Confocal imaging of chromatographic fouling under flow conditions

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 82:871–881 (2007)

Confocal imaging of chromatographicfouling under flow conditionsSun Chau Siu,1 Rihab Boushaba,1 Jonathan Liau,1 Rolf Hjorth2 andNigel J Titchener-Hooker1∗1The Innovative Manufacturing Research Centre (IMRC), University College London, Torrington Place, London WC1E 7JE, UK2GE Healthcare, Bio-Sciences AB, SE-75184, Uppsala, Sweden

Abstract

BACKGROUND: The fouling impact of selected fouling species was assessed by utilising confocal scanning lasermicroscopy (CSLM) to image a packed chromatographic bed during operation. A custom-made flow cell waspacked with Q Sepharose FF and loaded with partially clarified E. coli homogenate. Selective, multicolouredfluorescent dyes were used to label a bovine serum albumin (BSA) test protein (Cy5.5), dsDNA (PicoGreen)and host cell proteins (HCPs) (Cy3). The fouling caused by the various fluorescently labelled components wasvisualised as a result of the fluorescence emitted by the PicoGreen-labelled dsDNA and the Cy3-labelled proteinin the foulant stream, and by testing the adsorptive capacity of a test protein (BSA) onto the resin prior to andpost-fouling as well as following the application of a common CIP procedure.

RESULTS: Values for the effective diffusivity of BSA (De) were derived from the confocal images and the foulingimpact was assessed by comparing De values obtained from different fouling scenarios. Under the most extremeconditions examined, fouling caused a 20% reduction in capacity compared to a fresh bed. BSA diffusivity did notappear to be affected by the fouling conditions studied. Sequential CIP using 15 CVs of 1 mol L−1 NaCl then 15 CVsof 1 mol L−1 NaOH was shown to be effective in removing nucleic acids and HCPs. Subsequent BSA adsorptionshowed that the CIP regime successfully restored the column capacity to its original value. In contrast, 15 CVs of1 mol L−1 NaCl were ineffective in removing dsDNA but substantially removed HCPs.

CONCLUSION: CSLM was demonstrated to be a useful tool for visualising fouling mechanisms. Comparing theresults obtained by this technique using different modes of chromatographic operation provided insights into thefouling characteristics of finite baths versus packed beds. 2007 Society of Chemical Industry

Keywords: chromatographic fouling; confocal scanning laser microscopy; Escherichia coli; ion exchange; clean-in-place (CIP); packed bed; flow cell

NOTATION

C0 Feed protein concentration (mg mL−1)

De Effective diffusivity of solute (cm2 s−1)

D0 Free diffusivity in bulk fluid phase (cm2 s−1)

Dp Pore diffusion coefficient (defined on pore

sectional area basis) (cm2 s−1)

I1, I2 Integrals defined in Eqns (2) and (3)

kf External fluid film mass transfer coefficient (cm

s−1)

qs Saturation adsorbed-phase concentrations (par-

ticle volume basis) (mg mL−1)

Rf Radius of adsorption front (cm)

Rp Radius of adsorbent particles (cm)

t Time (s)

εp Inclusion porosity

η Ratio Rf/Rp

INTRODUCTIONFouling can have a serious, negative impact on theperformance of chromatography. Considerable effortis spent to prevent fouling species reaching the columnand in developing clean-in-place (CIP) protocols ofever-increasing complexity to mitigate their effects.Despite this, the knowledge of chromatographicfouling often seems anecdotal, with only a fewsystematic investigations currently reported in theliterature.1–4 Furthermore, conventional approachesto investigate chromatographic fouling only providean overall indication of the state of the matrixand do little to ascertain the mechanisms offouling.

The kinetic mechanisms underlying chromatog-raphy are reasonably well understood and havebeen discussed extensively in the literature for cleanchromatographic matrices.5 The adsorption of a soluteto a chromatographic bead is governed by threeprocesses: film mass transfer, pore diffusion and

∗ Correspondence to: Nigel J Titchener-Hooker, The Innovative Manufacturing Research Centre (IMRC), University College London, Torrington Place, LondonWC1E 7JE, UKE-mail: [email protected](Received 14 March 2007; accepted 3 May 2007)Published online 11 September 2007; DOI: 10.1002/jctb.1728

2007 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2007/$30.00

SC Siu et al.

adsorption kinetics. The kinetic mechanisms are pre-sumably far more complex in a scenario where foulingexists and this can directly influence the performanceof the column. Colloidal material may obstruct ornarrow bead pores, thus limiting intra-particle diffu-sion. Furthermore, specific binding of some foulingspecies to adsorption sites both on the bead surfaceand internal to the pore could alter the capacity of theadsorbent for the target protein and affect the positionof the breakthrough curve.2

The understanding of adsorption mechanisms ofmolecules within a porous support during chromato-graphic separations has been advanced via informa-tion gained from finite bath uptake6–8 and packedbed breakthrough experiments.9–11 Recently, the useof confocal scanning laser microscopy (CSLM) incombination with finite bath uptake has proven tobe a powerful tool for investigating transport andadsorption mechanisms within single chromatographicbeads.12–22 However, this approach does not allowreal-time, in situ observations within a packed bed.More recent publications describe the use of flow cellsin conjunction with CSLM to investigate adsorptionphenomena of clean materials in a packed bed and inreal time.23,24

Hubbuch et al.23 developed a flow cell that could bepacked with chromatography media and operated it asa fully functional mini-scale column. Using this flowcell in combination with CSLM, they visualised online,and in real time, the dynamics of protein adsorptionto porous stationary phases in packed bed mode.Specifically, they obtained adsorption profiles ofsingle- and two-component mixtures containing BSAand IgG2a during cation-exchange chromatography.The classical shrinking core model with pore diffusionas the dominant transport mechanism was foundto be adequate for describing the adsorption ofbovine serum albumin (BSA). Immunoglobulin G2a(IgG2a), on the other hand, appeared to exhibit adifferent transport mechanism in which an initialconcentration overshoot of bound IgG2a was detectedat the centre of the bead. For the two-componentsystem, adsorption was described as a superpositionof the transport adsorption mechanism of the single-component systems with a classical displacement of theweaker bound protein species. The authors observedthat even for adsorption to the same chromatographicmatrix, the uptake kinetics were a strong function ofthe actual protein being adsorbed. They also illustratedthe usefulness of the flow cell for in situ quantitativeinvestigations of protein adsorption dynamics within asingle chromatographic bead.

Dziennik et al.24 used CSLM to show that mecha-nisms other than diffusion may contribute to proteintransport in ion-exchange chromatography and thatthis may be exploited to achieve rapid uptake inprocess chromatography. The real-time studies wereperformed using a flow cell into which chromato-graphic particles were packed behind a microscopecover-slip. The design of the flow cell was different

from that of Hubbuch et al.23 but the method wassimilar in principle. The confocal images obtainedverified that protein diffusion at low ionic strengthconditions in all the adsorbents examined couldbe adequately described by classical shrinking corebehaviour but at relatively high salt concentrations(≥100 mmol L−1) diffuse adsorption fronts were seenin some adsorbents. Consequently, pore diffusivitiesfrom the confocal images were calculated from esti-mates of fractional uptake using linear regression ofthe finite volume solution of the shrinking core modelas described by Teo and Ruthven.25 This was basedon the radial position of the adsorption front and theassumption that the adsorption layer was saturated.

We recently demonstrated the effectiveness ofusing CSLM in combination with finite bath uptakeexperiments to visualise chromatographic fouling at asingle-bead level.26 The purpose of this paper is toapply the technique in order to visualise fouling ina packed bed, which is more relevant to industrialchromatographic processes. A flow cell, similar indesign to that used by Hubbuch et al.,23 was usedto visualise, in real time and in situ, the impact andthe degree of fouling caused by double-stranded DNA(dsDNA) and host cell proteins (HCPs) in a packedbed of Q Sepharose FF beads. By using the sameaggressive fouling stream of partially clarified E. colihomogenate as in a previous finite bath study,26

comparisons between the information gained underthe two modes of adsorption could be made.

MATERIALS AND METHODSChemicalsAll chemicals, unless specified otherwise, wereobtained from Sigma Chemical Co. Ltd (Poole, UK)and were of analytical grade. BSA (A-7030), wasof purity >98% Cy3 and Cy5.5 mono-reactivelabelling kits were obtained from GE Healthcare(Uppsala, Sweden). PicoGreen was obtained fromMolecular Probes Europe – Invitrogen (Leiden, TheNetherlands).

Chromatography matrixQ Sepharose Fast Flow, a strong anion exchanger, wasobtained from GE Healthcare. The mean particle sizewas ∼95 µm and the particle size range was 45–165 µmas quoted by the manufacturer.

Confocal flow cell and packingA flow cell of similar design to that used by Hubbuchet al.23 was used in the study. The design of theflow cell consisted of a Perspex block with precision-drilled inlets (45◦) on either side and an open channel(∼8 mm in length) grooved into the bottom of theblock. A window for observing under the microscopewas created by fixing a microscope cover-slip to theopen channel using epoxy glue (Ciba Araldite Rapid,Casco AB, Stockholm, Sweden). The window allowedobservations around the axial centre of the unit. The

872 J Chem Technol Biotechnol 82:871–881 (2007)DOI: 10.1002/jctb

Confocal imaging of chromatographic fouling under flow conditions

column had a total length of 22 mm, a cross-sectionalarea of 3 mm2 and a bed volume of 66 µL. The flow cellwas connected to a P-900 pump (GE Healthcare) forliquid-handling purposes. Polypropylene frits from GEHealthcare were placed at the ends of the flow channeland were held in place using conventional screwfittings from GE Healthcare (Uppsala, Sweden). Theflow cell was packed with equilibrated Q SepharoseFF by using a syringe and manually applying pressure.The packed flow cell was then equilibrated with 3 mLof 20 mmol L−1 Tris-HCl, pH 8.0, at a flow rate of0.2 mL min−1.

Confocal microscope systemCSLM experiments were performed with an invertedconfocal laser scanning microscope (Leica SP2DM IRBE, Leica Microsystems GmbH, Mannheim,Germany) equipped with helium/neon, krypton/argonand argon lasers. System control and data acquisitionwere achieved by the in-built Leica Control Software(version 2.5). A 63 × 1.2 water objective was used forthe experiments. A pinhole aperture of 114.48 µm wasused in all the measurements. The pixel depth was∼400 nm. BSA-Cy5.5, HCPs-Cy3 and PicoGreen-conjugated dsDNA were excited at 543 nm, 633 nmand 488 nm, respectively. Their emission detectionranges were set to 560–600 nm, 650–710 nm and505–535 nm, respectively. Images were recordedin succession to avoid any cross-talk (or bleed-through) between different detector channels whensimultaneously using the three fluorescent dyes.

Preparation of E. coli foulantThe foulant was prepared from a fermentation brothexpressing HumMAb4D5-8 Fab′ as an antibodyfragment from E. coli strain W3110 (pAC tAC4d5 Fab′). The fermentation was carried out inrepeated batch mode and grown on a defined mediumas described by Garcıa-Arrazola et al.27 The cellswere subsequently harvested and homogenised at500 bar(g) for five passes using a Lab-60 high-pressure homogeniser (APV Manton Gaulin, Everett,MA, USA). Cell homogenate was partially clarifiedby centrifugation in a J2-M1 laboratory centrifugewith a JA-10 rotor (Beckman Instruments Ltd,High Wycombe, UK) for 1 h at 10 000 rpm (RCF =13 000 × g). The final composition of the materialconsisted of 6.1 g L−1 proteins, 110 mg L−1 DNA and1.2 g L−1 solids. The supernatant was frozen at −20 ◦Cin 45 mL aliquots. A foulant aliquot was thawed onthe day of experimentation at room temperature. Gelelectrophoresis analysis revealed that the majority ofDNA fragments resulting from the high shear levelsencountered in the homogenisation step were in therange of 300–750 bp in size. Further details regardingthe E. coli foulant material used can be found in aprevious publication.26

Fluorescent labelling of BSA with Cy5.5BSA was dissolved in degassed conjugation buffer(100 mmol L−1 Na2CO3, pH 9.3) to a concentration

of 2 mg mL−1. 1 mL BSA conjugation solution wasadded to one vial of Cy5.5, pre-measured by themanufacturer and containing sufficient reactive dyeto label 1 mg protein. This mixture was incubatedfor 1 h. BSA test solution was made by adding 1 mLlabelled BSA to 9.0 mL unlabelled BSA dissolved indegassed 20 mmol L−1 Tris-HCl, pH 8.0. The molardye–protein labelling ratio (D/P) was measured as3. Free dye was reduced to a low level by subsequentdilution of the labelled fraction with unlabelled proteinas described by other authors.28 A series of controlexperiments were conducted to establish whether thereexist any interactions between the chromatographicmatrix and the free dyes or non-specific binding ofthe labelled BSA to the base, underivatised matrix(Sepharose CL-6B) and, in all cases, insignificantfluorescence was detected.26 Recent studies havesuggested that the labelling of proteins, under certainconditions, may alter binding behaviour and lead todisplacement effects during adsorption.28,29 For thesystem used in this study, we confirmed experimentallythat the labelled BSA had a similar retention time tothe unlabelled species by performing a test experimentin accordance with the methodology proposed byTeske et al.,28 whereby 25 µL pulses of Cy5.5-labelledBSA (D/P ratio of 3) and unlabelled BSA were injectedinto a 1 mL HiTrap Q Sepharose column. Our resultsshowed that, under the conjugation conditions weused, the residence time of Cy 5.5-labelled BSA andunlabelled BSA differed by <1% (refer to Fig. 1).

Multicolour fluorescent labelling of foulingmaterial to visualise HCPs and dsDNAMulticolour fluorescent labelling of biological samplesis routinely performed in fields such as proteomicsfor the purpose of identifying changes in proteinexpression with high sensitivity. In these applications,the capacity of these dyes to bind to the representativemajority of the protein population without significantly

Figure 1. Elution profiles of labelled and unlabelled BSA. Profileswere obtained by injecting 25 µL pulses of BSA containing aCy5.5-labelled fraction (D/P ratio of 3) and unlabelled BSA (2 mgmL−1) into a 1 mL HiTrap Q Sepharose column.

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SC Siu et al.

altering their behaviour is critical to the methods andinvestigations. In order to visualise fouling inside apacked bed under dynamic conditions, we appliedthe technique of multicolour fluorescent labelling totwo selected categories of known foulants: HCPs andDNA. Cy3 dye was used to label HCPs, whereasPicoGreen was the fluorophore of choice for dsDNAlabelling (due to its selectivity). It was reasonedthat as Cy dyes bind very generally through acharge-based mechanism, our approach ensures thata significant proportion of charged proteins, adequateto represent the HCPs population, will be labelled insuch an experiment. 0.5 mL of foulant solution wasincubated with 0.5 mL of 100 mmol L−1 Na2CO3,pH 9.3, degassed conjugation buffer for 1 h atroom temperature. The resultant 1.0 mL solution wasadded to pre-measured Cy3 dye (capable of labelling1 mg of protein) and incubated for a further 2 h atroom temperature. Foulant protein–Cy3 solution wasdiluted to a ratio of 1:20 (foulant protein–Cy3 tounlabelled foulant). To this Cy3-labelled mixture ofHCPs, PicoGreen was added at a concentration of10 µL mL−1 of foulant. The mixture was allowed toincubate for a further 1 h with frequent agitation. Itis important to note that carrying out the conjugationof PicoGreen to the foulant sample containing Cy3-labelled species does not compromise its binding todsDNA, because PicoGreen is selective for dsDNA. Inaddition, Cy dyes are not readily reactive with DNAas they only react with primary amine groups. Free Cydyes can only directly label amine-modified nucleicacids prepared via the post-label aminoallyl route.

Fouling of Q Sepharose FF bed in a flow cellA flow cell packed with Q Sepharose FF wasequilibrated with 20 mmol L−1 Tris-HCl, pH 8.0,for 20 min at a flow rate of 0.08 mL min−1, whichcorresponds to a linear velocity of 150 cm h−1.After equilibration, 5 column volumes (CVs) ofpartially clarified Fab′ E. coli homogenate preparedas previously described was loaded onto the packedbed at a flow rate of 0.08 mL min−1. Finally, the bedwas washed and re-equilibrated with 20 mmol L−1

Tris-HCl, pH 8.0, for 20 min at a flow rate of 0.08 mLmin−1 to remove unbound foulants.

BSA adsorption time series in a flow cellProtein adsorption time series studies were conductedon fresh, fouled and CIP-treated packed beds. Cy5.5-labelled BSA solution (2 mg mL−1) was loaded ontothe equilibrated bed at a flow rate of 0.08 mL min−1.Visualisation of the bed by CSLM was done in realtime and in situ, and confocal images were taken atpredetermined time intervals of 30, 60, 120, 180 and240 min.

CIP time seriesFlow cells packed with Q Sepharose FF were fouledas described above (‘Fouling of Q Sepharose FF bedin a flow cell’). After fouling and re-equilibrating,

the bed was first washed with 15 CVs of 20 mmolL−1 Tris-HCl, 1 mol L−1 NaCl, pH 8.0, at a flowrate of 0.08 mL min−1. During the wash, visualisationof the bed by CSLM was done in real time andin situ. Confocal images were taken at predeterminedtime intervals of 30, 60, 120, 180 and 240 min.After completing the first wash and prior to takinganother confocal scanning image, the bed was re-equilibrated with 5 CVs of 20 mmol L−1 Tris-HCl,pH 8.0, to ensure that the pH conditions under whichfluorescence emissions are recorded were identicalthroughout the runs. Subsequently, the bed waswashed with 15 CVs of 1 mol L−1 NaOH at a flowrate of 0.08 mL min−1. Similarly, visualisation of thebed by CSLM was done in real time and in situ andconfocal images were taken at predetermined timeintervals of 30, 60, 120, 180 and 240 min during thewash. After completing the NaOH wash, the bed wasre-equilibrated with 5 CVs of 20 mmol L−1 Tris-HCl,pH 8.0, to normalise the fluorescence signal, afterwhich another confocal image of the bed was taken.

Image analysisThe images of cross-sections through the centre of thebeads were analysed with the supplied Leica ControlSoftware (version 2.5) and recorded at a resolutionof 512 × 512 pixels. To reduce the backgroundfluorescence and noise, the images were generatedby averaging six scans per image. Further imageanalysis was done using ImageJ software (version1.31) (National Institutes of Health, Bethesda, MD,USA). The method of Hubbuch et al.23 was used tocalculate the amount of remaining labelled protein byintegrating the averaged radial intensity profiles to givea volumetrically averaged intensity.

Calculation of effective diffusivity from confocalimagesIf a shrinking core model can be assumed, then asingle lumped kinetic parameter or effective diffusivity,De, can be estimated directly from the confocalimages by using the position of the adsorption front.24

The approach assumes that the adsorption layer issaturated. The effective diffusivity is calculated fromthe linear regression of the infinite volume solutionof the model, which takes into account external masstransfer resistance25 as shown in Eqn (1):

Det

R2p

= εpDp

R2p

C0

qst =

[1 + εpDp

kfRp

]I2 − I1 (1)

where εp is the intraparticle porosity, Dp is the porediffusivity, C0 is the feed protein concentration, Rp isthe particle radius, Rf is the radial position of the front,kf is the external fluid film mass transfer coefficient,qs is the saturation adsorbed-phase concentrationand:

I1 =∫ η

1η dη = 1

/2(η2 − 1) (2)

I2 =∫ η

1η dη = 1

/3(η3 − 1) (3)



where η is the fractional uptake given by (Rf/Rp).No attempt was made to deconvolute the individual

parameters, such as Dp, εp and kf , in Eqn (1), asfurther independent experiments would have beenrequired to determine the values of some of theparameters. Physical complications that can developin the presence of fouling material can make this lessstraightforward. Instead, the effective diffusivity (De)value calculated from Eqn (1) was used as the onlyindication of any changes in the overall adsorption ratethat might have resulted from fouling of the packedchromatographic bed.

RESULTS AND DISCUSSIONHomogeneity of adsorptionAlthough it is reasonable to expect that the beadsurface facing the loading front may initially see ahigher concentration of adsorbent, the adsorptionprofile as determined by CSLM studies was relativelyhomogeneous throughout the adsorption process forthe conditions used. Interestingly, the effect of contactbetween neighbouring beads within the tightly packedbed did restrict adsorption close to the contactspots. This was seen for both protein and dsDNA,as clearly shown in Fig. 2. Hubbuch et al.23 alsonoticed this effect during the adsorption of BSA andβ-lactoglobulin to a packed bed of SP Sepharosebeads. They confirmed that the restricted transportin these areas leads not only to a reduction inprotein transport but also to a lower total amount ofprotein being adsorbed. This restricted transport wasmore pronounced early during adsorption, causingvisible dead zones, but the variation in intensitybecame less significant as the adsorption progressed.In contrast, our study showed that restricted transportof dsDNA remained significant even as the adsorptionprogressed: owing to its large size, DNA does nothave the ability to penetrate into the beads and diffuseto areas of more restricted transport (unlike protein).This feature could be particularly important whenusing packed bed columns to adsorb DNA as thecapacity of the column will be reduced. Such deadzones were not seen during finite bath experiments.26

Chromatography of Cy5.5-labelled BSA on afresh Q Sepharose FF bedThe CSLM image depicting adsorption of Cy5.5-labelled BSA to a single, fresh Q Sepharose FF beadin a packed bed under flow conditions is illustratedin Fig. 3(a). Classical shrinking core behaviour,as described by a number of authors,5,8,25,30 andvisualised by Linden et al.17 and Hubbuch et al.,23

was observed.Translation of the confocal images to fluorescence

intensity profiles over the particle diameter, as shownin Fig. 3(b), shows the expected flattening of theintensity profile over time; although even after240 min of adsorption, equilibrium was not completelyachieved, as indicated by the unsaturated bead core.

Figure 2. Dead zones of restricted transport at contact pointsbetween beads in a packed bed. A packed bed of Q Sepharose FFwas fouled with 5 CVs of partially clarified E. coli homogenate. ThedsDNA in the foulant was labelled with PicoGreen.

Fouling of the Q Sepharose FF packed bed andsubsequent chromatography of Cy5.5-labelledBSAThe objective of this section was to monitor thebinding of Cy5.5-labelled BSA onto a deliberatelyfouled chromatographic packed bed by means ofCSLM. In addition, the relative spatial distributions ofselected foulant species and the target protein (BSA)were mapped using this technique in conjunctionwith fluorescent multicolour labelling. Owing to thecomplexity and diversity of the biological materialsencountered in bioprocesses, attempting to track thespatial distribution of each contaminant or foulantspecies within the beads is infeasible. Instead, ourstudy aimed to circumvent this by selecting a numberof recognised and indicative fouling species; namelynucleic acids (in our case dsDNA) and host cellproteins (HCPs). We then selected a number of dyeswith different emission spectra with which to labelselectively these various categories of foulants, suchthat the trajectory of a representative proportion ofeach foulant species within the packed bed may bevisualised directly under dynamic flow conditions bymeans of CSLM. There is, however, a limitationin drawing conclusive conclusions from foulingdata obtained from labelled species in that native,unlabelled counterparts might behave differently ina real bioprocess scenario. Nevertheless, it is worthpointing out that only minute quantities of dye areused in these labelling reactions, precisely so as toavoid changes in protein properties. Several authorshave reported that fluorescent labelling has littleeffect on protein behaviour.16,17 The main quantitativeobjective of the study was therefore to compare thebinding profiles of BSA onto fresh, fouled and CIP-treated packed beds. Fluorescent labelling of thefoulant species was useful to our study in a qualitativeway as a convenient visualisation technique rather thana quantitative technique.


SC Siu et al.

(a)

(b)

30 min 60 min 120 min 180 min 240 min

Figure 3. Adsorption of BSA to a fresh bed. The bed was packed with Q Sepharose FF and BSA (2 mg mL−1) was loaded onto the bed at a flowrate of 150 cm h−1. (a) confocal images at various time intervals, (b) corresponding fluorescence intensity profiles at various time intervals: (a)30 min; (b) 60 min; (c) 120 min; (d) 180 min; (e) 240 min.

Q Sepharose FF is an anion exchanger withquaternary amine groups carrying a positive charge.Genomic dsDNA and HCPs (with pI values below8.0) in the E. coli homogenate, used as the challengingfouling stream in this study, would be expected toact as major fouling species. To investigate this,PicoGreen was used to label fluorescently dsDNAand Cy3 was used to label HCPs in the foulantmaterial before loading 5 CVs of the material ontothe bed. Figure 4(a) (0 min) shows the CLSM imagesillustrating the post-fouling spatial distributions ofthe PicoGreen-labelled dsDNA and the Cy3-labelledHCPs within single beads in the packed bed underflow conditions. The corresponding intensity profilesof the two fouling species within the selected bead areshown in Fig. 4(b). The results show that PicoGreen-labelled dsDNA adsorption only takes place at thebead exterior to a depth of approximately 3 µm. Similarobservations were seen when fouling of the beads withthe same fouling material in finite baths26 and have alsobeen reported for pure solutions of plasmid DNA byPrazeres et al.31 and Ljunglof et al.12 In contrast, Cy3-labelled HCPs absorbed virtually evenly throughoutthe bead, as confirmed by a relatively flat intensityprofile in Fig. 4(b). In contrast, in previous finite bathexperiments, the Cy3-labelled HCPs did not penetrateto the centre of the bead even under severe foulingfor a prolonged period of time, suggesting that other,unlabelled fouling species occupy the core region insuch operation modes.26

A solution of 2 mg mL−1 BSA containing a Cy5.5-labelled fraction was loaded onto the fouled bed,following the same procedure as that for the freshbed. Figure 4(a) shows the resulting CSLM images

for a series of adsorption time points, and thecorresponding fluorescence intensity profiles for BSAare shown in Fig. 5. The progressive adsorptionfront of the Cy5.5-labelled BSA depicted in theseimages did not appear to be hindered by the pre-adsorbed fouling species (namely Cy3-labelled HCPsand PicoGreen-conjugated dsDNA). The adsorptionfront appeared to follow a similar penetration patternto that observed in the fresh bed (Fig. 3). The imagesin Fig. 4(a) clearly show that Cy5.5-labelled BSAdid not occupy the outer layer where PicoGreen-labelled dsDNA had adsorbed but penetrated furtherand bound past this outer layer. Even after 240 min,Cy5.5-labelled BSA did not appear to displacecompletely the PicoGreen-conjugated dsDNA layer.This observation is consistent with that seen underfinite bath conditions.26

As expected, BSA partially displaced Cy3-labelledHCPs as the adsorption front moved towards thebead core, giving rise to a slight ‘hump’ in theintensity profile (Fig. 6) but eventually flattening out,indicating that the HCP concentration was relativelyconstant throughout the bead after 240 min. Therelative concentration of HCPs in the bead againsttime is plotted in Fig. 7, and shows a 50% decreasein HCP concentration after 240 min. Such classicalcompetitive adsorption behaviour is not always seenwith protein mixtures. For example, in the case ofthe competitive adsorption of a pure protein mixtureof hIgG and BSA to SP Sepharose FF, hIgG waspreferentially located in the centre of the bead, whileBSA was predominantly bound at the periphery.16 Inour case, HCPs and BSA both approached an evendistribution throughout the bead.



(a)

(b)

0 min 30 min 60 min

120 min 180 min 240 min

Figure 4. Adsorption of BSA to Q Sepharose FF beads in a fouled packed bed under flow conditions. A fresh bed was deliberately fouled with 5CVs of partially clarified E .coli homogenate. BSA (2 mg mL−1) comprising a Cy5.5-labelled fraction (red) was subsequently loaded onto the bed at150 cm h−1. dsDNA and HCPs in the foulant were labeled with PicoGreen (green) and Cy3 (blue), respectively. (a) CSLM images (the bead used forimage analysis is highlighted by an inserted box), (b) fluorescence intensity profiles of HCPs and dsDNA at time = 0 min.

Figure 5. Fluorescence intensity profiles for the adsorption of BSA toa bead in a fouled packed bed under flow conditions. A fresh QSepharose FF bed was fouled with 5 CVs of partially clarified E. colihomogenate. BSA (2 mg mL−1) containing a Cy5.5-labelled fractionwas subsequently loaded onto the bed at 150 cm h−1. The profilesshow the fluorescence intensity of BSA across the bead diameterthrough the bead centre over time: (a) 30 min; (b) 60 min; (c) 120 min;(d) 180 min; (e) 240 min.

CIP of the fouled Q Sepharose FF packed bedand subsequent chromatography ofCy5.5-labelled BSAThe ability to clean a column in situ without therequirement to reproducibly repeat the packingprocedure is an important consideration for theeconomics and validation purposes of a processinvolving packed bed chromatography. For thisexperiment, a column was deliberately fouled with5 CVs of foulant and subsequently cleaned using15 CVs of 1 mol L−1 NaCl followed by 15 CVsof 1 mol L−1 NaOH, both of which are commonlyused in industry. A Cy5.5-labelled BSA adsorptiontime series, following the same procedure as thatused for fresh beds, was performed on the CIP-treated bed. Fluorescence intensity profiles of thebound BSA layer are shown in Fig. 8. The BSAadsorption front advanced towards the centre ofthe CIP-treated beads in a similar pattern to thatobserved in the fresh bed (Fig. 3). These results seemto suggest that the initial CIP cleaning cycles which


SC Siu et al.

Figure 6. Intensity profiles for BSA and HCPs in a fouled bed. A fresh Q Sepharose FF bed was fouled with 5 CVs of partially clarified E. colihomogenate. BSA (2 mg mL−1) comprising a Cy5.5-labelled fraction was subsequently loaded onto the bed at 150 cm h−1. A classical displacementof HCPs is seen over time as the BSA adsorption front moves towards the bead core: (a) 30 min; (b) 60 min; (c) 240 min. ( . . . . . . . ) HCPs profile at2 min (initial); ( ) HCPs profile at the specified time; (- - - - ) BSA profile.

are applied to packed beds do not compromise thebinding profiles of the target protein in subsequentadsorption cycles. However, chromatographic beddeterioration would be expected to occur more rapidlywhen using extreme fouling conditions, followed byharsh CIP protocols, as indicated by finite bathexperiments.26

CSLM imaging was also used to monitor thecourse of CIP treatment with respect to clearanceof Cy3-labelled HCPs and PicoGreen-conjugateddsDNA from the beads (images not shown). Itwas observed that most of the Cy3-labelled HCPsappeared to be removed with loading 1 CV of 1 molL−1 NaCl and only a residual amount remained after15 CVs. However, even 15 CVs of 1 mol L−1 NaClappeared to be ineffective in removing PicoGreen-conjugated dsDNA. Although dsDNA is known tobind strongly to anion exchangers, the observation issurprising since previous studies in finite baths hadshown 1 mol L−1 NaCl to be effective in removingPicoGreen-conjugated dsDNA from fouled beads.In addition, Prazeres et al.31 have demonstrated thatNaCl concentrations of around 0.7 mol L−1 were able

Figure 7. Reduction of HCP concentration as BSA adsorptionprogresses over time. A classical displacement of HCPs is seen overtime as the BSA adsorption front moves towards the bead core. Afresh Q Sepharose FF bed was fouled with 5 CVs of partially clarifiedE. coli homogenate. BSA (2 mg mL−1) containing a Cy5.5-labeledfraction was subsequently loaded onto the bed at 150 cm h−1.

Figure 8. Fluorescence intensity profiles for the adsorption of BSA toa bead in a fouled then CIP-treated packed bed under flowconditions. A fresh Q Sepharose FF bed was fouled with 5 CVs ofpartially clarified E. coli homogenate. The fouled bed was thencleaned with a 15 CVs of 1 mol L−1 NaCl, followed by 15 CVs of 1 molL−1 NaOH. Subsequently, BSA (2 mg mL−1) containing aCy5.5-labelled fraction was loaded onto the bed at 150 cm h−1. Theprofiles show the fluorescence intensity of BSA across the beaddiameter through the bead centre over time: (a) 30 min; (b) 60 min;(c) 120 min; (d) 180 min; (e) 240 min.

to elute plasmid DNA from small-scale Q SepharoseFF columns loaded with a pure plasmid DNA solution.A combination of the complex fouling nature ofrealistic process streams combined with the dynamiceffects of a packed bed may be responsible for thisobservation. A higher concentration of NaCl may berequired to remove dsDNA from a fouled packedbed but further studies are required to prove this.Subsequently, 1 mol L−1 NaOH was used to furtherclean the bed and this proved to be an effective cleaningagent. All PicoGreen-conjugated dsDNA and residualCy3-labelled HCPs were removed after loading just 1CV of NaOH.

Comparison of Cy5.5-labelled BSA bindingcapacity between fresh, fouled and CIP-treatedQ Sepharose FF packed bedsThe adsorption curve of Cy5.5-labelled BSA to fresh,fouled and CIP-treated beds is displayed in Fig. 9



Figure 9. Comparison of dynamic BSA adsorption ratescorresponding to different matrix states of Q Sepharose FF packedbeds: (ž) fresh bed; (�) fouled bed; (�) fouled bed treated with CIPof 1 mol L−1 NaCl followed by 1 mol L−1 NaOH. (Error bars show 95%CI). A 20% reduction was experienced after fouling. The CIPtreatment was effective in regenerating the fouled bed to its freshstate.

and the approximate Cy5.5-labelled BSA bindingcapacities are listed in Table 1. It was observed thatthe binding capacity of Q Sepharose FF was reducedby 20% after fouling. Cleaning the fouled bed using 15CVs of 1 mol L−1 NaCl followed by 15 CVs of 1 molL−1 NaOH proved effective in restoring the capacity ofthe bed to its fresh state value. The high reduction incapacity might be accounted for by unlabelled foulantspecies occupying the binding sites. Some of thesefoulants might have stronger binding affinities towardQ Sepharose than BSA, such that they would not bedisplaced by bound BSA.

Comparison of Cy5.5-labelled BSA uptake rateand effective diffusivity within Q Sepharose FFpacked bedsThe confocal images suggest that the shrinkingcore model may be appropriate for describingthe adsorption of Cy5.5-labelled BSA onto QSepharose FF matrix for all chromatographic scenariosinvestigated in the present study (namely fresh matrix,severely fouled matrix and fouled then CIP-treatedmatrix). In all conditions examined, sharp adsorptionfronts that gradually moved from the periphery of thebead to the centre were seen. It was anticipated that thediffusivity of BSA within the chromatographic beads

Table 1. Comparison of BSA diffusivity and binding capacities for Q

Sepharose FF beads packed in a flow cell and subjected to different

fouling conditions of fouling and cleaning (errors represent 95% CI)

Beadcondition

De

(×10−10

cm2 s−1)

[1 + εpDp

kfRp

]

(intercept term)

Relative bindingcapacity

(arbitrary units)

Fresh beads 1.5 ± 0.9 1.1 ± 0.1 5.0 ± 0.5Fouled beads 1.5 ± 0.9 1.0 ± 0.1 4.0 ± 0.4CIP-treated beads 1.4 ± 0.9 1.1 ± 0.1 5.0 ± 0.5

may be influenced by fouling since the presence ofother bound species has been suggested to be a possiblelimiting mechanism to intra-particle diffusion.16 Inaddition, colloidal material may obstruct bead poresand alter the breakthrough curve.2 In order toinvestigate whether diffusivity of BSA was affected byfouling mechanisms, a single lumped kinetic parameteror effective diffusivity, De, was estimated directly fromthe confocal images by assuming that the adsorptionlayer is saturated as described above (‘Calculation ofEffective Diffusivity from Confocal Images’).

The derived De and intercept values from usingEqn (1) are listed in Table 1. The intercepts of theplots were in all cases close to but greater than unity,suggesting that external mass transfer resistance maybe significant but small. The effective diffusivities, De,for the three conditions were not significantly differentfrom each other, suggesting that fouling did not have asignificant impact on the overall protein mass transfer.

To validate our approach, Dp for fresh beads werecalculated using Eqn (1) by using the mean valueof the determined De and assuming values of εp =0.55 and qs = 137 mg mL−1 (determined empirically).The intra-particle diffusivity was determined to be1.9 × 10−8 cm2 s−1, which is reasonably close to thevalue of 5.6 × 10−8 cm2 s−1 quoted for an agarosematrix (the free diffusivity, D0, of BSA is reported tobe 3.6 × 10−7 cm2 s−1).32

Comparison of fouling in finite baths and packbed columnsComparing the effects of subjecting beads to the samefouling material for approximately the same contacttime in both finite baths and in packed beds has shedlight on how the degree of fouling can be affectedby the physical and hydrodynamic properties of thepacked bed. In the first case, beads were incubated infouling material for 5 min with mixing;26 and in thelatter case, 5 CVs of fouling material were loaded tothe packed bed at a flow rate of 150 cm h−1, giving anexposure time of 4.5 min. The capacity of the beadsfouled in the finite bath was reduced by 60%, whereasthose fouled in the packed bed only experienceda 20% reduction in capacity after fouling. Thesubsequent Cy5.5-labelled BSA adsorption patternsshowed even greater differences between these twooperation modes of chromatography, with respectto fouling impact. In the finite bath, Cy5.5-labelledBSA was unable to penetrate into the core of thefouled beads. In contrast, in the packed bed, Cy5.5-labelled BSA was able to penetrate the bead coreand exhibited classical shrinking core adsorptionbehaviour. Not unexpectedly, fouling was found tobe more pronounced in beads contacted in a finitebath mode than in a packed bed mode. Visualinspection of the fouled packed bed revealed thatmost of the cell debris in the foulant was trappednear the top of the bed. The top portion of thebed may have acted as a pre-filter for the remainingbed length. This observation is consistent with that


SC Siu et al.

reported elsewhere,33 where solids deposition leadingto band-broadening effects occurs near the very topof the column. It is reasonable to suspect that otherunlabelled fouling species may undergo preferentialbinding to the top portion of the bed, such thatthe beads in the middle and lower bed sections areexposed to an effectively lower concentration of foulingmaterials. However, this was not further investigatedowing to the design limitation of the flow cell, whichonly allowed observations around the axial centre ofthe packed bed. In comparison, the entire surfacearea of the bead would be exposed to the sameconcentration of fouling material in a well-mixed finitebath; hence such a spatial distribution would not bepossible in that mode of operation.

CONCLUSIONSCSLM provides a useful tool with which to studyfouling in packed beds. The interactions betweenvarious fouling species and the chromatographicmatrix can be imaged in real time using thishigh-resolution technique. In the present study, thefouling impact of fluorescently labelled HCPs anddsDNA were visualised by CSLM of Q SepharoseFF packed beds under dynamic flow conditions.The fouling impact was further quantified viathe comparative adsorption capacities for Cy5.5-labelled BSA, exhibited by fresh, fouled and CIP-treated matrices. The effectiveness of CIP procedureswere also evaluated for the packed-bed mode andcompared with those achieved with the finite bathmode, which were reported in a previous study.Furthermore, the adsorption of Cy5.5-labelled BSAto the chromatographic beads pre- and post-foulingprovided a comparative means of evaluating therelative degree of fouling for the different modes ofoperation.

In general, the fouling generated in a finite bathwas more severe than observed in a packed bed.The capacity of the beads fouled in the finite bathwas reduced by 60%, whereas those fouled in thepacked bed only experienced a 20% reduction incapacity after fouling. CSLM revealed the presenceof restricted protein and dsDNA adsorption closeto bead-to-bead contact moieties in the packed bed.Studies also showed that while a high salt wash (1 molL−1 NaCl) was able to remove dsDNA from fouledbeads in a finite bath, it was ineffective when used asa washing reagent in packed bed mode.

Confocal images for Cy5.5-labelled BSA adsorptionon fresh, fouled and CIP-treated columns were usedas the basis from which to determine a single lumpedkinetic parameter, the effective diffusivity, De. Theresults suggest that fouling, under the conditions usedin this study, did not have a significant impact onthe overall protein mass transfer; however, it mustbe noted that the design limitation of the flow cellonly allowed observations around the axial centre ofthe packed bed. Although this approach does not

allow the mechanistic discrimination of the intrinsicmass transfers parameters (Dp, kf and εp), it is stilluseful for comparisons when combined with CSLMimages.

ACKNOWLEDGEMENTSThe support of the Engineering and PhysicalSciences Research Council Life Science Interfaceand Innovative Manufacturing Research Centreinitiatives for the IMRC in Bioprocessing is gratefullyacknowledged. The IMRC is part of the AdvancedCentre for Biochemical Engineering, UCL, withcollaboration from a range of academic partners,biopharmaceutical and biotechnology companies.Special thanks to Anna Kjellgren, Maritha Mendel-Hartvig, Peter Swensson and all the other staff fromGE Healthcare (Uppsala, Sweden) who have providedinvaluable support and advice.

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Confocal imaging of chromatographic fouling under flow conditions