Ai

JAa

b

a

AA

KCIPS

1

giibctamomps

crndhrvdam

0d

Journal of Chromatography A, 1216 (2009) 2683–2688

Contents lists available at ScienceDirect

Journal of Chromatography A

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

daptation of hybridomas to protein-free media results in a simplified two-stepmmunoglobulin M purification process

eremy Leea, Anne Tscheliessniga,∗, Allen Chena, Yih Yean Leea, Gloria Adducia,ndre Chooa, Alois Jungbauerb

Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, Singapore 138668, 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 a b s t r a c t

rticle history:vailable online 21 October 2008

eywords:hromatography

An IgM antibody was purified from hybridoma supernatant containing serum using a three-step purifica-tion process comprising of tangential flow filtration, anion-exchange chromatography and size-exclusionchromatography. Recovery and purity were significantly improved upon adaptation of the hybridoma toserum-free media. The process could even be simplified by omitting the initial tangential flow filtrationstep. Even with a two-step purification process a purity of >98% and a recovery of >60% was obtained.

omcptofbupv[

isdspbint

gMrotein-freeerum

. Introduction

Different combinations of non-chromatographic and chromato-raphic methods have been introduced for the purification ofmmunoglobulin M (IgM) [1–9]. Isoelectric precipitation[8], precip-tation with ammonium sulfate [3] or polyethylene glycol [6] haveeen used as the initial concentration step. However, the precipitatean be difficult to dissolve and some activity may be lost due to pro-ein aggregation [3,6]. In contrast, chromatographic methods, suchs size-exclusion chromatography [1,2,6,8], ion-exchange chro-atography [3–5,9,10], hydroxyapatite chromatography [2,9,11,12]

r thiophilic adsorption [13] provide more selectivity. Affinity chro-atography using ligands such as mannan binding protein [14],

rotein L [15] or protein A [16] are usually only used in laboratorycale.

A significant drawback of chromatographic resins for the purifi-ation of IgM is the low dynamic binding capacity caused by theestricted diffusion of the large IgM molecules into the pores. Foron-hindered diffusive transport into pores, a pore to moleculeiameter ratio of 10:1 has been suggested [17]. For IgM, whichas a hydrodynamic diameter of 24 nm, pores of 240 nm would beequired, which is approximately twice the size of that found in con-

entional chromatographic resins [17]. One way to overcome thisrawback is to increase process time, which depresses productivitynd favors product degradation. A better option is the usage of chro-atographic resins which have been designed for the adsorption

∗ Corresponding author. Tel.: +65 6478 8909; fax: +65 6478 9561.E-mail address: anne tscheliessnig@bti.a-star.edu.sg (A. Tscheliessnig).

eto[preo

021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.10.067

© 2008 Elsevier B.V. All rights reserved.

f large molecules, such as perfusion media [4], membranes [18] oronoliths [19,20]. Membranes as well as monoliths have a mainly

onvection-controlled mass transfer, which is the transport of theroteins to the ligands as a result of the convective flow applied ontohe resin. The dynamic binding capacity is therefore independentf molecule size. Ion-exchange membranes have already been usedor IgM purification [21]. They are advantageous because they cane operated at a low backpressure and are available as disposablenits. However, the dead volume within the housings cause dis-ersion that depresses resolution [18,22]. Monoliths lack such deadolumes and therefore provide a higher resolution than membranes23].

Serum is a common supplement in hybridoma cultures provid-ng a ready source of hormones, growth factors and nutrients toustain the rapid cell division and improve the viability and pro-uctivity of the cell culture. The high concentration of albumin inerum also buffers against the detrimental effects of toxic wasteroducts and reduces shear stress on the cells during mixing in aioreactor [24]. However, purification is highly aggravated by the

ncreased amount of impurities as well as a higher viscosity. Recog-izing these complications, different methods have been employedo purify product from serum-containing supernatant. Jungbauert al. first removed large proteins from fresh serum-containing cul-ure media by ultrafiltration before commencing cell culture, andbserved its effects on the productivity of the hybridoma culture

25]. They found that although the product yield decreased, theurity improved. Alternatively, a more selective chromatographicesin can improve process performance. Recently, a monolith withthylenediamine binding sites which was able to resolve a mixturef human serum albumin and IgM was introduced [10]. As albumin

2 gr. A 1

ipibstwsa

os[spftapsacro

2

2

BYfowfbsepdw(tmtpaSfDgbrtThnr

2

(

(purmsGf

2

wSeptfia

2s

HsA(SCstteprf

ugorwwTe

2

ITppiwcs

2

684 J. Lee et al. / J. Chromato

s the main component of serum, this selectivity can facilitate theurification process even in the presence of serum. Despite such

mprovements, the best solution of process development woulde the adaptation of the hybridoma culture to chemically definederum-free or protein-free media. This would also meet regula-ory concerns regarding the safety of animal-derived products asell as significantly reduce processing costs. However, omission of

erum frequently results in lower maximum viable cell densitiesnd antibody titers [26–28].

At the Bioprocessing Technology Institute (BTI), a panel of mon-clonal IgMs that bind to surface markers on human embryonictem cells (hESC) but not differentiated cells has been generated29]. These IgMs are produced by hybridomas cultured in mediumupplemented with 10% fetal bovine serum. Here we present theurification of one of the IgMs raised against hESCs, mAb 84,rom clarified hybridoma culture. This antibody was purified fromhe culture supernatant sequentially by tangential flow filtration,nion-exchange chromatography using a monolithic stationaryhase and size-exclusion chromatography. Using serum-containingupernatant, this process did not result in a pure antibody. Afterdaptation of the hybridoma to serum-free media, the same purifi-ation process resulted not only in a higher purity but also betterecovery. The purification process could be further simplified bymitting the initial tangential flow filtration step.

. Materials and methods

.1. Cultivation conditions

Hybridoma 84 was cultured in a 20-l wave bioreactor (Waveiotech AG, Switzerland) using BD Cell MAb Medium Quantumield (BD Biosciences, San Jose, CA, USA) supplemented with 10%

etal bovine serum (Hyclone, Logan, UT, USA) at 37 ◦C. A mixturef 7% carbon dioxide and air was supplied continuously to theave bag. The rocker was set to 8◦ tilt amplitude with a rocking

requency of 16 rocks/min. The culture was harvested when the via-ility decreased to 60%. Alternatively, hybridoma 84 was adaptedtep-wise to a protein-free, chemically defined media (BTI propri-tary). A fed-batch hybridoma culture using protein-free media waserformed in a 5 l continuously stirred bioreactor (Sartorius Ste-im Biotech, Melsungen, Germany) at 37 ◦C. Bubble-less aerationas effected through the use of a silicone membrane tubing basket

B. Braun, Melsungen, Germany) and the dissolved oxygen concen-ration (DO) maintained at 30% of air saturation using an Air/N2

ix (early phase) or O2/Air mix (late phase) set at 1 l/min. Agita-ion rate was set at 120 rpm using a 3-blade segmented impeller.H in the culture was maintained at 7.20 using intermittent CO2ddition to the gas mix or 7.5% (w/v) NaHCO3 (Sigma–Aldrich,t. Louis, MO, USA) solution. The protein-free, chemically definedeed for the fed-batch cultures were formulated based on a 10×MEM/F12 (Sigma–Aldrich) and feeding was initiated to maintainlutamine above a preset glutamine set-point in the culture. Theioreactor was harvested when the viability reached 50%. For eithereactor, the harvested culture broth was clarified by centrifuga-ion at 4000 rpm (Beckman GS-6R, Palo Alto, CA, USA) for 10 min.he supernatant of the hybridoma culture in protein-free media isenceforth referred to as serum-free supernatant while the super-atant of the hybridoma culture in serum-containing media iseferred to as serum-containing supernatant.

.2. Chemicals

Chemicals of analytical grade were purchased from MerckDarmstadt, Germany) or BDH (Poole, UK). Bovine serum albumin

w0a

216 (2009) 2683–2688

BSA) and o-phenylenediamine dihydrochloride (OPD) tablets wereurchased from Sigma–Aldrich. All buffers were prepared usingltra-pure water (18.2 M� cm). Buffers used for the chromatog-aphy experiments were filtered using 0.22 �m nitrocelluloseembranes (Millipore, Carrigtwohill, Ireland). Sodium hydroxide

olutions were filtered using 0.2 �m nylon membranes (Sartorius,ottingen, Germany). Gel electrophoresis buffers were purchased

rom Invitrogen (Carlsbad, CA, USA).

.3. Tangential flow filtration

Ultrafiltration was performed using a 300-kDa moleculareight cutoff polyethersulfone membrane cassette on a Sartoflow

lice 200 unit (Sartorius). For 500 ml of clarified supernatant, anffective filtration area of 200 cm2 was used. The pump rate andinch valve on the retentate tubing were adjusted to achieve aransmembrane pressure of 0.7 bar and a flux of 2 × 10−5 m/s. Thelter cassette was regenerated using warm 1 M sodium hydroxidend stored in 0.1 M sodium hydroxide.

.4. Preparative anion-exchange chromatography andize-exclusion chromatography

An ÄKTA Explorer 100 chromatographic workstation (GEealthcare, Uppsala, Sweden) was used for both purification

teps. Protein was detected by monitoring absorbance at 280 nm.nion-exchange chromatography was performed using an 8 ml

45 mm × 15 mm I.D.) CIM-EDA tube (BIA Separations, Ljubljana,lovenia) at a flow rate of 8.0 ml/min if not specified otherwise. TheIM-EDA tube was equilibrated with equilibration buffer (30 mModium phosphate, 50 mM sodium chloride, pH 7.5). After loadinghe sample, unbound proteins were washed out with equilibra-ion buffer until a constant UV baseline was reached. IgM wasluted with a 50% step gradient using elution buffer (30 mM sodiumhosphate, 1 M sodium chloride, pH 7.5). The CIM-EDA tube wasegenerated with 10 column volumes (CVs) of 100% elution buffer,ollowed by 20 CVs of 1 M sodium hydroxide at 4.0 ml/min.

Preparative size-exclusion chromatography was performedsing a 600 mm × 35 mm I.D. BioPilot column Superdex 200 preprade (GE Healthcare). The column was equilibrated with 1.2 CVsf running buffer (30 mM sodium phosphate, 150 mM sodium chlo-ide, pH 7.0) at 3.0 ml/min. The eluate from the CIM-EDA columnas loaded onto the equilibrated column. Protein fractions of 5 mlere collected using a Frac-950 fraction collector (GE Healthcare).

he pooling decision was made after analysis by analytical size-xclusion chromatography.

.5. Analytical size-exclusion chromatography

Using a Shimadzu HPLC system (Kyoto, Japan), a 7.8 mm.D. × 30 cm G4000SWxl HPLC size-exclusion column (Tosoh,okyo, Japan) was equilibrated with running buffer (0.2 M sodiumhosphate, 0.1 M potassium sulfate, pH 6.0) at 0.6 ml/min. Sam-les were filtered using a 0.22 �m filter (Millipore) and 100 �l was

njected into the column and eluted with running buffer. Proteinas detected by measuring absorbance at 280 nm. The system was

alibrated using purified mAb 84, allowing the quantification ofamples in the range of 20–627 �g/ml.

.6. Enzyme-linked immunosorbent assay (ELISA)

A Maxisorb 96-well plate (Nunc, Roskilde, Denmark) was coatedith rabbit anti-mouse IgM (�-chain specific) antibody (5 �g/ml,

.5 �g/well, Open Biosystems, Huntsville, AL, USA) in PBS for 2 ht 37 ◦C. The plate was washed with washing buffer (0.1% Tween

gr. A 1216 (2009) 2683–2688 2685

2p((bcoposaic3spw

2

Ist

2

Bp2w4fstUg((rm(8pUwNi

FcTa

3

flfiptStbot((

3a

sr

TMs

H

H

J. Lee et al. / J. Chromato

0-PBS) and blocked with 3% BSA-PBS for 1 h at 37 ◦C. To pre-are a standard curve, 3.9–500 ng/ml of mouse IgM isotype controlSigma–Aldrich) was prepared by serial dilution with sample buffer1% BSA–0.1% Tween 20-PBS). Samples were likewise preparedy serial dilution with sample buffer. 100 �l of the diluted IgM-ontaining samples and standards were transferred into each wellf the washed plate and the plate was incubated for 1 h at 37 ◦C. Thelate was washed again and incubated for 1 h with 1:5000 dilutionf goat anti-mouse IgM (�-chain specific) peroxidase-conjugatedecondary antibody (Sigma–Aldrich) in sample buffer at 37 ◦C. Afterfinal wash, OPD substrate was prepared as per the manufacturer’s

nstructions and added to each well. The colorimetric reaction pro-eeded at room temperature and was stopped after 17 min withM hydrochloric acid. The intensity of the absorption was mea-

ured at 492 nm against a reference wavelength of 620 nm using alate reader (Tecan, Salzburg, Austria). The standards and samplesere measured in duplicates for experimental consistency.

.7. Total protein quantification

Total protein was quantified using the Bradford assay (Piercenstruments, Rockford, IL, USA) and measured against a BSA proteintandard (Pierce Instruments) according to the supplier’s instruc-ions.

.8. Reducing gel electrophoresis and immunoblotting

Samples were diluted and mixed with NuPAGE LDS Sampleuffer and NuPAGE Sample Reducing Agent according to the sup-lier’s instructions to give a final total protein concentration of5 �g/ml for silver staining and 10 �g/ml for immunoblotting. Theyere then incubated at 95 ◦C for 10 min and separated on precast–12% NuPAGE Bis–Tris gels (Invitrogen, Carlsbad, CA, USA) at 110 Vor 2 h. Total protein was visualized using the SilverQuest silvertaining kit (Invitrogen) according to the manufacturer’s instruc-ions and scanned using a densitometer (Bio-Rad, Hercules, CA,SA). For immunoblotting, the proteins were transferred from theel onto a 0.45 �m polyvinylidene difluoride (PVDF) membraneMillipore, Bedford, MA, USA) using a Mini Trans-Blot tank blotter80 mA, 1 h, Bio-Rad) containing NuPAGE transfer buffer (Invit-ogen) with 20% methanol (BDH, Poole, UK). After transfer, theembrane was blocked with 3% non-fat milk in washing buffer

0.1% Tween 20–PBS) for at least 1 h at room temperature. mAb

4 was detected with 1:5000 goat anti-mouse IgM alkaline phos-hatase conjugated secondary antibody (Calbiochem, La Jolla, CA,SA) in 1% non-fat milk in washing buffer. After 2 washing stepsith washing buffer for 10 min each, the IgM was visualized usingBT/BCIP substrate (Bio-Rad) as directed by the manufacturer’s

nstructions.

smtatr

able 1ass balance of the purification of mAb 84 from serum-containing and serum-free sup

tep/recovery process).

Feed

volume(ml)

mAb 84(�g/ml)

Total protein(mg/ml)

ybridoma supernatant with serum1. Tangential flow filtration 444.0 42 3.202. Anion-exchange chromatography 20.0 149 10.803. Size-exclusion chromatography 15.0 112 2.50

ybridoma supernatant without serum1. Tangential flow filtration 500.0 116 0.802. Anion-exchange chromatography 30.0 152 0.803. Size-exclusion chromatography 6.0 703 1.26

ig. 1. Analytical size-exclusion chromatograms of the TFF step for the serum-ontaining supernatant showing the starting material, the retentate and filtrate.he elution peaks of mAb 84 and fetal bovine serum albumin are indicated usingrrows.

. Results and discussion

A purification process comprising of centrifugation, tangentialow filtration (TFF), anion-exchange chromatography (AEC) andnally size-exclusion chromatography (SEC) was established forurification of mAb 84. We compared the efficiency of this purifica-ion procedure for serum-containing and serum-free supernatant.erum supplementation is usually required to achieve high productiters from a hybridoma culture; however, by employing a fed-atch process using our in-house protein-free media we did notbserve a decrease in product titers after adaptation; the productiter observed with protein-free media was similar or even higher49–116 �g/ml) compared to the titer in serum-containing media42 �g/ml).

.1. Comparison of mAb 84 purification from serum-containingnd serum-free supernatants

Application of the three-step purification procedure to theerum-containing supernatant resulted in a low purity (52%) andecovery (28%) (Table 1). While TFF successfully concentrated theupernatant 3.5-fold it was not able to completely remove smallerolecules such as albumin (Fig. 1). This is surprising, considering

hat the molecular weight cutoff of the membrane (300 kDa) is rel-tively large, about 5-fold larger than albumin (60 kDa). We assumehat either interactions of BSA with larger impurities or even IgMesulted in the retention of BSA. Additionally the charged surface

ernatants. Recovery is given for each step and for the overall process (recovery

Recovered Recovery (%) Purity (%)

volume(ml)

mAb 84(�g/ml)

Total protein(mg/ml)

83.0 149 10.80 66/66 116.0 112 2.50 60/41 415.0 78 0.15 70/28 52

253.0 152 0.80 66/66 196.4 703 1.26 99/65 56

25.0 106 0.11 63/41 96

2686 J. Lee et al. / J. Chromatogr. A 1216 (2009) 2683–2688

F nd (Bp

opnWeLoT

ucrbtaptsataeimm

iioemale

seestt(tt

mrpWptstasa

3t

cexchange the proteins to the anion-exchange equilibration buffer.However, as can be seen from Table 1, TFF had minimal benefiton the purity. In fact, ultrafiltration resulted in the loss of 30%of the total mAb 84 from both serum-containing and serum-freesupernatants. Therefore, it was evaluated if mAb 84 could directly

ig. 2. Analytical size-exclusion chromatograms of (A) the starting supernatants aeaks of mAb 84 and fetal bovine serum albumin are indicated using arrows.

f the PES membrane could impede unhindered transport of smallroteins. The charged surface of the membrane could also causeon-specific adsorption of IgM resulting in the observed losses.e found that increasing NaCl concentration increases the recov-

ry (data not shown) but impedes with the subsequent AEC step.ow-protein binding membranes with large molecular weight cut-ff (>100 kDa), e.g. regenerated cellulose, were not available for ourFF system.

When the TFF retentate was then loaded onto the CIM-EDA col-mn, the large concentration of serum proteins in the AEC loadompeted with mAb 84 for binding sites, resulting in the poorecovery and dynamic binding capacity (0.2 mg/ml) of mAb 84. Thisinding capacity is significantly lower than that obtained for par-ially purified IgM (40 mg/ml [23]) but this is not surprising for thedsorption of complex mixtures where different molecules com-ete for the available binding sites. Increasing the pH or decreasinghe conductivity of the feed might lead to an improved bindingtrength of mAb 84 to the monolith, but concomitantly it wouldlso have favored the adsorption of impurities. Considering thathe all impurities bind stronger than mAb 84 – mAb 84 elutes rel-tively pure in the first fraction (0.5 M NaCl) while the impuritieslute in the regenerate (1.0 M NaCl) – it is more likely that changesn pH or conductivity would result in lower binding capacities for

Ab 84. Additionally increasing the binding strength of mAb 84ight result in a decreased resolution leading to a less pure eluate.Cation-exchange chromatography would be another option, as

t can show lower binding of impurities. However, mAb 84 has ansoelectric point of approximately 6.0 which would require a pHf 4.0–4.5 to obtain sufficient high binding capacities on a cation-xchange resin. As we found in previous studies the capacity forAb 84 on the cation-exchange resin was not higher than for a

nion-exchange resin (data not shown) and due to the potentiallyower stability of mAb 84 at pH 4.0 we refrained from using cation-xchange chromatography for the purification.

The subsequent size-exclusion chromatography did not provideufficient resolution to remove the impurities present in the AECluate (Fig. 2A). A high purity would only have been possible at thexpense of recovery. In comparison, purification of mAb 84 fromerum-free supernatant gave a higher purity and recovery for the

wo chromatographic steps (Table 1). Due to the lower concentra-ion of impurities we observed a 2-fold higher binding capacity0.56 mg/ml) than in the presence of serum. This was still lowerhan the 1.7 mg/ml binding capacity reported by Brne et al. [10] butheir data was obtained for polyclonal IgM present in serum, which

Fe8

) the end-products of serum-containing and serum-free supernatant. The elution

ay explain the difference. The higher purity of the AEC eluate alsoesulted in a better resolution in the size-exclusion chromatogra-hy (Fig. 3) leading to a higher purity of the final product (Fig. 2B).ithout serum in the media, it was possible to reach a higher

urity (96%) and recovery (41%) using the same purification pro-ocol. Therefore, instead of introducing an additional purificationtep which would have reduced product recovery and increasedhe overall cost of the purification, adaptation of the hybridoma toprotein-free media resulted in the reduction of total protein in theupernatant permitting the successful purification of mAb 84 withhigh recovery.

.2. Comparison of mAb 84 purification with and withoutangential flow filtration from serum-free supernatant

Tangential flow filtration was chosen as the initial step to con-entrate the sample, remove low molecular-weight impurities and

ig. 3. Chromatogram of the preparative size-exclusion chromatography of the AECluate from serum-containing and serum-free supernatant. The elution peak of mAb4 is indicated using an arrow.

J. Lee et al. / J. Chromatogr. A 1216 (2009) 2683–2688 2687

Table 2Mass balance of the three-step and two-step purification for serum-free hybridoma supernatant. Recovery is given for each step and for the overall process (recoverystep/recovery process).

Feed Recovered Recovery (%) Purity (%)

volume(ml)

mAb 84(�g/ml)

Total protein(mg/ml)

volume(ml)

mAb 84(mg/ml)

Total protein(mg/ml)

Three-step process1. Tangential flow filtration 225.0 49 0.38 214.0 43 0.20 84/84 212. Anion-exchange chromatography 120.0 43 0.20 7.4 523 0.82 75/63 643. Size-exclusion chromatography 6.9 523 0.82 25.0 123 0.14 85/53 88

Two-step process1. Anion-exchange chromatography 120.0 49 0.38 15.0 308 0.62 79/79 502. Size-exclusion chromatography 14.5 308 0.62 25.0 137 0.14 77/60 98

F um-fr(

bau

i

w

Fa

ig. 4. Analytical size-exclusion chromatograms of mAb 84 purifications from serTFF). The elution peak of mAb 84 is indicated using an arrow.

e captured from serum-free supernatant without adjusting the

ntibody concentration or ionic strength of the supernatant byltrafiltration.

One aliquot of serum-free supernatant was buffer exchangednto anion-exchange equilibration buffer by ultrafiltration before it

li2o

ig. 5. Silver stain of reduced electrophoresis gels comparing the purified fractions of thend light chain (LC) of mAb 84 are indicated by arrows.

ee supernatant (A) with and (B) without diafiltration by tangential flow filtration

as loaded onto the CIM-EDA column. Another aliquot was directly

oaded onto the CIM-EDA column without adjusting the pH andonic strength. Each eluate was finally purified using the Superdex00 prep grade size-exclusion column. The two-step protocol,mitting ultrafiltration, gave a similar purity and recovery as the

(A) three-step and (B) two-step process for serum-free supernatant The heavy (HC)

2 gr. A 1

t(maStW(bcpno

4

aooUflcmoafrtfstlsiaDc

A

A

R

[

[[[[[

[

[[[[

[

[[[[

688 J. Lee et al. / J. Chromato

hree-step protocol (Table 2). From the SEC-HPLC chromatogramFig. 4) it can be seen that the TFF step removed only the low

olecular-weight impurities from the supernatant which couldlso be achieved by anion-exchange chromatography alone. TheEC-HPLC chromatograms of the CIM-EDA eluates are almost iden-ical for either purification process. Moreover, the silver stain and

estern blot of the reduced electrophoresis gels of the three-stepFig. 5A) and the two-step process (Fig. 5B) also give a compara-le profile for the AEC eluate as well as final product. Therefore weoncluded that the TFF step is not required to obtain the requiredurity. Adaptation of the hybridoma culture to protein-free mediaot only resulted in a higher purity but also allowed simplificationf the purification process.

. Conclusion

Due to their high complexity and relatively low solubility IgMre often considered difficult to purify. We found that the majorbstacle in our purification process was not the biochemistry ofur IgM but rather the presence of serum in the supernatant.sing a three-step purification process comprising of tangentialow filtration, anion-exchange chromatography and size-exclusionhromatography we found that it was impossible to obtain pureAb 84. This was surprising considering that size separation meth-

ds such as size-exclusion chromatography have been frequentlypplied to purify IgM. By adapting hybridoma 84 culture to protein-ree media we removed the serum proteins from the supernatant,educing the amount of impurities while maintaining the mAb 84iter. The same three-step purification process using the serum-ree supernatant yielded a pure product. We could even furtherimplify our purification process by omitting tangential flow filtra-ion, a step that led to no significantly higher purity but rather highosses. Comparing this two-step process with the previous three-

tep process we found no influence on the final purity and almostdentical profiles of the size-exclusion chromatography eluate innalytical size-exclusion chromatography or gel electrophoresis.ue to the omission of serum from the media the overall productionosts could be reduced.

[[[[

216 (2009) 2683–2688

cknowledgement

This work was supported by the Biomedical Research Council of*STAR (Agency for Science, Technology and Research), Singapore.

eferences

[1] G. Coppola, J. Underwood, G. Cartwright, T.W. Hearn, J. Chromatogr. 476 (1989)269.

[2] A.J. Henniker, K.F. Bradstock, Biomed. Chromatogr. 7 (1993) 121.[3] V.P. Knutson, R. Ann Buck, R.M. Moreno, J. Immunol. Methods 136 (1991) 151.[4] E. McCarthy, G. Vella, R. Mhatre, Y.P. Lim, J. Chromatogr. A 743 (1996) 163.[5] S.H. Neoh, C. Gordon, A. Potter, H. Zola, J. Immunol. Methods 91 (1986) 231.[6] D. Roggenbuck, U. Marx, S.T. Kiessig, G. Schoenherr, S. Jahn, T. Porstmann, J.

Immunol. Methods 167 (1994) 207.[7] X. Santarelli, F. Domergue, G. Clofent-Sanchez, M. Dabadie, R. Grissely, C. Cas-

sagne, J. Chromatogr. B 706 (1998) 13.[8] F. Steindl, A. Jungbauer, E. Wenisch, G. Himmler, H. Katinger, Enzyme Microb.

Technol. 9 (1987) 361.[9] I. Tornoe, I.L. Titlestad, K. Kejling, K. Erb, H.J. Ditzel, J.C. Jensenius, J. Immunol.

Methods 205 (1997) 11.10] P. Brne, A. Podgornik, K. Bencina, B. Gabor, A. Strancar, M. Peterka, J. Chromatogr.

A 1144 (2007) 120.11] Y. Yamakawa, J. Chiba, J. Liquid Chromatogr. 11 (1988) 665.12] K. Aoyama, J. Chiba, J. Immunol. Methods 162 (1993) 201.13] T.W. Hutchens, J.S. Magnusson, T.T. Yip, J. Immunol. Methods 128 (1990) 89.14] J.R. Nevens, A.K. Mallia, M.W. Wendt, P.K. Smith, J. Chromatogr. 597 (1992) 247.15] B.H.K. Nilson, L. Loegdberg, W. Kastern, L. Bjoerck, B. Akerstroem, J. Immunol.

Methods 164 (1993) 33.16] S. Ghose, M. Allen, A. Hubbard, C. Brooks, S.M. Cramer, Biotechnol. Bioeng. 92

(2005) 665.17] A. Jungbauer, J. Chromatogr. A 1065 (2005) 3.18] R. Gosh, J. Chromatogr. A 952 (2002) 13.19] G. Iberer, R. Hahn, A. Jungbauer, LC–GC N. Am. 17 (1999) 998.20] A. Zoechling, R. Hahn, K. Ahrer, J. Urthaler, A. Jungbauer, J. Sep. Sci. 27 (2006)

819.21] M.J. Jacobin, X. Santarelli, J. Laroche-Traineau, G. Clofent-Sanchez, Hum. Anti-

bodies 13 (2004) 69.22] U. Gottschalk, Biotechnol. Prog. 24 (2008) 496.23] P. Gagnon, F. Hensel, R. Richieri, Biopharm Int. 26 (March (Suppl.)) (2008) 26.24] B. Griffiths, Trends Biotechnol. 4 (1986) 268.25] A. Jungbauer, E. Wenisch, F. Steindl, G. Himmler, S. Reiter, F. Rueker, K. Wagner,

H. Katinger, Dev. Biol. Stand. 66 (1987) 87.26] F. Chua, S.K.W. Oh, M. Yap, W.K. Teo, J. Immunol. Methods 167 (1994) 109.27] C. Heath, R. Dilwith, G. Belfort, J. Biotechnol. 15 (1990) 71.28] G.M. Lee, A. Varma, B.O. Pallson, Biotechnol. Bioeng. 38 (2004) 821.29] 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 26 (2008) 1454.