Synthesis and Application of a Novel Ligand for Affinity Chromatography BasedRemoval of Endotoxin from Antibodies

Laura A. McAllister, Mark S. Hixon, Roberta Schwartz, Diane S. Kubitz, and Kim D. Janda*

Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, and Worm Institute for Research andMedicine (WIRM), The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037.Received September 22, 2006; Revised Manuscript Received December 22, 2006

Endotoxin or lipopolysaccharide (LPS) contamination in proteins expressed by Gram-negative bacteria is a majordrawback associated with protein expression. Endotoxin intoxication in humans and animals above a certainthreshold level can result in a fatal immune response. Reduction in endotoxin levels is therefore essential beforeproteins can be used in in vivo studies or sold as pharmaceutical products. Affinity chromatography employingthe peptide Polymyxin B (PMB) as an affinity ligand is one way in which endotoxin contamination has beenaddressed; this is, however, a costly process. We describe the synthesis of a novel affinity ligand based on thestructure of the drug pentamidine, which can be applied effectively in endotoxin removal. The synthetic route tothis ligand is straightforward and inexpensive, while the ligand can be readily immobilized onto activated sepharosebeads. Thus, we demonstrate that these pentamidine affinity beads bind endotoxin/LPS with comparable capacityto PMB affinity systems, that the beads can be recycled efficiently and economically without loss of bindingcapacity, and application of the functionalized beads for endotoxin removal in an authentic contaminated antibodysample.

INTRODUCTION

Endotoxin or lipopolysaccharide (LPS) (Figure 1) is the majorconstituent of the outer membrane of Gram-negative bacteria,composed of a nontoxic polysaccharide portion and the biologi-cally active portion known as lipid A (1). Lipid A is a bis-phosphorylated disaccharide bearing several hydrophobic acylchains (Figure 2). The presence of endotoxin in blood, knownas Gram-negative sepsis, is the leading cause of death inintensive care units (2). Endotoxin/LPS induces an exaggeratedresponse by the immune system resulting in uncontrolledproduction of inflammatory mediators, which can lead to fatalshock syndrome and eventually multiple organ failure (3).

Proteins expressed by Gram-negative bacteria are oftencontaminated with endotoxin; this contamination must bereduced sufficiently before a protein can be used in in vivostudies within animal models or sold as pharmaceutical products,for human use, such as vaccines. The FDA recommends thatno more than 5.0 EU per kilogram of body weight be introducedparenterally into a human or an animal (10 EU is approximately1 ng ofE. coli LPS). Endotoxin removal is an expensive, time-consuming, and laborious part of the protein purification process,which often results in poor recovery of the protein (4). Affinitychromatography, whereby a ligand with a high binding affinityfor LPS is immobilized on a solid support, is the gold standardfor endotoxin removal. Polymyxin B (PMB) is a cycliclipopeptide produced byBacillus polymyxathat binds LPS withhigh affinity. Agarose-supported Polymyxin B is used in affinitychromatography for endotoxin removal (Pierce, product no.20344, Detoxi-Gel AffinityPak Prepacked Columns (5× 1 mL,currently $95); however, we note that, while this ligand-supportsystem is exceptional for LPS removal, it is also prohibitivelyexpensive. Furthermore, Polymyxin B is a synthetically chal-lenging molecule and is therefore obtained exclusively bybacterial expression.

The anti-Pneumocystis cariniidrug, pentamidine (Figure 2),has been reported to bind to lipid A (the toxic constituent ofLPS) with a comparable affinity to PMB (5, 6). The high affinityof this drug for lipid A has been attributed to the terminalamidine groups of the drug being an optimal distance apart fortheir simultaneous interaction with the two anionic phosphategroups of lipid A. We reasoned that a ligand based on thestructure of pentamidine could be applied to affinity-basedremoval of endotoxins. We sought to unveil an economical,synthetically accessible alternative to Polymyxin B whichdisplays similar endotoxin binding ability. Such a moleculecould be readily synthesized on a large scale at low cost andwould offer a less expensive alternative to the use of PMB inthe endotoxin removal process. Herein, we report an efficient,facile synthesis of a tethered derivative of pentamidine that canbe immobilized onto a sepharose support. Chemical synthesisof this ligand is straightforward and inexpensive, and wedemonstrate that this support can remove endotoxin withcomparable effectiveness to PMB affinity systems. The penta-midine-based affinity system can be recycled repeatedly usinginexpensive reagents with no significant reduction in perfor-mance and results in greater protein recovery than the PMBsystem.

* Corresponding author. K. D. Janda. E-mail: kdjanda@scripps.edu.Tel: 858 784 2516. Fax 858 784 2595.

Figure 1. Schematic representation of the structure of bacterialendotoxin/LPS.

559Bioconjugate Chem. 2007, 18, 559−566

10.1021/bc0602984 CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 02/22/2007

EXPERIMENTAL PROCEDURES

General Considerations.All synthetic reactions were per-formed under an atmosphere of Ar or N2, using anhydroussolvents unless otherwise stated. Reactions were carried outusing oven-dried glassware. THF was distilled from sodium/benzophenone, and CH2Cl2 and MeOH were distilled fromCaH2. Anydrous DMF, toluene, and ethanol were purchasedfrom Aldrich. Reagents were purchased from commercialsources and used without further purification.1H NMR and13CNMR were recorded on Bruker AMX-400, Bruker DRX-500,or Bruker DRX-600 instruments, with chemical shift valuesbeing reported in parts per million relative to residual chloroform(δH ) 7.27 or δC ) 77.2) as an internal standard. High-resolution mass spectrometry was carried out at The ScrippsResearch Institute using an IonSpec Ultima high-resolutionFTMS instrument. Reactions were monitored by thin-layerchromatography (TLC) carried out on 0.25 mm E. Mercksilicagel plates (60F-254) visualizing by UV light and KMnO4

stain. Flash chromatography was carried out using Merck silicagel 60 (230-400 mesh). Reagent-grade solvents for chroma-tography were obtained from Fisher Scientific.

A BODIPY-TR cadaverine probe was purchased fromInvitrogen. LPS fromE. coli 0111:B4, LPS fromE. coli 0111:B4 FITC conjugate, pentamidine isethionate, Polymyxin B, andagarose-supported Polymyxin B were all purchased from Sigma.The Reacti-Gel CDI support was purchased from Pierce.Fluorescence measurements were carried out on a SpectraMaxGeminiEM fluorescence plate reader. Corning nonbindingsurface 96-well plates obtained from Fischer were used for allfluorescence assays. 1 mL Detoxi-Gel columns were obtainedfrom Pierce. TheLimulusAmebocyte Lysate (LAL) QCL-1000assay kit was purchased from Cambrex bioscience.

Dimethyl 3-(tert-Butyldimethylsilyloxy)pentanedioate (1b).To a solution of dimethyl 3-hydroxy-glutarate (1a) (2.92 g, 16.6mmol, 1 equiv) in DMF (10 mL) was added imidazole (1.24 g,18.3 mmol, 1.1 equiv) and TBSCl (2.74 g, 18.3 mmol, 1.1equiv). The reaction was allowed to stir at room temperature(rt) for 16 h. The reaction was diluted with 50% EtOAc/Hxand the organic layer washed with H2O. The organic layer wasthen dried (MgSO4) and concentrated in vacuo to give dimethyl3-(tert-butyldimethylsilyloxy)pentanedioate (1b) (4.80 g, 16.5mmol, 99.5%) as a clear oil which was used without furtherpurification.

1H NMR (CDCl3, 500 MHz)δ 0.06 (6H, s, (CH3)2Si), 0.84(9H, s, (CH3)3Si)), 2.55 (4H, d,J ) 7.0 Hz, 2× CH2), 3.69(6H, s, 2× CH3O), 4.55 (1H, t, CHO).

13C NMR (CDCl3, 125 MHz) δ -5.00 (2 × CH3), 17.9(C(CH3)3), 25.6 (CH3)3C), 42.4 (2× CH2), 51.5 (2× CH3O),66.3 (CHO), 171.3 (CdO).

MS (ESI-TOF): [M + H]+, C13H27O5Si requires 291.1622,found 291.1620.

3-(tert-Butyldimethylsilyloxy)pentane-1,5-diol (2). To asolution of dimethyl 3-(tert-butyldimethylsilyloxy)pentanedioate

(1b) (7.75 g, 26.7 mmol, 1 equiv) in CH2Cl2 (150 mL) at 0°Cwas added DIBALH (71 mL of 1.5 M solution in toluene, 106.8mmol, 4 equiv). The reaction was allowed to stir at 0°C for 3h and then quenched with an aq sat solution of potassium sodiumtartrate and allowed to stir overnight. The reaction mixture wasthen extracted several times with CHCl3. Combined organiclayers were dried (MgSO4) and concentrated in vacuo to give3-(tert-butyldimethylsilyloxy)pentane-1,5-diol (2) (5.78 g, 24.7mmol, 93%) as a clear oil which was used without furtherpurification.

1H NMR (CDCl3, 600 MHz)δ 0.09 (6H, s, (CH3)2Si), 0.88(9H, s, (CH3)3Si)), 1.57 (2H, broad s, 2× OH), 1.75 (2H, m,CH2), 1.83 (2H, m, CH2), 3.71 (2H, m, CH2O), 3.79 (2H, m,CH2O), 4.14 (1H, m, CHO).

13C NMR (CDCl3, 150 MHz) δ -4.70 (2 × CH3), 17.9(C(CH3)3), 25.8((CH3)3C), 38.4 (2× CH2), 59.8 (2× CH2O),69.6 (CHO).

MS (ESI-TOF): [M + H]+, C11H27O3Si requires 235.1724,found 235.1721.

4,4′-(3-(Tert-butyldimethylsilyloxy)pentane-1,5-diyl)bis-(oxy)dibenzonitrile (3a). To a solution of 3-(tert-butyldimeth-ylsilyloxy)pentane-1,5-diol (2) (5.78 g, 24.7 mmol, 1 equiv) inTHF (150 mL) was added 4-hydroxybenzonitrile (8.8 g, 74.1mmol, 3 equiv), DIAD (19.1 mL, 98.8 mmol, 4 equiv), andPPh3 (25.8 g, 98.8 g, 4 equiv). The reaction was allowed to stirat rt for 18 h. EtOAc was added, and the reaction mixture waswashed with 1 M NaOH, dried (MgSO4), and concentrated invacuo. The crude product mixture was purified by flashchromatography on silica gel (30% EtOAc/Hx) to give 4,4′-(3-(tert-butyldimethylsilyloxy)pentane-1,5-diyl)bis(oxy)dibenzoni-trile (3a) together with an impurity derived from DIAD. Thismixture was used in the next step without further purification.

1H NMR (CDCl3, 600 MHz) δ -0.01 (6H, s, 2× CH3Si),0.88 (9H, s, (CH3)3Si), 2.01 (4H, m, 2× CH2), 4.10 (4H, t,J) 6.6 Hz, 2× CH2O), 4.20 (1H, m, CHO), 6.93 (4H, d,J )9.0 Hz, 4× ArH), 7.58 (4H, d,J ) 9.0 Hz, 4× ArH).

13C NMR (CDCl3, 150 MHz) δ -4.65 (CH3)2Si, 17.9(C(CH3)3), 21.6 (2× CH2), 25.8 ((CH3)3Si), 64.5 (CH2O), 65.8(CHO), 103.9 (2× ArC), 115.1 (4× ArCH), 119.2 (2× CN),134.0 (4× ArCH), 162.1 (2× ArCO).

MS (ESI-TOF): [M+ H]+, C25H33N2O3Si requires 437.2255,found 437.2255.

4,4′-(3-Hydroxypentane-1,5-diyl)bis(oxy)dibenzonitrile (3b).4,4′-(3-(tert-butyldimethylsilyloxy)pentane-1,5-diyl)bis(oxy)-dibenzonitrile (3a) from the previous step was dissolved inMeOH (250 mL). Conc HCl (6 drops) was added and thereaction allowed to stir at rt for 1 h. The reaction mixture wasconcentrated in vacuo, and the crude product mixture waspurified by flash chromatography on silica gel (30% EtOAc/Hx) to give 4,4′-(3-hydroxypentane-1,5-diyl)bis(oxy)dibenzoni-trile (3b) (3 g, 9.3 mmol, 38% over 2 steps) as a white solid.

Figure 2. Structure of lipid A and the anti-Pneumocystis cariniidrug, pentamidine.

560 Bioconjugate Chem., Vol. 18, No. 2, 2007 McAllister et al.

1H NMR (CDCl3, 600 MHz)δ 2.00 (2H, m, CH2), 2.06 (2H.m, CH2), 4.18 (3H, m, CHO and CH2O), 4.26 (2H, m, CH2O),6.96 (4H, d,J ) 9.0 Hz, 4× ArH), 7.57 (4H, d,J ) 9.0 Hz,4 × ArH).

13C NMR (CDCl3, 150 MHz) δ 36.5 (CH2), 65.5 (CH2O),66.6 (CHO), 104.1 (2× ArC), 115.1 (4× ArCH), 119.1 (2×CN), 134.0 (4× ArCH), 161.9 (2× ArCO).

MS (ESI-TOF): [M+ H]+, C19H19N2O3 requires 322.1390,found 322.1395.

tert-Butyl 2-(1,5-Bis(4-cyanophenoxy)pentan-3-yloxy)ac-etate (4). To a solution of 4′-(3-hydroxypentane-1,5-diyl)bis-(oxy)dibenzonitrile (3b) (32 mg, 0.10 mmol, 1 equiv) in toluene(2 mL) was added 25% aq NaOH and tetrabutylammoniumhydrogen sulfate (4 mg). The reaction was allowed to stirvigorously at rt for 4 h. The reaction mixture was then extractedwith CH2Cl2. The combined organic layers were dried (MgSO4)and concentrated in vacuo. The crude product mixture waspurified by flash chromatography on silica gel (30% EtOAc/Hx) to give tert-butyl 2-(1,5-bis(4-cyanophenoxy)pentan-3-yloxy)acetate (4) (34 mg, 0.08 mmol, 77%) as a clear oil.

1H NMR (CDCl3, 600 MHz)δ 1.39 (9H, s, (CH3)3C), 2.07(4H, q,J ) 7.2 Hz, 2× CH2), 3.84 (1H, t,J ) 7.2 Hz, CHO),3.97 (2H, s CH2CO2tBu), 4.14 (2H, m, CH2O), 4.24 (2H, m,CH2O), 6.94 (4H, d,J ) 8.5 Hz, 4× ArH), 7.56 (4H, d,J )9.0 Hz, 4× ArH).

13C NMR (CDCl3, 150 MHz) δ 28.0 (CH3)3C), 33.7 (2×CH2), 64.6 (2× CH2O), 67.7 (CHO), 74.8 (CH2CO2tBu), 81.7(C(CH3)3), 104.0 (2× ArC), 115.2 (4× ArCH), 119.2 (2×CN), 133.9 (4× ArCH), 162.0 (2× ArCO), 169.3 (CdO).

MS (ESI-TOF): [M+ Na]+, C25H28N2O5Na requires 459.1890,found 459.1883.

tert-Butyl 6-(2-(1,5-Bis(4-cyanophenoxy)pentan-3-yloxy)-acetamido)hexylcarbamate (5).To a solution of tert-butyl2-(1,5-bis(4-cyanophenoxy)pentan-3-yloxy)acetate (4) (262 mg,0.61 mmol, 1 equiv) in CH2Cl2 (20 mL) was added TFA (4mL), and the reaction was allowed to stir at rt for 1 h. Thereaction was then concentrated in vacuo to givetert-butyl 6-(2-(1,5-bis(4-cyanophenoxy)pentan-3-yloxy)acetamido)hexylcar-bamate which was used without further purification.tert-Butyl6-(2-(1,5-bis(4-cyanophenoxy)pentan-3-yloxy)acetamido)hexy-lcarbamate (0.61 mmol, 1 equiv) was dissolved in CH2Cl2 (20mL). NEt3 (0.43 mL, 3.05 mmol, 5 equiv), BOP (322 mg, 0.73mmol, 1.2 equiv), andN-Boc-1,6-diaminohexane (158 mg, 0.73mmol, 1.2 equiv) were added and the reaction allowed to stirat rt for 4 h. The reaction mixture was washed with aq sat NaCl,dried (MgSO4), and concentrated in vacuo. The crude productmixture was purified by flash chromatography on silica gel(EtOAc) to givetert-butyl 6-(2-(1,5-bis(4-cyanophenoxy)pentan-3-yloxy)acetamido)hexylcarbamate (5) (264 mg, 0.46 mmol,75% over 2 steps).

1H NMR (MeOD, 600 MHz)δ 1.26 (4H, m, 2× CH2), 1.41(13H, s and m, 2× CH2 and (CH3)3C of NHBoc), 2.11 (4H,m, 2 × CH2), 2.99 (2H, m, CH2N), 3.15 (2H, m, CH2N), 3.92(1H, t, J ) 6.0 Hz, CHO), 3.99 (2H, s, CH2), 4.21 (4H, m, 2×CH2O), 7.05 (4H, d,J ) 9.0 Hz, 4× ArCH), 7.63 (4H, d,J )8.4 Hz, 4× ArCH).

13C NMR (MeOD, 150 MHz) δ 27.5 (2 × CH2), 28.8((CH3)3C), 30.5 (CH2), 30.9 (CH2), 34.6 (2 × CH2), 39.9(CH2N), 41.4 (CH2N), 66.1 (2× CH2O), 69.7 (CH2O), 76.4(CHO), 79.5 (Cquat of NHBoc), 104.8 (2× ArC), 116.6 (4×ArCH), 120.1 (2× CN), 135.2 (4× ArCH), 158.6 (CdO),163.8 (2× ArCO), 172.3 (CdO).

MS (ESI-TOF): [M+ Na]+, C35H42N4O6Na requires 601.2996,found 601.2298.

N-(6-Aminohexyl)-2-(1,5-bis(4-carbamimidoylphenoxy)-pentan-3-yloxy)acetamide (6).To a solution oftert-butyl 6-(2-(1,5-bis(4-cyanophenoxy)pentan-3-yloxy)acetamido)hexylcar-

bamate (5) (70.6 mg, 0.12 mmol, 1 equiv) in anhydrous ethanolwas added NEt3 (0.14 mL, 0.98 mmol, 8 equiv) and anhydrousNH2OH (68 mg, 0.98 mmol, 8 equiv). The reaction mixturewas placed in a sealed tube and heated overnight at 90°C. Thereaction mixture was then concentrated in vacuo to remove theethanol solvent. The residue was dissolved in EtOAc and washedwith water. The organic layer was then dried (MgSO4) andconcentrated to give the bis-aldoxime. A solution of the bis-aldoxime in MeOH (30 mL) was placed under an argonatmosphere. 10% Pd/C (10 mg) was added and the reactionmixture placed under an atmosphere of hydrogen at balloonpressure. After 18 h, the hydrogen atmosphere was removed,and the reaction mixture was filtered through celite andconcentrated in vacuo to givetert-butyl 6-(2-(1,5-bis(4-cy-anophenoxy)pentan-3-yloxy)acetamido)hexylcarbamate (62 mg,0.10 mmol, 84% over 2 steps). Finally,tert-butyl 6-(2-(1,5-bis-(4-cyanophenoxy)pentan-3-yloxy)acetamido)hexylcarbamate wastreated with 25% TFA in CH2Cl2 (20 mL). The reaction wasallowed to stir at rt for 4 h and then concentrated in vacuo togive theN-(6-aminohexyl)-2-(1,5-bis(4-carbamimidoylphenoxy-)pentan-3-yloxy)acetamide (6) in quantitative yield, which wasused in the immobilization step without further purification.

1H NMR (MeOD, 600 MHz)δ 1.33 (4H, m, 2× CH2), 1.47(2H, m, CH2), 1.60 (2H, m, CH2), 2.11 (4H, m, 2× CH2), 2.87(2H, t, J ) 7.5 Hz, CH2N), 3.16 (2H, t,J ) 7.2 Hz, CH2N),3.92 (1H, m, CHO), 3.98 (2H, s, CH2O), 4.22 (4H, m, 2×CH2O), 7.11 (4H, d,J ) 8.4 Hz, 4× ArH), 7.76 (4H, d,J )9.0 Hz, 4× ArH).

13C NMR (MeOD, 150 MHz)δ 27.0 (CH2), 27.4 (CH2), 28.5(CH2), 30.4 (CH2), 34.8 (2× CH2), 39.8 (CH2N), 40.7 (CH2N),66.2 (2× CH2O), 69.9 (CH2O), 76.4 (CHO), 116.3 (4× ArCH),121.2 (2× ArC), 131.1 (4× ArCH), 165.1 (2× ArCO), 167.6(CdNH), 172.5 (CdO).

MS (ESI-TOF): [M+ H]+, C27H41N6O4 requires 513.3184,found 513.3187.

Immobilization of Ligand (6) onto Activated SepharoseBeads.Endotoxin-binding ligandN-(6-aminohexyl)-2-(1,5-bis-(4-carbamimidoylphenoxy)pentan-3-yloxy)acetamide (6) wasattached to carbonyldiimidazole-activated Sepharose beads(Reacti-Gel CDI support, Pierce) according to the manufactur-er’s directions. Briefly, a 5 mg/mL solution of ligandN-(6-aminohexyl)-2-(1,5-bis(4-carbamimidoylphenoxy)pentan-3-yloxy)-acetamide in 100 mM borate buffer, pH 9, was prepared. Reacti-Gel beads were added to a polypropylene cartridge, acetonesolvent was drained off, and 1 mL of 5 mg/mL ligand solutionwas added to the cartridge per milliliter of Reacti-Gel beads.The mixture was allowed to shake for 24 h at rt. The solutionof ligand was then drained from the beads. 50 mM Tris buffer,pH 9.2, was added (1 mL of buffer per mL of bead volume)and the mixture allowed to shake overnight in order to quenchunreacted CDI groups. Finally, excess Tris buffer was drained,and the beads were washed with PBS buffer (pH 7.4) and storedin a suspension of PBS until use. Control beads were preparedanalogously by treatment of Reacti-Gel beads with 100 mMborate buffer containing no ligand, followed by blocking reactivegroups with Tris buffer.

Fluorescein Displacement Assay for Quantifying BindingAffinity to LPS. The BODIPY-cadaverine (BC) displacementassay was used to quantify the binding affinity of ligand6 toLPS. This assay was performed according to a publishedprocedure (7). Briefly, 10 mM stock of ligand in 50 mM Tris(pH 7.4) was prepared. A stock solution of 5 mg/mL LPS fromE. coli 0111:B4 (Sigma) in 50 mM Tris (pH 7.4) and a stocksolution of 500µM BC (BODIPY-TR cadaverine, obtainedfrom Invitrogen)-50 mM Tris (pH 7.4) were prepared. 0.1 mLof each of the LPS and BC stocks were mixed and the mixturediluted to a final volume of 10 mL using 50 mM Tris buffer to

Novel Affinity System for Removal of Endotoxin Bioconjugate Chem., Vol. 18, No. 2, 2007 561

give an LPS-BC mixture composed of 50µg/mL LPS and 5µM BC. Corning nonbinding surface 96 well plates were usedin this assay.

To the first well of a 96 well plate was added 40µL of 10mM ligand stock solution and 40µL of 50 mM Tris buffer.Twofold serial dilution across the plate was carried out, andthen 40µL of Tris was added to each well to give 80µL volumein each well. 80µL of LPS-BC (50 µg/mL-5µM) mixturewas added to each well. Each experiment was repeated intriplicate. Fluorescence measurements were made at 25°C ona SpectraMAX GeminiEM fluorescence plate reader. BCexcitation wavelength of 580 nm was used; emission wasmeasured at 620 nm.

The Kd(app) values for interaction of ligand6, PMB, andpentamidine with LPS were calculated. Curves were fitted tothe functionF ) Fmax[L]/( Kd + [L]) + c usingWinCurVeFitsoftware. In the case of ligand6 and pentamidine, fluorescencemeasurement values used in the curve fit were corrected toaccount for a concentration-dependent linear background fluo-rescence effect.

Binding Assays of Sepharose-Ligand 6 Beads with FITC-Labeled LPS. Sepharose-ligand 6 beads were tested for theability to bind endotoxin using FITC-labeled LPS. A 50 mg/mL concentrated stock solution ofE. coli 0111:B4 FITCconjugate (purchased from Sigma) in 50 mM Tris buffer, pH7.4, was prepared. A standard curve of fluorescence at variousconcentrations of LPS-FITC was prepared (excitation at 495nm, emission at 525 nm).

Sepharose-ligand6 beads, control beads, and commerciallyavailable agarose-supported Polymyxin B (purchased fromSigma) were tested concurrently for ability to bind LPS-FITC.The experiments were carried out as follows.

100 mg of dried beads was weighed into an Eppendorf tube,and 400µL of 50 mM Tris buffer, pH 7.4, was added to thetube. A known quantity of concentrated LPS-FITC stock (50mg/mL) was added to each tube, and the tubes were allowed toshake 30 min. The mixtures were then centrifuged; and 100µLof the supernatant was removed and placed in a microplate well,and the fluorescence (ex 495 nm, em 525 nm) was measured.Following the fluorescence measurement, the sample wasreturned to the appropriate Eppendorf tube, and a further aliquotof concentrated LPS-FITC stock was added. This process wasrepeated until a final LPS-FITC concentration of 8000µg/mLhad been reached. [LPS-FITC] free in the supernatant afterincubation was calculated and plotted against the original [LPS-FITC] in the tube.

Curves were fitted to the following function usingKaleida-graph:

whereM1 ) Kd andM2 ) total binding sites.Endotoxin Removal and Protein Loss Following Purifica-

tion of Samples of Endotoxin-Contaminated Antibody.Sepharose-ligand6, control beads, and Pierce Detoxigel beads(agarose-bound PMB) were poured into columns of 1 mLvolume. Using pyrogen-free solutions, each column was equili-brated with 3× column volume washes of 1% sodium deoxy-cholate soln, followed by 5× the column volume of nanopure,filtered water. Solutions of antibody (of known concentration,volume, and endotoxin content) buffered with 0.1-0.5 M NaClwere loaded onto each column. Columns were capped off andallowed to incubate for 1 h. Samples were then eluted from thecolumns, and the columns were then rinsed with an addition 1mL of water to clear the void space. Samples were collected,

the OD at 280 nm was measured, and the protein concentrationwas calculated. Endotoxin levels in each sample were then testedusing the LAL (LimulusAmebocyte Lysate) endotoxin test kit,purchased from Cambrex Bioscience (cat. no. 50-647U).

The LAL endotoxin test uses modifiedLimulusamebocytelysate and a chromogenic peptide substrate. Bacterial endotoxincatalyzes the conversion of a pro-enzyme in theLimulusamebocyte lysate to an active enzyme. The active enzyme thencatalyzes the release ofp-nitroaniline from a colorless peptidesubstrate. After a given time period, the reaction is stopped byaddition of acid, and thep-nitroaniline released can be quan-titated photometrically by measuring absorbance at 405-410nm. The amount ofp-nitroaniline released is proportional tothe endotoxin concentration present and can be calculated froma standard curve.

The LAL assay was carried out according to the manufac-turer’s directions as described in the instruction manual. Briefly,samples were diluted to various strengths, mixed with the lysatereagent supplied in the kit, and incubated at 37°C for 10 min.The chromogenic peptide substrate was then added and thesamples allowed to incubate for a further 6 min. Finally, thereaction was stopped by the addition of 25% glacial acetic acid.Absorbance at 405-410 nm was measured, and these valueswere used to calculate endotoxin content in the samples.

RESULTS AND DISCUSSION

Synthetic Route to Tethered Pentamidine-Derived Ligand.Synthesis of tethered pentamidine ligand6 is shown in Scheme1. The alcohol functionality of commercially available dimethyl3-hydroxyglutarate1a was protected as the TBS ether and theterminal ester groups were subsequently reduced with DIBALHto give diol2 in good yield. Mitsonobu reaction of diol2 with4-hydroxybenzonitrile followed by removal of the siliconprotecting group afforded bis-nitrile3b. Alkylation of thesecondary alcohol in3b with tert-butyl bromoacetate wasachieved under mild conditions using a biphasic system of THF/H2O in the presence of 25% NaOH and a phase-transfer catalystto give tert-butyl ester4. Deprotection of ester4 by treatmentwith TFA, followed by BOP coupling of the resulting acid withN-Boc-1,6-diaminohexane provided amide5. Finally, the nitrilegroups were converted into amidines in a two-step process. Bis-nitrile 5 was first treated with hydroxylamine, which had beendried by azeotroping with toluene prior to the reaction. Ifhydrated hydroxylamine is used in this step, then a significantamount of the nitrile is converted to the corresponding amide.This unwanted byproduct is difficult to separate from the desiredbis-amidine6, such that HPLC purification is required. However,use of anhydrous hydroxylamine results exclusively in theformation of the desired bis-aldoxime intermediate, which canthen be converted to pure bis-amidine in good yield by catalytichydrogenation. Ultimately, removal of the Boc group from theterminal nitrogen gave pentamidine-derived ligand6.

Binding Affinity of Pentamidine-Derived Ligand (6) toLPS. Prior to immobilization of ligand6 onto sepharose support,we examined its binding affinity to an LPS solution. Althoughpentamidine itself is reported to bind strongly to lipid A, wefelt it was important to confirm that incorporation of a tetherfunctionality did not have a detrimental effect on bindingaffinity. It has been postulated that modifying the structure ofpentamidine such that it incorporates a hydrophobic chain atan appropriate position may indeed improve its affinity for LPS/lipid A due to stabilizing interactions with the acyl chains oflipid A (6).

A fluorescent probe displacement assay employing BO-DIPY-TR cadaverine (BC) was used to test the binding affinityof ligand 6 with LPS, and for comparison, pentamidine andPolymyxin B were also examined (7). BC interacts strongly with

LPSfree ) LPStotal -

[(LPStotal+ M1+ M2 - x(LPStotal+ M1 + M2)2 - 4LPStotalM2)

2 ]

562 Bioconjugate Chem., Vol. 18, No. 2, 2007 McAllister et al.

LPS, and upon binding, its fluorescence emission intensity at620 nm is significantly reduced. In the presence of compoundswhich bind LPS, BC is displaced, and an increase in emissionintensity at 620 nm is observed. This assay provides aconvenient method of calculating apparent dissociation constantsfor the binding of molecules to LPS.

The BODIPY fluorescent displacement assay curves forligand6 and for PMB are shown in Figure 3. It was found thatligand6 binds to LPS withKd(app) of 180µM ((60), which issimilar to the affinity of pentamidine at 190µM ((40) (curvenot shown). Therefore, it could be concluded that incorporationof a tether group at this position in the molecule does notadversely affect the binding affinity. In our control,Kd(app) ofPolymyxin B was found to be 2µM ((0.6), which is consistentwith published results (8). It is noteworthy that David et al.have reported PMB and pentamidine to bind lipid A with similaraffinities, havingKd(apps) of 0.37 and 0.12µM, respectively(5). In our studies, where we examined relative affinities to LPS,we discovered that PMB has an appreciably greater affinity forLPS than pentamidine. However, the affinity of pentamidineand its derivatives, such as ligand6, is still significant in viewof the simplicity of their molecular structures.

Immobilization of Ligand 6 onto CDI-Activated Sepharose,Reacti-Gel. Ligand 6 was immobilized onto commerciallyavailable carbonyldiimidazole-activated sepharose beads (Reacti-Gel, Pierce) as shown in Scheme 2. Reacti-Gel is an activatedform of sepharose particularly suitable for immobilizing smallorganic molecules and peptides bearing free amino groups. Asolution of the ligand in basic aqueous buffer was allowed toreact with the Reacti-Gel beads to form a carbamate linkage tothe support; unreacted groups were then blocked by incubationwith Tris buffer.

Binding Studies of FITC-Labeled LPS with Sepharose-Supported Ligand 6.The ability of our affinity beads to removeLPS from solutions was assessed using LPS fromE. coli 0111:B4, which had been labeled with fluorescein (LPS-FITC). Thechange in fluorescence of a solution after incubation with thebeads was measured. Thus, modified sepharose beads7 werefiltered to remove excess buffer, and 100 mg samples of7 wereweighed for use. 50 mM Tris buffer (400µL) was added to7,and aliquots of a concentrated LPS-FITC stock (50 mg/mL)

were added successively to the suspension of beads. After theaddition of each aliquot, the mixture was allowed to shake for30 min. Following centrifuging to separate7 from the LPS-FITC solution, a 100µL aliquot from the supernatant was

Scheme 1. Synthetic Route to Ligand 6a

a (a) TBSCl, imdazole, DMF, rt, 100%; (b) DIBALH, CH2Cl2, 0 °C, 93%; (c) 4-hydroxybenzonitrile, PPh3, DIAD, THF; (d) HCl, MeOH, 38%over 2 steps; (e)tert-butyl-2-bromoacetate, NaHSO4, 25% NaOH, toluene, rt, 77%; (f) 1. 25% TFA in CH2Cl2; 2. N-Boc-1,6-diaminohexane, BOP,NEt3, CH2Cl2, 75%; (g) 1. NH2OH‚HCl (anhydrous), NEt3, EtOH, sealed tube; 2. H2, Pd/C, AcOH, MeOH, 84% over 2 steps; 3. 25% TFA inCH2Cl2.

Figure 3. Displacement of LPS bound BC by ligand6 (top).Displacement of LPS bound BC by Polymyxin B (bottom).

Novel Affinity System for Removal of Endotoxin Bioconjugate Chem., Vol. 18, No. 2, 2007 563

removed and its fluorescence measured. This experiment wasalso performed concurrently on control beads (Reacti-Gel whichhad not been treated with ligand6 but had been treated withTris buffer to block reactive groups) and on commerciallyavailable agarose-supported Polymyxin B. A standard curve offluorescence versus concentration of FITC-LPS was prepared(λex ) 496 nm andλem ) 525 nm) and used to calculated freeLPS concentration in the supernatant following incubation witheach type of bead. Free [LPS-FITC] in the supernatant wasplotted against the total [LPS-FITC] for both7 and agarose-PMB beads and curves fitted to the following function:

whereM1 ) Kd andM2 ) total binding sites. Figure 4 showsthe relationships between [LPS-FITC]free and [LPS-FITC]total. We were surprised to find that both7 and agarose-PMB beads performed similarly, particularly at lower [LPS-FITC] concentrations. However, we note that theKd values forboth ligands appear to be significantly greater when they areattached to the support than theirKd values in solution.Sepharose-bound ligand6 displays aKd of 596µM ( 120 and

agarose-bound PMB shows aKd of 948µM ( 340, indicatingthat the resin-bound versions of both these ligands do not interactas strongly with endotoxin as they do in solution. However,both systems can effectively reduce endotoxin concentration byapproximately 50% at the lower concentrations. From this data,we were also able to estimate a maximal binding capacity forboth affinity beads. Agarose-PMB resin had a maximal bindingcapacity of 24( 5 µg [LPS-FITC]/mg resin, while resin7had a maximal binding capacity of 17( 2 µg [LPS-FITC]/mg resin. The apparently higher maximal binding capacity forthe agarose-PMB system may account for its improvedperformance in reducing [LPS-FITC] levels at higher concen-trations.

These results suggested that our affinity system can effectivelyremove LPS from contaminated solutions. Subsequently, wewanted to assess if this support could be successfully recycled.Table 1 shows the results of recycling with various reagents.Following the incubation of each sample of beads with 500µg/mL LPS-FITC solution, the beads were washed with a solutionof the recycling reagent. The beads were then reincubated witha 500µg/mL solution of LPS-FITC, and the reduction in LPS-FITC concentration was measured by the fluorescence methoddescribed (vide supra). In most cases, the beads could berecycled with only a slight reduction in LPS binding capabilityafter the first round of recycling. We found that 2% Triton X-100was very effective at restoring LPS binding capability of thebeads even after the third round of recycling.

Application of Sepharose-Bound Ligand 6 Beads inAffinity Chromatographic Purification of Endotoxin-Con-taminated Antibodies.Finally, we have applied affinity beads7 in the chromatographic purification of endotoxin-contaminatedsamples of several different antibody samples of various proteincontamination levels and endotoxin contamination levels. Proteinloss during affinity-based purification procedures is a significantshortcoming associated with endotoxin removal. We thereforewanted to ensure that our system could effectively reduceendotoxin levels without resulting in unacceptable levels ofprotein loss. The antibody samples were each passed through a1 mL column of affinity beads7, Pierce Detoxi-Gel beads (i.e.,

Scheme 2. Immobilization of Ligand 6 onto Reacti-Gel Beads

Figure 4. Binding of LPS-FITC by sepharose-ligand 6 and agarosePMB affinity beads.

Table 1. Recycling of Affinity Beads

recycling reagent

sepharose-ligand6(% reduction in concn

of LPS-FITC soln)

agarose-PMB(% reduction in concnof LPS-FITC soln)

before recycling 43 ((3) 57 ((1)1% sodium deoxycholate 34 ((1) 46 ((4)methanol 1st round: 39 ((8) 1st round: 45 ((1)

2nd round: 25 ((4) 2nd round: 45 ((1)1 M NaOH then methanol 35 ((3) 47 ((3)2% Triton X-100 1st round: 37 ((6) 1st round: 60 ((2)

2nd round: 27 ((1) 2nd round: 60 ((1)3rd round: 34 ((4) 3rd round: 55 ((4)

LPSfree ) LPStotal -

[(LPStotal+ M1 + M2 -x(LPStotal+ M1 + M2)2 - 4LPStotalM2)

2 ]

564 Bioconjugate Chem., Vol. 18, No. 2, 2007 McAllister et al.

agarose-bound PMB), and control beads. Following elution fromthe column, the samples were tested for endotoxin concentrationusing the LAL (LimulusAmebocyte Lysate) assay (9) and forprotein content. The results of the endotoxin testing and proteincontent tests are shown in Table 2 and Figure 5, respectively.

Table 2 displays the results of LAL endotoxin testing on theantibody samples following affinity chromatography. AntibodyA7R34 was contaminated with 10-25 EU/mL prior to affinitychromatography. When a sample loading of 1 mL of A7R34was applied to 1 mL chromatography columns, both sepharose-ligand6 and agarose-PMB columns reduced endotoxin levelsto a level lower than the detection limit of the LAL assay, whilethe control column did not reduce endotoxin content. At a highersample loading volume of 4 mL of A7R34 (and therefore higherendotoxin load), the sepharose-ligand 6 column was alsoeffective in reducing endotoxin level completely. Each affinitycolumn was regenerated, and antibody KA 417F4 contaminatedwith 1.25 EU/mL was applied. Gratifyingly, the endotoxin levelin KA 417F4 was also reduced to a negligible level by thesepharose-ligand6 affinity system. ELISA studies on purifiedKA 417F4 against the original BSA-hapten conjugate to whichit was raised confirmed that the antigen binding capability ofthe antibody had not been diminished during the purificationprocess and that no cross contamination had occurred (i.e., thatKA 417F4 had not become contaminated with A7R34).Antibodies GNL 23A6 and FTB 8E9 both had considerablyhigher protein concentrations and endotoxin contamination

levels. In both cases, affinity chromatography with the agarose-PMB system was slightly more effective at reducing endotoxinlevels. This is possibly due to a greater number of binding sites;however, affinity beads7 did give a significant reduction inendotoxin level in the case of both antibodies. These resultsconfirmed that sepharose-ligand 6 beads could reduce endot-oxin levels as effectively as the agarose-PMB systems at lowerendotoxin loadings and that regeneration of the columns waseffective and did not result in cross contamination betweenprotein samples.

Figure 5 shows the percentage of protein loss from differentantibody samples following chromatography using each typeof affinity system. With the exception of antibody GNL 23A6,protein loss following chromatography with the sepharose-ligand 6 system was comparable to that found with the PMBsystems. There does not appear to be a strong correlationbetween antibody concentration and protein loss; for example,2.7 mg/mL AR734 and 11 mg/mL FTB 8E9 have the mostcomparable protein loss of any of the antibodies tested despitea large difference in antibody concentration. The differences inpercentage of protein loss between the different antibodies maybe attributed to the differences in charge on the protein itself.Ansprach (4) has reported significant differences betweenprotein recoveries of negatively charged BSA and positivelycharged lysozyme when endotoxin-contaminated solutions ofeach of these proteins were subjected to identical endotoxinremoval affinity columns. Ansprach proposed that the negativelycharged protein competes with endotoxin for ligand binding siteson the affinity column, and thus protein recovery is reduced. Itwas also observed that different affinity sorbents displayeddifferent degrees of protein loss depending on the degree ofspecificity of the ligand for endotoxin over the negativelycharged protein. The high protein loss on GNL 23A6 for thesepharose-ligand6 column may be attributed to the charge ofthe antibody.

CONCLUSION

In conclusion, we have described a simple synthetic route toa novel ligand which binds endotoxin. We have demonstratedthat sepharose beads bearing this ligand can be used in affinitychromatography for the removal of endotoxin from contaminatedsolutions. These affinity beads can be recycled using a varietyof simple reagents. In addition, we have verified that thisinexpensive system does not result in any greater loss in proteinthan more expensive commercially available PMB systems. Byacknowledging the crucial role of endotoxin removal in proteinpurification, this system provides an efficient, economicalalternative to the PMB systems.

ACKNOWLEDGMENT

This work was supported by The Skaggs Institute forChemical Biology.

LITERATURE CITED

(1) David, S. A. (2001) Towards a rational development of anti-endotoxin agents: novel approaches to sequestration of bacterialendotoxins with small molecules.J. Mol. Recognit. 14, 370-387.

(2) Gashe, Y., Pittet, D., and Sutter, P. (1995) Outcome and prognosisfactors in bacteremic sepsis. InClinical trials for treatment of sepsis,pp 35-51, Springer-Verlag, Berlin.

(3) Bone, R. C. (1993) Gram-negative sepsis: a dilemma of modernmedicine.Clin. Microbiol. ReV. 6, 57-68.

(4) Anspach, F. B., and Hilbeck, O. (1995) Removal of endotoxins byaffinity sorbents.J. Chromatogr., A 711, 81-92.

(5) David, S. A., Bechtel, B., Annaiah, C., Mathan, V. I., and Balaram,P. (1994) Interaction of cationic amphiphilic drugs with lipid A:

Table 2. Endotoxin Removal from Antibody Samples

Results of LAL Endotoxin Test (EU/ml)affinity column

antibodysepharose-

ligand6 controlagarose-

PMB

1 mL of 2.7 mg/mLA7R34 (10-25 EU/mL)a

NDb >10 NDb

4 mL of 2.7 mg/mLA7R34 (10-25 EU/mL)a

NDb >10 NDb

4 mL of 3.48 mg/mLKA 417F4 (1.25 EU/mL)a

NDb 5-10 NDb

1.5 mL of 10 mg/mLGNL 23A6 (125-250 EU/mL)a

250-500 500-1000 5-10

2 mL of 11 mg/mLFTB 8E9 (500-1000 EU/mL)a

50 125-250 25-50

a Values in paretheses indicate initial endotoxin contamination level priorto affinity chromatography.b ND indicates that endotoxin was not detected;levels were below sensitivity of detection in LAL assay, i.e.,e0.1 EU/mL.

Figure 5. Graph representing % protein loss from samples of antibodyfollowing affinity chromatography.

Novel Affinity System for Removal of Endotoxin Bioconjugate Chem., Vol. 18, No. 2, 2007 565

implications for development of endotoxin antagonists.Biochim.Biophys. Acta 1212, 167-175.

(6) David, S. A., Mathan, V. I., and Balaram, P. (1995) Interactionsof linear dicationic molecules with lipid A: structural requisites foroptimal binding affinity.J. Endotoxin Res. 2, 325-336.

(7) Wood, S. J., Miller, K. A., and David, S. A. (2004) Anti-endotoxinagents. 1. Development of a fluorescent probe displacement methodoptimized for the rapid identification of lipopolysaccharide-bindingagents.Comb. Chem. High Throughput Screening 7, 239-249.

(8) Wood, S. J., Miller, K. A., and David, S. A. (2004) Anti-endotoxinagents. 2. Pilot high-throughput screening for novel lipopolysac-charide-recognizing motifs in small molecules.Comb. Chem. HighThroughput Screening 7, 733-747.

(9) LimulusAmebocyte Lysate QCL-1000 instruction manual, cat. no.50-647.U, Cambrex Bioscience, Walkersville, MD.

BC0602984

566 Bioconjugate Chem., Vol. 18, No. 2, 2007 McAllister et al.