Ring-opening metathesis polymerization-derived, lectin-functionalized monolithic supports for affinity separation of glycoproteins

J. Sep. Sci. 2013, 36, 1169–1175 1169

Rajendar Bandari1Jurgen Kuballa2

Michael R. Buchmeiser1,3

1Lehrstuhl fur MakromolekulareStoffe und Faserchemie, Institutfur Polymerchemie, UniversitatStuttgart, Stuttgart, Germany

2Galab Laboratories GmbH,Geesthacht, Germany

3Institut fur Textilchemie undChemiefasern (ITCF),Denkendorf, Germany

Received November 13, 2012Revised December 27, 2012Accepted December 27, 2012

Research Article

Ring-opening metathesispolymerization-derived, lectin-functionalizedmonolithic supports for affinity separationof glycoproteins†

Lectin-functionalized monolithic columns were prepared within polyether ether ketone(PEEK) columns (150 × 4.6 mm id) via transition metal-catalyzed ring-opening metathe-sis polymerization of norborn-2-ene (NBE) and trimethylolpropane-tris(5-norbornene-2-carboxylate) (CL) using the first-generation Grubbs initiator RuCl2(PCy3)2(CHPh) (1, Cy =cyclohexyl) in the presence of a macro- and microporogen, i.e. of 2-propanol and toluene.Postsynthesis functionalization was accomplished via in situ grafting of 2,5-dioxopyrrolidin-1-yl-bicyclo[2.2.1]hept-5-ene-2-carboxylate to the surface of the monoliths followed by reac-tion with �,�-diamino-poly(ethyleneglycol). The pore structure of the poly(ethyleneglycol)-derivatized monoliths was investigated by electron microscopy and inverse-size exclusionchromatography, respectively. The amino-poly(ethyleneglycol) functionalized monolithiccolumns were then successfully used for the immobilization of lectin from Lens culinarishemagglutinin. The thus prepared lectin-functionalized monoliths were applied to the affin-ity chromatography-based purification of glucose oxidase. The binding capacity of Lens culi-naris hemagglutinin-immobilized monolithic column for glucose oxidase was found to be2.2 mg/column.

Keywords: Bioseparation / Glycoproteins / Lectin affinity chromatography / Mono-lith / ROMPDOI 10.1002/jssc.201201042

1 Introduction

Glycoproteins play a pivotal role in many biological pro-cesses including immune defense, fertilization, cell–cell ad-hesion, and inflammation [1]. They are also key componentsof cell membranes and important tools for pharmaceuticalsand biomarker development [2]. Currently, glycoprotein pu-rification is accomplished via lectin affinity chromatographyon lectin-modified agarose [3–5]. Generally, the principle ofaffinity separation relies on the reversible complex formationbetween a polymer-bound selector and specific sites of theglycoprotein and can be tuned by pH, the ionic strength anddwell time. Other biomolecules present in the sample, whichlack the complementary-binding sites, are eluted in courseof several washing steps. Finally, the purified glyocprotein isdesorbed from the support by rinsing with concurring lig-

Correspondence: Prof. Michael R. Buchmeiser, Lehrstuhl furMakromolekulare Stoffe und Faserchemie, Institute of PolymerChemistry, University of Stuttgart, Pfaffenwaldring 55, D-70569Stuttgart, GermanyE-mail: [email protected]: +49-0-711-68564050

Abbreviations: ISEC, inverse-size exclusion chromatography;GOX, glucose oxidase; LCH, Lens culinaris hemagglutinin;PEEK, polyether ether ketone; SP, swelling propensity

ands of higher affinity or alteration of the buffer system [6].Until now, numerous ligand-adsorbant systems and formatshave been reported [6]. Among these, lectin-based systemshold a strong position. Lectines are proteins or glycoproteins,usually consisting of 2–4 subunits, offering one or more spe-cific carbohydrate recognition domains [7]. Nowadays, a broadvariety of substrates for lectin immobilization is available,whether based on agarose [5,8], cellulose [9], silica [5,10–12],or polymeric media [5,11,13–15]. Nonetheless, fast separationsystems, which allow for the work-up of large volumes withinshort times, are still rare. During the last decade, the uniqueproperties of polymeric monolithic media have attracted con-siderable attention in the field of separation science and het-erogeneous catalysis [16–21]. Polymeric monoliths consist ofone single unitary piece of a highly porous cross-linked poly-meric material whose particular structure is designed in a waythat meets the requirements for the fast separation of macro-molecules, e.g. proteins and enzymes [16, 17, 19]. Recently,we reported on miniaturized poly(methacrylate)-based mono-lithic systems prepared via electron beam-initiated free rad-ical polymerization and a simple one-step surface function-alization with amine-containing polymers for lectin affinity

†This paper is included in the virtual special issue Monolithsavailable at the Journal of Separation Science website.

C© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1615-9314/homepage/virtual_special_issue__monoliths.htm

1170 R. Bandari et al. J. Sep. Sci. 2013, 36, 1169–1175

chromatography in the 96-tip, high-throughput format [22].Here, we report on lectine-functionalized monoliths in thecolumn format prepared via ring-opening metathesis poly-merization and their successful use for the selective extrac-tion of glucose oxidase via affinity chromatography.

2 Experimental

2.1 Chemicals and reagents

Trimethylolpropane triacrylate (TMPTA), dicyclopentadi-ene, methanol, toluene, 2-propanol (2-PrOH), N-hydroxysuc-cinimide, norborn-2-ene, 5-norbornene-2-carboxylic acid,(endo/exo mixture), CH2Cl2, DMSO, DMF, ethyl vinylether (EVE), THF, �,�-diaminopoly(ethyleneglycol) (�,�-dia-mino-PEG, Mn = 3400 g/mol), and the first-generationGrubbs initiator RuCl2(PCy3)2(CHPh) (1) were pur-chased from Sigma-Aldrich (Munich, Germany). Trimethy-lolpropane-tris-(5-norbornene-2-yl-carboxylate) (CL) was pre-pared via a published protocol [23]. Polystyrene (PS)standards 800 < Mw < 2 000 000 g/mol used for inverse-sizeexclusion chromatography (ISEC) were purchased from Poly-mer Standards Service (PSS, Mainz, Germany). Lens culinarishemagglutinin (LCH) and the adsorption buffer for LCH,Bis-Tris (pH 6.0) were obtained from Galab Technologies(Geesthacht, Germany). Manganese(II)chloride tetrahydrate,methyl �-D-mannopyranoside, NaN3, zinc chloride, Bis-Tris,glucose oxidase from Aspergillus niger (type VII, lyophilizedpowder, ≥100 000 units/g, without added oxygen) were pur-chased from Sigma-Aldrich (Munich, Germany). CaCl2 waspurchased from VWR International (Darmstadt, Deutsch-land). NMR data were obtained at 250.13 MHz for protonand 62.90 MHz for carbon in the indicated solvent at 25�Con a Bruker Spectrospin 250 and are listed in parts per mil-lion downfield from tetramethylsilane for proton and car-bon. SEMs were recorded on a Zeiss Auriga applying 1.74 or2.40 kV.

2.2 HPLC system

Analytical-scale separations were carried out on an AgilentTechnology HPLC-system (Boblingen, Germany). The HPLCsystem consisted of a binary HPLC pump, a diode array UV-Vis detector, an auto sampler, a column oven and a samplethermostat. Glucose oxidase was detected at � = 280 nm.

2.3 Preparation of buffers

Adsorption buffer A: the content of a 1.00 L bottle of Bis-Tris(pH 6.0) adsorption buffer (Galab technologies) was dissolvedin 1 L of deionized water, then NaN3 (0.02 wt%) was addedand stored at 4�C. Elution buffer B was prepared by diluting amixture of 10 mM Bis-Tris solution (3.45 g, 16.4 mmol), 150mM NaCl solution (4.4 g, 75.2 mmol), 1 mM CaCl2 solution(50 mg, 340 �mol), 1 mM MnCl2 solution (100 mg, 507 �mol),and 200 mM methyl �-D-mannopyranoside solution (19.4

g, 100 mmol) to 500 mL using HPLC water. The pH wasadjusted to 6.0 using HCl and finally, 100 mg of sodiumazide were added and the solution was stored at 4�C.

2.4 Preparation of 2,5-dioxopyrrolidin-1-yl-bicyclo

[2.2.1]hept-5-ene-2-carboxylate (2)

To bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (7.5 g, 54.2mmol) and N,N′-dicyclohexyl carbodiimde (13.43, 65 mmol)in anhydrous THF (50 mL) was added N-hydroxysuccinimide(7.496 g, 65.1 mmol) at 0�C. The reaction was stirred at roomtemperature for 16 h; then the reaction mixture was filteredthrough a bed of celite. The THF was removed under re-duced pressure. The crude product was dissolved in CH2Cl2and filtered through a bed of silica and washed with another20 mL of CH2Cl2. Finally, the CH2Cl2 fractions were mixedand the solvent was removed in vacuo under reduced pres-sure. A white solid was obtained. Yield; 10.9 g (85%). 1H-NMR(250 MHz, CDCl3) � = 1.32–1.35 (m, 1H), 1.58 (m, 2H),2.20 (m, 1H), 2.81–2.85 (s, 4H), 2.99 (m, 1H), 3.27 (m, 1H),3.40–3.42 (m, 1H), 6.10–6.24 (m, 2H); 13C-NMR (250 MHz,CDCl3) � = 25.5, 29.5, 29.7, 40.5, 42.4, 46.4, 49.6, 132.1, 138.1,169.2, 169.9. IR (ATR mode): 3535 (m), 3067 (m), 2993 (s),2868 (s), 1735 (s), 1714 (s), 1450 (s), 1427 (s), 1212, (s), 1069(s), 1008 (s), 946 (s), 897 (s), 776 (s), 734 (s), 711 (s) cm−1;GC-MS (EI) calculated for C12H13NO4 m/z = 235.08; found235 (M·+).

2.5 Polymerization of 2,5-dioxopyrrolidin-1-yl-bicyclo

[2.2.1]hept-5-ene-2-carboxylate

The polymerization procedure was as follows: A solution of1 (4 mg, 0.0048 mmol) in 1 mL of CH2Cl2 was added to asolution of 2 (110 mg, 0.468 mmol) in 4 mL of CH2Cl2. Themixture was stirred for 2 h at 40�C. After this time, ethylvinyl ether (0.6 mL) was added and the mixture was stirredfor another 30 min. The solvent removed in vacuo, then10 mL of methanol was added. The white polymer that formedwas filtered off and dried in vacuo. Isolated yield: 90%. Poly-(2): 1H-NMR (250 MHz, CDCl3) � = 1.2–1.6 (b, 1H), 1.87(bs, 2H), 2.17–2.18 (bm, 1H), 2.63–2.80 (bs, 6H), 3.19 (b,1H), 5.3–5.68 (m, 2H); 13C-NMR (250 MHz, CDCl3) � = 15.6,25.4, 35.6, 39.1, 42.1, 44.3, 45.5, 46.7, 66.1, 128.4, 132.4, 136.0,169.1, 170.1; IR (ATR mode): 3495–3535 (b), 3067 (m), 2993(b), 2868 (b), 1735 (b), 1451 (b), 1427 (s), 1213 (b), 1069 (b),1008 (s), 830 (s), 734 (s) cm−1

. GPC traces in (THF); calcu-lated Mw for poly-2 = 21 000; found (Mw) = 28 000 g/mol,PDI = 1.7.

2.6 Preparation and functionalization of monoliths

PEEK columns (4.6 × 150 mm id) were consecutively washedwith acetone, water, and ethanol and dried at 40�C for 2 h.The PEEK-HPLC columns were closed at one end with endfittings and placed in an ice-cold water bath. Two differentsolutions (A, B) were prepared and cooled to –15�C. Solution


J. Sep. Sci. 2013, 36, 1169–1175 Liquid Chromatography 1171

A consisted of 21.5 wt% of cross-linker (CL), 21.5 wt% ofNBE, and 47.5 wt% of 2-PrOH, while solution B consisted of0.4 wt% of initiator 1 in toluene. Both solutions were mergedat –15�C and mixed for approximately 20 s. The column wasfilled with the polymerization mixture, sealed with Tefloncaps and kept at 0�C for 15 min. After rod formation wascomplete, the column was removed from the ice bath andstored at room temperature for 8 h. In order to remove non-reacted monomers, the columns’ open ends were cleanedand closed with end fittings and attached to the HPLC sys-tem, then the columns were flushed with 10 mL of a 1,2-dichloroethane at a flow rate of 0.1 mL/min. Polymerizationwas reinitiated by addition of 2. For these purposes, a 3.0 wt%solution of 2 in 1,2-dichloroethane was introduced into themonoliths at a flow rate of 0.5 mL/min. Functionalizationwas allowed to proceed for 2 h at 40�C. After functionaliza-tion, the monolith was end caped with a solution of EVE/DMSO/THF (20:30:30 vol.%). Afterwards, monoliths werewashed with 10 mL of 1,2-dichloroethane, then a solutionof �,�-diamino-poly(ethyleneglycol) (150 mg, 42.6 �mol in2 mL of 1,2-dichloroethane) was introduced into the mono-lith. Monolith were sealed at both ends and kept at roomtemperature for 16 h. Next, the monoliths were flushedwith 15 mL 1,2-dichloroethane to remove any unattached�,�-diamino-poly(ethyleneglycol). Finally, monoliths werewashed with10 mL of ethanol and stored in a 50:50 mixture(vol/vol) of ethanol and water.

2.7 Immobilization of lectin

LCH 2.5 mg/mL was dissolved in adsorption buffer A,3 mL were injected into the column and immobilization was

allowed for 16 h at 4�C. Then, the monolith was washed with20 mL of adsorption buffer at a flow rate of 0.2 mL/min.Finally, the monolith was tested for affinity chromatographyof glucose oxidase (GOX).

2.8 Characterization of monoliths

ISEC [24, 25] measurements were carried out according to apublished protocol.

3 Results and discussion

3.1 Synthesis and functionalization of monoliths

Monolithic columns were synthesized from NBE andtrimethylolpropane-tris-(5-norbornene-2-yl-carboxylate) (CL)according to a published procedure [23]. 2-PrOH served asmacroporogen, toluene served as microporogen. 0.4 wt% ofthe initiator RuCl2(PCy3)2(CHPh) (1, Cy = cyclohexyl) wereused throughout. For the functionalization of monoliths, 2,5-dioxopyrrolidin-1-yl-bicyclo[2.2.1]hept-5-ene-2-carboxylate (2,Scheme 1) was grafted to the monolith’s surface using a“grafting-from” approach. Optimum conditions for graftingwere determined by homopolymerizing 2 with initiator 1 inCH2Cl2 at 40�C. Termination with ethyl vinyl ether/DMSOensured for a complete removal of the Ru-based initiator aschecked by the inductively coupled plasma-optical emissionspectroscopy. The final Ru-contentin the monolithic rod was>28 ppm (below the LOD of the inductively coupled plasma-optical emission spectroscopy).

Next, monoliths were treated with �,�-diamino-poly(ethyleneglycol) to increase the hydophilicity of the

Scheme 1. Synthesis and func-tionalization of monoliths.



monoliths and to react the amino groups with the surface-bound active ester moieties (Scheme 1). Finally, monolithswere washed with 30 mL of 1,2-dichloroethane to remove anyunreacted �,�-diamino-poly(ethyleneglycol).

3.2 Preparation of LCH-modified columns

LCH (7.5 mg) was dissolved in 3 mL of adsorption buffer Aand the solution was injected onto the column (M1). Immo-bilization was allowed to proceed for 16 h at 4�C. Next, themonolith was washed with 8 mL of adsorption buffer at a flowrate of 0.2 mL/min. RP-HPLC was performed for the evalua-tion of the amount of immobilized LCH. For comparison, anonfunctionalized column (4.6 × 150 mm id peek column)prepared from the same recipe as applied to the synthesis ofmonolith M1 was used. The following mobile phases wereused: A – 0.1% TFA in water and B – 0.1% TFA in ACN. Thegradient was 0–100% B within 10 min, the flow rate was set to1.0 mL/min. The amount of immobilized LCH was estimatedby subtracting the total integral absorbance at � = 254 nmof the effluent and washes from the one of the original LCHsolution. Accordingly, 5.7 mg of LCH were immobilized onmonolith M1.

3.3 Characterization of monoliths

Slices of the monolithic matrix were subjected to SEM, whichrevealed average microglobule diameters in the range of 4–5 �m and interconnected flow through channels (voids) inthe range of approximately 12 �m. Moreover, SEM imagesshowed a homogeneous porous structure obtained after thefunctionalization with �,�-diamino-poly(ethyleneglycol) withno visible coating formation on the microglobules, whichwould result in a reduction in micro- and meso porosity(Fig. 1).

For the determination of the total porosity and pore sizedistribution, monolith M1 was characterized in wet condi-tions by ISEC [24, 25]. Such measurements are essentialsince one wants to avoid immobilization of major fractionsof valuable-binding sites inside pores in the range of 10–50 nm that are hardly accessible by the analyte of inter-est. In such case, synthesis would have to be adapted. Formeasurements, a series of narrowly distributed poly(styrene)(PS) standards with molecular weights between 800 < Mw <

2 000 000 g mol−1 as well as toluene (Mw = 92 g/mol)were applied using CHCl3 as the mobile phase. For thedetermination of the exact elution volumes, 5 �L samplesof the individual PS standards and toluene (0.25 mg/mL)dissolved in chloroform were injected applying a flowrate of 0.6 mL/min. Elution volumes were determined at254 nm from the peak maximum and corrected for the ex-tra column volume of the equipment. All further calcula-tions were done according to a published protocol [24, 25].Figure 2 reports the pore-size distributions of monolithM1. According to these measurements, monolith M1 pos-

Figure 1. SEM images of monoliths M1 (A) before and (B) afterfunctionalization of the monoliths.

Figure 2. Logarithm of the average pore diameters (log�average,in A) versus their relative abundance (R, in %) for monolith M1.

sessed a total porosity (g t) of 64 vol% with a volume frac-tion of pore volume of 12%. The pore volume was ca.400 �L/g. A major fraction of pores (ca. 60%) is found inthe micropore region, while there are virtually no mesopores.The macropore region accounts for roughly 40%.

3.4 Column permeability

For the evaluation of the mechanical strength of monolithM1, the pressure drop was measured for water, CH3OH,THF, and ACN at different flow rates. Highly linear plots



Figure 3. Pressure drops for monolith M1 applying water,methanol, acetonitrile, and THF at various linear velocities.

(Fig. 3) with 0.006 < R2 < 0.999, were obtained. The back-pressure of monolith M1 was only 210 MPa applying wateras mobile phase and a linear flow of 5 mm/s. According toDarcy’s law, the flow of a liquid through a porous medium isproportional to the pressure drop over a given distance. Usingthis relationship, the permeability of the monolithic columnwas calculated according to:

B0 = L�/P (1)

where B0 is the column permeability, stands for the linearvelocity, L is the column length; � is the solvent viscosityand P is the column backpressure. According to Eq. (1), thepermeability of M1 was found to be B0 = 3.2 × 10−13 m2.Next, the swelling propensity (SP) was calculated. The SP isa criterion for the shrinkage and swelling of the material indifferent solvents. The closer the SP-value is to zero, the lesspronounced swelling and shrinking are. The SP was deter-mined by measuring the pressure drop along the columnand found to be 0.20, 0.17, and 0.10, respectively, for THF,CH3OH, and ACN. In view of these values, which are quiteclose to zero, the monoliths can be considered mostly rigidwith a very low swelling propensity, at least for the solventsinvestigated.

3.5 Affinity separation of GOX

The separation performance of the LCH-modified monolithM1 was tested by injecting a model mixture of BSA and glu-cose oxidase. Prior to analysis, monolith M1 was equilibratedwith 10 mM Bis-Tris buffer solution (pH 6.0) containing150 mM NaCl, 1 mM CaCl2, and 1 mM MnCl2 (bindingbuffer A). Then glucose oxidase and BSA (1 mg/mL) were dis-solved in adsorption buffer A and 50 �L of this solution wereinjected onto monolith M1, followed by consecutive washingwith adsorption buffer A for 3 min and with 0–100% of elutionbuffer B within 10 min applying a flow rate of 1.0 mL/min.This analysis demonstrated that the unbounded protein BSAwas not retained on the column M1 (Fig. 4A) while GOX was

Figure 4. Affinity separation of glucoseoxidase on monolith M1.PEEK column (150 × 4.6 mm id; adsorption buffer A: 10 mMBis-Tris buffer solution (pH 6.0) containing 150 mM NaCl, 1 mMCaCl2, and 1 mM MnCl2; elution buffer B: 10 mM Bis-Tris buffersolution (pH 6.0) containing 150 mM NaCl, 1 mM CaCl2 and 1 mMMnCl2 and 200 mM of methyl �-D-mannopyranoside; flow rate1.0 mL/min; gradient: 1–10 min; 0–3 min 100% of A, then 3–10min 0–100% of B. UV: 280 nm.

specifically bounded to LCH and quantitatively eluted witha 200 mM solution of methyl �-D-mannopyranoside (elu-tion buffer B). Next, various amounts (20–100 �L) of a GOXsolution (5 mg/mL) were injected onto M1, followed by con-secutive washing with adsorption buffer A for 3 min and with0–100% elution buffer B within 10 min applying a flow rateof 1.0 mL/min. Gradient elution rested in symmetric peaksat � = 280 nm, intensities increased with increasing amountsof GOX (Fig. 4B).

3.6 Binding capacity of LCH-functionalized

monoliths

The column-binding capacity was determined from the break-through curves of GOX. Briefly, the monolithic columnM1 was equilibrated with binding buffer A. Then, a GOX(1 mg/mL) solution was passed through the column at aflow rate of 1.0 mL/min and the effluent was monitoredvia UV-absorption at � = 280 nm. The void volume of thecolumn was determined by injecting water while washingwith binding buffer A. Once breakthrough was observed, the



Figure 5. Breakthrough curves for GOX on the monolithic col-umn M1. Conditions for 5 (A): GOX (1.0 mg/mL) was dissolvedin buffer A and pumped through the column at a flow rate of 1.0mL/min from 0–15 min; conditions for 5 (B): eluted GOX using100% of elution buffer B from 0–10 min, flow rate 1.0 mL/min, UV(280 nm).

LCH-functionalized column was washed with adsorptionbuffer for 10 min at a flow rate of 1.0 mL/min. Then elu-tion buffer B was pumped through the column at 1.0 mLmin for 10 min and the UV-absorption was recorded at � =280 nm. Finally, the column was reequilibrated and reequili-brated with adsorption buffer A. This procedure was repeatedthree times. The column capacities were calculated accordingto the following equation:

column capacity (Q, �eq/mL) = C0(VL − Vo − Ve) (2)

Where C0 is the concentration of GOX (1 mg/mL), VL

is the volume loaded up to the breakthrough point; V0 is thevoid volume of the column and Ve is the extra column volumeof the column. According to these calculations, (Fig. 5), theaverage loading capacity was estimated to be 2.2 mg/columnfor monolith M1, corresponding to 2.4 mgGOX/gpolymeric support.This value obtained for monolith M1 in the column formatis one order of magnitude lower than the one previouslyreported for columns in the spin-tip format [22].

3.7 Reproducibility of affinity performance

The reproducibility of the affinity separation of GOX waschecked by preparing two identical LCH-functionalizedcolumns. The batch-to-batch reproducibility was determinedby calculating the binding capacities for GOX, whichwas found to be 2.2 ± 0.2 mg/column. These resultsclearly indicate that the presented method for the prepa-ration of LCH-functionalized monolithic columns is highlyreproducible.

4 Conclusions

A versatile method for the preparation and functionalizationof hydrophilic polymeric monoliths by ring-opening metathe-sis polymerization has been developed. The prepared PEG-amine-monoliths were successfully used for the immobi-lization of lectin i.e. LCH and successfully tested for theirperformance in the separation of glucose oxidase using affin-ity chromatography.

This work was generously supported by the Federal Ministryof Education and Research (BMBF, contract nr. 0315333B).

The authors have declared no conflict of interest.

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Ring-opening metathesis polymerization-derived, lectin-functionalized monolithic supports for affinity separation of glycoproteins