Journal of Chromatography A, 1216 (2009) 78517864

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Engineering of a two-step purication strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells
Anne Tscheliessnig a, , Danny Ong a , Jeremy Lee a , Siqi Pan a , Gernalia Satianegara a , Kornelia Schriebl a , Andre Choo a,b , Alois Jungbauer c
a

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

a r t i c l e

i n f o

a b s t r a c t
A two-step purication strategy comprising of polyethylene glycol (PEG) precipitation and anionexchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.57.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its signicantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purication strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be puried to >95% purity with the exception of two which gave a lower nal purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purication of up to 2 l hybridoma supernatants. 2009 Elsevier B.V. All rights reserved.

Article history: Received 10 August 2009 Received in revised form 17 September 2009 Accepted 23 September 2009 Available online 26 September 2009 Keywords: IgM PEG Precipitation Anion-exchange chromatography Benzonase Adsorption isotherm Adsorption kinetics Number of binding sites

1. Introduction Various purication strategies have been developed for the purication of immunoglobulin M (IgM) antibodies from different sources, such as serum, ascites or cell culture supernatants. Most of those strategies comprise techniques based on size separation, such as size-exclusion chromatography (SEC) [111] or polyethylene glycol (PEG) precipitation [3,12]. Additionally, highly selective techniques such as afnity chromatography using a range of different ligands [1,1323], anion-exchange chromatography (AEC) [13,5,7,11,2427] as well as hydroxyapatite chromatography [4,2831] have been applied. In particular, the combination of AEC with a subsequent SEC step [1,2,5,7,11] was successful for the purication of IgM from ascites and cell culture supernatants. PEG precipitation is advantageous as an initial enrichment step for large proteins such as IgM (960 kDa). The predominant mechanism 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). 0021-9673/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.09.059

suspension volume which is taken up by dissolved PEG (excluded volume) [32,33]. Differently sized proteins can be easily and selectively separated depending on the PEG size and concentration [3436]. While excluded volume is the major effect, it has been shown that other parameters, such as temperature, pH or ionic strength can affect the solubility signicantly [35]. In particular, conditions that favor the self-association of proteins are advantageous, e.g. a pH close to the isoelectric point [37]. Therefore, design of an efcient PEG precipitation step not only includes selection of PEG size and concentration but also the screening for other parameters such as pH or salt concentration. Due to their size, IgM antibodies also carry many charged groups, which enable adsorption to ion-exchange chromatography resins even at moderate pH, thereby reducing the potential for degradation during purication [30,38]. In particular, anionexchange chromatography has proven to be efcient in the purication of IgM from cell culture supernatant [7,30]. Its major disadvantage, however, is the adsorption of most other host cell proteins leading to a reduced capacity for IgM. In contrast, cationexchange chromatography, while having the advantage of minimal host cell protein adsorption, is less generic for adsorption of IgM.

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Often the pH of the supernatant has to be adjusted to allow adsorption [30]. Besides the selection of the chromatographic mode, it is also important to consider the pore geometry of the resins [39]. Conventional porous resins provide a large surface area. However, the pore diameter might not be sufciently wide to allow large molecules, such as IgM, to diffuse to the binding site. In contrast, not only will the transport be slow due to hindered diffusion but adsorbed molecules could potentially block the pore preventing diffusion of further molecules into the pore. It has been suggested that for unhindered diffusion, the pore diameter should be at least 10 times the molecule diameter [39]. For IgM with a hydrodynamic radius of 20 nm [7] this would require a pore of 200 nm diameter. However, the average pore diameter for porous resins is 50100 nm [39]. Interestingly we found that for one resin, Fractogel EMD, the ratio of protein to pore size is not a constraining factor [40]. The adsorption kinetics of proteins in their unfolded state (20 nm, hydrodynamic diameter) were only 1/3 to 2/3 that of the native proteins (610 nm, hydrodynamic diameter) when using the Fractogel EMD [40]. For other resins, e.g. Toyopearl, the uptake was severely impeded. The mechanism of the mass transfer of the unfolded proteins in the Fractogel is not understood but we would expect a similar fast mass transfer for the IgM. Alternatively, convection driven resins, such as membranes and monoliths have been recommended for the purication of IgM [7,30]. While the binding capacity is reduced due to a lower surface area, their advantage is the fast mass transfer that allows processing of large volumes in short times. Gagnon et al. [30] reported ow rates between 2.5 and 12.0 column volumes (CVs)/min for a separation of an IgM from cell culture supernatant on a cation-exchange monolith. At BTI we have raised a panel of hybridoma producing IgM antibodies selectively targeting surface markers on undifferentiated human embryonic stem cells (hESC) [41]. One of the antibodies, mAb 84, not only binds but also kills hESC within 30 min of incubation [41]. Initially all hybridoma clones were cultured in media containing 10% fetal bovine serum (FBS). A purication strategy, developed for mAb 84, comprising tangential ow ltration (TFF), anion-exchange chromatography (AEC) and sizeexclusion chromatography (SEC) was only minimally successful in obtaining puried mAb 84 [7]. After adaptation of the hybridoma clone to serum-free media we not only obtained highly pure mAb 84 but could also simplify the purication process by omitting the initial TFF without affecting the nal purity. However, the developed purication strategy had only a low concentration factor (2.7) [7]. Therefore we decided to modify the purication strategy by replacing the nal SEC step that resulted in high sample dilution, with an initial enrichment step using PEG precipitation. For AEC we compared different chromatographic resins, such as Fractogel EMD, Toyopearl and Capto Q for their adsorption equilibrium and kinetics. With DNA being one of the major contaminants, a DNA removal step using benzonase was also evaluated. 2. Material and methods

trophoresis buffers were purchased from Invitrogen (Carlsbad, CA, USA). 2.2. Cultivation conditions Hybridoma were cultured in a continuously stirred 5 l or 3 l bioreactor (Sartorius Stedim Biotech, Melsungen, Germany). All hybridoma clones had been adapted step-wise to a protein-free, chemically dened media (proprietary to BTI). Temperature was kept at 37 C and a silicon membrane tubing basket (B. Braun, Melsungen, Germany) for bubble-less aeration was used. The dissolved oxygen concentration (DO) was maintained at 30% of air saturation using an Air/N2 mix (early phase) or O2 /Air mix (late phase) set at 1 l/min. pH in the culture was adjusted to 7.10 using intermittent CO2 addition to the gas mix or 7.5% (w/v) NaHCO3 (SigmaAldrich) solution. The protein-free, chemically dened feed was based on a 10 DMEM/F12 (Sigma). For fed-batch cultures (5 l) feeding was set to maintain glutamine above a preset glutamine set-point in the culture. The culture broth was harvested when the viability reached 60% and claried by centrifugation at 4000 g (Sigma 8k, Sartorius Stedim Biotech, Goettingen, Germany) for 10 min followed by ltration using a 0.2-m depth lter (Sartorius). 2.3. PEG precipitation For the solubility experiments, 4 ml of a 535% (w/v) PEG 6000 (BDH Prolabo) was added to an equi-volume of hybridoma 84 supernatant and the solution was incubated at 4 C for up to 4 h. The suspension was then centrifuged (5900 g) and the collected precipitate dissolved in 2 ml AEC equilibration buffer (30 mM sodium phosphate, 50 mM NaCl, pH 7.5). mAb 84 present in the dissolved PEG precipitate and claried supernatant was quantied using ELISA. For measurement of the precipitation kinetics a similar experimental set-up was used: 100 ml of a 20% (w/v) PEG 6000 (BDH Prolabo) was added to an equi-volume of hybridoma mAb 84 supernatant. In one set-up the solution was mixed to obtain homogeneity and then incubated at 4 C without further mixing. Alternatively, to evaluate the inuence of mixing during precipitation, the same mixture was continuously mixed at 4 C using a magnetic stirrer. At indicated times, samples (4 ml) were drawn, centrifuged and the precipitate obtained dissolved in 2 ml AEC equilibration buffer. The concentration of mAb 84 in the dissolved PEG precipitate was measured by ELISA. The small-scale and large-scale PEG precipitation was performed similar to the kinetics experiments: An equi-volume of 20% (w/v) PEG 6000 (BDH Prolabo) was mixed with hybridoma supernatant and the solution stirred using a magnetic stirrer at 4 C for a minimum of 2 h. The precipitate was then harvested by centrifugation (5900 g, Sigma 8k) and dissolved in 25% of the suspensions volume using AEC equilibration buffer. The dissolved PEG precipitate was ltered using a 0.2-m depth lter (Sartorius Stedim Biotech) to remove particulate matter. 2.4. Anion-exchange chromatography

2.1. Chemicals Chemicals of analytical grade were purchased from Merck (Darmstadt, Germany) or BDH Prolabo (Poole, UK). Bovine serum albumin (BSA) and o-phenylenediamine dihydrochloride (OPD) tablets were purchased from SigmaAldrich (St. Louis, USA). All buffers were prepared using ultra-pure water (18.2 M cm). Buffers used for the chromatography experiments were ltered using 0.2 m nitrocellulose membranes (Millipore, Carrigtwohill, Ireland). Sodium hydroxide solutions were ltered using 0.2 m nylon membranes (Sartorius, Goettingen, Germany). Gel elecThe anion-exchange chromatography was performed on the kta Explorer 100 (GE Healthcare, Uppsala, Sweden) using Fractogel EMD DEAE (M), a weak anion-exchange media with a particle diameter of 4090 m and pore diameter of approximately 80 nm (Merck). For linear gradient elution a Tricorn 5/50 (GE Healthcare) was packed with approximately 1 ml of Fractogel EMD DEAE (M). The ow rate was 1 ml/min if not specied otherwise. The column was rst equilibrated with AEC equilibration buffer followed by loading of 100200 ml of dissolved PEG precipitate at 1 ml/min. Unbound

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proteins were washed out with AEC equilibration buffer until a constant UV (280 nm) and conductivity baseline were observed. The proteins were then eluted using a linear gradient from 0% to 100% AEC elution buffer (30 mM sodium phosphate, 1000 mM NaCl, pH 7.5) over 20 column volumes (CVs) and the eluate collected in fractions of 1 ml. The column was regenerated with 10 CVs AEC elution buffer, followed by 0.5 M NaOH until a steady UV (280 nm) baseline was observed for at least 10 CVs. The fractions were analyzed for IgM and total protein concentration. For the step gradient elution either a Tricorn 5/50 (GE Healthcare, Uppsala, Sweden) or a Tricorn 10/100 (GE Healthcare, Uppsala, Sweden) were packed with approximately 1 ml or 8 ml Fractogel EMD DEAE (M), respectively. The ow rate was either 1 ml/min for the 1 ml column or 4 ml/min for the 8 ml column. 1001000 ml of claried dissolved PEG precipitate was loaded onto the column and unbound proteins washed out using AEC equilibration buffer. IgM was, depending on the respective mAb, eluted with 2327% AEC elution buffer and fractions of 1 ml were collected. The column was regenerated using 100% AEC elution buffer, followed by 0.5 M NaOH until a constant UV (280 nm) baseline was observed for at least 10 CVs. The fractions were analyzed for IgM and total protein concentration. 2.5. Benzonase treatment For measurement of the kinetics of the benzonase, 1 ml of supernatant was mixed with benzonase (purity grade II, >90%, Merck) to give a nal concentration of 10 U/ml, 50 U/ml or 100 U/ml. The samples were mixed at 4 C, 37 C and room temperature and after 15, 30, 45, 60 and 120 min the benzonase activity was inhibited by addition of 1 ml of 400 mM EDTA. For all samples, the DNA concentration was analyzed as given below. For the comparison of the purication with and without benzonase treatment, benzonase was added to 200 ml of hybridoma 84 supernatant to a nal concentration of 50 U/ml and mixed for 1 h at room temperature. The benzonase-treated supernatant was ltered through a 0.2-m depth lter (Sartorius Stedim Biotech) and subjected to PEG precipitation and AEC as described previously. For the control, the hybridoma 84 supernatant was also stirred for 1 h at room temperature and thereafter ltered using a 0.2-m depth lter (Sartorius Stedim Biotech) subject to PEG precipitation and AEC. For large scale purications, benzonase treatment was slightly modied: To the hybridoma 84 supernatant, benzonase was added to a nal concentration of 25 U/ml. After mixing for 30 min at room temperature, additional benzonase was added to give a nal concentration of 50 U/ml and the supernatant mixed for another 30 min at room temperature. The suspension was then ltered and subject to PEG precipitation and AEC as described previously. 2.6. Adsorption isotherms The particulate resins, Fractogel EMD DEAE (M) (Merck), Fractogel EMD DEAE (S) (Merck), Toyopearl DEAE-650 M (Tosoh, Tokyo, Japan), Toyopearl DEAE-650S (Tosoh) and Capto Q (GE Healthcare) were pre-equilibrated in AEC equilibration buffer. Using the MediaScout ResiQuot (Atoll GmbH, Weingarten, Germany) with the corresponding perforated plate, 7.8 l of resin were aliquoted into a 96-well micro plate. Different concentrations of 0.10.5 ml puried mAb 84 were added and the suspension incubated for approximately 6 h on a rotator (SB 3, Barloworld Scientic, Stone, UK). The 96-well micro plate was then centrifuged and the supernatant collected and analyzed. From the mass balance, the amount of mAb 84 adsorbed onto the media could be calculated: q= c0 V0 cV Vg (1)

with c0 and V0 as the mAb 84 concentration and volume in the beginning and c and V as the nal mAb 84 concentration and the total volume of the suspension. Vg is the settled media volume in the 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 of the kta explorer 100. Using the sample pump P960, 2.02.9 ml of mAb 84 at different concentration were circulated over the column at 0.2 ml/min for 5 h. After washing the disk until a constant UV baseline (280 nm) was obtained, bound mAb 84 bound was eluted using AEC elution buffer. The mAb 84 content in the collected fraction was quantied using ELISA. The obtained data points, c and q, were then tted to the Langmuir isotherm using TableCurve 2D software to give the apparent maximum adsorption capacity, qm and the apparent adsorption equilibrium parameter, KA : q= qm KA c 1 + KA c (2)

2.7. Adsorption kinetics The adsorption kinetics were measured using the method of shallow bed [42]. In short, approximately 20 l of a 1:2 slurry of pre-equilibrated resin was placed into a Tricorn 5/20 column (GE Healthcare) and mixed with 200 l of Sephadex G25 to emulate conditions of a xed bed. The column was connected to an kta Purier 10 (GE Healthcare) and equilibrated with AEC loading buffer at 4 ml/min. Using the sample pump P960, 90450 ml of puried mAb 84 (80 g/ml) as well as dissolved PEG precipitate from benzonase treated and untreated hybridoma 84 supernatant (each 80 g/ml mAb 84) was circulated over the column at 4.0 ml/min. At specied times, loading was stopped, the column washed until a constant UV (280 nm) baseline was obtained before bound proteins are eluted using AEC elution buffer. The collected fraction was analyzed for DNA, IgM and total protein concentration. To evaluate the adsorption kinetics of the EDA (monolith), a 34l CIM EDA MiniDisk (gift from BIA Separations) was used. The ow rate had to be reduced to 12 ml/min to accommodate for the pressure increase during loading. 2.8. Number of binding sites Measurement of the number of binding sites was performed according to Yamamoto and Ishihara [43]. A Tricorn 5/100 (GE Healthcare) packed with the respective resin or a CIM Disk housing (BIA Separations) loaded with 4 EDA CIM Disks (BIA Separations) were connected to an kta Explorer 100. The ow rate throughout the experiment was 1 ml/min. The column was rst equilibrated with 2.5 CV of AEC loading buffer followed by loading 1 mg of mAb 84 (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 AEC elution buffer with slopes of different steepness (3090 CVs). The elution of mAb 84 was observed by measurement of the absorption at 280 nm. Then, correlating the normalized gradient slope, , to the NaCl concentration of the peak maximum of the elution peaks, the number of binding sites (z) can be calculated according to =
R CM
(z +1)

A(z + 1)

(3)

with = (Vt Vv ) (4)

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is the gradient slope and v the interstitial mobile phase velocity. R is the modier concentration at the peak maxima and parameter CM A is dened as A = Ke
B

(5) as the total

with Ke as the equilibrium association constant and ion-exchange capacity. 2.9. Analytical methods

2.9.1. Enzyme-linked immunosorbent assay (ELISA) The IgM content was quantied using a high-sensitivity sandwich enzyme-linked immunosorbent assay (ELISA). First, a microtiter plate (Immuno 96 MicroWell Plates, MaxiSorb, NUNC, Roskilde, Denmark) was coated with rabbit anti-mouse IgM (-chain specic) antibody (0.005 mg/ml, Open Biosystems, Huntsville, USA) in 0.05 M sodium carbonate, pH 9.6 for 2 h at 37 C. The plates were then blocked with 3% bovine serum albumin (BSA, SigmaAldrich) in PBS/Tween (137.0 mM NaCl, 2.7 mM KCl, 10.0 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 0.1% Tween 20, pH 7.4) for 1 h at 37 C. For each plate, a duplicate standard curve (0.5000.003 g/ml) was prepared by serial dilution (1:2) using an in-house standard (protein Apuried mAb 84, 0.5 g/ml in PBS) in 1% BSA in PBS/Tween. The samples were diluted likewise and after transferring samples and standards to the microtiter plate, it was incubated for 1 h at 37 C. After incubation with a goat anti-mouse IgM (-chain specic) antibody horseradish peroxidase conjugate (HRP) (1:5000, SigmaAldrich) in 1% BSA in PBS/Tween for 1 h at 37 C, O-phenylenediamine dihydrochloride (Sigma FastTM OPD tablets, SigmaAldrich) was added for staining. The enzymatic reaction was stopped by adding 3 M HCl to each well. The absorbance at 492 nm was read using a Tecan Sunrise plate reader (Tecan, Salzburg, Austria) using a reference wavelength of 620 nm. Using the decimal logarithm of the IgM concentration of the standards and their measured absorbance a linear regression was performed. Only data points which gave a t with an R2 larger 0.99 were considered. The IgM concentration of the samples was determined using the obtained calibration curve. For each sample duplicates were performed where a minimum of 3 dilutions had to have a concentration deviating less than 10% from the average. A limit of quantitation (LOQ) was 0.019 g/ml and the limit of detection (LOD) was 0.009 g/ml. 2.9.2. Analytical size-exclusion chromatography A G4000SWxl size exclusion column (Tosoh, Tokyo, Japan) was connected to the HPLC system (Shimadzu, Kyoto, Japan) and equilibrated with SEC running buffer (0.2 M sodium phosphate, 0.1 M potassium sulfate, pH 6.0) at 0.6 ml/min. 100 l of ltered (0.2 m, Millipore, Carrigtwohill, Co. Cork, Ireland) sample were injected and the protein detected by measuring the absorbance at 280 nm at the column outlet. mAb 84 present in supernatant and puried samples could be quantied in the range of 20627 g/ml using a calibration curve of protein A-puried mAb 84. 2.9.3. Total protein quantication Absorbance was measured at 280 nm using either the UVvisible photometer UV-1601 (Shimadzu, Kyoto, Japan) or the NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, USA). The concentration of total protein was calculated using an extinction coefcient of 1.2 (AU ml/cm mg), which has been suggested for IgM [30]. 2.9.4. Reduced SDS-PAGE electrophoresis and immunoblotting Reduced SDS-PAGE was performed using 412% NuPAGE BisTris gels (Invitrogen) according to the manufacturers instructions. The samples were diluted with NuPAGE LDS Sample Buffer

(Invitrogen), NuPAGE Reducing Agent (Invitrogen) and RO-water to give a maximum load 0.250.30 g IgM for Coomassie staining and 0.830.99 g IgM for Western blot. After incubation at 95 C for 10 min, the samples were loaded onto precast 412% NuPAGE BisTris gels (Invitrogen, Carlsbad, CA, USA) and separated for 35 min using NuPAGE MES buffer (Invitrogen) and a Novex XCell SureLock Mini-Cell PAGE system (Invitrogen) with the voltage set to 200 V. For Coomassie staining, the gels were incubated in 100 ml Coomassie Blue staining solution (0.1% (w/v) Coomassie brilliant blue R-250, 30% (v/v) absolute ethanol, 10% (v/v) glacial acetic acid) for approximately 30 min. The gel was then incubated in the destaining solution (10% (v/v) absolute ethanol and 10% (v/v) glacial acetic acid) until a clear background was observed. For immunoblotting, the proteins were transferred from the gel onto a 0.2-m polyvinylidene diuoride (PVDF) membrane (Millipore) 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 at least 1 h at room temperature. IgM was detected using a goat antimouse IgM alkaline phosphatase conjugated secondary antibody (1:5000, Calbiochem, La Jolla, CA, USA) in 1% non-fat milk powder in washing buffer. After 2 washing steps with washing buffer for 10 min each, the IgM was visualized using NBT/BCIP substrate (Bio-Rad) as directed by the manufacturers instructions. 2.9.5. DNA quantication DNA was quantied using the Quant-iT PicoGreen dsDNA kit (Invitrogen) in microplate format. All buffers and reagents were prepared according to the manufacturers instructions. Using a 96-well microplate the samples and standards (diluted to either 200 or 1000 g/ml Lambda DNA standard) were 1:2 serial diluted with TE-buffer. 100 l of sample were transferred into a black 96-well Polystyrene Microplate (Greiner-Bio, Frickenhausen, Germany) 100 l of the Quant-iT PicoGreen dsDNA reagent added and the solutions gently mixed. The uorescence intensity was measured using the Tecan Innite M200 (excitation: 480 nm with 9 nm bandwidth, emission: 520 nm with 20 nm bandwidth, automatic gain, 10 reads with 20 s integration time). A linear calibration curve was tted to the standard dilutions and used for calculation of DNA concentration of the standard. 2.9.6. Isoelectric focusing All samples were rst buffer-exchanged into 0.05 M NH4 HCO3 using PD-10 Desalting Columns (GE Healthcare) according to the manufacturers instructions and then concentrated using Vivaspin 500 (MWCO 100 kDa, Sartorius Stedim Biotech) to a concentration of approximately 5 mg/ml. IsoGel Agarose IEF Plates, pH 310 (Lonza, Rockland, USA) were set up in the Multiphor (GE Healthcare) according to the manufacturers instructions. 10 g of sample (2 l of 5 mg/ml), 1 l of the Broad Range marker (pI 4.459.60, BioRad) and 5 l of the High Range marker (pI 5.010.5, GE Healthcare) were loaded and the gel run for 6090 min with the initial settings of 25 W, 1000 V. The gel was xed and Coomassie stained according to the manufacturers instructions. 2.9.7. Flow cytometry For all clones, supernatants and puried IgM were brought to a concentration of 25 g/ml, either by concentrating using a Vivaspin 500 (Sartorius Stedim Biotech) with a MWCO of 10 kDa or by dilution with PBS for supernatant or 1% BSA/PBS for puried IgM. The assay was performed as described elsewhere [41]. In short, 2 105 HES-3 cells, harvested as single cell suspension using trypsin, were resuspended in 10 l of 1% BSA/PBS and 200 l of the puried IgM or supernatant added. After incubation for 30 min

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Fig. 1. IEF gel for the panel of clones. The pI of each clone can be estimated from comparison to the Broad Range marker (BR marker) and High Range marker (HR marker).

on ice, the cells were washed with cold 1% BSA/PBS and incubated for another 15 min (4 C) with a goat anti-mouse antibody uorescein isothiocyanate conjugate (FITC) (1:500, DAKO, Glostrup, Denmark). After washing with 1% BSA/PBS, the cells were resuspended in 200 l 1% BSA/PBS and 1.25 mg/ml propidium iodide (pI, SigmaAldrich) and analyzed using FACScan (Becton, Dickinson and Company, Franklin Lakes, USA). An anti SSEA-1 (IgM) was used as negative control. 2.9.8. Cytotoxicity assay for mAb 84 and mAb 85 Hybridoma 84 and hybridoma 85 supernatant as well as puried mAb 84 and mAb 85 were brought to 25 g/ml by dilution with PBS (supernatant) or 1% BSA/PBS (puried IgM). The assay was performed as described elsewhere [41]. In short, 2 105 HES-3 cells, harvested as single cell suspension using trypsin, were resuspended in 10 l of 1% BSA/PBS and 150 l of the supernatant or puried IgM added. After incubation for 45 min on ice the cells were washed and resuspended in 1% BSA/PBS and 1.25 mg/ml pI using FACScan (Becton, Dickinson and Company). To validate the results obtained using pI exclusion assays, viability for each sample was also determined using tryphan blue exclusion. 3. Results and discussion Our aim was to develop a purication strategy for a panel of monoclonal IgM antibodies from hybridoma. The IgM in the panel have different isoelectric points (pI), ranging from approximately 5.5 for the most acidic IgM (mAb 5) to around 7.7 for the more basic IgM (mAb 14) (Fig. 1). The majority of the IgM have a range of different isoforms with isoelectric points spreading over around one pH unit, only mAb 5 and mAb 14 have a narrow isoform spectrum (Fig. 1). Recently we have developed a purication strategy for an IgM from this panel using anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC) [7]. While we were able to obtain a high purity, the concentration factor of this purication was low; therefore we decided to modify this strategy: the nal SEC step was replaced by an initial enrichment step using PEG precipitation and for the AEC step, a panel of resins were screened to identify one with high binding capacity. 3.1. PEG precipitation We used PEG 6000 which is a common precipitant for IgG and IgM [3,12]. First we determined the PEG concentration required for maximum precipitation by mixing equal volumes of hybridoma 84 supernatant and PEG 6000 to obtain a nal concentration of 17.5%

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 that prevents smaller precipitates to settle during centrifugation. The formation of small precipitates might have been due to the lack of mixing during precipitation. Thus in later experiments all samples were mixed, if not mentioned otherwise. Adjustment of the pH closer to the isoelectric point has been suggested to improve the precipitation yield [37]. For mAb 84 (pI 6.57.0) adjusting the pH to pH 5.0 and pH 6.0 resulted in a higher yield at lower PEG concentrations (Fig. 3). Only as little as 7.5% PEG were sufcient for high yield of mAb 84 for both either pH 6.0 or pH 5.0. From the pI it would be expected that mAb 84 has a positive net charge at pH 5.0 and pH 6.0 as a negative net charge at pH 7.4. Therefore, one would expect a similar precipitation yield at a positive and negative charge. However, from the results, we could assume that the surface charge distribution between the positively and negatively charged IgM is sufciently different to give this different precipitation behavior. Either there is more attraction between the mAb 84 molecules at pH 5.0 and 6.0 or less repulsion leading to precipitation at lower PEG 6000 concentrations. We did not implement the pH adjustment in the purication strategy even though the results were favorable as there is product loss (pH 6.0). In addition an additional clearance step is required to remove impurities precipitated by the pH shift.

Fig. 2. Precipitation of mAb 84 from serum-free hybridoma supernatant using different nal concentrations of PEG 6000.

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Fig. 5. AEC chromatogram for linear gradient elution of mAb 84. The fractions comprising mAb 84 and the impurities are indicated. Fig. 3. Comparison of the precipitation yield of mAb 84 from serum-free hybridoma supernatant which has been adjusted to pH 5.0 (black), pH 6.0 (light grey) or has been used without prior adjustment, pH 7.5 (dark grey).

Considering our reduced yield observed for unmixed precipitated samples, we studied the inuence of mixing on precipitation kinetics at a larger scale. We incubated sets of precipitated samples with and without mixing. As can be seen from Fig. 4, for the rst 15 min there is little inuence from mixing on the precipitation kinetics. This is likely so because the precipitate particles are still small enough to be unaffected by mixing. For particles smaller than 1 m diameter, it has been suggested that precipitation is mainly governed by diffusion, meaning the precipitation kinetics are inuenced by the viscosity of the solution, particle size and diffusivity. This is usually referred to as the perikinetic aggregation [44]. When particles become larger than 1 m in diameter, uid motion will cause particles to collide and aggregate. At this stage, mixing becomes pivotal to obtaining a high recovery; this is referred to as orthokinetic precipitation [44]. We observed that for mAb 84, precipitation particles around 1 m diameter are formed after 1530 min [45] which is in good agreement with the data presented here: between 15 and 60 min, the yield of the suspension which was mixed while incubation increases signicantly whereas the suspension which was not mixed during incubation showed only little increase in yield over time. This can most likely be explained by the formation of larger precipitate particles upon mixing which results in a higher yield during centrifugation.

We selected the following conditions for the PEG precipitation as the initial enrichment step for all hybridoma supernatants: continuous mixing of the hybridoma supernatant at a nal PEG concentration of 10% (w/v) with a magnetic stirrer for a minimum of 2 h at 4 C. 3.2. Anion-exchange chromatography (AEC) The AEC step was developed based on a previous purication strategy for mAb 84 [7]. From measurement of the breakthrough curves of the dissolved PEG precipitate for different AEC resins (data not shown), we selected Fractogel EMD DEAE (M) instead of the previously used EDA disk (monolith) due to the higher capacity observed. First, we performed a linear gradient elution (Fig. 5) to obtain the conditions needed for the step gradient elution. mAb 84 eluted in the rst peak was already at a high purity (data not shown) because the impurities were mostly found in a later eluting peak. For the rst batch of hybridoma 84 supernatant, the amount of impurities was low, as can be seen from Fig. 5. For the following batches of mAb 84 as well as all other clones the amount of impurities was signicantly higher than the amount of mAb 84 present; it is not clear why the level of impurities was so much lower for the rst fed-batch run. For the step elution of mAb 84, 24% elution buffer corresponding to approximately 280 mM NaCl (Fig. 6A) was used. Only minor amounts of mAb 84 were found in the owthrough and regenerate (data not shown) and as can be seen from the analytical SEC (Fig. 6B). mAb 84 of high purity can be obtained using this two-step purication strategy. The same process, linear gradient followed by step gradient elution, was performed for the other IgMs. For some IgMs, the concentration of the elution buffer in the AEC step had to be adjusted (Table 1). Table 1 gives the mass balance showing the concentration of IgM and total protein in the supernatant as well as the IgM concentration, purity and yield for the PEG precipitation and AEC step elution. For mAb 95, mAb 375 and mAb 432, the low yield in the PEG precipitation can be explained by the solubility of the IgM at 10% PEG 6000. Assuming a similar solubility for all IgMs, we can use the solubility of mAb 84 (12 g/ml) for some simple calculations: When mixing an equi-volume of supernatant with a 20% PEG, the mAb 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 solubility of 1.5 g/ml, a theoretical yield of only 50% is possible for mAb 95. This is in agreement with the data observed. The same is true for mAb 375 and mAb 432. For these IgMs, PEG precipitation would be more efcient if an initial concentration step is introduced

Fig. 4. Precipitation kinetics for mAb 84 from hybridoma supernatant at a nal concentration of 10% (w/v) PEG 6000. The suspension was incubated with mixing (inverse triangle) and without mixing (full circle). Samples were drawn for quantication at specied time steps.

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Fig. 6. (A) AEC chromatogram of purication of mAb 84 with step gradient. The fractions comprising mAb 84 and the impurities are indicated. (B) The analytical SEC chromatogram of the hybridoma 84 supernatant (solid line), PEG precipitate (black dashed line) and AEC eluate (grey dashed-dotted line) shows that the majority of impurities can be removed during PEG precipitation while AEC leads to further purication and concentration of mAb 84.

or if the PEG is at a higher initial concentration, e.g. 50%. For mAb 5 and mAb 14, which also have yields <80%, the initial concentration is sufciently high so we would assume that their signicantly different isoelectric points as compared to the other IgM resulted in the lower precipitation efciency. At pH 7.5, both IgMs (mAb 5 and mAb 14) would be strongly charged hence intra-molecule repulsion results in hindered precipitation. For such cases, adjustment of the pH closer to its isoelectric point might increase the yield signicantly. The recovery for the AEC step is around 7993% with the exception of mAb 375 and mAb 529 which have a recovery of 5561%.

This is due to the strong tailing of the elution peak for mAb 375 and mAb 529 which was not collected to avoid excessive dilution of the puried mAb. We feel that such losses will be less pronounced at a larger scale because due of the observed higher concentrations of IgM in the eluate a less conservative peak collection could be applied. From Table 1 we can also see that most mAbs have a purity greater than 90%, with the exception of mAb 5 and mAb 375 which have a purity of 85.0% and 46.6%, respectively. The analytical SEC for mAb 5 does not show signicant presence of other proteins, a discrete symmetrical peak at the elution time of an IgM is observed (data not shown). It is possible that the presence of DNA affected the nal purity, as DNA will contribute to UV absorbance at 280 nm. For mAb 375, the analytical SEC showed the presence of other proteins besides mAb 375. An additional purication step will be needed to obtain the required purity. We found that hydroxyapatite gave good results in this case: mAb 375 is rst diluted to a phosphate concentration of 10 mM and then eluted with 300 mM phosphate in the presence of 10% (w/v) PEG 600 (data not shown). This method was suggested by Gagnon for the removal of antibody fragments and aggregates [29]. The difference in the purication efciency between mAb 375 and other antibodies with a similar pI, e.g. mAb 529 or mAb 95, is surprising but could be explained by differences in the surface charge distribution. Comparing the concentration of NaCl needed for elution, it can be seen that mAb 375 actually requires the highest NaCl concentration (300 mM NaCl) for elution, suggesting that the binding of mAb 375 to the AEC resin is stronger than for the other mAbs. As we noticed, the impurities usually have a stronger binding to the AEC resin, we assumed that they would be co-eluting with mAb 375 at the selected NaCl concentration. Besides adding an additional step, the purication could be improved by changes of pH or by switching to different resin chemistries, e.g. cation-exchange chromatography. For all IgMs, the supernatant and nal puried IgM after AEC were loaded onto SDS-PAGE (412%) and visualized using Coomassie staining (Fig. 7) or immunoblotting (Fig. 8). The protein transfer during immunoblot was not efcient, in particular for small-molecular weight proteins, which can be seen from the missing light chain (Fig. 8). Additional bands below 50 kDa can be observed for some of the AEC puried IgM in the Coomassie stain. These are most likely fragments visualized in the immunoblot. For mAb 375, the bands below 50 kDa on the Coomassie stain have no corresponding bands in the immunoblot. This corresponds to the observations 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 except mAb 84 by comparing the binding to hESC before and after purication using ow cytometry. As can be seen from Fig. 9, no signicant difference was found between the mAbs present in the supernatant

Table 1 The mass balance is given for the small-scale purication including hybridoma supernatants (SN) using PEG precipitation (PEG) and anion-exchange chromatography (AEC). The recovery is always given as step recovery/overall recovery. For AEC, the NaCl concentration needed for elution is given for each mAb. mAb 5 SN Volume [ml] IgM [g/ml] Total protein [mg/ml] PEG IgM [g/ml] Purity [%] Recovery [%] AEC NaCl for elution [mM] IgM [g/ml] Purity [%] Recovery [%] 190 57.7 2.90 205.2 30.0 71.1/71.1 288 1122.4 85.0 79.0/56.2 mAb 14 200 60.4 1.80 213.0 63.1 79.3/79.3 288 1563.5 >99.0 82.0/65.0 mAb 63 200 50.7 4.35 91.0 35.0 89.7/89.7 278 2605.0 93.0 79.3/71.1 mAb 84 100 93.0 0.62 229.2 35.0 91.4/91.4 278 1233.1 >99.0 87.0/79.5 mAb 85 100 139.5 5.55 356.4 52.8 89.9/89.9 278 1080.3 >99.0 93.1/83.7 mAb 95 155 6.9 1.78 23.4 17.6 50.5/50.5 278 62.4 >99.0 55.7/28.1 mAb 375 200 13.8 3.23 28 13.5 73.2/73.2 307 955.6 46.6 61.3/44.9 mAb 432 200 23.0 2.84 55.3 36.4 78.8/78.8 297 1008.1 95.5 84.2/66.3 mAb 529 200 78.9 2.26 282.7 57.5 85.3/85.3 278 2531.3 >99.0 67.1/57.3

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Fig. 7. Coomassie stain of SDS-PAGE (412%) of supernatant (SN) and AEC eluate (AEC) of all clones (M1, See Blue Plus2; M2, Mark 12).

and corresponding puried mAb solutions. As mAb 84 kills hESC within 30 min, no signal can be obtained by ow cytometry [41]. Instead we compared the killing efciency of the puried mAb 84 to that of the supernatant. mAb 85 binds to the same epitope as mAb 84 but has no effect on cell viability and is therefore used as a negative control (isotype control). We found that our purication strategy did not affect the activity of either mAb: mAb 84 had the same cytotoxic effect when used in supernatant or in its puried form while neither fraction of mAb 85 exhibited cytotoxic activity (data not shown). 3.3. Benzonase treatment of hybridoma supernatant A challenge of this purication process was the presence of high concentrations of DNA in the hybridoma supernatant. This was particularly obvious in an mAb 84 purication performed at larger scale where DNA precipitation was observed during PEG precipitation (data not shown). During this step, a large amount of mAb 84 (30%) was lost, which we assume was due to adsorption to the precipitated DNA. Additionally we found that part of the DNA forms a soluble precipitate that is co-puried with proteins and contaminates the dissolved precipitate. In the following AEC step, the DNA binds to the resin, resulting in a lower binding capac-

ity as well as a lower purity of the eluate. We therefore proposed treating of the hybridoma supernatant with DNAase prior to further purication. Benzonase is a genetically engineered endonuclease originating from Sewatia murcescens that can hydrolyze single- or doublestranded, linear, circular or supercoiled DNA as well as RNA resulting in the formation of oligonucleotides of 35 bases length [46]. The enzyme is active between pH 6.010.0 and from 0 C to 40 C but its activity can be affected by the presence of phosphate (>100 mM) or monovalent cations (>150 mM). Low concentrations of Mg2+ is crucial for its activity. The enzyme can be inactivated by addition of EDTA (>5 mM). It is necessary to test the optimum cleavage conditions, time and temperature, for the respective solution. Preliminary studies showed that addition of 50 U benzonase per ml of supernatant reduced the DNA level from 15 g/ml to 2 g/ml within 1 h when incubated at room temperature (data not shown). At 4 C the nal level was only reduced to 7 g/ml. Lower concentrations (10 U/ml) were not as efcient whereas higher concentrations (100 U/ml) showed no signicant improvement (data not shown). The conditions for this step were therefore set to 50 U benzonase per ml of hybridoma supernatant and incubation for 1 h at room temperature with stirring.

Fig. 8. Immunoblot of SDS-PAGE (412%) of supernatant (SN) and AEC eluate (AEC) of all clones (M1, See Blue Plus2; M2, Mark 12).

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Fig. 9. Flow cytometry assay for evaluating the binding efciency of the different IgM on hESC. The shaded peak gives the auto-uorescence signal of the hESC (negative control). The signal for the puried mAb (solid line) is in all cases similar if not slightly stronger than for mAb present in the supernatant (dotted line).

We compared the purication of antibodies from hybridoma 84 supernatant which has been treated with benzonase to the purication of one without benzonase (Table 2). During incubation with benzonase, the supernatant turned turbid; this precipitate was not removed and we assume that it formed the majority of the non-dissolvable precipitate observed after PEG precipitation. The dissolved PEG precipitate was ltered prior to AEC. However, due to the large amount of non-dissolvable precipitates present after benzonase treatment, a lter with a larger surface area was needed. The loss of 30 ml of dissolved PEG precipitate obtained from
Table 2 Comparison of the mass balance of a mAb 84 purication from benzonase-treated and untreated hybridoma supernatant (SN). The recovery during PEG precipitation (PEG) and anion-exchange chromatography (AEC) is given as step recovery/overall recovery. mAb 84 No benzonase SN Volume [ml] IgM [g/ml] Total protein [mg/ml] DNA [ng/ml] PEG Volume [ml] IgM [g/ml] Total protein [mg/ml] DNA [ng/ml] Purity [%] Recovery [%] AEC Volume [ml] IgM [g/ml] Total protein [mg/ml] DNA [ng/ml] Purity [%] Recovery [%] Column capacity [mg/ml] 200 121.2 0.71 18222.6 17.1 96.4/96.4 7.00 1192.0 1.8 15796.9 67.3 40.0/38.5 1.1 With benzonase 400 62.6 5.14 20980.5 175 124.8 0.28 1924.9 45.1 88.2/88.2 8.00 1982.0 2.0 312.9 99.1 70.5/62.2 1.7

the benzonase-treated supernatant can be ascribed to the use of a larger lter area (Table 2). Comparison of the ltered dissolved PEG precipitate with and without benzonase treatment (Fig. 10A) using analytical size-exclusion chromatography showed a significant difference: after benzonase treatment, all high-molecular weight impurities were removed. This suggests that these impurities are actually proteins bound to co-precipitating DNA resulting in apparent high-molecular weight proteins. For the AEC, the same volume of dissolved PEG precipitate was loaded, in both cases the column was overloaded and mAb 84 was found in the owthrough resulting in a low step recovery (Table 2). Fig. 10B shows the analytical size-exclusion chromatography of both AEC steps. Though the AEC eluate from the benzonase-treated supernatant showed presence of fragments and/or small-molecular weight impurities, it is most likely that these are also present in the AEC eluate in the untreated supernatant although they are less obvious because of the lower signal. Due to the adsorption behavior of IgM and the impurities present in the dissolved precipitate, overloading of the column leads to the replacement of IgM by impurities resulting in a lower purity of the eluate. This is the main reason for the lower purity of the purication without benzonase treatment (67.3%). Due to the lower amount of impurities present in the dissolved PEG precipitate from the benzonase-treated supernatant, the column is not being overloaded and the purity of the AEC eluate is signicantly improved (99.1%). The AEC eluate from the purication including the benzonase treatment has also a 50 reduction in DNA content and a higher nal concentration. The latter is due to the higher binding capacity of the column (1.5-fold). From these results, we decide to include benzonase treatment to our purication process for all mAbs. 3.4. Adsorption isotherms and kinetics A purication process can also be improved by selecting a chromatography resin with high capacity and mass transfer [39]. Initially we selected the Fractogel EMD DEAE (M) resin, due to our experience with the purication of unfolded proteins [40]. The Frac-

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Fig. 10. Comparison of the chromatograms of analytical size-exclusion chromatography of (A) the PEG precipitates and (B) the AEC puried mAb 84 obtained from benzonase treated (dotted line) or untreated (solid line) hybridoma 84 supernatant.

togel is a grafted resin where 10 nm long polymer chains carrying the ligands have been grafted on the inner and outer surfaces of the resins particles [47]. The ligands are therefore more exible resulting in the higher binding capacity, in particular for larger proteins [47]. We also selected Capto Q, a strong anion-exchange resin, with a high-ow agarose matrix as backbone with dextrans grafted onto the particles surface. This resin has been recommended for high volume process scale as well as for the purication of viruses. For the purication of large proteins, convection driven resins such as membranes and monoliths have also been suggested frequently [26,47,48]. We found that membranes have, at least at small scale, large extra-column effects, which we felt disadvantageous as it leads to diluted eluate peaks. Therefore, we selected a monolith for representing convection driven chromatography resins. The EDA CIM MiniDisk is a weak anion-exchange resin with a ligand that

has a higher selectivity for IgM than for IgG or BSA [27]. We have used this resin successfully in our previous purication of mAb 84 [7]. Finally, we included Toyopearl, a resin with the same backbone as Fractogel EMD while the surface of Fractogel is covered with polymer chains carrying the ligands, called tentacles the ligands for Toyopearl are directly immobilized onto the surface of the resin. For Fractogel and Toyopearl, different particle diameters were tested, the medium size (6090 mm) and the small size (2040 mm). This was to evaluate if the larger outer surface of the smaller particles would lead to an increased adsorption capacity. First, we evaluated the adsorption equilibrium for puried mAb 84, see Fig. 11 and Table 3. We selected an incubation time of 6 h before evaluating the concentration of bound and unbound IgM. As shown later for the adsorption kinetics for puried mAb 84 (Fig. 13) 6 h was sufcient to reach equilibration for most resins. We found that Fractogel EMD DEAE (M) and (S) had the highest capacity (6066 mg/ml) and steepest isotherm (KA > 68 ml/mg), followed by Capto Q with a capacity of 55 mg/ml and a KA 56 ml/mg. Toyopearl resins had the lowest capacity with only 1516 mg/ml. The observed difference in the KA value of the Toyopearl DEAE650 M and DEAE-650S is unclear. However, it might be due to non-equilibrium conditions for the Toyopearl DEAE-650 M. This would be in agreement with data on the adsorption kinetics shown later. The results for the porous resins are not very surprising due to the size of IgM as accessibility to the binding sites is hindered resulting in a smaller capacity compared to smaller proteins, e.g. BSA (100 mg/ml, Fractogel EMD DEAE (M)). The difference between the grafted resins, Fractogel EMD and Capto Q, and the non-grafted resin, Toyopearl can be traced back to the specic properties of grafted resins [47,49]. Also notable is that there is no difference if smaller or larger particles are used, the maximum capacity for Fractogel EMD DEAE (M) and (S) or Toyopearl DEAE-650 M or DEAE650S are almost the same. This suggests that IgM not only adsorbs on the outer surface of the particle, but rather diffuses into the porous network of the particles. The EDA CIM disk had a capacity (24 mg/ml) higher than Toyopearl but lower than Fractogel and Capto Q resins. The EDA CIM disk has a surprisingly low KA (3 ml/mg) despite the high afnity for IgM reported by Brne et al. [27]. However, in this work, a polyclonal IgM was used and the shallow isotherm slope might be specic for our mAb 84. From later kinetic studies, we found out that the selected 6 h of incubation time might be insufcient for porous resins, in particular for Toyopearl DEAE-650C (M) and Capto Q; therefore slight changes in the 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 of binding sites (1113) were found. The differences in the intercept of Toyopearl 650C DEAE (M) and Toyopearl 650C DEAE (S) could be due to variations of the effective total ion-exchange capacity,

Fig. 11. Adsorption isotherms for puried mAb 84 on different chromatographic resins: (A) EDA CIM disk and Capto Q, (B) Toyopearl DEAE-650 M and Toyopearl DEAE-650S and (C) Fractogel EMD DEAE (M) and Fractogel EMD DEAE (S). The obtained data points were tted to the Langmuir isotherm (Eq. (1)); the t (solid line) and the 95% condence interval (dashed lines) are given.

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Table 3 Particle diameter, pore diameter, apparent adsorption isotherm parameters and apparent number of binding sites and equilibrium constant A for the different AEC resins investigated. Particle diameter Dp [m] Fractogel EMD DEAE (M) Fractogel EMD DEAE (S) Toyopearl DEAE-650M Toyopearl DEAE-650S Capto Q CIM EDA (monolith) 4090 2040 4090 2050 90 (d50v ) Not applicable Pore diameter dp [nm] 80 80 100 100 Not given 1500 [48] Apparent adsorption isotherm parameters qm [mg/ml] 66.14 59.62 16.43 15.03 55.20 24.34 1.31 1.07 0.39 0.54 3.16 3.20 KA [ml/mg] 85.98 68.45 19.47 6.73 56.59 3.21 32.53 9.37 2.78 1.17 11.84 1.04 Number of binding sites z [] 17.1 18.6 13.7 11.4 13.4 15.1 1.3 1.7 1.4 0.8 1.0 1.0 A [] 6.6 1.1 (1010 ) 4.1 9.2 (1011 ) 9.9 2.1 (1013 ) 6.6 1.3 (1012 ) 4.4 2.5 (1010 ) 4.9 4.1 (105 )

between the resins rather than differences in the adsorption behavior [43]. For Fractogel, both resins gave similar number of binding sites as well as similar intercepts. Interestingly, we found that for the elution of mAb 84 from the EDA CIM disk, a higher NaCl concentration was required than for all the other resins. As the number of binding sites is within the range of the other resins (15) and the equilibration association constant from the isotherm is actually the lowest for all resins, we did not expect the need for such a stringent elution condition. Possible reasons could be a high effective total ion-exchange capacity ( ) or strong interactions between the sites involved in adsorption of the ligand (ethylene diamine) and mAb 84. The latter could explain the higher afnity for the EDA to the IgM as compared to other resins. From our measurements of the adsorption capacity and number of binding sites, we found that Fractogel EMD DEAE is a good choice for the purication of mAb 84 as well as the other IgMs in our panel (Table 1). The results might be different if there is competitive adsorption of impurities present in the dissolved PEG precipitates. Also the issue of mass transfer has to be considered to nd the resin with the optimum dynamic binding capacity. We performed uptake kinetics using the method of shallow bed with puried mAb 84 and dissolved PEG precipitate with and without benzonase treatment. Although the shallow bed method is convenient for the measurement of adsorption kinetics, it should be noted that even slight variances in the resin volume (10 l) can lead to signicant errors in the nal data. The curves should therefore rather be seen as indicative of a trend of the adsorption kinetics. We performed an empirical t for better visualization of the data. Fig. 13A gives the uptake curves for mAb 84 on the EDA CIM MiniDisk. The uptake curve of the puried mAb 84 showed the advantage of a monolith for adsorption of large molecules. This

R Fig. 12. The ionic strength of the elution peak (CM ) was plotted against the respective normalized gradient slope ( ) and the data points tted according to Yamamoto and Ishihara [43] (solid line). From the slope the number of binding sites (z) and from the intercept the binding strength can be evaluated.

is apparent at the initial time point (5 min) where the maximum capacity (4 mg/ml) is obtained. Longer incubation times do not affect the binding capacity thereafter. The slight increase after 240 min of incubation could be ascribed to aggregation on the column or multi-layer adsorption. However, looking at the dissolved PEG precipitate without benzonase treatment, we nd an extremely low capacity for mAb 84. The DNA and other impurities are probably adsorbing at a similar rate as the mAb 84 and are most likely at a higher afnity. For the dissolved PEG precipitate after benzonase treatment, low incubation times (5 min) result in a capacity similar to the puried mAb 84 but upon longer incubation, capacity for IgM decreases. We found from the analytical SEC that this is due to the impurities present in the dissolved PEG precipitate which seem to bind with a higher afnity than IgM (data not shown). A similar displacement effect can be observed for all porous resins; the capacity for mAb 84 present in the dissolved pellet increases initially but after 23 h incubation, it drops to relatively low values. This suggests that for these resins, loading times for AEC should be kept below 2 h or IgM might be replaced by impurities resulting in low recovery. We found that for Capto Q, the presence of impurities and DNA led to a signicant decrease in capacity (Fig. 13B). For dissolved PEG precipitate from benzonase-treated supernatant, the capacity was still comparable to the monolith (5 mg/ml) while for the precipitate from the untreated supernatant, it was below 1 mg/ml. Comparing the kinetics of Toyopearl DEAE 650 M (Fig. 13C) and Toyopearl DEAE 650S (Fig. 13D) we found, that the saturation of the bigger particles took longer than for the smaller particles. After 6 h the Toyopearl DEAE 650C (M) was still not in equilibrium with the feed. This was probably due to the bigger diameter of the (M) particles (4090 m) as compared to the (S) particles (2050 m). Interestingly for both resins, the dissolved PEG precipitate with and without benzonase treatment showed an identical uptake behavior as the puried mAb 84 in the initial adsorption phase. After 13 h, the amount of mAb 84 adsorbed from either dissolved precipitates to Toyopearl dropped again to a low level. Besides the monolith, Fractogel resins seem to have the fastest adsorption kinetics. Approximately 80% of the capacity of Fractogel EMD DEAE (M) (Fig. 13E) for puried mAb 84 is reached after about 1 h while it takes about 2 h for Capto Q or Toyopearl resins. This is surprising as the pore diameter of the Fractogel EMD is smaller (80 nm) than for the Toyopearl resins (100 nm). It is most likely that the mass transfer mechanism for the diffusion of the mAb 84 into Fractogel EMD is different from that of a conventional porous resin [47]. For the dissolved pellet, the adsorption kinetics are slightly slower. Even for Fractogel EMD DEAE it takes about 23 h to reach the maximum capacity for mAb 84. Displacement effects are probably an inuencing factor in this case. Besides the IgM concentration, we also monitored the total protein and DNA content adsorbed from the dissolved precipitates to the resin (data not shown). For both, DNA as well as total protein, we observed that after 6 h of incubation the equilibrium is not yet reached. This slow adsorption kinetics is probably not a factor of

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Fig. 13. The adsorption kinetics for puried mAb 85 (solid square), and dissolved PEG precipitate obtained from benzonase-treated (inverse triangle) and untreated (solid circle) mAb 84 hybridoma supernatant. For easier visualization the data points for (A) EDA CIM Disk, (B) Capto Q, (C) Toyopearl DEAE-650 M, (D) Toyopearl DEAE-650S, (E) Fractogel EMD DEAE (M) and (F) Fractogel EMD DEAE (S) were approximated by an empirical t.

diffusivity but rather due to the replacement of adsorbed proteins such as mAb 84 by DNA or stronger binding proteins. With long loading times, it would be expected that mAb 84 as well as other weaker bound proteins are displaced from the column and only strong binding proteins and DNA are retained.

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

Table 4 Mass balance of purication of 2 l mAb 84. For the PEG precipitation step (PEG) the conditions of the hybridoma supernatant (SN) as well as the dissolved PEG precipitate (DP) and for the anion-exchange chromatography step (AEC) also the dissolved PEG precipitate (DP) and nal eluate containing the mAb 84 peak (EL) are given. Prior to PEG precipitation the hybridoma supernatant was treated with benzonase for DNA removal. The recovery is given as step recovery/overall recovery. Volume [ml] PEG SN DP AEC DP EL 2000.0 780.0 770.0 24.0 IgM [g/ml] 70.0 160.0 160.0 4075.0 Total protein [mg/ml] 4.42 0.25 0.25 4.26 DNA [ng/ml] 20081.8 581.8 581.8 1256.3 Purity [%] Recovery [%]

64.0

89.1/89.1

95.6

79.4/70.7

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Fig. 14. (A) Coomassie stain and (B) immunoblot for 2 L purication of mAb 84 showing the hybridoma 84 supernatant (SN), the supernatant of the PEG precipitate (SN2), the dissolved PEG precipitate (DP), the AEC owthrough (FT), the AEC eluate (EL) and the AEC regenerate (RE). The positive control (pos. ctrl.) is afnity-puried mAb 84, the negative control (neg. ctrl.) is cell culture media and the marker is See Blue Plus2 (Invitrogen).

particle but rather diffuses into the pores where it adsorbs. The diffusion is probably hindered due to bound IgM (20 nm), which decreases the free pore diameter We up-scaled our purication strategy to process a total of 2 l of hybridoma 84 supernatant (Table 4). A highly pure and concentrated mAb 84 could be obtained with our strategy outlined above (Fig. 14). The DNA removal was not as efcient as for the smallscale purications. In particular, the AEC step concentrated not only the mAb 84 but also the DNA present. If a higher DNA clearance is required, an additional purication step could be implemented. 4. Conclusion We developed a two-step purication strategy for a panel of monoclonal IgM from hybridoma with a pI of 5.57.7 comprising of PEG precipitation and anion-exchange chromatography (AEC). The process was characterized and optimized with regards to precipitation kinetics, resin selection and loading time. While the whole characterization of the purication was quite time-consuming, it gave us valuable information on the presence and effect of impurities, e.g. the competitive adsorption of impurities on the AEC resins at longer loading times. This information can be used to obtain a highly efcient and rugged purication process. Nomenclature

Vg Vt Vv z

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

Acknowledgements This work was supported by the Biomedical Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. The authors would also like to thank Yu Hosokai and Dr. Lee Yih Yean 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. Lgdberg, W. Kastern, L. Bjrck, B. Akerstrm, 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.

A c c0
R CM KA Ke

q qmax V V0

=Ke z gradient slope (M/ml) concentration of antibody in suspension (mg/ml) concentration of antibody in stock solution (mg/ml) normalized gradient slope (=L/v) (M) ionic strength at peak maximum (M) apparent adsorption equilibrium parameter (ml/mg) equilibrium association constant total ion exchange capacity (mequiv./ml) concentration of antibody bound to chromatography resin (mg/ml) apparent maximum adsorption capacity (mg/ml) volume of suspension (ml) volume of antibody stock solution (ml)

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A. Tscheliessnig et al. / J. Chromatogr. A 1216 (2009) 78517864 [39] A. Jungbauer, J. Chromatogr. A 1065 (2005) 3. [40] A. Tscheliessnig, B. Kanatschnig, R. Hahn, A. Jungbauer, 6th European Symposium 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 Symposium 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. Mller, J. Chromatogr. A 1006 (2003) 229. [48] A. Jungbauer, R. Hahn, J. Sep. Sci. 27 (2004) 767. [49] E. Mller, Chem. Eng. Technol. 28 (2005) 1295.

[26] X. Santarelli, F. Domergue, G. Clofent-Sanchez, M. Dabadie, R. Grissely, C. Cassagne, 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. Torne, 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.