WO2014109704A1

WO2014109704A1 - New material for use in high performance liquid chromatography

Info

Publication number: WO2014109704A1
Application number: PCT/SE2014/050025
Authority: WO
Inventors: Elinor MEIBY; Malin M. ZETTERBERG; Sten Ohlson; Katarina Edwards
Original assignee: Transientic Interactions Ab
Priority date: 2013-01-14
Filing date: 2014-01-13
Publication date: 2014-07-17

Abstract

A material adapted to interact with a mobile phase suitable for high performance liquid chromatography, comprising a solid sorbent material with immobilized lipid bilayer discs, The lipid bilayer discs comprise at least one polar lipid, at least one lipid conjugated to polyethylene glycol and at least one lipid conjugated to polyethylene glycol having functional groups bound to functional groups of the solid sorbent material. The specification also discloses methods of studying interactions between the material and analytes.

Description

New material for use in high performance liquid chromatography Field of invention

The invention relates to a material comprising a solid sorbent material with immobilized lipid bilayer discs suitable for interaction with mobile phases in high performance liquid chromatography and methods using the material exemplified by drug partition

chromatography and weak interaction chromatography.

Background of the invention Lipid-based model membranes, that accurately reflect the structure and properties of biological membranes, constitute essential tools for studies in various biological, analytical and pharmaceutical areas. Liposomes constitute one of the most frequently used model membranes, and have been used in combination with a number of chromatographic techniques for the purpose of membrane interaction studies. Although liposomes certainly have proven useful as model membranes in numerous studies, their use in interaction studies is associated with some potential complications. First, since liposomes are closed bilayer structures comprising an inner aqueous core, only the lipids in the outer bilayer leaflet stand in direct contact with the surrounding bulk media. This, together with the fact that liposome preparations normally contain a fraction of bi- and multilamellar structures, means that a substantial, and typically unknown, fraction of the lipids initially are shielded from interaction with analytes dissolved in the bulk media. The presence of an effectively hidden lipid fraction may slow down or hamper analyte equilibration and thereby prevent reliable and reproducible collection of interaction data. Another drawback is that when reconstituting membrane proteins into liposomes, a fraction of the protein as a rule incorporates with the active site facing towards the liposome interior, thus being inaccessible for interaction with potential ligands in the bulk solution. Finally, conventional liposomes have a rather limited shelf life and, over time, tend to aggregate and fuse into larger, less well- defined structures.

PEG-stabilized bilayer disks, referred to as lipodisks or lipid bilayer discs, have emerged as an interesting alterative to liposomes for the use as model membranes in interaction studies, see Johansson E et al . Biochim Biophysi Acta, 2007, 1768: 1518-1525. Lipodisks are obtainable by mixing lipids that spontaneously form bilayers with lipids that have a large polyethylene glycol (PEG) chain covalently attached to their head group. The lipodisks generally are flat circular lipid aggregates consisting of a lipid bilayer surrounded by a highly curved rim Similar to the case with liposomes, membrane spanning, as well as peripheral, membrane proteins can be incorporated into the lipodisks. However, in contrast to the case with liposomes, the open structure of the disks ensures that both lipid bilayer leaflets are readily available for interaction with analytes present in the bulk aqueous phase. Further, the heavy PEGylation of the disks protects them against fusion and ensures excellent long term stability Taken together the lipodisks possess properties that make them highly interesting for use as model membranes in interaction studies. Lipodisks functionalized with biotin have also been successfully immobilized to streptavidin-covered sensor surfaces and employed in studies based on the surface plasmon resonance, see A Lundquist et al in Anal Biochem, 2010, 405: 153-159. It would accordingly be desirable to obtain stable solid phase systems, for example as employed in conventional high performance liquid chromatography systems, including this type of lipid bilayer discs serving as model membranes in other highly efficient screening technologies for the purpose of for example studying drug partitioning and to study protein- ligand interactions with weak affinity chromatography, thereby extending the methodology to studies of poorly soluble and sensitive membrane proteins.

Description of the invention

In a first aspect, the invention relates to a material adapted to interact with a mobile phase suitable for high performance liquid chromatography. The material comprises a solid sorbent material with immobilized lipid bilayer discs, wherein the lipid bilayer discs comprise at least one polar lipid, at least one lipid conjugated to polyethylene glycol, and at least one lipid conjugated to polyethylene glycol having functional groups bound to functional groups of the solid sorbent material.

In this context a sorbent material is a material conventionally used with high performance liquid chromatography, exemplified by porous silica materials with a suitable surface area and defined pore size. Many such silica materials are established among practitioners and the materials with the trade names Nuclesosil and POROS as exemplified in this specification serve as illustrating examples.

Also in this context, "polar lipid" has the conventional meaning of having polar head groups and possessing surface activity, such as phospholipids or glycolipids and cover as a class amphiphatic or amphiphilic lipid classes and bilayer and non-bilayer forming classes.

Further in this context, the term lipid conjugated to polyethylene glycol means that there is a covalent bond established between the lipid and the polyethylene glycol.

Still further in this context, the term "functional group" bound to " functional groups" may include covalent bonds between the groups as can be established between a number of functional groups such as amines, thiols, carboxylic group etc. with aldehydes, hydroxyl groups, epoxy groups etc. The term "functional group" may also include such groups that establish strong non-covalent bonds that establish sufficient immobilization of the lipid discs to a sorbent material, as exemplified by bonds between biotin and streptavidin.

In one aspect of the invention the material comprises at least one lipid bilayer discs having an incorporated bioactive protein or peptide fragment thereof. The term "incorporated" would here have the general meaning that the protein is associated with the lipid bilayer discs in sufficiently permanent way that it can be can adequately studied how it interacts with a the components of a mobile phase in conventional high performance liquid chromatography conditions. In one aspect of the invention, the bioactive protein or fragment thereof is a membrane protein or fragment thereof. In the present context, the term "membrane protein" is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. "Membrane proteins" conventionally are divided in classes like integral membrane proteins, peripheral membrane proteins and lipid anchored proteins which all are encompassed in the term.

In one aspect of the invention, the functional groups of the polyethylene glycol are amine groups and the functional groups of the sorbent material are aldehyde groups. In another aspect of the invention the functional groups of the polyethylene glycol are biotin groups and the functional groups of the sorbent support material are streptavidin groups.

In one aspect of the invention, the material comprises bilayer discs with a size that is essentially less the than the pore size of the sorbent material. The lipid bilayer discs can have size of from about 8 to about 200 nm and the pore size of the sorbent material can be between 300 and 10000 A. In one aspect of the invention the lipid bilayer discs can have size of from about 8 to about 20 nm. In one aspect of the invention, the bilayer discs comprise polar lipids selected from at least one of 1,2-dipalmitoyl -sw-gly cero-3 -phosphocoline (DPPC), 1 -palmitoyl-2-oleoyl-.w?- glycero-3-phosphocoline (POPC), soy L-a-phosphatidylethanolamine (Soy PE), 1,2-disteroyl- s«-glycero-3 -phosphocoline (D SPC), 1 -palmitoyl-2-oleoyl-sft-gly cero-3 - phosphoethanolamine (POPE), 1,2-dioleylpalmitoyl -sft-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-sw-glycero-3-[phosphor-L-serine] (sodium salt) (POPS), 1,2- dimyristoyl -sn-gly cero-3 -phosphatidylcholine (DMPC), phosphatidylcholine (EPC), 2- hydroxy-sn-glycerol-3-phosphatidylcholine (MSPC), L-a-phosphatidylinositol, a

sphingomyelin, and a cerebroside. In one aspect of the invention lipid bilayer discs comprise lipids conjugated to polyethylene glycol selected from at least one of N-palmitoyl-sphingosine-1- {succinyl[methoxy (polyethylene glycol)2000]} (Ceramide-PEG2ooo), N-palmitoyl- sphingosine-l-{succinyl[methoxy (poly ethylene glycol)5000]} (Ceramide-PEGsooo), 1,2- distearoyl-OT-gly cero-3 -phosphoethanolamine-N-[biotinyl (poly ethylene glycol)-2000] (DSPE- PEG2000), l,2-distearoyl-5n-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-5000] (DSPE- PEG₅₀oo), 1.2-dimyristoyl -sw-glycero-3- phosphatidylcholine(polyethylene glycol 2000) (DMPC- PEG2₀₀₀), and cholesterol conjugated to polyethylene glycol. In one aspect of the invention, the lipid bilayer discs comprise at least one of the polyethylene conjugated lipids are provided with amine groups or biotin as functional groups, preferably the lipid is l,2-distearoyl-^«-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2ooobiotin) or l,2-distearoyl-5«-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG2oooamine). The lipid bilayer discs of the invention can further comprise cholesterol, or another suitable equivalent sterol. Alternatives or complements to cholesterol can be selected among lanosterol sitosterol and stigmasterol.

In one aspect of the invention, the lipid bilayer discs comprise POPC, soy PE, cholesterol, ceramide-PEG(2000) and DSPE-PEG(2000) functionalized with amine or biotin. In this aspect, the material comprises 1 to 10 μηιοΐ lipids per mL sorbent material. The invention further relates to a method of studying the interaction between a material comprising any of the defined lipid bilayer discs and a composition of analytes. The method comprises providing a stationary phase of the material, transporting the composition of analytes to said stationary phase and establishing interaction conditions between the analytes and the stationary phase; and determining how each analyte interacts with the lipid bilayer discs of the material.

In one aspect, the method comprises determining partitioning of each analyte, wherein the lipid bilayer discs are model membranes in a drug partitioning study. In one aspect of the invention, the method comprising of screening the binding to a biological target with transient interactions, for example under conditions outlined by Duong-Thi M-D, et al. in Anal Biochem. 201 1 414: 138-146. In this aspect, the method comprises the steps of providing a stationary phase from the material, having a bioactive protein such as a membrane protein , or a peptide fragment thereof, incorporated in any of the defined lipid discs, providing a composition of ligands with a concentration of each ligand that is less than 0.1 mM, transporting the ligand composition to said stationary phase, thereby establishing weak affinity interactions between the ligands and the biological target in the range of 0.001 to 10 mM expressed as dissociation constant (KD), and detecting said ligands in compositions arriving from said stationary phase to discriminate between different ligand affinities in order to estimate the affinity and the dynamics of each ligand/target weak affinity interaction. Also, in this aspect, it is preferred that the bioactive protein is a membrane protein. Detailed and exemplifying part of the description

In the following examples, lipid bilayer discs (lipodisks) were successfully immobilized onto two different HPLC support materials by either reductive amination (lipodisks of amine functionality) or streptavidin-biotin binding (lipodisks of biotin functionality). Production of a HPLC column with covalently immobilized lipodisks resulted in an efficient HPLC system that showed high stability, and generated data with excellent reproducibility. MS detection enabled high throughput analysis of analytes in mixtures. A HPLC-MS system including the solid material according to the invention is demonstrated to represent a new and improved technique for the determination of drug substance partition behavior and for studying of the interaction between a membrane protein and ligands.

Description of attached figures

Fig. 1 is a schematic illustration of the cross-section of a lipodisk.

Fig. 2 is a Cryo-TEM image of lipodisks composed of POPC/Soy PE/cholesterol/Ceramide- PEG2ooo DSPE-PEG2oooamine (30:28: 17:21 :4 mol%). The arrow and arrow head indicate lipodisks observed edge-on and face-on, respectively. Scale bar = 100 nm.

Fig. 3 shows examples of analysis of a mixture of 7 compounds on the lipodisk column using ammonium acetate buffer as mobile phase. (A) UV detection 214 nm, (B) TIC of SIM positive mode, (C) EICs of individual analytes in SIM positive mode (1. theophylline, 2. naproxen, 3. prednisolone, 4. pindolol, 5. diclofenac, 6. indomethacin 7. propranolol). The void time was 0.63 min.

Fig. 4 shows retention times of 15 drug compounds during analysis on the lipodisk column and the reference column with ammonium acetate as mobile phase. Error bars represent the standard deviation (n=3). Ibuprofen was detected at a sample concentration of 0.1 mM, whereas the other analytes at 10 μΜ. Fig. 5 shows comparison of log K_s values for uncharged (circles), positively (squares), and negatively (triangles) charged drugs obtained using covalently immobilized lipodisks in PBS compared to ammonium acetate buffer. Fig. 6 sows COX-l/lipodisks association isotherm. R_es represents the effective associated COX-1 dimer/lipid mol ratio. [COX]_eq is the equilibrium bulk concentration of the protein. Error bars represent the standard error from three repetitions of the experiment. The data at [COX]eq = 3 and 4 μg mL^"1 represent single experiments. The solid line represents the fitting of the data according to the Langmuir isotherm (i?_eff = K i?_eff,max [COX]_eq (l+ [COX]_eq)^"

Example 1

Preparation and Characterization of Lipodisks 1.1 Chemicals

Sephadex G-50 was purchased from GE Healthcare Lifescience (Uppsala, Sweden). Dry powder of l-palmitoyl-2-oleoyl-s«-glycero-3-phosphocoline (POPC), soy L-a- phosphatidylethanolamine (Soy PE), N-palmitoyl-sphingosine-1- {succinylfmethoxy (polyethylene glycol)2000]} (Ceramide-PEG2ooo), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE- PEG₂ooobiotin) and 1 ,2-distearoyl-s«-glycero-3 -phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG₂oooamine) were purchased from Avanti Polar Lipids (Alabaster, USA). Ovine cyclooxygenase I (COX-1) was purchased from Cayman Europe (Talinn, Estonia). Cholesterol, octyl β-D-glucopyranoside (OG), alprenolol, pindolol, lidocaine, promethazine, propranolol, theophylline, diclofenac, ibuprofen, indomethacin, naproxen, warfarin, cortisone, hydrocortisone, prednisolone, corticosterone, periodic acid, sodium cyanoborohydride, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), sodium sulfite, sodium metabi sulfite, ammonium molybdate, 4-amino-3 -hydroxy- 1 -nap htalenesulfonic acid, dimethyl sulfoxide (DMSO), sodium dodecyl sulphate (SDS), diethyldithiocarbamate (DDC) and streptavidin were purchased from Sigma Aldrich Chemical (Steinheim, Germany). The analytes for HPLC were dissolved in ethanol (Kemetyl, Haninge, Sweden) at 5 mM and were further diluted in water to working concentrations. 1.2 Preparation of lipodisks

The lipodisks used in this study were composed of POPC, Soy PE, cholesterol, Ceramide- PEG2₀₀₀ and DSPE-PEG₂₀oobiotin or DSPE-PEG₂₀ooamin in the molar ratio 30:28: 17:21 :4. The lipids were dissolved in chloroform and thereafter dried under a gentle stream of nitrogen gas. Remaining chloroform was then removed under vacuum overnight. The lipid film was dissolved in an OG-solution and the solution was allowed to equilibrate for at least 4 h with intermittent vortex mixing. The lipid to detergent molar ration was 1 : 10 (21.5 mM extra OG, i.e. corresponding to the OG critical micelle concentration (CMC) was added to the sample. The solution was run on a Sephadex G-50 column (35 x 1.9 cm). As mobile phase, 0.1 M sodium phosphate, 0.15 M sodium chloride buffer, pH 7.0 (coupling buffer), was used and the flow rate was approximately 0.7 mL/min. The lipodisks and the detergent were eluted from the column as two well separated fractions. The lipodisks were then concentrated on a Miniplus concentrator (Millipore, Billerica, MA, USA) and stored at 4°C until further use.

1.3 Cryo-Transmission Electron Microscopy

The lipodisks were characterized using cryogenic transmission electron microscopy (cryo- TEM) using a Zeiss EM 902A Transmission Electron Microscope (Carl Zeiss NTS,

Oberkochen, Germany). Observations were made in zero loss bright-field mode at an accelerating voltage of 80 kV. Digital images were recorded under the low dose conditions with a BioVision Pro-SM Slow scan CCD camera (ProScan, Miinster, Germany). An underfocus of 1-2 μηι was used to enhance the image contrast.

The sample preparations were performed in a custom-built climate chamber at 25 °C and

>99% relative humidity. First a small drop (~ 1 uL) of the lipodisk solution was deposited on a copper grid covered with a carbon reinforced holey polymer film. Thin sample films were prepared by blotting the grid with a filter paper. The liquid film was vitrified by immediately plunging it into liquid ethane kept just above its freezing point. Samples were kept below -165 °C and protected against atmospheric pressure throughout the analysis.

1.4 Dynamic Light Scattering The aggregate size in the lipodisk preparations was assessed using dynamic light scattering (DLS). The experimental setup consisted of a Uniphase He-Ne laser (Milpitas, CA) emitting vertically polarized light with a wavelength of 632.8 nm operating at 25 mW. The scattered light was detected at 90° scattering angle using a Perkin Elmer (Quebec Canada) diode detector connected to an ALV-5000 multiple digital autocorrelator (ALV-laser;

Vertriebsgesellschaft, Germany).

Example 2

2.1 Immobilization of lipodisks on HPLC support materials Two different HPLC support materials (Nucleosil silica ; 10 μηι in diameter, 1000 A pore size; Macherey-Nagel, Diiren, Germany) which had been silanized into diol-substituted silica according to standard procedures [7] (25 mg samples) and POROS® AL Self Pack® media with aldehyde functionality (20 μιη in diameter, 500 - 10 000 A pore size; Applied

Biosystems, Carlsbad, USA; 14 mg samples) were suspended in MilliQ water and

ultrasonicated for a few minutes. The diol silica was oxidized into aldehyde silica by 0.5 mL 0.1 g/mL periodic acid at ambient temperature for 2 h. The materials were washed with coupling buffer (5 mL).

For immobilization of lipodisks of biotin functionality (DSPE-PEG₂ooobiotin), 1.25 mg streptavidin dissolved in 1.25 mL coupling buffer was added to the material samples and sodium cyanoborohydride was added to a final concentration of 9 mg/mL. The samples were incubated at ambient temperature for 20 h and washed with coupling buffer. The eluates from washing of the samples were collected and the amount of immobilized streptavidin was determined indirectly from the protein concentration of the eluates and of the applied sample, as measured by absorbance readings at 280 nm. Lipodisks with biotin functionality (345 μΕ, 60 mM lipid) were mixed with the HPLC support materials with immobilized streptavidin. For immobilization of lipodisks with amine functionality (DSPE-PEG2oooamin;106 mM lipid) they were mixed with samples of Nucleosil aldehyde silica (215 μΐ_^ lipodisk solution in each sample) and POROS material (170 μΐ_^ lipodisk solution in each sample). Sodium

cyanoborohydride was added to the samples to a final concentration of 9 mg/mL. Samples of HPLC media and lipodisks were incubated for 67 h at ambient temperature and washed with MilliQ water (5 mL) to remove all phosphate from the coupling buffer. Immobilized lipodisks were dissolved from the materials by incubation in 1 mL 121.5 mM octylglycosid for 18 h. The dissolved lipids were quantified by phosphorous analysis as described by Bartlett, GR in J Biol Chem, 1959, 234:466-468. 2.2 Analysis by HPLC-MS

2.2.1 Preparation of Nucleosil silica with immobilized lipodisks

Immobilization of lipodisks with amine functionality on 990 mg Nucleosil silica was performed as described above. As a control of passive immobilization of lipodisks to the silica, 30.5 mg silica was taken out after the oxidation step and mixed with lipodisks (72.5 μΙ_. 63 mM lipid). The remaining part of the silica was mixed with funtionalized lipodisks (1800 μΐ., 63 mM lipid). Sodium cyanoborohydride was added to a final concentration of 9 mg/mL and the samples were incubated for 68 h at ambient temperature. The silica was washed with coupling buffer. Silica samples from active (samples incubated with sodium

cyanoborohydride) and passive immobilization were taken out for phosphorous analysis. The samples were washed with MilliQ water and incubated in 1 mL 173 mM SDS for 16 h at ambient temperature. Phosphorous analysis was used to quantify the dissolved lipids. 2.2.2 Preparation of aldehyde Nucleosil silica

Aldehyde silica was prepared to pack a reference column. Nucleosil diol silica was suspended in MilliQ water and ultrasonicated for 8 min. The diol silica was oxidized into aldehyde silica by incubation in 1.25 ml 0.1 g/mL periodic acid at ambient temperature for 2 h and washed with MilliQ water.

2.2.3 Packing of columns

The Nucleosil silica with immobilized lipodisks was used to pack two 35 x 2.1 mm stainless steel columns (column 1 and 2) and the aldehyde silica was used to pack one 35 x 2.1 mm stainless steel column (column 3) with an air-driven liquid pump (Haskel, Burbank, USA) at 300 bar for 15 min. PBS pH 7.4 (0.01 M sodium phosphate, 0.15 M sodium chloride) (lipodisk column) and MilliQ water (aldehyde silica column) were used as mobile phase during packing. The columns were stored in PBS pH 7.4 or ammonium acetate buffer (20 mM) pH 6.8 -7.0 at 4°C between analyses.

2.2.4 Analysis of drug compounds on a lipodisk HPLC column

Screening was performed on an Agilent 1200 series capillary HPLC system equipped with a diode-array detector (DAD) and a single quadropole mass spectrometry detector (MSD; Agilent Technologies, Santa Clara, USA). On MS detection, analytes were ionized by electrospray at atmospheric pressure (API-ES) in positive mode. Drying nitrogen gas flow was 12 L/min at 350°C. The nebulizer pressure was 50 psig and the capillary voltage was 3000 V. MS signal acquisition was set at selected ion monitoring (SIM) mode on sample target masses. The [M+l]⁺ ion was monitored for each analyte. The fragmentor was set to 100 V. On UV detection, analytes were detected at a wavelength of 214±4 or 254±4 nm with a reference wavelength of 360±50 nm. Retention times were based on peak apexes of the extracted ion chromatogram (EIC). Chromatograms were analyzed with the Agilent

ChemStation version B.04.01 chromatography data system.

15 analytes of various charges were analyzed in triplicates on one of the lipodisk columns (column 1) and the reference column packed with aldehyde silica (column 3). Screening was performed with an injection volume of 1 uL and a flow rate of 0.2 mL/min. The column temperature was 22°C. Analysis was performed isocratically with PBS pH 7.4 or ammonium acetate buffer (20 mM) pH 6.8 - 7.0 as mobile phase. The analyte concentration was 10 μΜ (0.2% ethanol). The analytes were analyzed as single injections in both mobile phases. During analysis in ammonium acetate buffer, the analytes were also studied in mixtures in sets of 6 or 7 analytes in each mixture (1.2 or 1.4% ethanol). The retention time of ibuprofen was determined from a single injection at a sample concentration of 0.1 mM (2% ethanol) in order to facilitate detection during analysis on the lipodisk column with ammonium acetate as mobile phase and during analysis on the aldehyde column using PBS as mobile phase. The void time of the column with immobilized lipodisks was determined from the retention time of an injection of water, as detected by a negative peak by the DAD at 200±2 nm. Since the MSD is located after the DAD, the void time to the MSD is slightly longer. The difference in void times between the detectors was determined by single injections of theophylline. The void time of the DAD and the difference in retention times of theophylline between the two detectors were used to determine the void time of the MSD. For the reference column, the void time was determined from the retention time of 0.05% DMSO (214 nm).

2.2.5 Data Analysis

The drug partitioning was evaluated from the retention time on the lipodisk column. The normalized capacity factor (M^"1) was calculated for each analyte according to F Beigi et al in Int J Pharm 164: 129-137:

_ ( V R.lipodisk— Reference V F

s^_ A

(1) where ^ 'R,ii_podisk is the adjusted retention time on the lipodisk column, t 'preference the adjusted retention time on the reference column, the flow rate during analysis and .4 the amount of lipids (mol) on the column. The adjusted retention times were calculated by subtraction of the void time from the retention times of the analytes.

2.2.6. Production of a COX-1 column by in situ incorporation COX-1 was incorporated in situ into immobilized lipodisks of one of the lipodisk columns (column 2). Similar conditions were used as during incorporation of COX-1 into liposomes according to MirAfzali et al. [30], The column was equilibrated with mobile phase (80 mM Tris-HCl buffer pH 8.0). COX-1 (94 μg ml) dissolved in 80 mM Tris-HCl buffer pH 8.0, 0.019% Tween 20, 300 μΜ DDC was incorporated into the immobilized lipodisks on the column by 14 x 100 injections with a flow rate of 0.1 mL/min. DDC acts as a reductive agent and a conservative for the protein. The flow rate was stopped for 2.25 min between each injection. The column temperature was 37°C and 1 h was required to incorporate all protein. The column was rinsed with mobile phase. The material of the lipodisk column and the COX- 1 column was taken out and washed with MilliQ water. The amount of immobilized lipids on the columns was determined by phosphorous analysis. The amount of incorporated protein on the COX-1 column was determined by amino acid analysis. Example 3

QCM-D analysis

The binding behavior of COX-1 to the lipodisks was followed using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). A QCM-D D300 (Q-Sense,

Gothenburg, Sweden) instrument thermostated at 21°C was employed for all measurements.

A QCM-D gold sensor was cleaned with hot piranha solution (3 : 1 sulfuric acid:hydrogen peroxide), rinsed with MilliQ water and absolute ethanol, and then incubated overnight in 1 mM 1-mercaptoundecanoic acid (MUA) dissolved in ethanol. Before use, the sensor was rinsed with absolute ethanol and dried under a gentle nitrogen flow. After mounting of the sensor, the system was equilibrated with MilliQ water until a stable baseline was obtained. The surface was then activated for 10 minutes with a freshly prepared 0.1 M NHS : 0.4 M EDC 1 : 1 mixture. A suspension of amine functionalized lipodisks (50 μΜ total lipid concentration) in 80 mM acetate buffer (pH 4.5) was then loaded. The amine groups bind to the active surface, resulting in a layer of immobilized lipodisks. After rinsing with the acetate buffer to remove any non-bound lipodisks, the remaining active surface was inactivated by addition of 1 M ethanolamine. After a 10 minutes inactivation period, the system was finally equilibrated with the working buffer (80 mM Tris-HCl pH 8.0). In order to determine the COX-1 binding isotherms, solutions with increasing concentrations of the protein were sequentially loaded into the system. Before each protein addition the system was rinsed with the working buffer in order to remove any non-specifically bound material. The binding of the protein to the lipodisks is recorded as negative shifts in the oscillation frequency of the quartz crystal. Quantitative results were obtained by fitting the obtained frequency and dissipation shifts with the viscoelastic model described by Voinova et al. (1999) Phys Scr 59:391-396. As Tween 20 is present in the COX-1 solution, analysis of the results assume that the recorded mass changes arise from the binding and partition of the protein and the detergent in the same weight proportions in which they are found in the original solution (32:68 COX- l :Tween 20). Therefore, the results provided represent the lower limit for the binding of COX-1 to the lipodisks. Results and Discussion

Lipodisk characterization

Lipodisks were produced by the detergent depletion method, which enables preparation of lipodisks with a small diameter suitable for immobilization into the pores of the HPLC support materials. Cryo-TEM was used in combination with DLS to determine the size and shape of the lipid structures in the preparations. The cryo-TEM investigation showed that the lipodisk samples contained mainly disk shaped aggregates. No structural difference was observed between the samples prepared with biotinylated PEG and those prepared with amine functionalized PEG. A representative micrograph is shown in Figure 2. Note that due to the poor contrast of the polymer, the PEG-chains are invisible in the micrograph. By studying a large amount of micrographs and measuring more than 500 structures the apparent radius of the amine functionalized lipodisks was determined to 6.4 ± 2.2 nm. By adding 3.5 nm for the length of the PEG chains the full disk radius was calculated to 9.9 nm. According to DLS analysis the hydrodynamic radius (R_h) of the same disks was 9.3 ± 1.3 nm. For the biotinylated disks the corresponding values were 8.6 ± 2.3 nm (with PEG) from cryo-TEM and 8.9 ± 1.2 nm from DLS. The hydrodynamic radius obtained with DLS is determined based on the assumption that all particles are spherical. The hydrodynamic radius can be recalculated into the radius of a lipodisk via a model described by NA Mazer et al. in Biochem, 1990, 19, 601-615 In order to do this accurately the thickness of the disks must be known. Since the thickness of the lipodisks used in this study is unknown and not easily estimated no recalculation of the hydrodynamic radius into the disk radius was done here.

Immobilization of lipodisks on HPLC support materials

As a first step, immobilization of lipodisks by various coupling methods, and on different HPLC materials, was evaluated. Lipodisks with amine and biotin functionalities were immobilized onto Nucleosil silica and POROS media. For immobilization of lipodisks with biotin functionality, streptavidin was immobilized by reductive amination coupling of mainly the lysine side chains of the protein to the aldehyde groups of the materials. The coupling yield was close to 100% (0.45 μιηοΐ protein/mL) on the Nucleosil silica and 55% (0.31 μιτιοΐ protein/mL media) on the POROS media. The amount of immobilized lipids on each material was determined by phosphorous analysis. Coupling by reductive amination of lipodisks resulted in 12 μηιοΐ lipids/mL Nucleosil silica and 19 μιηοΐ lipids/mL POROS media. Coupling by streptavidin-biotin binding of lipodisks with biotin functionality resulted in 8.2 μιηοΐ lipids/mL Nucleosil silica and 9.3 μηιοΐ lipids/mL POROS media. No lipodisks were found in the reference samples, representing passive coupling.

Although the amount of immobilized lipodisks on the POROS media was slightly higher compared to that on the Nucleosil silica, Nucleosil silica was chosen for further studies, as this material is more commonly used for HPLC. Since coupling via reductive amination is more straightforward and also more cost effective than streptavidin-biotin binding, we opted to use this method in our further studies based on HPLC analysis. Furthermore, non-specific interactions between analytes and immobilized streptavidin may interfere when studying membrane protein-ligand interactions.

During a second immobilization of lipodisks with amine functionality on Nucleosil silica, 8.2 μιηοΐ lipids/mL silica was immobilized. Similar to in the previous immobilization experiment, lipodisks were added to the silica material in great excess (coupling yields corresponding to 2 and 13%, respectively). Since the amount of immobilized lipodisks was about the same in both experiments, it is likely that ~ 10 μιηοΐ lipids/mL silica is the maximum amount of lipid that can be immobilized in the form of lipodisks on the limited surface area of the HPLC support materials. Choosing the optimal HPLC support material for immobilization of lipodisks is a tradeoff between pore size and surface area, since the pores must the big enough to harbor the disk, while the available surface area decreases dramatically with increasing pore size.

HPLC analysis of drug-disk interactions

The performance of one of the lipodisk columns (column 1) packed with Nucleosil silica with immobilized lipodisks was tested by analysis of 15 drug compounds and the reproducibility and stability of the column was evaluated. Figure 3 demonstrates typical chromatograms from analysis of a mixture of 7 compounds on the lipodisk column using ammonium acetate buffer as mobile phase, showing both the chromatogram from UV detection (λ=214 nm), the total ion chromatograms (TICs) of SIM positive mode and the extracted ion chromatograms (EICs) of individual analytes in the mixture. The retention time of the analytes on the lipodisk column, corrected for the retention time on the reference column, indicates the extent of interaction with immobilized lipodisks. Analysis on the lipodisk column resulted in reproducible retention times with low relative standard deviations of 0.5 - 1.5%. No differences in retention times were observed for the analytes throughout the study (a few weeks), which indicates that the immobilized lipodisks were stable and no leakage of the column occurred.

The analytes were analyzed both as single injections and in mixtures on the lipodisk column using ammonium acetate as mobile phase and UV detection in combination with MS detection. The differences in retention times during analysis in mixtures compared to single injections were very small showing that the partition behavior of individual drugs was not affected by the presence of other drugs in the mixture. Figure 4 shows retention times of the 15 drug compounds on the lipodisk column (analysis as singles and in mixtures) and the reference column (analysis in mixtures). Retention times on the reference column were short for all analytes. Hence, interactions with the silica matrix or remaining aldehyde groups did not significantly contribute to the overall retention on the lipodisk column.

The amount of lipids immobilized on the lipodisk column was determined to 1.18 μπιοΐ (9.8μπιο1/ηιΕ silica) by phosphorous analysis. Analysis of the 15 drug compounds on the lipodisk column using PBS instead of ammonium acetate buffer as mobile phase resulted in small differences in obtained log Ks, values for charged compounds (Table 1, below and

Figure 5). As expected, electrostatic effects between the negatively charged DSPE-PEG-lipids and the analytes becomes more apparent in the ammonium acetate buffer due to the considerably weaker ionic strength as compared to the PBS. The influence of electrostatic effects on the interaction between charged analytes and lipodisks has previously been reported and discussed by Johansson et al. in Biochim Biophysi Acta 2007, 1768: 1518-1525.

]

Taking the above-mentioned electrostatic effects into account, it can be concluded that the log Ks values obtained using the two different mobile phases correlated well. Moreover, there was no difference in the drug retention times obtained from single injections and mixtures. These results suggest that individual drug compounds present in mixtures can be analyzed with high throughput using ammonium acetate buffer as mobile phase in combination with MS detection. COX-1 incorporation into immobilized lipodisks

In the present Examples, a COX-1 column was produced by in situ incorporation of COX-1 into lipodisks immobilized by reductive amination to the silica material (see Example 2.2.6 for details). This strategy will work well for peripheral proteins, such as COX-1, that can be added to the column after lipodisk immobilization and deactivation of remaining aldehyde groups. However, for transmembrane proteins that need to be incorporated into the lipodisks prior to immobilization, streptavidin-biotin coupling might be preferable in order to avoid amine coupling of lysine side chains of the protein to the support material. The COX-1 column (column 2) was emptied and the content was analyzed by amino acid and phosphorous analysis. The amount of incorporated COX-1 on the column was determined to 0.6 nmol (5.0 nmol COX-l/mL Nucleosil silica) and the amount of lipids to 1.32 μηιοΐ (10.9 μιηοΐ lipid/mL silica). Hence, the number of lipids on the column for each COX-1 dimer was about 4400.

Specific binding of COX-1 to immobilized lipodisks was validated by interaction studies by QCM-D. Lipodisks from the same batch as was used for HPLC-MS experiments were successfully immobilized onto the QCM-D sensor (data not shown). Addition of COX-1 resulted in negative frequency shifts. Blank measurements performed on a modified gold sensor surface that had been inactivated with ethanolamine prior to protein addition showed that the non-specific binding of the protein to the surface is negligible. Therefore, it is safe to assume that the observed shifts result from binding of the protein (and associated Tween 20) to the lipodisks. The binding isotherm shown in figure 6 was obtained from the experiments. Given that the associated protein to lipid ratio is rather low, it is unlikely that more than one COX-1 dimer is located on each lipodisk at saturation conditions. Furthermore, the amount of immobilized lipodisks was kept comparably low (calculated to -300 ng/cm², in contrast to the maximum obtained coverage at long immobilization times of -900 ng/cm²). The system can then be treated as an array of separated binding sites. Each lipodisk constitutes an independent binding site to which only the binding of a single protein is possible. Also, given the low degree of coverage, the lipodisks can be assumed to be immobilized with some distance to each other, implying that the binding of COX-1 to one lipodisk will not affect the properties of other lipodisks. Hence, the Langmuir association isotherm can be employed as an approximation to describe the binding behavior of COX-1 to immobilized lipodisks. The effective associated protein to lipid ratio at saturation ( ?^ _max) can therefore be estimated from the experiments. According to the QCM-D results, at saturation, one COX-1 dimer is found for every 2200- 2900 lipid molecules (R_eff,max = 3 9^χ 10^"4 ± 5.5 ^χ 10^"5). This finding correlates reasonably well with the results obtained from the amino acid analysis of the COX-1 column. The difference in protein to lipid ratio obtained from the QCM-D and amino acid analysis may partly be due to the fact that the former analysis was carried out at 21°C, whereas incorporation of COX-1 in situ on the lipodisk column was performed at 37°C. Further, it is possible that the accessibility of the disks for protein binding is somewhat compromised in the narrow pores of the Nucleosil silica.

These QCM-D results also support that COX-1 was bound specifically to immobilized lipodisks on the column rather than non-specifically to other parts of the column. An indication of the affinity of the protein for the lipodisks would be given by the association equilibrium constant K (M^"1), which can be obtained by generation of a binding hyperbola on basis of the experimental data. However, given the limited number of measurement points, the experimentally determined value of K can only be determined with rather large error margins (0.78 ± 0.38 mL^g = (1.1 ± 0.5)x l0⁸ M^"1). Nonetheless, it is safe to state that equilibrium concentrations slightly above 10 μg/mL (7.1 nM) are enough to achieve over 90 % saturation of the lipodisks with the COX-1 protein. The QCM-D experiments thus indicate that the lipodisks are saturated with bound COX-1 at significantly lower protein concentrations than what was used during the in situ incorporation of COX-1 on the lipodisk column.

Results of the present Examples show moreover that COX-1 is stably bound to the immobilized disks via a straightforward protocol for in situ incorporation of the protein. Provided full activity of the protein, the COX-1 column produced in the present is useful to detect COX-1 binders with sub-μΜ affinities if the system dimensions are optimized. Further optimization of the system in terms of mobile phase conditions such as pH and temperature is also feasible within the context of the present invnetion. Accordingly, the material and the methods outlined according to the present invention are useful to study protein-ligand interactions. Table 1. Log K_s values obtained using covalently immobilized lipodisks composed of POPC/Soy PE/cholesterol/Ceramide-PEG₂ooo/DSPE-PEG₂oooamine (30:28: 17:21 :4 mol%).

log Ks

D_m„ Charge at Ammonium acetate buffer

pH 7-7.4 _single PBS

. . . mixtures

injections

Alprenolol + 3.10 3.09 2.74

Lidocaine + 1.78 1.76 1.96

Pindolol + 2.40 2.35 1.50

Promethazine + 4.15 4.14 3.79

Propranolol + 3.59 3.55 3.20

Theophylline + 0.70 0.59 0.51

Diclofenac - 2.79 2.81 3.05

Ibuproten - 1.65 1 .65 1.81

Indomethacin - 2.81 2.81 3.06

Naproxen - 1.42 1.49 1.60

Warfarin - 1.86 1 .88 1.93

Corticosterone 0 2.25 2.25 2.28

Cortisone 0 2.30 2.31 2.34

Hydrocortisone 0 2.53 2.56

Prednisolone 0 2.06 2.05 2.11

Claims

1. A material adapted to interact with a mobile phase suitable for high performance liquid chromatography, comprising a solid sorbent material with immobilized lipid bilayer discs, wherein the lipid bilayer discs comprise:

at least one polar lipid;

at least one lipid conjugated to polyethylene glycol; and

at least one lipid conjugated to polyethylene glycol having functional groups bound to functional groups of the solid sorbent material.

2. A material according to claim 1, having a bioactive protein incorporated in at least one lipid bilayer disc.

3. A material according to claim 2, wherein the bioactive protein is a membrane protein.

4. A material according to claim 1 or 3, wherein the functional groups of the

polyethylene glycol are amine groups and the functional groups of the sorbent material are aldehyde groups.

5. A material according to any of claims 1 or 3, wherein the functional groups of said polyethylene glycol are biotin groups and the functional groups of the sorbent support material are streptavidin groups.

6. A material according to any of claims 1 to 5, wherein the size of the bilayer discs is essentially less the than the pore size of the sorbent material.

7. A material according to claim 5 comprising lipid bilayer discs have size of from about 8 to about 200 nm and the pore size of the sorbent material is between 300 and 10000

A.

8. A material according to any of claims 1 to 7, wherein the bilayer discs comprise at least one of 1,2-dipalmitoyl -OT-glycero-3-phosphocoline (DPPC), 1 -palmitoyl-2- oleoyl-s«-glycero-3-phosphocoline (POPC), soy L-a-phosphatidylethanolamine (Soy PE), l,2-disteroyl-OT-glycero-3-phosphocoline (DSPC), l-palmitoyl-2-oleoyl-^- glycero-3-phosphoethanolamine (POPE), 1,2-dioleylpalmitoyl -sn-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-.sw-glycero-3-[phosphor-L- serine] (sodium salt) (POPS), 1,2-dimyristoyl -sft-glycero-3 -phosphatidylcholine (DMPC), phosphatidylcholine (EPC), 2-hydroxy-sn-glycerol-3-phosphatidylcholine (MSPC), L-a-phosphatidylinositol, a sphingomyelin, and a cerebroside.

9. A material according to any of claims 1 to 8, wherein the lipid bilayer discs comprise at least one of N-palmitoyl-sphingosine-l-{succinyl[methoxy(polyethylene glycol)2000]} (Ceramide-PEG2ooo), N-palmitoyl-sphingosine-1- {succinyl[methoxy (polyethylene glyco 1)5000]} (Ceramide-PEGsooo), 1,2-distearoyl- ^«-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE- PEG2000), l,2-distearoyl-5w-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-5000] (DSPE- PEG5000), 1.2-dimyristoyl -sw-glycero-3- phosphatidylcholine(polyethylene glycol 2000) (DMPC- PEG2000), and cholesterol conjugated to polyethylene glycol.

10. A material according to any of claims 1 to 9, comprising at least one of the

polyethylene conjugated lipids according to claim 9 comprising an amine group or biotin as a functional group, preferably the lipid is l,2-distearoyl-^-glycero-3- phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2ooobiotin) or l,2-distearoyl-i-«-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)- 2000] (DSPE-PEG₂oooamine).

11. A material according to any of claims 1 to 10, comprising a sterol, such as cholesterol.

12. A material according to any of claims 8 to 1 1, comprising lipid bilayer discs

comprising POPC, soy PE, cholesterol, ceramide-PEG(2000) and DSPE-PEG(2000) functionalized with amine groups or biotin.

13. A material according to claim 12, comprising 1 to 10 μηιοΐ lipids per mL sorbent material.

14. A method of studying the interaction between a material according to any of claims 1 to 13 and a composition of analytes, comprising providing a stationary phase of the material; transporting the composition of analytes to said stationary phase and establishing interaction conditions between the analytes and the stationary phase; and determining how each analyte interacts with the lipid bilayer discs of the material.

15. A method according to claim 14, comprising determining partitioning of each analyte, wherein the lipid bilayer discs are model membranes in a drug partitioning study.

16. A method according to claim 14 comprising of screening the binding to a biological target with transient weak interactions, comprising the steps of:

(a) providing a stationary phase from the material according to any of claims 1-13 having a bioactive protein or peptide, preferably a membrane protein or any fragment thereof, incorporated in the lipid discs;

(b) providing a composition of ligands with a concentration of each ligand that is less than 0.1 mM,

(c) transporting the ligand composition to said stationary phase, thereby establishing weak affinity interactions between the ligands and the biological target in the range of 0.001 to 10 mM expressed as dissociation constant (K_D); and

(d) detecting said ligands in compositions arriving from said stationary phase to

discriminate between different ligand affinities in order to estimate the affinity and the dynamics of each ligand/target weak affinity interaction.