitate from the benzonase-treated super-natant, the column is not being overloaded and the purity of theAEC eluate is significantly improved (99.1%). The AEC eluate fromthe purification including the benzonase treatment has also a 50×reduction in DNA content and a higher final concentration. The lat-ter is due to the higher binding capacity of the column (1.5-fold).From these results, we decide to include benzonase treatment toour purification process for all mAbs.

3.4. Adsorption isotherms and kinetics

A purification process can also be improved by selecting achromatography resin with high capacity and mass transfer [39].Initially we selected the Fractogel EMD DEAE (M) resin, due to ourexperience with the purification of unfolded proteins [40]. The Frac-

7860 A. Tscheliessnig et al. / J. Chromatog

Frb

ttri[atv

sqsalE

Fac

ig. 10. Comparison of the chromatograms of analytical size-exclusion chromatog-aphy of (A) the PEG precipitates and (B) the AEC purified mAb 84 obtained fromenzonase treated (dotted line) or untreated (solid line) hybridoma 84 supernatant.

ogel is a grafted resin where ∼10 nm long polymer chains carryinghe ligands have been grafted on the inner and outer surfaces of theesin’s particles [47]. The ligands are therefore more flexible result-ng in the higher binding capacity, in particular for larger proteins47]. We also selected Capto Q, a strong anion-exchange resin, withhigh-flow agarose matrix as backbone with dextrans grafted onto

he particle’s surface. This resin has been recommended for higholume process scale as well as for the purification of viruses.

For the purification of large proteins, convection driven resinsuch as membranes and monoliths have also been suggested fre-

uently [26,47,48]. We found that membranes have, at least at smallcale, large extra-column effects, which we felt disadvantageouss it leads to diluted eluate peaks. Therefore, we selected a mono-ith for representing convection driven chromatography resins. TheDA CIM MiniDisk is a weak anion-exchange resin with a ligand that

ig. 11. Adsorption isotherms for purified mAb 84 on different chromatographic resins: (nd (C) Fractogel EMD DEAE (M) and Fractogel EMD DEAE (S). The obtained data poinonfidence interval (dashed lines) are given.

r. A 1216 (2009) 7851–7864

has a higher selectivity for IgM than for IgG or BSA [27]. We haveused this resin successfully in our previous purification of mAb 84[7].

Finally, we included Toyopearl, a resin with the same backboneas Fractogel EMD while the surface of Fractogel is covered withpolymer chains carrying the ligands, called tentacles the ligands forToyopearl are directly immobilized onto the surface of the resin. ForFractogel and Toyopearl, different particle diameters were tested,the medium size (60–90 mm) and the small size (20–40 mm). Thiswas to evaluate if the larger outer surface of the smaller particleswould lead to an increased adsorption capacity.

First, we evaluated the adsorption equilibrium for purified mAb84, see Fig. 11 and Table 3. We selected an incubation time of 6 hbefore evaluating the concentration of bound and unbound IgM. Asshown later for the adsorption kinetics for purified mAb 84 (Fig. 13)6 h was sufficient to reach equilibration for most resins.

We found that Fractogel EMD DEAE (M) and (S) had the highestcapacity (60–66 mg/ml) and steepest isotherm (KA > 68 ml/mg), fol-lowed by Capto Q with a capacity of ∼55 mg/ml and a KA ∼56 ml/mg.Toyopearl resins had the lowest capacity with only 15–16 mg/ml.The observed difference in the KA value of the Toyopearl DEAE-650 M and DEAE-650S is unclear. However, it might be due tonon-equilibrium conditions for the Toyopearl DEAE-650 M. Thiswould be in agreement with data on the adsorption kinetics shownlater. The results for the porous resins are not very surprising due tothe size of IgM as accessibility to the binding sites is hindered result-ing in a smaller capacity compared to smaller proteins, e.g. BSA(∼100 mg/ml, Fractogel EMD DEAE (M)). The difference betweenthe grafted resins, Fractogel EMD and Capto Q, and the non-graftedresin, Toyopearl can be traced back to the specific properties ofgrafted resins [47,49]. Also notable is that there is no differenceif smaller or larger particles are used, the maximum capacity forFractogel EMD DEAE (M) and (S) or Toyopearl DEAE-650 M or DEAE-650S are almost the same. This suggests that IgM not only adsorbson the outer surface of the particle, but rather diffuses into theporous network of the particles. The EDA CIM disk had a capac-ity (24 mg/ml) higher than Toyopearl but lower than Fractogeland Capto Q resins. The EDA CIM disk has a surprisingly low KA(3 ml/mg) despite the high affinity for IgM reported by Brne et al.[27]. However, in this work, a polyclonal IgM was used and theshallow isotherm slope might be specific for our mAb 84. Fromlater kinetic studies, we found out that the selected 6 h of incuba-tion time might be insufficient for porous resins, in particular forToyopearl DEAE-650C (M) and Capto Q; therefore slight changes inthe isotherm could be expected.

We also compared the number of binding sites for all resins(Fig. 12): For Toyopearl resins as well as Capto Q, similar number ofbinding sites (11–13) were found. The differences in the interceptof Toyopearl 650C DEAE (M) and Toyopearl 650C DEAE (S) couldbe due to variations of the effective total ion-exchange capacity, �

A) EDA CIM disk and Capto Q, (B) Toyopearl DEAE-650 M and Toyopearl DEAE-650Sts were fitted to the Langmuir isotherm (Eq. (1)); the fit (solid line) and the 95%

A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864 7861

Table 3Particle diameter, pore diameter, apparent adsorption isotherm parameters and apparent number of binding sites and equilibrium constant A for the different AEC resinsinvestigated.

Particle diameter Pore diameter Apparent adsorption isotherm parameters Number of binding sites

Dp [�m] dp [nm] qm [mg/ml] KA [ml/mg] z [–] A [–]

Fractogel EMD DEAE (M) 40–90 80 66.14 ± 1.31 85.98 ± 32.53 17.1 ± 1.3 6.6 ± 1.1 (×1010)Fractogel EMD DEAE (S) 20–40 80 59.62 ± 1.07 68.45 ± 9.37 18.6 ± 1.7 4.1 ± 9.2 (×1011)

16. 13

15.55.24.

bisttbeleii8I

bcicptppbvntrW

Ma

Ftat

Toyopearl DEAE-650M 40–90 100Toyopearl DEAE-650S 20–50 100Capto Q 90 (d50v) Not givenCIM EDA (monolith) Not applicable 1500 [48]

etween the resins rather than differences in the adsorption behav-or [43]. For Fractogel, both resins gave similar number of bindingites as well as similar intercepts. Interestingly, we found that forhe elution of mAb 84 from the EDA CIM disk, a higher NaCl concen-ration was required than for all the other resins. As the number ofinding sites is within the range of the other resins (∼15) and thequilibration association constant from the isotherm is actually theowest for all resins, we did not expect the need for such a stringentlution condition. Possible reasons could be a high effective totalon-exchange capacity (�) or strong interactions between the sitesnvolved in adsorption of the ligand (ethylene diamine) and mAb4. The latter could explain the higher affinity for the EDA to thegM as compared to other resins.

From our measurements of the adsorption capacity and num-er of binding sites, we found that Fractogel EMD DEAE is a goodhoice for the purification of mAb 84 as well as the other IgMsn our panel (Table 1). The results might be different if there isompetitive adsorption of impurities present in the dissolved PEGrecipitates. Also the issue of mass transfer has to be consideredo find the resin with the optimum dynamic binding capacity. Weerformed uptake kinetics using the method of shallow bed withurified mAb 84 and dissolved PEG precipitate with and withoutenzonase treatment. Although the shallow bed method is con-enient for the measurement of adsorption kinetics, it should beoted that even slight variances in the resin volume (10 �l) can leado significant errors in the final data. The curves should thereforeather be seen as indicative of a trend of the adsorption kinetics.

e performed an empirical fit for better visualization of the data.Fig. 13A gives the uptake curves for mAb 84 on the EDA CIM

iniDisk. The uptake curve of the purified mAb 84 showed thedvantage of a monolith for adsorption of large molecules. This

ig. 12. The ionic strength of the elution peak (CRM

) was plotted against the respec-ive normalized gradient slope (�) and the data points fitted according to Yamamotond Ishihara [43] (solid line). From the slope the number of binding sites (z) and fromhe intercept the binding strength can be evaluated.

43 ± 0.39 19.47 ± 2.78 13.7 ± 1.4 9.9 ± 2.1 (×10 )03 ± 0.54 6.73 ± 1.17 11.4 ± 0.8 6.6 ± 1.3 (×1012)20 ± 3.16 56.59 ± 11.84 13.4 ± 1.0 4.4 ± 2.5 (×1010)34 ± 3.20 3.21 ± 1.04 15.1 ± 1.0 4.9 ± 4.1 (×105)

is apparent at the initial time point (5 min) where the maximumcapacity (∼4 mg/ml) is obtained. Longer incubation times do notaffect the binding capacity thereafter. The slight increase after240 min of incubation could be ascribed to aggregation on thecolumn or multi-layer adsorption. However, looking at the dis-solved PEG precipitate without benzonase treatment, we find anextremely low capacity for mAb 84. The DNA and other impuri-ties are probably adsorbing at a similar rate as the mAb 84 and aremost likely at a higher affinity. For the dissolved PEG precipitateafter benzonase treatment, low incubation times (5 min) result in acapacity similar to the purified mAb 84 but upon longer incubation,capacity for IgM decreases. We found from the analytical SEC thatthis is due to the impurities present in the dissolved PEG precipi-tate which seem to bind with a higher affinity than IgM (data notshown).

A similar displacement effect can be observed for all porousresins; the capacity for mAb 84 present in the dissolved pelletincreases initially but after 2–3 h incubation, it drops to relativelylow values. This suggests that for these resins, loading times for AECshould be kept below 2 h or IgM might be replaced by impuritiesresulting in low recovery. We found that for Capto Q, the pres-ence of impurities and DNA led to a significant decrease in capacity(Fig. 13B). For dissolved PEG precipitate from benzonase-treatedsupernatant, the capacity was still comparable to the monolith(∼5 mg/ml) while for the precipitate from the untreated super-natant, it was below 1 mg/ml.

Comparing the kinetics of Toyopearl DEAE 650 M (Fig. 13C) andToyopearl DEAE 650S (Fig. 13D) we found, that the saturation ofthe bigger particles took longer than for the smaller particles. After6 h the Toyopearl DEAE 650C (M) was still not in equilibrium withthe feed. This was probably due to the bigger diameter of the (M)particles (40–90 �m) as compared to the (S) particles (20–50 �m).Interestingly for both resins, the dissolved PEG precipitate with andwithout benzonase treatment showed an identical uptake behavioras the purified mAb 84 in the initial adsorption phase. After 1–3 h,the amount of mAb 84 adsorbed from either dissolved precipitatesto Toyopearl dropped again to a low level.

Besides the monolith, Fractogel resins seem to have the fastestadsorption kinetics. Approximately 80% of the capacity of FractogelEMD DEAE (M) (Fig. 13E) for purified mAb 84 is reached after about1 h while it takes about 2 h for Capto Q or Toyopearl resins. Thisis surprising as the pore diameter of the Fractogel EMD is smaller(80 nm) than for the Toyopearl resins (100 nm). It is most likely thatthe mass transfer mechanism for the diffusion of the mAb 84 intoFractogel EMD is different from that of a conventional porous resin[47]. For the dissolved pellet, the adsorption kinetics are slightlyslower. Even for Fractogel EMD DEAE it takes about 2–3 h to reachthe maximum capacity for mAb 84. Displacement effects are prob-ably an influencing factor in this case.

Besides the IgM concentration, we also monitored the total pro-tein and DNA content adsorbed from the dissolved precipitates tothe resin (data not shown). For both, DNA as well as total protein,we observed that after 6 h of incubation the equilibrium is not yetreached. This slow adsorption kinetics is probably not a factor of

7862 A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864

F EG prc (A) EDF empi

dslws

TM(p

ig. 13. The adsorption kinetics for purified mAb 85 (solid square), and dissolved Pircle) mAb 84 hybridoma supernatant. For easier visualization the data points forractogel EMD DEAE (M) and (F) Fractogel EMD DEAE (S) were approximated by an

iffusivity but rather due to the replacement of adsorbed proteinsuch as mAb 84 by DNA or stronger binding proteins. With longoading times, it would be expected that mAb 84 as well as other

eaker bound proteins are displaced from the column and onlytrong binding proteins and DNA are retained.

able 4ass balance of purification of 2 l mAb 84. For the PEG precipitation step (PEG) the cond

DP) and for the anion-exchange chromatography step (AEC) also the dissolved PEG precirecipitation the hybridoma supernatant was treated with benzonase for DNA removal. T

Volume [ml] IgM [�g/ml] Total protein [m

PEGSN 2000.0 70.0 4.42DP 780.0 160.0 0.25

AECDP 770.0 160.0 0.25EL 24.0 4075.0 4.26

ecipitate obtained from benzonase-treated (inverse triangle) and untreated (solidA CIM Disk, (B) Capto Q, (C) Toyopearl DEAE-650 M, (D) Toyopearl DEAE-650S, (E)

rical fit.

Again we concluded that the selected resin, Fractogel EMD DEAE(M) is a good choice for the purification of our IgM. Although theadsorption kinetics is slower than with the monolith, it has a highercapacity even at short incubation times. The slow adsorption kineticsuggests that IgM does not only adsorb on the outer surface of the

itions of the hybridoma supernatant (SN) as well as the dissolved PEG precipitatepitate (DP) and final eluate containing the mAb 84 peak (EL) are given. Prior to PEGhe recovery is given as step recovery/overall recovery.

g/ml] DNA [ng/ml] Purity [%] Recovery [%]

20081.8581.8 64.0 89.1/89.1

581.81256.3 95.6 79.4/70.7

A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 7851–7864 7863

F wingt d then nvitro

pdd

ot(str

4

mPptcgtah

N

Aˇcc�CKK�q

qVV

[

[

[[[

[[

[

[[

ig. 14. (A) Coomassie stain and (B) immunoblot for 2 L purification of mAb 84 shohe dissolved PEG precipitate (DP), the AEC flowthrough (FT), the AEC eluate (EL) anegative control (neg. ctrl.) is cell culture media and the marker is See Blue Plus2 (I

article but rather diffuses into the pores where it adsorbs. Theiffusion is probably hindered due to bound IgM (20 nm), whichecreases the free pore diameter

We up-scaled our purification strategy to process a total of 2 lf hybridoma 84 supernatant (Table 4). A highly pure and concen-rated mAb 84 could be obtained with our strategy outlined aboveFig. 14). The DNA removal was not as efficient as for the small-cale purifications. In particular, the AEC step concentrated not onlyhe mAb 84 but also the DNA present. If a higher DNA clearance isequired, an additional purification step could be implemented.

. Conclusion

We developed a two-step purification strategy for a panel ofonoclonal IgM from hybridoma with a pI of 5.5–7.7 comprising of

EG precipitation and anion-exchange chromatography (AEC). Therocess was characterized and optimized with regards to precipi-ation kinetics, resin selection and loading time. While the wholeharacterization of the purification was quite time-consuming, itave us valuable information on the presence and effect of impuri-ies, e.g. the competitive adsorption of impurities on the AEC resinst longer loading times. This information can be used to obtain aighly efficient and rugged purification process.

omenclature

=Ke�z

gradient slope (M/ml)concentration of antibody in suspension (mg/ml)

0 concentration of antibody in stock solution (mg/ml)normalized gradient slope (=ˇL/v) (M)

RM ionic strength at peak maximum (M)A apparent adsorption equilibrium parameter (ml/mg)e equilibrium association constant

total ion exchange capacity (mequiv./ml)

concentration of antibody bound to chromatographyresin (mg/ml)

max apparent maximum adsorption capacity (mg/ml)volume of suspension (ml)

0 volume of antibody stock solution (ml)

[[[[[[

the hybridoma 84 supernatant (SN), the supernatant of the PEG precipitate (SN2),AEC regenerate (RE). The positive control (pos. ctrl.) is affinity-purified mAb 84, thegen).

Vg volume of settled chromatography resin in slurry (ml)Vt total column volume (ml)Vv column void volume (ml)z number of binding sites

Acknowledgements

This work was supported by the Biomedical Research Council ofA*STAR (Agency for Science, Technology and Research), Singapore.The authors would also like to thank Yu Hosokai and Dr. Lee YihYean for their help with the cell culture.

References

[1] F.-M. Chen, G.S. Naeve, A.L. Epstein, J. Chromatogr. 444 (1988) 153.[2] P. Clezardin, G. Bougro, J.L. McGregor, J. Chromatogr. 354 (1986) 425.[3] G. Coppola, J. Underwood, G. Cartwright, T.W. Hearn, J. Chromatogr. 476 (1989)

269.[4] A.J. Henniker, K.F. Bradstock, Biomed. Chromatogr. 7 (1993) 121.[5] H.H. Hwang, M.C. Healey, A.V. Johnston, J. Chromatogr. 430 (1988) 329.[6] V.P. Knutson, R.A. Bruck, R.M. Moreno, J. Immunol. Methods 136 (1991) 151.[7] J. Lee, A. Tscheliessnig, A. Chen, Y.Y. Lee, G. Adduci, A. Choo, A. Jungbauer, J.

Chromatogr. A 1216 (2009) 2683.[8] P. Novales-Li, Biomed. Chromatogr. 9 (1995) 42.[9] J.R. Pattison, J.E. Mace, J. Clin. Pathol. (Lond.) 28 (1975) 670.10] D. Roggenbruck, U. Marx, S.T. Kiessig, G. Schoenherr, S. Jahn, T. Porstmann, J.

Immunol. Methods 167 (1994) 207.11] Y. Shimada, T. Goto, S. Kawamoto, T. Kiso, A. Katayama, Y. Yamanaka, T. Aki,

K.-C. Chiang, T. Nakano, S. Goto, C.-L. Chen, N. Ohmori, K. Ono, S. Sato, Biomed.Chromatogr. 22 (2008) 13.

12] A.W. Cripps, S.H. Neoh, I.J. Smart, J. Immunol. Methods 57 (1983) 197.13] G.K. Ehrlich, H. Michel, H.P. Chokshi, A.W. Malick, J. Mol. Recognit. 9999. (2008).14] M. Hakoda, N. Kamatani, S. Hayashimoto-Kurumada, G.J. Silverman, H.

Yamanaka, C. Terai, S. Kashiwazaki, J. Immunol. (1996) 2981.15] E. Johnson, L. Miribel, P. Arnaud, K.Y. Tsang, Immunol. Lett. 14 (1987) 159.16] C.L. Jones, G.M. Georgiou, K.J. Fowler, P.I. Wajngarten, D.M. Roberton, J.

Immunol. Methods 104 (1987) 237.17] A. Nethery, R.L. Raison, S.B. Easterbrook-Smith, J. Immunol. Methods 126 (1990)

57.18] J.R. Nevens, A.K. Mallia, M.W. Wendt, P.K. Smith, J. Chromatogr. 597 (1992) 247.19] B.H.K. Nilson, L. Lögdberg, W. Kastern, L. Björck, B. Akerström, J. Immunol.

Methods 164 (1993) 33.

20] G. Palombo, A. Verdoliva, G. Fassina, J. Chromatogr. B 715 (1998) 137.21] E.H. Sasso, G.J. Silverman, M. Mannik, J. Immunol. 142 (1989) 2778.22] N. Shibuya, J.E. Berry, I.J. Goldstein, Arch. Biochem. Biophys. 267 (1988) 676.23] M.A. Vidal, F.P. Conde, J. Biochem. Biophys. Methods 4 (1981) 155.24] M. Abdullah, R.J.H. Davies, J.A. Hill, J. Chromatogr. 347 (1985) 129.25] E. McCarthy, G. Vella, R. Mhatre, Y.-P. Lim, J. Chromatogr. A 743 (1996) 163.

7 matog

[

[

[[[[

[[[[[

[[

[[

[

[[[[

864 A. Tscheliessnig et al. / J. Chro

26] X. Santarelli, F. Domergue, G. Clofent-Sanchez, M. Dabadie, R. Grissely, C. Cas-sagne, J. Chromatogr. B 706 (1998) 13.

27] P. Brne, A. Podgornik, K. Bencina, B. Gabor, A. Strancar, M. Peterka, J. Chromatogr.A 1144 (2007) 120.

28] K. Aoyama, J. Chiba, J. Immunol. Methods 162 (1993) 201.29] P. Gagnon, J. Immunol. Methods 336 (2008) 222.30] P. Gagnon, F. Hensel, R. Richieri, Biopharm. Int. (2008) 26.31] I. Tornøe, I.L. Titlestad, K. Kejling, K. Erb, H.J. Ditzel, J.C. Jensenius, J. Immunol.

Methods 205 (1997) 11.32] D. Knoll, J. Hermans, J. Biol. Chem. 258 (1983) 5710.33] H. Mahadevan, C.K. Hall, AIChE J. 36 (1990) 1517.

34] D.H. Atha, K.C. Ingham, J. Biol. Chem. 256 (1981) 12108.35] I.R.M. Juckles, Biochim. Biophys. Acta Prot. Struct. 229 (1971) 535.36] A. Polson, G.M. Potgieter, J.F. Largier, G.E.F. Mears, F.J. Joubert, Biochim. Biophys.

Acta Gen. Subjects 82 (1964) 463.37] S.I. Miekka, K.C. Ingham, Arch. Biochem. Biophys. 191 (1978) 525.38] A.C. Davis, M.J. Shulman, Immunol. Today 10 (1989) 118.

[

[[[

r. A 1216 (2009) 7851–7864

39] A. Jungbauer, J. Chromatogr. A 1065 (2005) 3.40] A. Tscheliessnig, B. Kanatschnig, R. Hahn, A. Jungbauer, 6th European Sym-

posium on Biochemical Engineering Science (ESBES), Salzburg, Austria,2006.

41] A.B. Choo, H.L. Tan, S.N. Ang, W.J. Fong, A. Chin, J. Lo, L. Zheng, H. Hentze, R.J.Philp, S.K.W. Oh, M. Yap, Stem Cells (Miamisburg) 26 (2008) 1454.

42] A.T.A.Z.A.J.R. Hahn, Chem. Eng. Technol. 28 (2005) 1241.43] S. Yamamoto, T. Ishihara, J. Chromatogr. A 852 (1999) 31.44] D. Bell, M. Hoare, P. Dunnill, Downstream Processing, 1983, p. 1.45] A. Tscheliessnig, D. Ong, S. Pan, A. Choo, A. Jungbauer, 28th International Sym-

posium on the Separation of Proteins, Peptides and Polynucleotides (ISPPP),

Baden-Baden, Germany, 2007.

46] J.M. Moreno, J.M. Sanchez-Montero, J.V. Sinisterra, L.B. Nielsen, J. Mol. Catal. 69(1991) 419.

47] E. Müller, J. Chromatogr. A 1006 (2003) 229.48] A. Jungbauer, R. Hahn, J. Sep. Sci. 27 (2004) 767.49] E. Müller, Chem. Eng. Technol. 28 (2005) 1295.