Journal of Membrane Science 318 (2008) 311–316

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Journal of Membrane Science

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

Fractionation of monoclonal antibody aggregates usingmembrane chromatography

ilton

Abs)sirabeparaggrerapi

ath-1ur hymor

Lu Wang, Raja Ghosh ∗

Department of Chemical Engineering, McMaster University, 1280 Main Street West, Ham

a r t i c l e i n f o

Article history:Received 24 January 2008Received in revised form 20 February 2008Accepted 22 February 2008Available online 2 March 2008

Keywords:Monoclonal antibodyAggregatesCampath-1HMembrane chromatographyHydrophobic interaction

a b s t r a c t

Monoclonal antibodies (mis a common though undeanalysis and preparative sticularly for higher order a(HIMC) based method forwas able to resolve Campalso strongly supported oaggregation, i.e. a dimer is

1. Introduction

Monoclonal antibodies (mAbs) comprise an important class ofbiopharmaceuticals [1] and are widely used as diagnostic agents

[2]. MAbs, being recombinant molecules which do not occur nat-urally are less stable compared to serum antibodies. Consequentlythey show significant tendency to self-associate into aggregates [3].Several factors such as high concentration [4], extremes of pH [5],high shear rates [6] and thermal stress [7] are known to encour-age aggregate formation. In a biopharmaceutical manufacturingprocess mAb aggregates can be formed during fermentation [8],purification [5] and storage [8]. Clinically used mAbs should beaggregate free since aggregates are immunogenic and are knownto cause adverse reactions in patients [9].

Protein-A and protein-G based affinity chromatography whichare widely used for mAb purification do not distinguish betweenthe antibody monomer and its aggregates [8,10] since these lig-ands recognize and bind onto the Fc region of antibodies whichare unaffected by the aggregation process. Aggregates are thereforeseparated from monomers by preparative size exclusion chro-matography (SEC) while the aggregate content of mAb samplesis determined either by analytical SEC or by native polyacry-lamide gel electrophoresis (PAGE) [8]. SEC is time-consumingand cannot resolve higher order aggregates. Native PAGE is able

∗ Corresponding author. Tel.: +1 905 525 9140x27415; fax: +1 905 521 1350.E-mail address: rghosh@mcmaster.ca (R. Ghosh).

0376-7388/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.memsci.2008.02.056

, Ontario L8S 4L7, Canada

comprise an important class of biopharmaceuticals. Aggregation of mAbsle occurrence. Size exclusion chromatography (SEC) which is used for bothtion of mAb aggregates is slow and results in poorly resolved peaks, par-

gates. We describe a hydrophobic interaction membrane chromatographyd and efficient separation and analysis of mAb aggregates. Our methodH monomer, dimer, trimer, tetramer and pentamer. The results obtainedpothesis that the hydrophobicity of mAb increases with the degree of

e hydrophobic than a monomer, a trimer more than a dimer, and so on.© 2008 Elsevier B.V. All rights reserved.

to resolve higher order aggregates but is extremely slow. In arecent paper we discussed the separation of hIgG1-CD4 monomerfrom its dimer using a hydrophobic interaction membrane chro-matographic (HIMC) technique [11]. This technique which utilizedthe difference in hydrophobicity between the mAb monomerand its dimer was fast and gave significantly better resolutionof peaks than SEC. In a more recent paper the use of column

based hydrophobic interaction chromatography for monoclonalantibody aggregate removal has been discussed [12]. Our currentwork attempts the separation of higher order mAb aggregatessuch as trimer, tetramer and pentamer using HIMC. This isbased on our hypothesis that the hydrophobicity of mAb wouldincrease with degree of aggregation since aggregate formationis known to take place through interactions between the rel-atively hydrophilic Fab regions of the IgG1 type mAbs [10].Researchers have shown that the Fc region is more hydrophobicthan the Fab regions [13]. The Fc region of the mAb is neitherinvolved in aggregate formation nor is affected by the process[10].

The model mAb used in our current study is Campath-1H (alsoknown as Alemtuzumab). This is a humanized IgG1 type mAbagainst human leukocyte antigen CD52 [14].Campath-1H is syn-thesized by grafting hypervariable loops specific for CD52 onto ahuman antibody framework. Campath-1H is used for the treatmentof multiple sclerosis, organ transplant rejection [8] and severaltypes of leukemia [15]. We examined four types of polyvinylidinefluoride (PVDF) membranes for their mAb binding properties andselected the most appropriate among these for the HIMC experi-

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ments. The working principle was that the mAb and its aggregatedforms would bind reversibly on the selected membrane in the pres-ence of high anti-chaotropic salt concentration and these would beeluted out in order of increasing hydrophobicity on application of anegative salt concentration gradient. The fractionated samples thusobtained were analyzed by SEC and native PAGE to determine theiridentity. The separation process was optimized for high resolution.

2. Materials and methods

2.1. Materials

Humanized monoclonal antibody Campath-1H monomer(batch no. 44) and Campath-1H dimers (batch no. 23) (note:the term dimers denotes a mixture of mAb dimer andhigher order aggregates; this term is commonly used in theindustry) were kindly donated by the Therapeutic Antibody Cen-tre, University of Oxford, UK. Polyvinylidene fluoride (PVDF)microfiltration membranes (hydrophilic 0.1 �m pore size, cata-logue # VVLP09050; hydrophilic 0.22 �m pore size, catalogue# GVWP14250, hydrophilic 0.65 �m pore size, catalogue #DVPP04700; and hydrophobic 0.1 �m pore size, catalogue #VVHP04700) used as chromatographic media were purchased fromMillipore, USA. All samples and mobile phases used in the chro-matographic experiments were prepared using 20 mM sodiumphosphate buffer (pH 7.0) which in turn was prepared using ultra-pure water (18.2 M� cm) obtained from a Diamond Nanopurewater purification unit (Barnstead, USA). All chemicals used in theexperiments, e.g. sodium phosphate (mono- and di-basic), ammo-nium sulfate and sodium chloride were purchased from SigmaAldrich, Canada.

2.2. Contact angle measurement

The suitability of the different PVDF membranes for carryingout HIMC was assessed by contact angle measurement. The contactangles obtained with water and 1.5 M ammonium sulfate solutionin water were determined by using a DSA1 Drop Shape Analysissystem (Kruss, USA) under sessile drop measurement mode, thedrop volume being 0.02 mL. The data obtained was recorded byDSA1 Drop Shape Analysis software.

2.3. mAb Binding and elution experiments

The suitability of the PVDF membranes for HIMC was furtherassessed using mAb binding and elution experiments carried outusing an AKTA Prime liquid chromatography system (GE HealthcareLife Sciences, Canada). Two membrane discs (effective diame-ter = 18 mm) were housed in a custom designed membrane module[16] which were then integrated with the AKTA Prime system. Theabsorbance at 280 nm wavelength and conductivity of the efflu-ent from the module were continuously measured and the datawas logged into a computer using Prime View (GE Healthcare LifeSciences, Canada). The mAb was bound to the membrane from apulse of injected sample in the presence of 1.5 M ammonium sulfatecontaining buffer and eluted using ammonium sulfate free buffer.These experiments were carried out at 3 mL/min flow rate. Theamounts of protein bound on the membrane and that subsequentlyrecovered were determined from the area under the curve data forthe unbound and eluted peaks, based on appropriate calibration.

2.4. Non-reducing native PAGE

Samples were tested for presence of mAb aggregates by acidicnon-reducing native polyacrylamide gel electrophoresis [8] (PAGE)

ne Science 318 (2008) 311–316

(10% gel) using a Hoefer MiniVE vertical electrophoresis unit (GEHealthcare Life Sciences, Canada). The acidic pH ensured that themAb and its aggregates were all positively charged and the gelswere run with reversed polarity at 140 V (∼40 mA current). Thegels were stained with Coomassie blue dye to visualize the proteinbands.

2.5. Reducing SDS-PAGE

Campath-1H monomer and dimers were analyzed by reducingSDS-PAGE (12.5% gel) using a Hoefer MiniVE vertical electrophore-sis unit (GE Healthcare Life Sciences, Canada) after treatment withdithiothreitol (DTT) to break the disulfide bonds (–S–S–). These gelswere run with normal polarity at 120 V (∼35 mA current). Proteinbands were visualized by staining with Coomassie blue dye.

2.6. Protein-A chromatographic analysis

Campath-1H dimers were analyzed by protein-A affinity chro-matography using a HiTrapTM rProtein-A FF affinity column (GELife Sciences, Canada). The binding buffer used in this experimentwas 20 mM sodium phosphate (pH 7.0) and while the eluting bufferwas 100 mM sodium citrate (pH 3.0). Five hundred microliters ofdimers sample was injected and the separation was carried out at1 mL/min flow rate.

2.7. Separation of aggregates by HIMC

The analytical HIMC experiments were carried out using thesame experimental set up as used for testing mAb binding andelution. Two membrane discs were used in these experiments.The binding buffer contained 1.5 M ammonium sulfate and theseparation was carried out at 3 mL/min flow rate. Five hundredmicroliters of mAb dimers (i.e. aggregate mixture) prepared inbinding buffer and having a total content of 0.12 mg were injectedinto the membrane module. The bound proteins were eluted outwith ammonium sulfate free buffer using a negative salt gradient,going from 0% to 100% eluting buffer over 90 mL. The preparativeHIMC experiments were carried out using a stack of 5 membranediscs housed in the same membrane module. Five milliliters ofdimers solution prepared in the binding buffer and containing1.05 mg of total protein were injected for fractionation of different

components. The effect of gradient length on efficiency of separa-tion was examined. Samples corresponding to each of the elutedpeaks were collected and analyzed by SEC, non-reducing nativePAGE to identify the components present in these. Prior to SEC andnative PAGE, these samples were concentrated and desalted usingcentrifugal ultrafilters (Amicon Ultra 30 kDa MWCO, Millipore, USA,catalogue # UFC903008).

2.8. Size exclusion chromatography

The Campath-1H dimers samples fractionated by HIMC wereanalyzed by SEC with a SuperdexTM 200 gel filtration column(10 mm i.d., 300 mm length, GE Life Sciences Canada, catalogue #17-5175-01) using a HPLC system (Prepstar 218, Varian Canada).Two hundred and fifty millimolar sodium chloride solution wasused as the mobile phase at a flow rate of 0.2 mL/min. A 100 �L loopwas used for sample injection. The molecular weight calibrationfor SEC was performed using High Molecular Weight Gel Filtrationcalibration kit (GE Life Sciences, Canada, catalogue # 28-4038-42).The SEC data was analyzed using Varian Star 6.2 workstation soft-ware.

embrane Science 318 (2008) 311–316 313

Fig. 2. Binding and elution of Campath-1H on different PVDF membranes (elut-ing buffer: 20 mM sodium phosphate buffer, pH 7.0; binding buffer: elutingbuffer + 1.5 M ammonium sulfate).

on the contact angle measurements. The hydrophilic PVDF mem-brane with 0.22 �m pore size was used in all subsequent HIMCexperiments.

The Campath-1H dimers sample was analyzed by polyacry-lamide gel electrophoresis. Fig. 3 shows the acidic native PAGE (A)run with reversed polarity and the reducing SDS-PAGE (B) obtainedwith the mAb dimers. Five distinct bands can be seen on the stainednative gel, the lowest corresponding to the Campath-1H monomer,and the degree of aggregation increasing toward the top of the gel.The SDS-PAGE was run after treating the sample with dithiothre-itol (DTT) to break the disulfide bonds (–S–S–) between the mAblight chains and heavy chains. The stained gel shows two bands, onecorresponding to a molecular weight of 55 kDa (heavy chain) and

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Fig. 1. Results obtained from contact angle experiments carried out with differ-ent PVDF membranes at two solution conditions: in the absence and presence ofammonium sulfate. The ammonium sulfate concentration used was 1.5 M.

3. Results and discussion

PVDF is inherently hydrophobic but can be hydrophilizedusing appropriate techniques [17]. It is common practice in themembrane industry to refer to such hydrophilized hydrophobicmembranes as hydrophilic membranes. Hydrophilic PVDF mem-branes have been shown to be suitable to carrying out HIMCsince they demonstrate hydrophobic properties in the presenceof high anti-chaotropic salt concentration, i.e. can bind proteins,but are quite hydrophilic in the absence of such salts, i.e. canrelease the bound proteins [18]. Fig. 1 shows the results of thecontact angle experiments carried out with the different PVDFmicrofiltration membranes at two solution conditions, i.e. withand without ammonium sulfate. The addition of anti-chaotropicsalts like ammonium sulfate result in the removal of the structuredwater layer adjacent to the membrane surface and accentuate theirhydrophobic properties. However, as soon as the salt is removedthe water layer recovers and the surface becomes hydrophilic. Thehydrophobic PVDF membrane with 0.1 �m pore size was studiedmainly for comparison. The contact angle results indicate that itshydrophobicity did not change significantly with salt concentra-tion, i.e. it was hydrophobic at both solution conditions. With thehydrophilic PVDF membranes, the contact angles increased quitesignificantly due to the presence of 1.5 M ammonium sulfate, clearlyindicating that these became hydrophobic. The biggest change incontact angle was observed with the one with 0.22 �m pore size.

This membrane had the smallest contact angle at the salt free con-dition i.e. it was the most hydrophilic. However, in the presence ofsalt, its contact angle was greater that the two other hydrophilicPVDF membranes examined. These results seemed to suggest thatthe membrane with 0.22 �m pore size was most suitable for HIMC.

Fig. 2 shows the results of the mAb binding and elutionexperiments carried out with the different PVDF membranes. Inthese experiments the membranes were challenged with 5 mL of0.3 mg/mL Campath-1H monomer solution prepared using bind-ing buffer i.e. having 1.5 M ammonium sulfate concentration. Thehydrophobic PVDF membrane bound all the mAb from the injectedsample but the binding was irreversible in nature as evident fromthe lack of an elution peak. Among the hydrophilic PVDF mem-branes, the one with 0.22 �m pore size adsorbed the maximumamount of mAb (based on the area of the unbound peak). Based onthe amount of Campath-1H eluted, the reversible binding capac-ity of this membrane was estimated to be around 20 mg/mL ofmembrane bed volume. The monoclonal antibody recovery fromthe membrane was approximately 97%, this data being obtainedby material balance. The results obtained in the mAb binding andelution experiments were consistent with the expectations based

the other 25 kDa (light chain). The SDS-PAGE results prove that the

Fig. 3. Polyacrylamide gel electrophoresis of Campath-1H dimers (A) acidic nativePAGE run with reversed polarity (B) reducing SDS-PAGE.

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Fig. 4. Protein-A affinity chromatogram obtained with Campath-1H dimers (elut-ing buffer: 20 mM sodium phosphate buffer, pH 7.0; binding buffer: elutingbuffer + 1.5 M ammonium sulfate).

bands observed on the native gel were due to the mAb and its aggre-gates alone and no other proteins were present in the Campath-1Hdimers sample at detectable concentration.

Fig. 4 shows the protein-A affinity chromatogram obtained withCampath-1H dimers. All components present in the dimers, i.e.the mAb and its aggregates bound to the affinity column at condi-tions favorable for binding (pH 7.0) and were eluted out as a singlepeak at acidic pH conditions. Protein-A binds antibodies throughtheir Fc region. Therefore the Fc region of the mAb aggregates wereunaffected by the aggregation process. These results are consis-tent with reports by earlier workers [8,10] that protein-A affinitychromatography could not be used to separate mAb from theiraggregates. Protein aggregation has been shown to be initiated by

Fig. 5. Analytical HIMC chromatogram obtained with Campath-1H dimers (numberof membrane discs: 2; sample volume: 500 �L; amount injected: 0.12 mg; flow rate:3 mL/min; gradient length: 90 mL; eluting buffer: 20 mM sodium phosphate buffer,pH 7.0; binding buffer: eluting buffer + 1.5 M ammonium sulfate).

Fig. 6. Preparative HIMC separation of Campath-1H dimers (number of membranediscs: 5, sample volume: 5 mL, amount injected: 1.05 mg, flow rate: 3 mL/min, gra-dient length: 90 mL, 100 mL and 130 mL).

specific interactions between folding and unfolding intermediates[19]. Earlier workers have reported that Campath-1H dimer forma-tion takes place due to Fab–Fab interaction [10]. The Fab regionsof the mAb have lower microstability [20] compared to that ofthe Fc region, making them more prone to unfolding and refold-ing and hence more likely responsible for aggregate formation. TheFab regions of the mAb molecules involved in aggregate formationwould therefore be present towards the inside of these complexes.Consequently the Fc regions of these molecules are more likely tobe more exposed to the outside. In a recent study it was shown that

Fig. 7. SEC chromatograms obtained with the dimers (feed) and fractionated sam-ples obtained by preparative HIMC using 90 mL negative salt gradient.

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when an IgG molecule bound to a PVDF membrane via hydropho-bic interaction, it still retained the ability to bind its antigen [21].

This clearly showed that the antigen binding Fab regions were notinvolved in the membrane binding process which presumably tookplace through the more hydrophobic Fc region. Nagaoka et al. [13]have also shown that IgG binding onto hydrophobic polymer sur-faces took place through the Fc region [13] and concluded that thisregion was more hydrophobic than the Fab region. Based on theseobservations we hypothesized that as the degree of mAb aggrega-tion increased, a greater proportion of the external surface area ofthese complexes would be occupied by the Fc region. As a resultthe hydrophobicity would increase with increase in degree of mAbaggregation. To test this hypothesis, we carried out HIMC experi-ments with the dimers.

Fig. 5 shows the analytical HIMC chromatogram obtained witha stack of two hydrophilic PVDF membrane discs (having 0.22 �mpore size) by injecting 500 �L of Campath-1H dimers (having atotal protein content of 0.12 mg). The purpose of this experimentwas to test the feasibility of resolving the different mAb aggregatebased on hydrophobicity difference: the more hydrophobic the pro-tein, the later it would appear on the negative salt gradient. Thechromatogram shows 5 distinct peaks and a shoulder, possibly cor-responding to the monomer, dimer, trimer, tetramer, pentamer and

Fig. 8. Acidic, non-reducing native PAGE results obtained with dimers and HIMC resolved4: lane H and peak 5: lane G).

ne Science 318 (2008) 311–316 315

hexamer respectively. The amount of protein injected in the ana-lytical HIMC experiment was quite low and this did not allow for

sufficient protein samples to be collected from each of the peaksfor identity determination. In order to do this, preparative HIMCexperiments were carried out using the same membrane modulewith more membrane discs.

Fig. 6 shows the preparative HIMC chromatograms obtainedwith three different negative salt gradients. In each of these exper-iments 5 mL of mAb dimers (containing 1.05 mg of total protein)prepared in binding buffer was injected into the membrane mod-ule which contained five membrane discs. This was followed bythe application of the appropriate negative salt gradient to elutethe mAb and its aggregates in order of increasing hydrophobicity.In each of the chromatograms, five distinct peaks presumable dueto monomer, dimer, trimer, tetramer and pentamer respectivelycan be observed. The retention times of the peaks increased butthe peak resolution did not get significantly better with increase ingradient length. In fact the first peak became quite broad with the130 mL gradient, this being a shallow gradient. The peaks obtainedwith the 90 mL gradient were quite well resolved and thereforecollected for identity determination using SEC and native PAGE.

Fig. 7 shows the SEC chromatograms obtained with the feed(dimers) and samples from each of the resolved peaks obtained

peaks. (Dimers: lanes B, C and F, peak 1: lane A, peak 2: lane D, peak 3: lane E, peak

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Table 1Estimation of molecular weight of the HIMC peak components

HIMC peak # SEC Retention time (min) Predicted molecular weight

1 57.1 208 kDa2 47.5 489 kDa3 43.5 644 kDa4 39.3 Out of calibration range5 38.7 Out of calibration range

Calibration molecular weight range 10–669 kDa. Calibration equation:Mw = 0.7611t2 − 108.98t + 3947.2, where t is retention time (min) and Mw ismolecular weight (kDa), R2 = 0.985.

from the preparative HIMC experiment with 90 mL negative saltgradient. Quite clearly SEC was able to separate the mAb monomerfrom its aggregates (see the feed chromatogram). However, eachaggregate type could not be resolved by SEC. In fact, the dimerschromatogram shows only three overlapping aggregate peaks andnot four as could be expected based on the preparative HIMCresults. The SEC retention times obtained with the HIMC fraction-ated samples showed an inverse relationship with the HIMC peakretention times, i.e. the molecular weight of the HIMC peak com-ponents increased with increase in retention time. This providedpreliminary evidence supporting our hypothesis, i.e. hydrophobic-ity increased with degree of aggregation. In order to verify thatthe fractionated samples indeed contained the monomer, dimer,trimer, tetramer and pentamer respectively, the SEC peak reten-tion times were compared with molecular weight calibration data(see Table 1). Based on the calibration equation shown in the tablethe molecular weights of the proteins present in the first, sec-ond and third HIMC peaks were estimated to be 208 kDa, 489 kDaand 684 kDa respectively. The molecular weight of IgG1 type mAbmonomer is in the 150–160 kDa range and therefore the dimerand trimer should have molecular weights in the 300–320 kDa and450–480 kDa ranges respectively. The main reason for this discrep-ancy is that the calibration proteins were globular while mAb andits aggregates had irregular shapes which made it harder for them topenetrate the pores of the SEC medium [3]. Consequently they hadlower retention times than globular proteins having similar molec-ular weights. The molecular weights of the mAb and its aggregateswere therefore overestimated by SEC. It is therefore safe to say thatthe first three HIMC peaks were due to mAb monomer, dimer andtrimer respectively. The fourth and fifth HIMC peaks were presum-ably due to tetramer and pentamer but this could not be verifiedby SEC on account of their retention times being out of range.

Fig. 8 shows the acidic non-reducing native-PAGE resultsobtained with the dimers and samples from the fractionate peaksobtained by preparative HIMC. The dimers sample was loadedon lanes B, C and F. The samples from HIMC peaks 1–5 wereloaded in lanes A, D, E, H and G respectively. The dimers sam-ple showed five distinct peaks corresponding to the monomer,dimer, trimer, tetramer and pentamer respectively. Quite clearly themajor components present in lanes A, D, E, H and G were the mAbmonomer, dimer, trimer, tetramer and pentamer respectively. TheHIMC technique was therefore able to resolve Campath-1H fromits aggregates. Each individual aggregate type from dimer to pen-tamer was resolved. These results provide further evidence that thehydrophobicity of mAb increased with degree of aggregation.

4. Conclusion

The HIMC technique discussed in this paper was more efficientthan SEC both in terms of speed and resolution. It was able toresolve Campath-1H monomer, dimer, trimer, tetramer and pen-tamer as separate peaks. This study also clearly demonstrated thathydrophobicity of mAb increased with degree of aggregation, i.e. a

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ne Science 318 (2008) 311–316

trimer was more hydrophobic than a dimer and so on. A method forselecting appropriate membrane for HIMC based on contact anglemeasurement was described. The conclusions drawn based on con-tact angle measurements were in excellent agreement with theresults obtained from the mAb binding and elution experiments.

Acknowledgements

We thank Drs. Geoff Hale, Pru Bird, Mark Frewin and other mem-bers of the Therapeutic Antibody Centre, Oxford University, UKfor donating the Camapth-1H monomer and dimers samples. WeiChen of Chemical Engineering Department, McMaster Universityis acknowledged for helping with the contact angle measurementexperiments. We thank Natural Science and Engineering ResearchCouncil of Canada (NSERC) for funding this study. LW thanks ShellCanada and China National Scholarship Council for personal schol-arships. RG holds the Canada Research Chair in BioseparationsEngineering.

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