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haracterisation of grafted weak anion-exchange methacrylate monoliths
ida Frankovic a, Ales Podgornika,∗, Nika Lendero Krajnca, Franc Smrekara,eter Krajncb, Ales Strancara
BIA Separations d.o.o., Teslova 30, SI-1000 Ljubljana, SloveniaUniversity of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova 17, SI-2000 Maribor, Slovenia
r t i c l e i n f o
rticle history:eceived 29 September 2007eceived in revised form 4 August 2008ccepted 11 August 2008vailable online 14 August 2008
A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate mono-lith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. Thethickness of the grafted layer was determined by measuring permeability and found to be approximately90 nm. The effects of different buffer solutions on the pressure drop were examined and indicated theinfluence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC)were found to be flow-unaffected up to a linear velocity of 280 cm/h. A comparison of the BC for thenon-grafted and grafted monolith was performed using �-lactoglobulin, bovine serum albumin (BSA),thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-foldhigher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and17 mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1 MNaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form
DNA-Factor
was detected. Protein z-factors were equal for the non-grafted and grafted monolith indicating that thesame number of binding sites are available although elution from the grafted monolith occurred at higher
d moA fro
dltaimdsto
dmiMd
ionic strengths. The graftebaseline separation of pDN
. Introduction
CIM ion-exchange methacrylate monolithic chromatographicupports (BIA Separations, Ljubljana, Slovenia) are becoming theaterial of choice for the fast separation and purification of larger
iomolecules, like peptides, DNA, or even viruses [1,2]. They exhibitow-unaffected resolution and dynamic binding capacity [1,3] as aonsequence of their particular structure. CIM monoliths consist ofighly interconnected channels showing a bimodal pore size dis-ribution (contributes to the low pressure drop at high flow-rates)ith a relatively high specific surface area [4] results in a high bind-
ng capacity for very large molecules like pDNA or viruses [5]. Beingptimized for those kinds of molecules, their capacity for smallerolecules, e.g. medium or small size proteins is lower when com-
ared to conventional porous particulate supports. In fact, it washown that the maximization of the capacity is obtained by adjust-
ng the pore size of particles to the molecular size of the solutes6,7].
Capacity is especially important for the purification ofiomolecules on the laboratory and preparative scale where pro-
uctivity is essential. In the case of methacrylate monoliths oneogical approach to increase capacity for smaller proteins would beo decrease the channel size, which results at constant porosity inn increased surface area. However, a reduced channel size resultsn higher pressure drop [8] and also reduced permeability. This
ight, because of equipment pressure limitations on a large scale,ecrease the applicable flow rate. In addition, Bencina et al. demon-trated that small channel sizes limit access of DNA molecules tohe entire surface area, and might even cause molecule degradationr column clogging [9].
A possible alternative to resolving the problem of low capacityue to a small surface area is the use of grafted functional poly-ers. Grafting is a chemical reaction initiated by radicals resulting
n a covalent bond between monomers and the matrix [10–12].onomers grafted onto the polymeric matrix form polymer chains
enoted as brushes or tentacles [13–15]. The most important bene-t of the tentacle chemistry is a large amount of sterically accessible
igands for the binding of biomolecules and resulting in higher bind-ng capacity. This approach was first applied to porous particles
13]. Müller prepared an acrylamide tentacle type anion-exchangery grafting ion-exchange groups containing vinyl monomers ontoydrophilic polymer beads. This resulted in the formation of lin-ar functionalized polymer chains over the entire pore surface ofhe beads. The obtained material exhibited improved selectivity
ue to reduced non-specific interactions of proteins with theatrix and a two to threefold increase in binding capacity when
ompared to the same matrix modified by direct attachment ofon-exchange groups. The increase in capacity was attributed tohe presence of hydrophilic tentacles on the surface which formedthree dimensional hydrogel. Protein binding in such a hydrogelas assumed to occur in multilayers. Effectiveness of the tentacleolymer chains for holding various proteins has been investigatedy Janzen et al. [16]. Adsorption isotherms for BSA were foundo follow the Hill type of isotherm confirming multilayer adsorp-ion. It was also found that the higher degree of grafting increasesrotein-binding capacity [17]. However, at a very high degreef grafting, protein capacity decreased along with the perme-bility which was explained by significant cross-linking betweenhe individual tentacles and consequently lower accessibility forroteins [17].
Also, the effect of the mobile phase composition was extensivelytudied. Tsuneda et al. demonstrated that increasing the salt con-entration or pH of the protein buffer solution resulted in higherermeability and decreased capacity [18]. Gebauer et al. ascer-ained that during the elution of protein from grafted cellulose
embrane with 1 M NaCl, the pressure was about 50% lower thanith the equilibration buffer also demonstrating higher permeabil-
ty. They concluded that this is due to the shrinkage of the graftedayer [19].
More recently, grafting was applied to different types of sup-orts like membranes [15,18,20–24] and also monoliths [14,25–28].here were several studies performed with porous hollow fiberembranes grafted with polymer chains containing weak anion-
xchange groups [15,18,20–22]. Also in these cases, higherelectivity and a significant increase in capacity was observedxceeding the respective monolayer values by 11-fold [15]. Fromhe analysis of the breakthrough curves on the grafted mem-ranes, Gebauer et al. concluded that the transport of proteins
nto the grafted layer is the rate-limiting step [19]. However, Kub-ta et al. and Müller and Baurmeister found that BC remainedow-unaffected even for extremely high linear velocities [21,29].significant increase in capacity was achieved on monolithic sup-
orts as well. For cryogels, a fourfold increase in capacity for BSAnd a threefold for lysozyme was reported [14]. Another cryogelpplication with a much higher capacity for BSA and even pDNA waseported by Voss and Flaschel [28]. They found that the BSA capac-ty on grafted cryogel was a hundred times higher as comparedo the non-grafted one and the plasmid capacity was 3.5 timesigher. Methacrylate-based monoliths were also grafted to form
on-exchangers [25–27] exhibiting significantly higher capacityompared to the non-grafted monoliths [25]. However, no informa-ion about the effect of linear velocity on BC of grafted monolithsas reported.
In the present study, the chromatographic properties of graftedEAE monolith and the commercially available CIM DEAE mono-
ith were investigated and compared. The effect of the flow rate onhe separation of test proteins and binding capacity was explored.ifferent mobile phases were used to investigate the behavior ofrafted DEAE tentacles for the determination of permeability. Fur-hermore, the effect of grafting on z-factor, capacity and recoveryor different sizes of proteins, and pDNA was examined.
. Experimental
.1. Materials
All experiments were carried out on a 0.34 ml (3 mm thick and2 mm diameter) conventional CIM DEAE monolith with an average
tlp
t
gr. A 1207 (2008) 84–93 85
ore radius of 650 nm (referred further as a non-grafted monolith;-g); a DEAE methacrylate monolith with an average pore radiusf around 160 nm; a grafted DEAE methacrylate monolith (referredurther as the grafted monolith; g) packed into a CIM disk housing,rovided by BIA Separations (Ljubljana, Slovenia). The grafted DEAEonolith was prepared from a CIM OH monolith (BIA Separations,
hloride, tris(hydroxymethyl)aminomethane (Tris), ammonium-ulfate, hydrochloric acid fuming (HCl) were obtained from MerckDarmstadt, Germany). Ethylenediaminetetraacetic acid disodiumalt dehydrate (EDTA) was obtained from Kemika (Zagreb, Croatia).mmonium cerium(IV) nitrate, nitric acid 65%, glycidyl methacry-
ate 97% (GMA), albumin from bovine serum (BSA), myoglobin,onalbumin from chicken egg white, soybean trypsin inhibitorSTI), thyroglobulin from porcine thyroid and �-lactoglobulinrom bovine milk were purchased from Sigma (St. Louis, MO,SA), ethanol was obtained from Pharmachem (Ljubljana, Slove-ia). E. coli strain containing 4.7 kbp plasmid DNA (pEGFP-N1)ere purchased from Clontech Laboratories (Mountain View, CA,SA 94043). pDNA conformations were analyzed on agarose gellectrophoresis (Bio-Rad, Richmond, CA, USA). 1 kb Plus DNAadder and SYBR Safe DNA gel stain were obtained from Invit-ogen (Eugene, OR, USA). Agarose gel was made of SeaKemE Agarose (Lonza Group, Basel, Switzerland) and deionizedater.
All solutions were prepared with deionized water purified by aatek IWA 80 roi (Ledec nad Sázavon, Czech Republic) water purifi-
ation system and analytical grade reagents. The pH values of theris–HCl buffer solutions were adjusted with HCl. Buffer solutionsere filtered through a 0.45-�m filter made of Sartolon polyamide
Sartorius, Goettingen, Germany).
.2. Methods
.2.1. Grafting procedure and DEAE modificationThe grafting was performed according to Müller [17]. Briefly,
MA was grafted on the monolith using an ammonium cerium(IV)itrate solution as a redox initiator. The epoxy groups present onrafted tentacles were modified into DEAE groups by reacting themith a 50% solution of diethylamine in ethanol. Finally, the monolithas conditioned by pumping through a 1 M solution of NaOH andeionized water.
.2.2. Pore size distributionPore size distribution was measured by a Pascal 440 (Thermo-
uest Italia, Rodano, Italy) mercury porosimeter with a range of–10 000 nm. Approximately 0.1 g of dried monolith samples (CIMH, and grafted CIM DEAE) were measured.
.2.3. Permeability determinationThe pressure drop on the monolithic columns were measured
sing a differential manometer (Mid-West Instruments, Sterlingeights, MI, USA). Different mobile phases (deionized water, 20 mMris solutions, pH range from 7.4 to 9; 20 mM Tris, pH 7.4, with NaCloncentration range from 0.1 to 1 M; 20 mM Tris, 10 mM EDTA, pH.4; 50 mM Tris, 10 mM EDTA, pH 7.2; 20 mM Tris, 1 M NaCl, pHange from 7.4 to 9 and 50 mM Tris, 10 mM EDTA, 1.5 M NaCl, pH.2) were separately pumped through the empty CIM housing and
hen, CIM housing containing either a grafted or non-grafted mono-ith. The influence of different mobile phases on pressure drop andermeability were analyzed.
Flow rates from 1 to 3 ml/min were applied. This data was usedo determine the permeability of the monolith according to Darcy’s
8 omato
l
wcp
2
de
�
w
a
ttaaf
svε
ε
I
T
d
F
2
m2
22dK�KMT
2mfwfiamtttpawf
K
˚atctc
2fnMcvracl
k
wpof
2iwTtfl
l7(st
dvwtt
6 V. Frankovic et al. / J. Chr
aw:
�p
L= u�
B0(1)
here B0 is permeability, �p pressure drop on monolith (Pa), L theolumn length (m), u is superficial velocity (m/s) and � is mobilehase viscosity (Pa s).
.2.4. Determination of grafted layer thicknessFor the calculation of the grafted layer’s thickness, the pressure
rop on the monolith was assumed to follow the Kozeny–Carmanquation:
P = 72k1u�L
d2
(1 − ε)2
ε3(2)
here d is particle diameter (m), ε is porosity and k1 is a constant.Combining Eqs. (1) and (2), the permeability can be expressed
s
1B0
= 72k11d2
(1 − ε)2
ε3(3)
Although d2 in Eq. (2) stands for particle diameter, it was shownhat for methacrylate monoliths the square relation holds even ifhe pore diameter is used [30]. Since the structure of the graftednd non-grafted monolith is very similar, k1 can be considered asconstant. Based on this assumption, the ratio of the permeability
or grafted and non-grafted monolith becomes:
BG
BNG= (1/d2
NG)((1 − εNG)2/ε3NG)
(1/d2G)((1 − εG)2/ε3
G)(4)
Further assuming that the thickness of the grafted layer is con-tant, than the volume of the grafted layer decreases the poreolume and that pores have cylindrical shape, the relation betweenNG and εG is
G =(
dG
dNG
)2
εNG (5)
nserting Eq. (5) into Eq. (4) after some rearrangement gives:
BG
BNG=
(dG
dNG
)8 (1 − εNG)2
((1 − (dG/dNG)2)εNG)2
(6)
ransforming this equation into the explicit form we obtain Eq. (7):
G = dNG
√√√√√
ε2NG(BG/BNG) + 4
√BG/BNG(1 − εNG) − εNG
√BG/BNG
2(1 − εNG)(7)
rom Eq. (6) or Eq. (7) dG can be calculated.
.2.5. Nitrogen determinationNitrogen determination was performed with a PerkinElmer ele-
ental analyzer for carbon, hydrogen and nitrogen (CHN), type PE400 Series II Elemental analyzer (Waltham, MA, USA).
.2.6. Chromatographic experiments
.2.6.1. Hardware. All experiments were performed using a gra-ient Knauer HPLC system (Berlin, Germany) consisting of two
nauer Type 64 analytical pumps, an injection valve with a 20-l sample loop and a Knauer UV–vis absorbance detector model-2500 with a 10 mm optical path cell. The pH meter was frometrel (Horjul, Slovenia) with an InLab 412 electrode from Metler
oledo (Urdof, Switzerland).
q
˚fic
gr. A 1207 (2008) 84–93
.2.6.2. Ionic capacity. The ionic capacity was estimated using theethod described by Lendero et al. [31]. Measurements were per-
ormed by using the 20 mM Tris–HCl buffer with (buffer B) andithout 1 M NaCl (buffer A), both at pH 7.4. The columns wererst equilibrated with a high ionic strength buffer until reachingn absorbance at 200 nm where the pH is stabilized. After that, theobile phase was switched to the buffer with low ionic strength
o form the pH transient. The measurement was completed whenhe absorbance and pH of the effluent reached the value of the ini-ial buffer solution. The time between the switching of the mobilehases and 50% of the breakthrough was used as a measure for lig-nd density. The relative elution volume, K, is linearly correlatedith the total ionic capacity [31,32] and was calculated using the
ollowing equation:
= (t50% − td) × �
Vc(7)
represents the flow rate (ml/min), t50% is the time where the finalbsorbance value reached the 50% of the breakthrough curve, td ishe time where the buffer with low ionic strength flows out of theolumn and Vc is the total column volume (0.34 ml). Value K washen used as a relative measure for the total ionic capacity of theolumn.
.2.6.3. Determination of z-factor. The numbers of binding sites (z-actor) for �-lactoglobulin, BSA and thyroglobulin on grafted andon-grafted monoliths were estimated using an isocratic method.obile phases of 20 mM Tris–HCl buffer, pH 7.4 with different NaCl
oncentrations ranging from 0.20 to 1 M were used. The injectionolume was 20 �l, the absorbance was set to 280 nm, and the flowate was 1 ml/min. The retention time of each elution was measurednd the capacity factor (k′) at each mobile phase composition wasalculated. The z-factor was determined from Eq. (8) by plottingogarithmic values of k′ vs. z and fitted them with a straight line:
′ = N × c−z (8)
here N is a constant (M−1), c is the concentration of salt in mobilehase (M), z is a stoichiometric parameter related to the numberf the moles of the salt needed for salvation and k′ is the capacityactor.
.2.6.4. Binding capacity determination. The protein binding capac-ty was determined with frontal analysis experiments. Monoliths
ere first equilibrated with a standard buffer solution of 20 mMris–HCl, pH 7.4 and loaded with a protein solution (1 mg/ml BSA,hyroglobulin and �-lactoglobulin in 20 mM Tris–HCl, pH 7.4) at aow rate of 1 ml/min. The absorbance at 280 nm was measured.
For the pDNA binding capacity, the monoliths were first equi-ibrated with a buffer containing 50 mM Tris, 10 mM EDTA, pH.2 and two different NaCl concentrations (0.1 and 0.2 M). PurifiedA260/280 was 2.05) 4.7 kbp pDNA was dissolved in the same bufferolution, obtaining a concentration of 69 �g/ml, was loaded ontohe column. The absorbance at 260 nm was measured.
To determine the effect of the flow rate on BSA binding capacity,ifferent flow rates 1, 3, and 5.3 ml/min (corresponding to linearelocity up to 280 cm/h or 15.5 column volumes/min (CVs/min))ere applied. Binding capacity (q) was calculated at 10 and 50% of
he final absorbance value of the breakthrough curve by followinghe equation:
(˚ × t − V ) × c
= 10 or 50% d 0
Vc(9)
represents the flow rate (ml/min), t10 or 50% is the time where thenal absorbance value reached the 10 or 50% of the breakthroughurve, Vd is the dead volume of the system (ml), c0 is an initial
matogr. A 1207 (2008) 84–93 87
p(
2gdeflT
2CnTTmlsfpn(a
2
ggaSs
3
vtttiorfta
opcwScmsOTsfltsdgg
Fd
flilmstrated that the effective pore diffusivity for proteins in a dextran-grafted matrixes is 4–10 times higher than in the free solution.
Data of the effect of linear velocity on the binding capacity andseparation efficiency are presented in Fig. 2. The results demon-strate that similar to other reports both the capacity and separation
Fig. 2. Effect of the flow rate on chromatographic properties of grafted DEAE mono-liths. (A) On binding capacity for non-grafted CIM DEAE (n-g) and grafted DEAE
V. Frankovic et al. / J. Chro
rotein concentration (mg/ml) and Vc the total column volume0.34 ml).
.2.6.5. Protein separations. Separations of protein mixture (myo-lobin, 0.5 mg/ml; conalbumin, 1.5 mg/ml; and STI, 2 mg/ml)issolved in 20 mM Tris–HCl buffer solution was performed usinglution in a linear sodium chloride gradient (0–0.7 M) at differentow rates (1, 3 and 5.3 ml/min). The absorbance was set at 280 nm.he sample injection volume was 20 �l.
.2.6.6. pDNA purification. pDNA lysate was purified on an 8 ml CIM4 (HIC) tube monolithic column (BIA Separations, Ljubljana, Slove-ia). The eluted plasmid was dissolved in 2.0 M (NH4)2SO4, 50 mMris, 10 mM EDTA buffer, pH 7.2 with a concentration of 50 �g/ml.he eluted sample was loaded afterwards onto a DEAE 8 ml tubeonolithic column (BIA Separations). The column was first equi-
ibrated with buffer A (50 mM Tris, 10 mM EDTA, pH 7.2). Afterample loading, the plasmid was eluted with a gradient (30 CVs)rom 0 to 100% of buffer B (50 mM Tris, 10 mM EDTA, 1.1 M NaCl,H 7.2). To achieve a better separation of RNA and pDNA, a combi-ation of stepwise (0–33% buffer B) and linear (33–100%) gradient30 CVs) were used. 500 �l were injected at a flow rate of 2 ml/minnd absorbance was monitored at 260 nm.
.2.7. Gel electrophoresisAnalysis of pDNA degradation was performed with horizontal
el electrophoresis. Samples were loaded onto a 0.7% (w/v) agaroseel at a voltage of 80 V for 60 min. 1 kbp Plus DNA Ladder was useds the molecular weight marker. The gel was stained with SYBRafe DNA gel stain and than visualized and photographed underhort-wavelength UV light.
. Results and discussion
One of the frequently used grafting procedures is to graft a singleinyl monomer onto a polymeric matrix. In the present investiga-ion, GMA was chosen as the monomer. The matrix onto whichhe grafting occurs should contain hydroxyl groups that can beransformed to free radicals [33]. In such a case GMA monomers covalently bound to the base matrix. The result is the formationf grafted layer which, after the modification of active groups intoespective ion-exchanger functionality, results in a higher capacityor different size molecules [6]. However, as Müller demonstrated,he degree of grafting can significantly affect the column perme-bility [17].
In contrast to conventional porous particles, the morphologyf CIM support is characterized by a single monolithic block ofolymer that contains pores, open on both sides (channels). Thesehannels are highly interconnected forming a flow-through net-ork and the whole mobile phase is forced to run through them.
ubstantial grafting might therefore decrease their diameter andonsequently significantly increase pressure drop (decrease per-eability). As it can be seen from Fig. 1, in our case there was no
ignificant difference in pore size distribution between basic CIMH and grafted monolith, as measured by mercury porosimetry.his might be an indication that no substantial decrease of poreize during the grafting procedure occurred. However, while suitedor pore size determination of non-grafted methacrylate mono-ith, the mercury intrusion porosimetry does not necessarily show
he contribution of hydrated surface polymer on the apparent poreize, because in the dry state the grafted hydrogel exists as a dehy-rated flat layer and experiments under operating conditions mightive different results. Therefore, we further investigated chromato-raphic properties for grafted DEAE monolith.
(pSpta
ig. 1. Pore size distribution curves of the CIM OH, and grafted DEAE monolith asetermined by mercury porosimetry.
It was demonstrated several times that the monoliths exhibitow independent chromatographic properties [34–36] and we
nvestigated whether this behavior is present also on grafted mono-iths. This is very probably because it was already demonstrated for
embranes [21,29], and since recently Stone and Carta [37] demon-
g) monolith; 1 mg/ml BSA dissolved in 20 mM Tris–HCl, pH 7.4 was loaded; (B) onrotein separation; sample: myoglobin (0.5 mg/ml), conalbumin (1.5 mg/ml) andTI (2 mg/ml) dissolved in mobile phase A (20 mM Tris–HCl buffer, pH 7.4); mobilehase B, 1 M NaCl in buffer A, pH 7.4; injection volume 20 �l; linear gradient from 0o 70% buffer B. Conditions—flow rates: 1 (−), 3 (- -), 5.3 (. . .) ml/min; UV detectiont 280 nm.
88 V. Frankovic et al. / J. Chromato
Fn7
r(rta82ile
pcimfdtoistwavfboTcTgbdtcdcpD
fuNfc
rggwtoip7avp
i7m(agtltstpfiwasb3g
tm7wradius of the grafted monolith was calculated according to Eq. (6)and Eq. (7) decreased to 560 nm in Tris–HCl, indicating the layerthickness of nearly 90 nm and the pore size reduction of about 14%.One should keep in mind however, that this estimate assumes that
ig. 3. Effect of the flow velocity on the pressure drop on grafted DEAE (g) andon-grafted (n-g) CIM DEAE monolith. The mobile phase was 20 mM Tris–HCl, pH.4.
emain flow-unaffected at least up to a linear velocity of 280 cm/h15.5 CVs/min). This range was investigated since it covers valuesecommended for commercial CIM supports [36]. More impor-antly, from the breakthrough curves, it can be seen that there issignificantly higher capacity of grafted monolith being around
0 mg/ml in comparison to commercial CIM values of around5 mg/ml [34], meaning more than a threefold increase. Such an
ncrease is comparable to other reports of grafting [17,25–27]. Theong tail of the breakthrough curves is most likely due to the pres-nce of di- and multimers of BSA [38] and not to the flow profile.
During these experiments, the measurements revealed that theressure drop of the grafted monolith is substantially higher as oneould conclude from the mercury porosimetry data. From the datan Fig. 3 obtained by using Eq. (1), it can be calculated that the per-
eability of the non-grafted monolith was 9.8 × 10−15 m2 whileor the grafted one it was only 1.6 × 10−15 m2, indicating a sixfoldecrease. To obtain more general information about the effect ofhe mobile phase on the permeability, we investigated the effectf the pH value and salt concentration. There are several reportsn the literature describing the effect of the mobile phase compo-ition. Hautojärvi et al. studied the effect of the ionic strength onhe permeability of poly(vinylidene fluoride) membranes graftedith acrylic acid. They found that there were higher permeability
t higher salt concentrations and elevated pH [39]. The same obser-ation was reported by Kontturi et al. [40] and by Gebauer et al. [19]or the effect of ionic strength. This phenomenon can be explainedy polyelectrolyte theory [41] describing swelling and shrinkingf polyelectrolytes dissolved in solutions of different composition.he structure of the grafted layer has significant impact not only onolumn permeability but also on the adsorption of the molecules.suneda et al. demonstrated on grafted membranes bearing DEAEroups that with increasing salt concentration or pH of the proteinuffer solution, the graft chains shrank and BSA binding capacityecreased [18]. Wittemann et al. also found out that the adsorp-ion of BSA on polyelectrolyte tentacles was higher at lower saltoncentrations [42]. However, one should be aware that theecrease of capacity is not necessarily only a consequence of ahange in the thickness of the grafted layer, but also of the com-etitive adsorption resulting in a capacity decrease for non-graftedEAE matrices [34].
In our case, we selected mobile phases most frequently applied
or the purification of various proteins and pDNA on DEAE col-mn. Before measurement, the columns were regenerated with 1 MaOH. Fig. 4 demonstrates that the calculated permeability for dif-
erent buffer solutions for grafted and non-grafted monoliths. Onean see that using deionized water or a Tris–HCl buffer, pH 9 after
Fos
gr. A 1207 (2008) 84–93
egeneration with NaOH, the permeability of the grafted and non-rafted monolith is high. On the other hand, permeability for therafted monolith in buffer solutions with pH 8 or lower and even inater, after conditioning with Tris–HCl, pH 7.4, was decreased dras-
ically. Interestingly, it can be seen that the increase of ionic strengthf the buffer solution did not significantly impact the permeabil-ty of the grafted nor of the non-grafted monolith (for example:ermeability for the grafted monolith with 20 mM Tris–HCl, pH.4 is slightly lower – 1.6 × 10−15 m2 – in the same buffer with theddition of 1 M NaCl or 10 mM EDTA, 2.6 × 10−15 m2). Those obser-ations were also confirmed by a stepwise change of the mobilehases with and without salt (data not shown).
The dependence of permeability on pH and salt concentrationn Tris–HCl buffer are presented in Fig. 5. Increasing the pH from.4 to 9 only slightly increased the permeability of the non-graftedonoliths while for the grafted monolith the increase is significant
Fig. 5A). This observation is consistent with reported data [39–40]nd is probably a consequence of the lower association of DEAEroups. Because of that, more DEAE groups are not charged so theentacles can move freely without any repulsive forces and presentower resistance for the mobile phase. On the other hand loweringhe pH, more and more DEAE groups become charged which reflectstronger repellent interactions that cause the adjacent tentacleso be as far away from each other as possible so they extend androduce high resistance for the mobile phase. This is further con-rmed with the constant capacity of the monolith in the pH rangehere chains are charged (pH 7.4 and 8) and substantial decrease
t pH 9. In contrary to other reports, however, the effect of the ionictrength was found to be almost negligible. Possible reason mighte the high density of hydroxyl groups estimated on approximately.8 mmol/g of monolith [43] and consequently the high density ofrafted chains.
Based on the permeability data, it is possible to estimate thehickness of the grafted layer under operating conditions. The per-
eability data of non-grafted and grafted monolith in Tris–HCl (pH.4) and water were used. Since the initial pore radius was estimatedith mercury porosimetry at 650 nm and a porosity of 60%, the pore
ig. 4. Effect of mobile phase composition on monolith permeability. Permeabilityf grafted DEAE (g) and non-grafted (n-g) CIM DEAE monolith when different bufferolutions were pumped through them.
V. Frankovic et al. / J. Chromatogr. A 1207 (2008) 84–93 89
Fig. 5. Effect of mobile phase salt concentration and pH value on monolith perme-a(N
tlmfpttpam
ttosbstapltagrp
wgw
Table 1Relative ionic capacity and nitrogen content for a grafted and non-grafted monolith
Stationaryphase
Relativeioniccapacity
% N Estimated liganddensity (mmol/gdry support)
Normalized DEAEgroup “activity”
Grafted 25 1.72 1.23 ≈0.3Non-grafted
165 3.13 2.24 1
Estimated ligand density was calculated assuming that each nitrogen atom formsocg
Tmmncts[tomosgglanit3“tis
fs5e[eiTstligiacdinftverge with the increase of molecular size to values slightly above 2.
bility. Permeability data for grafted DEAE (g), non-grafted (n-g) CIM DEAE monolithA) as a function of pH value of 20 mM Tris–HCl buffer solution; (B) as a function ofaCl concentration dissolved in 20 mM Tris–HCl, pH 7.4.
he pores are uniform, which is not the case for methacrylate mono-iths and that the grafted layer is totally impermeable to flow which
ight not always be the case. Nevertheless, if the same procedureor thickness estimation is performed, obtained values can be com-ared. The calculation of the layer thickness in water results in ahickness of only 13 nm, which represent only 2% in pore size reduc-ion. One should take this difference into account when designingurification processes since the data from the often uses water asmobile phase [25–26,44–46] and different values of permeabilityight occur when different mobile phases are applied.Since there was a substantial difference in the permeability of
he grafted and non-grafted monolith, one might wonder whathe contribution of grafting was on the increased capacity increasebserved. A reduction in pore size commonly means that a higherurface area and consequently a higher capacity. To discriminateetween these two effects, a non-grafted DEAE monolith with theame permeability as the grafted one was prepared. In this case,he lower permeability was due to the smaller channel size with anverage radius of around 160 nm (determined by mercury intrusionorosimetry) having the same porosity of the non-grafted mono-
ith with larger pores. The capacity of such monoliths was foundo be slightly higher in comparison to non-grafted monolith beinground 40 mg/ml but still twice lower than the capacity of therafted monolith. This result indicates that grafting was the maineason for a capacity increase and that the higher capacity at a givenermeability can be achieved by grafting.
To better understand the mechanism of molecule interactionith the grafted layer, the ionic capacity of grafted and non-
rafted monoliths was investigated. The relative ionic capacityas determined using a method based upon pH transient [31,47].
Iica
ne DEAE group. Normalized “activity” was calculated by dividing relative ionicapacity by an estimated ligand density and normalized it with the value of non-rafted monolith.
he estimated relative ionic capacities for grafted and non-graftedonoliths were 25 and 165, respectively (Table 1). The graftedonolith exhibited 6.6 times lower ionic capacity compared to a
on-grafted monolith despite having a significantly higher proteinapacity. This result confirms the conclusion that the higher pro-ein capacity is not related to the ionic capacity, but rather to thepace distribution of charges as already demonstrated by others16]. However, one cannot directly correlate relative ionic capaci-ies of grafted and non-grafted ion exchangers since the activitiesf the ion exchange groups might be different [48]. To get the infor-ation about the amount of DEAE groups, an elemental analysis
f nitrogen was performed. Data are shown in Table 1. There is aignificant difference in the nitrogen content. Ligand density onrafted monolith is 1.8-fold lower when compared to the non-rafted monolith. It is also interesting to compare ionic activity withigand density. If ionic capacity is divided by ligand density an aver-ge “activity” of the group is obtained. When values for grafted andon-grafted monolith are further divided by average group “activ-
ty” for non-grafted monolith (normalized) it becomes obvious thathe average “activity” of DEAE groups on grafted monoliths in only0% of the “activity” of non-grafted groups. Differences in groupactivity” indicate the different nature of DEAE groups present onhe surface and therefore we can consider that this should resultn different trends of binding capacity for molecules of differentizes.
The effect of molecular size on capacity was estimated for dif-erent proteins and for pDNA. Three proteins of different sizes withimilar isoelectric points were used: �-lactoglobulin (18.4 kDa; pI.2), BSA (67 kDa; pI 5.6) and thyroglobulin (660 kDa; pI 4.3). Theirstimated radius of gyration is 1.6 nm [7], 2.8 nm [7] and 6.4 nm49], respectively. In addition, 4.7 kbp pDNA (3055 kDa) with anstimated radius of gyration of 94 nm (assuming that it is predom-nantly in CCC form) [50] was tested. The data are summarized inable 2. It can be seen that in all cases the binding capacity wasignificantly higher for the grafted monolith when compared tohe non-grafted one. The protein capacity for non-grafted mono-ith was similar, while it was considerably lower for pDNA. Thisndicates a restricted access to certain specific surfaces. For therafted monolith, a different trend was obtained. The highest bind-ng capacity was achieved for the smallest protein (�-lactoglobulin)nd decreased with enlarging the molecular size. This somehowonfirms the assumption that the number of layers should beependent on the molecular size since the grafted layer thickness
s constant. An interesting graph is obtained when the ratio ofon-grafted and grafted monolith binding capacity is plotted as a
unction of the molecule’s radius of gyration (Fig. 6). It can be seenhat for the smallest protein this value is 3.5 and it seems to con-
t is interesting that this is also true for pDNA despite having a sim-lar size as the estimated grafted layer thickness. The same trend ofapacity for different size proteins (�-lactoglobulin, BSA and ure-se) was also reported by Kugoma et al. [23]. The exception was
90 V. Frankovic et al. / J. Chromatogr. A 1207 (2008) 84–93
Ftu
eiea
diIawameamippm
tmvsTbabimwAcbtf
Fig. 7. Agarose gel electrophoresis of plasmid pEGFP (4.7 kbp). M, marker (1 �l); L,lDps
bbacltrlttcfet
tnoDtclose to 100%, while conditions without NaCl indicated lower recov-
TB
S
GN
ig. 6. Binding capacity ratio between grafted and non-grafted monolith as a func-ion of molecular radius of gyration. Binding capacity values from Table 2 weresed.
xtremely high capacity for urease on grafted membrane resultingn a tenfold higher number of layers in comparison to BSA. How-ver, it might be that another mechanism was responsible for suchhigh capacity.
The high capacity of the grafted monolith caused a higherensity of the molecules on the surface, especially for proteins,
ncreases agglomeration and consequently lowers the recovery.n addition, if the grafted layer would act like a uniform spacebove the skeleton, it can be speculated that the bound moleculesould be surrounded by tentacles resulting in a multipoint inter-
ction between the molecules and the tentacles. Again, recoveryight be lower due to the higher strength of the bond. However,
xperimental data shows that in all cases recovery was above 85%nd comparable within the experimental error for the non-graftedonolith (Table 2). Furthermore, electrophoresis analysis of pDNA
ndicates also that the open circular and supercoiled form ratio isreserved. Therefore, we can conclude that no degradation of thelasmid occurred during the purification of pDNA on the graftedonolith (Fig. 7).To further investigate the nature of molecule–ligand interaction,
he z-factor representing the number of binding sites was deter-ined. The isocratic method was used in all cases. Logarithmic
alues of retention time vs. salt concentration for tested proteins arehown in Fig. 8 and the corresponding calculated z-values in Table 2.he z-factor for pDNA was not determined since it is expected toe very high [51] and the accuracy of its determination is question-ble. Under the tested conditions the numbers of binding sites onoth grafted and non-grafted monoliths are identical. This result
s somehow unexpected since one would expect that when theolecule is surrounded by tentacles the number of interaction sitesould increase due to the availability of the entire molecule surface.lso, the theoretical prediction for non-charged grafted layer indi-
ates that when the molecule begins to penetrate the layer, voidehind the molecule fills quickly [52]. It is also not the case thathe measured number of binding sites is the maximum value sinceor e.g. free BSA in solution with pH 7.4 its charge is estimated to
e0nw
able 2inding capacity, recovery and number of binding sites for different proteins on a grafted
tationary phase Binding capacity on 10% and 50% (mg/ml)/recovery (%)
oading sample (8 �l); n-g, pDNA (2 �l) eluted after saturation on non-grafted CIMEAE (n-g); g, pDNA (2 �l) eluted after saturation on grafted DEAE (g). Conditions:DNA samples were analyzed on 0.7% agarose gel, voltage 80 V for 60 min. Gel wastained with SYBR Safe.
e around 10, therefore significantly higher [53]. The same num-er of binding sites could indicate that the grafted layer does notlways behave like a uniform space, but rather like a set of tenta-les to which the molecule is attached. In this scenario, the graftedayer would actually increase the surface area provided by the ten-acle chains. This conclusion is in agreement with the experimentalesults from Kusumo et al. [54]. They investigated the effect of chainength and density on the binding capacity for BSA and found thathere was a constant ratio of 120 monomer units per bound pro-ein molecule regardless of the tentacle length and density. If theharged grafted chains act like tentacles, one can also speculate theacilitation of diffusion as reported by Stone and Carta [37]. How-ver, before any final conclusion is drawn, further experiments haveo be performed.
Measuring BC and recovery for pDNA at different salt concen-rations indicated another similar behavior between grafted andon-grafted monolith. Both monoliths exhibited the same trendf capacity as a function of NaCl concentration and the recovery.ata are summarized in Table 3. Recovery of pDNA for both sys-
ems with added salt (for grafted and non-grafted monolith) was
ries. We can see that the maximal capacity was obtained at around.1 M NaCl and a similar trend was reported by Tianinen et al. onon-grafted QA agarose resins where the maximum pDNA capacityas obtained in the presence of 0.2 M NaCl [55].
and non-grafted monolith
Number of binding sites, z factor
hyroglobulin �-Lactoglobulin BSA Thyroglobulin
0% 50% recovery
1 71 97 7 5 21 35 95 7 5 2
V. Frankovic et al. / J. Chromatogr. A 1207 (2008) 84–93 91
Fig. 8. Logarithmic values of retention time vs. salt concentration for �-lactoglobulin (A), BSA (B) and thyroglobulin (C). Mobile phases: 20 mM Tris–HCl, pH 7.4 with additionof proper amount of NaCl; sample injection: concentration, 1 mg/mL; volume, 20 �l 1 mg/ml; UV detection at 280 nm, flow rate 1 ml/min. Insets show raw data.
92 V. Frankovic et al. / J. Chromatogr. A 1207 (2008) 84–93
Table 3Binding capacity and recovery for pDNA measured at different salt concentrations in the loading buffer solution on a grafted and non-grafted monolith
Stationary phase Binding capacity for pDNA (mg/ml) at different salt concentrations/recovery (%)
To investigate the practical applicability of grafted monoliths,he purification of pDNA was performed on grafted and non-grafted
onoliths. Both chromatograms are shown in Fig. 9. Similar tohe slope of the breakthrough curves also in the case of separa-ion broader peaks are obtained indicating higher dispersion ofhe grafted monolith. Another important difference is that RNAnd pDNA were both eluted at 0.05 M NaCl higher ionic strength,ndicating stronger interaction with active groups. Higher ionictrength was required also for the test protein separation on the
rafted monolith (data not shown). We have to mention that thisata is not in contradiction with the equal z-factor for grafted andon-grafted monoliths. Ionic strength at which the molecule isluted is the sum of the number of binding sites and their strength
aite
ig. 9. Purification of pDNA by ion-exchange chromatography on grafted (g) and non-graf0 mM EDTA pH 7.2; buffer B: 50 mM Tris–HCl, 10 mM EDTA, 1.1 M NaCl, pH 7.2. Gradientradient from 0 to 33% to 100% buffer B. Flow rate 2 ml/min. UV detection at 260 nm. Inje
100 14.1 16.1 100100 6.0 6.4 100
f interaction. Therefore, the same number of binding sites canive different ionic strengths of elution or different number ofinding sites can result in the same ionic strength of elution [51].igher ionic strength of elution for the grafted monolith coulde explained with high flexibility of grafted chains; therefore the
nteractions between the molecule and the active group can betronger.
From the practical point of view, we can see that in both casesase line separation was achieved and elution times between RNA
nd pDNA were comparable. Because of that and the high bind-ng capacity, grafted monoliths can be advantageous especially inhe processes for purification of large molecules like proteins andspecially pDNA [56,57].
ted (n-g) monolith at different gradients. Mobile phase—buffer A: 50 mM Tris–HCl,s were as followed: (A) linear gradient from 0 to 100% buffer B; (B) stepwise-linearction volume was 500 �l.
mato
4
rbct
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V. Frankovic et al. / J. Chro
. Conclusions
An increase in the binding capacity in loading experiment andetention in an elution mode with reduced permeability, and aroader and more symmetrical peak with the grafted material areonsistent with the contribution of the grafted layer of increasedhickness to the retention of proteins.
The results of this study demonstrate that grafted monolithsxhibit at least twofold higher binding capacity over a broad rangef molecular sizes including pDNA. Although the introduction ofolymer chains onto the surface of a methacrylate monolith led todecrease of permeability, their capacity is still significantly higher
hen non-grafted monolith having the same permeability underperating conditions. Since the flow-unaffected properties werereserved, grafted methacrylate monoliths may become a resin ofhoice for the downstream processing of various macromolecules.
cknowledgments
Support from the Ministry of Education, Science and Sport andhe Ministry of the Economy of the Republic of Slovenia throughhe project L2-7080 is gratefully acknowledged. We also thank Dr.othar Britsch for careful reading of the manuscript and valuableuggestion.
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