Patent Description:
The functionalized substrates, methods of preparing said ligand-linker functionalized substrates and the linker length optimisation thereof. More specifically, the ligand linker functionalized substrates include a solid substrate, which has been modified to provide binding ligand groups covalently bound via a linker length, used to increase the binding rate and the dynamic binding capacity of receptor compound of interest to substrate.

Isolation and purification of target molecules, such as biomacromolecules, are important for therapeutic purposes and in biomedical research. The general industrial purification process often includes a number of unit operations, like extraction, filtration, precipitation, as well as anion- and cation-exchange chromatography, and affinity purification.

Most current capture or affinity purification chromatography is done via conventional resin based column techniques. Porous beaded chromatography resins have high surface area to mass ratio bearing interactive affinity ligand groups, and have therefore been used in protein affinity purification processes. These techniques are time consuming and provide severe bottlenecking issues in downstream purification, due to binding rates dependent on mass transport by diffusion. This also limits the chromatography techniques applicability in high throughput screening and for rapid downstream processing.

Filter, membrane, nano-fibre and nano-particles -based technologies, especially in disposable format, are becoming increasingly important in biopharmaceutical and vaccine manufacturing processes. Membranes have been used in passive, molecular size-based separation and in active filtration.

Functionalized membranes having enhanced process flow properties (including functional polymer-bearing membranes) have typically suffered from relatively low biomaterial binding capacities due to low surface area to mass ratio, which in general have limited their use in certain large-scale purification processes.

However, membranes and filters have high productivity potential (product mass/volume/time) due to the possible high process stream velocities and efficient mass transport by convection. Today membranes and filters are mainly used in polishing processes to remove impurities to very low levels in product streams. These materials, membranes, filters and porous bead resins, could be improved further. Also, bead resin-based materials could be more economical applicable by optimising the dynamic binding kinetic between ligand and receptors. The resulting membrane, filter or bead processes will be even faster and more efficient and make it possible to have downstream purification unit operations with smaller foot print, higher capacity and provide less waste water and consumption of process buffer fluids.

Standard ligand-functionalized substrates, such as ligand-functionalized membranes, are widely used for the separation and affinity purification of various target molecules. For example, ligand-functionalized membranes can be used to purify or separate a target molecule based on an affinity interaction, or based on the formation of a covalent bond.

<CIT> discloses substrates having azlactone-functional surfaces, adduct substrates, and methods of preparing both.

<CIT> discloses guanidinyl ligand-functionalized polymers, methods of making the same, and substrates bearing a grafted coating of the ligand-functional polymers.

<CIT> discloses ligand functionalized substrates, methods of making ligand functionalized substrates, and methods of using functionalized substrates.

<CIT> discloses ligand functionalized substrates with enhanced binding capacity.

<CIT> discloses a process for purification of a target biomolecule, comprising the steps: (a) contacting (i) a target biomolecule, (ii) a dual affinity polypeptide (DAP), and (iii) a solid substrate comprising a catching ligand; and (b) recovering the target biomolecule by elution, wherein the target biomolecule and the dual affinity polypeptide are contacted in solution before the mixture is contacting the solid substrate.

<CIT> discloses a process for purification of a target molecule, comprising the steps: (a) contacting a target molecule, and a population of target binding polypeptides (TBP), in solution for a sufficient time to allow complex formation; and (b) isolating the target from the complex from (a) by subsequent purification steps, wherein (i) the target binding polypeptides have at least two binding functionalities; a first binding functionality towards the target and a second binding functionality towards a catching ligand comprised in a solid substrate; and (ii) the first binding functionality comprises at least two binding sites for the target, and the target comprises at least two binding sites for the TBP.

<NPL> discloses affinity ligands for protein purification.

The dual affinity polypeptide (DAP) described in <CIT> have the same functionality as the target binding polypeptides (TBP) described in <CIT> and the two terms DAP and TBP is used herein interchangeably to describe the same compound.

While a number of ligand-functionalized substrates exist, there is a need in the art for more optimal ligand-linker functionalized substrates that can operated at a high binding rate and with high dynamic binding capacity to provide high product throughput - and thereby high productivity to reduce manufacturing equipment investment, operating cost and environmental foot print.

It is an object of embodiments of the invention to provide a use of a ligand-linker functionalized substrate for purification of a target biomolecule that allows a fast binding rate to the receptor (compound of interest) without compromising a high dynamic binding capacity.

It has been found by the present inventors that by providing a ligand-functionalized substrate wherein the ligand is bound to the substrate via a linker of a specific length and preferably a non-bendable structure, a considerably increased dynamic binding capacity as well binding rate between the ligand and a receptor of interest may be obtained.

The invention relates to a use as defined in the claims.

According to the invention, the linker comprises one, two, or three of the groups defined by:
<CHM>.

In the present context the length of the linker is defined by the number of bonds, wherein bonds are C-C, C-N, C-N(H), C-C(O) and/or C-O bonds. <FIG> further illustrates how the number of linker bonds is determined. It is noted that biotin by nature contains a lipophilic linker. The linker used in accordance with the present invention contains amide and partly sterically hindered groups, which prevents the linker from being fully flexible.

It is to be understood that a linker refers to a chain of at least <NUM> N, C, S or O atoms linking the substrate and ligand-functional group of the invention. Typically, the linker is a linear sequence of at least <NUM> N, O or C atoms, wherein the length of the linker is defined by the total amount of bonds in the linear sequence of atoms (see <FIG>). Bonds may include C-C, C-N, C-N(H), C-C(O) and/or C-O.

It should be understood that the practical length of the linker depends on the actual lengths of the individual chemical bonds, the surrounding functionalities, the angles, the rotational flexibility and the conformation in the solvent. For example, the average C-C, C-O and C(O)-N(H) bond are about <NUM>, <NUM> and <NUM>Å, respectively. The C-O bond can partly rotate, whereas the amide bond is not as free to rotate.

The linker length is defined as the number of chemical bonds in the linker. That is from the ligand functional group, such as a biotin to the substrate. It is noted that the terminal bond to the substrate is also counted. Accordingly, when counting the length of a linker as shown herein having a terminal -ene, the bond from the terminal carbon atom to the substrate should also be counted. If the linker as shown herein has a terminal amino groups (-NH2), the bond to this amino groups will be counted as the last bond. One should be aware, that the biotin has naturally C5 linker between the ring structure, the ureido and tetrahydrothiophene groups, and the carboxyl group.

The biotin-binding domain in avidin/streptavidin molecules are buried about <NUM>Å below the protein surface and hence. The presence of bulky groups in the vicinity of the biotin-binding pocket may create steric hindrances and reduce the binding efficiency.

It should be understood that the functionality and the length of the linker is associated with the dynamic binding capacity and the binding rates of the DAP/TBP molecules to the surface.

As used herein a "dual affinity polypeptide" or "DAP" used interchangeably with "target binding polypeptides" or "TBP" refers to a recombinant protein comprising at least one binding domain capable of binding to the target biomolecule with the desired binding specificity as described herein, as well as at least one binding domain capable of binding to the ligand-functionalized substrate according to the invention. Suitable DAP molecules are described in <CIT> and suitable TBP molecules is described in <CIT> includes in a preferred embodiment a streptavidin monomer fused in a fusion protein to four protein A binding domains.

The present invention relates to a use of a ligand-functionalized substrate, said ligand-functionalized substrate comprising: a) A substrate having carboxylic (-COOH), hydroxy (-OH), thio (-SH), amino groups (-NH2) C-C double bonds (-ene) or C-C triple bonds (-yne) at the surface thereof; b) Covalently bound to said carboxylic (-COOH), hydroxy (-OH), thio (-SH), amino groups (-NH2), C-C double bonds (-ene) or C-C triple bonds (-yne) of said substrate a linker having a linker length of <NUM>-<NUM>, such as <NUM> -<NUM> bonds, wherein bonds are C-C, C-N, C-N(H), C-C(O) and/or C-O; and c) A ligand-functional group bound to the surface of the substrate via said linker.

In an embodiment of the ligand-functionalized substrate according to the invention the substrate is a solid substrate, such as in the form of sheets, membranes, beads, saddles or granulates. With both smooth surfaces and large inner surfaces due to porosity.

Optionally, the ligand-functionalized substrate according to the invention the substrate is a solid substrate which can be grafted by radical initiated vinyl polymerization. In an embodiment of the ligand-functionalized substrate according to the invention the substrate is selected from the group consisting of agarose, kieselguhr, silica gel, cellulose, cellulose ethers, carboxymethyl cellulose, degenerated cellulose, agarose or paper based membranes, nitrocellulose, nitrocellulose mixed esters, silicas, and controlled pore glasses, polyamides, poly sulfone ether, polyvinylalcohols, polycarbonate, polyurethane, polyethersulfone, polysulfone, polyethylene terephthalate, polyvinylidene fluoride, polystyrene or polypropylene, polyethylene and co-block polymers, blends and combinations hereof. In an embodiment the substrate is in the form of particles, films, woven or nonwoven webs, sponges, fibres, sheets, beads, filter, or a membrane. The substrate used according to the invention is usually porous for increased surface area and optimal liquid flow properties and can be both organic and inorganic, including polymeric.

The synthesis methods of stepwise elongating peptides by stepwise coupling protected amino acids or other multifunctional building blocks together is well-described from for example classical Merrifield solid phase peptide synthesis or in solution. The various chemo selective and orthogonal protection groups include Boc, Fmoc, Benzyl or t-But among others. The amide formation is done using for example carbodiimides, efficient active ester, HATU/HOAt or similar reactions. A summary of SPPS reagents and methods can be found in for example Academic Press: San Diego, CA, <NUM>; pp <NUM>-<NUM> or in <NPL>. and in more general for conjugation and cross-linking in <NPL>.

Also, the use of <NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone (VDMA) and derivatives for elongating and grafting linkers are well known. For example described in <NPL>, <CIT>, and other references cited herein.

According to the invention, in the ligand-functionalized substrate, the ligand functional group is biotin.

The present inventors have surprisingly found that the length and nature of the linker has a distinct influence on both the dynamic binding capacity (DBC) and binding rate of the resulting use thereof for binding a receptor molecule like e.g. dual affinity polypeptide (DAP) or target binding protein (TBP) and efficiently removing it from solution, which is a requirement for the purification of a target biomolecule. The linker should preferably be semi flexible in water at pH <NUM>, to allow a stretched configuration and thereby higher binding efficiency. The linker between the substrate and the ligand-functional group, such as biotin, should be extended in water, but not be completely flexible or free to rotate, or bend in all directions.

Further, the inventors have found that the optimal length for reaching into a ligand functional group being a biotin binding pocket require guidance to the optimal dynamic binding efficiency. The various linker optimizations described in the prior art are typically based on steady state equilibrium, static binding conditions or from experiments with small molecules or from anion- and cation-exchange chromatography.

The use according to the invention is for purification of a target biomolecule, comprising the steps:.

In some embodiments, the method comprises a step of washing away impurities, such as host cell proteins (HCP), DNA, viruses, selectively immobilizing or removing the dual affinity polypeptide from target product solution.

In an embodiment thereof the target biomolecule is an antibody.

It is to be understood that the term antibody as used herein may refer to any immunoglobulin molecule, such as an immunoglobulin molecule with binding affinity to Protein A, such as any immunoglobulin molecule comprising an Fc region, or a Fab region, such as from IgG of the human VH3 gene family.

The following examples illustrate the present invention. In summary, a number of linkers according to the invention was prepared and the dynamic binding capacities and binding rates were measured.

In more detail, biotin was functionalized with an amin. By stepwise synthesis using VDMA, the linkers were constructed with either an acrylic or amine end group. The same method was used to synthesis longer versions of the linker. In an alternative synthesis route, the linkers of various lengths were synthesized using step wise amide couplings, similar to SPPS methods.

The various linkers were coupled or grafted to the substrates and tested for dynamic binding capacity and binding rates. The measured dynamic capacity and rates depended on the linker lengths.

Flash column chromatography was performed on a Teledyne ISCO, Combi Flash Rf+ Lumen using RediSepRf alumina basic or SiliCycle SiliaSep silica columns.

Analytical HPLC-MS was performed using an Agilent <NUM> series Liquid Chromatograph/Mass Selective Detector (MSD) (Single Quadropole) equipped with an electrospray interface and a UV diode array detector. Analyses were performed with methods using either an ACE <NUM> C8 (<NUM> x <NUM>) column with a gradient of acetonitrile in <NUM>% aqueous TFA over <NUM> and a flow of <NUM>/min, or an Xbridge C18 (<NUM> x <NUM>) column with a gradient of acetonitrile in <NUM> ammonium bicarbonate over <NUM> and a flow of <NUM>/min.

Intermediate NMR spectra were recorded on a Nanalysis NMReady 60e at <NUM>. Final NMR spectra were recorded on a Bruker <NUM> instrument at <NUM> °C using D6-DMSO as solvent and TMS as reference. Commercial names or trivial names were used for commercial materials and reagents.

A: Synthesis of aminated biotin: N-(<NUM>-aminoethyl)-<NUM>-(<NUM>-oxo-<NUM>,<NUM>,3a,<NUM>,<NUM>,6a-hexahydrothieno[<NUM>,<NUM>-d]imidazol-<NUM>-yl)pentanamide.

Biotin was refluxed (<NUM> gram, Aldrich) in methanol (<NUM>) and TsOH (<NUM>, Aldrich) overnight, then the reaction volume was reduced by gentle distillation in vacuum in rotary evaporator to produce a clear oil of the biotin methyl ester. Then <NUM> of <NUM>,<NUM> diamino ethane (Aldrich) in ethanol (<NUM>) were added and the mixture was stirred overnight and then reduced by gentle distillation in vacuum in rotary evaporator to yield a light yellow semicrystalline oil. The aminated biotin was pure according to H-NMR, LC-MS and thin layer chromatography (TLC) and was used without further purification for the addition of VDMA (<NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone, <NPL>) in water in the next step.

B: To a solution of N-(<NUM>-aminoethyl)-<NUM>-(<NUM>-oxo-<NUM>,<NUM>,3a,<NUM>,<NUM>,6a-hexahydrothieno[<NUM>,<NUM>-d]imidazol-<NUM>- yl)pentanamide (<NUM>, <NUM> mmol) in water (<NUM>) was added VDMA , <NUM>, <NUM> mmol). The reaction was stirred for <NUM> and then freeze dried. The crude (<NUM>) was purified by silica chromatography column (<NUM> silica, liquid injection in methanol, DCM/MeOH, <NUM> % <NUM> CV, <NUM> to <NUM> % in <NUM> CV, <NUM> to <NUM> % in <NUM> CV, <NUM> % for <NUM> CV). The pooled fractions were concentrated. The residue was dissolved in water and freeze dried to yield Biotin-C10-ene as a white solid (<NUM>, <NUM> mmol, yield: <NUM> %). [M+H] + <NUM> Da.

See <FIG> illustrating the synthesis of Biotin-C10-ene.

The Biotin-C10-ene was used for the radical initiated co-grafting experiment on solid supports like membranes by co-polymerization with an acrylamide and could be used for coupling of the Biotin-C-<NUM>-ene to aminated substrates by Michael addition to the terminal vinyl bond.

To Biotin-C10-ene (<NUM>, <NUM> mmol) was added a <NUM> N solution of ammonia in water (<NUM>, <NUM>, <NUM> mmol). The reaction mixture was stirred at <NUM> for <NUM> in a closed pressure resistant vessel. The solution was freeze dried to obtain <NUM> of Biotin-C10-NHz. The compound was fully characterized by H-NMR, C-NMR and LCMS.

See <FIG> illustrating the synthesis of Biotin-C10-NH<NUM>.

<NUM>, (<NUM> mmol) VMDA was added to a solution of Biotin-C10-NH2, synthesized as in example <NUM>, (<NUM>, <NUM> mmol) in water (<NUM>). The reaction was stirred for <NUM> and then freeze dried. The crude solution (<NUM>) was purified by silica chromatography column (<NUM> silica column, liquid injection in methanol, DCM/MeOH <NUM> % <NUM> CV, <NUM> to <NUM> % in <NUM> CV, <NUM> to <NUM> % in <NUM> CV, <NUM> % <NUM> CV). The fractions containing the product and hydrolysed VDMA were pooled and concentrated by gentle distillation in vacuum in rotary evaporator. The material was further purified by basic alumina chromatography column (<NUM> alumina basic column, liquid injection in methanol, DCM/MeOH <NUM> to <NUM> % in <NUM> CV). The pooled fractions were concentrated by gentle distillation in vacuum in rotary evaporator. The residue was dissolved in water and freeze dried to yield Biotin-C17-ene as a white solid (<NUM>, <NUM> mmol, yield: <NUM> %). [M+H] + <NUM> Da.

See <FIG> illustrating the synthesis of Biotin-C17-ene.

Biotin-C17-ene was used for the radical initiated co-grafting experiment on membranes by co-polymerization with an acrylamide and used for coupling of the Biotin-C-<NUM>-ene to aminated substrates by Michael addition to the terminal vinyl double bond of Biotin-C17-ene.

The linker was also used in a separate example for a further extension with another VDMA unit, creating an even longer linker derivative.

This example illustrates the synthesis route for the Biotin-linkers of different lengths (C-<NUM>, C-<NUM> and C-<NUM>). Two types of affinity ligands (series A: terminal NH<NUM> amino and series B: terminal vinyl -ene) with different linker length (C10, C17 and C24) were synthesized using peptide coupling reactions by HATU/DIPEA, and orthogonal N-terminal Fmoc and Boc deprotection by piperidine and <NUM> HCl. The final steps are the N-terminal coupling of Biotin as active N-hydroxy succinimide (NHS) ester to the peptide linker molecules and final N-Boc deprotection for the synthesis of the N-terminal amino Biotin linker molecules Biotin-C10-NH<NUM>, Biotin-C17-NH<NUM> and Biotin-C-<NUM>-NH<NUM> (series A).

The Biotin-linker molecules Biotin-C10-ene, Biotin-C17-ene, Biotin-C24-ene with terminal - ene groups (series B) have been synthesized by <NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone VDMA coupling onto the terminal amino group of Biotin-EDA-NH<NUM>, Biotin-C10-NHz and Biotin-C17-NH<NUM> in water in order to achieve a C7 elongated linker molecule.

The first type, named Biotin-C10-NHz, Biotin-C17-NH<NUM> Biotin-C24-NH<NUM>, have a terminal amino group (series A). The second type, named Biotin-C10-ene, Biotin-C17-ene,Biotin-C24-ene, have a terminal vinyl -ene group (series B).

For each type below, three compounds were required with respectively n=<NUM>, <NUM> and <NUM>.

The approach used peptide coupling reactions. The chain of the linker is composed of three different components: Ethylene diamine EDA and the two amino acids: α-methyl alanine and β-alanine.

The linker chain composed of ethylene diamine EDA, α-methyl alanine α-MeAla and β-alanine β-Ala.

As an example, for the series B synthesis, shows <FIG> adding <NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone VDMA to Biotin-C10-NHz will give the C7 elongated Biotin-C17-ene affinity ligand.

To a solution of Fmoc-C) (<NUM>, <NUM> mmol) in dichloromethane (<NUM>) cooled with ice bath, was added dropwise a solution of tert-butyl N-(<NUM>-aminoethyl) carbamate (Boc-EDA) (<NUM>, <NUM> mmol) in <NUM> DCM. The reaction was stirred at room temperature. After <NUM>, a white precipitate formed. <NUM> of MeOH was added and the solution became homogenous. The mixture was washed with water (<NUM> x <NUM>). The organic phase was dried over magnesium sulphate, filtered and concentrated. The solid was washed with diethyl ether and dried under vacuum to yield Fmoc-EDA-Boc (<NUM>, <NUM> mmol, yield: <NUM> %). To a solution of Fmoc-EDA-Boc (<NUM>, <NUM> mmol) in <NUM>,<NUM>-dioxane (<NUM>) was added a <NUM> solution of HCl in <NUM>,<NUM>-dioxane (<NUM>, <NUM>, <NUM> mmol). The reaction mixture was stirred for <NUM> hour. The solid was filtered and washed with diethyl ether to yield Fmoc-EDA-HCl (<NUM>, <NUM> mmol, yield: quantitative) as a white solid.

To a solution of N,N'-disuccinimidyl carbonate (<NUM>, <NUM> mmol) and biotin (<NUM>, <NUM> mmol) in N,N-dimethylformamide (<NUM>) was added triethylamine TEA (<NUM>, <NUM> mol). The reaction mixture was stirred for <NUM> hours. The solid was filtered, washed with diethyl ether and dried under vacuum to yield Biotin-NHS (<NUM>, <NUM> mol, yield: <NUM> %) as a white solid and used without further purification.

To Fmoc-EDA-MeAla-NHBoc or Fmoc-EDA-NHBoc in <NUM>,<NUM>-dioxane was added a <NUM> solution of HCl in <NUM>,<NUM>- dioxane (<NUM> eq). The reaction mixture was stirred for <NUM> hour and then concentrated. The crude material was used without further purification.

To a solution of Boc-α-methylalanine (<NUM> eq) and HATU (<NUM> eq) in acetonitrile (concentration ca <NUM>) was added DIPEA (<NUM> eq). The mixture was stirred for <NUM>. To this mixture was added the N-terminal amino compounds (<NUM> eq) and the resulting solution was stirred for <NUM>. When the product precipitated, the solid was filtered, washed with diethyl ether and dried under vacuum. Otherwise, the solution was diluted with ethyl acetate, washed twice with a <NUM> solution of HCl in water, washed twice with water and washed once with brine. The organic phase was then dried over magnesium sulphate, filtered and concentrated.

To a solution of Boc-β-alanine (<NUM> eq) and HATU (<NUM> eq) in acetonitrile (concentration ca <NUM>) was added DIPEA (<NUM> eq). The mixture was stirred for <NUM>. To this mixture was added the N-terminal amino compound (<NUM> eq) and the resulting solution was stirred for <NUM>. When the product precipitated, the solid was filtered, washed with diethyl ether and dried under vacuum. Otherwise, the solution was diluted with ethyl acetate, washed twice with a <NUM> solution of HCl in water, washed twice with water and washed once with brine. The organic phase was then dried over magnesium sulphate, filtered and concentrated. In both cases, the crude material was used without further purification.

To Fmoc-ethylenediamine-NHBoc was added a <NUM> % solution of piperidine in acetonitrile. The reaction mixture was stirred for <NUM> minutes and then concentrated. The solution was concentrated, and the crude was dried <NUM> hours under vacuum. The solid was triturated three time with diethyl ether. The solid was filtered, washed with diethyl ether and dried under vacuum. The crude material was used without further purification.

To a solution of NH2-ethylenediamine-NHBoc in acetonitrile and water (concentration ca <NUM>) was added Biotin-NHS (<NUM> eq). The reaction mixture was stirred <NUM> and check by LCMS. If needed additional <NUM> eq of Biotin-NHS was added and the mixture was stirred for <NUM> hour. The mixture was concentrated in rotary evaporator and purified by silica chromatography (liquid injection in methanol; DCM <NUM> CV, DCM/MeOH <NUM> to <NUM> % in <NUM> CV).

To Biotin-Ethylenediamine-NHBoc was added a <NUM> solution of HCl in water. The reaction mixture was stirred overnight, and concentrated. The crude material was re-dissolved in methanol and concentrated. The solid was dried under vacuum.

Biotin-C10-NHz (<FIG>) has been synthesized by a series of peptide coupling reaction coupling reactions starting from HATU coupling of Boc-MeAla and N-Fmoc protected ethylene diamine Fmoc-EDA in acetonitrile to form FmocNH-EDA-MeAla-NHBoc. The terminal N-Boc protecting group was removed by <NUM> HCl to for the N-terminal amino group Fmoc-EDA-MeAla-NH<NUM>. The Fmoc-EDA-MeAla-NH<NUM> was coupled to the BocNH-β-alanine to form the Fmoc-EDA-MeAla-β-Ala-NHBoc. The terminal Fmoc group was removed by <NUM>% piperidine in acetonitrile to form the terminal amino group. Then Biotin-NHS was coupled onto the NH<NUM>-EDA-MeAla-β-Ala-NHBoc in acetonitrile-water at room temperature and finally the NHBoc group was removed with <NUM> HCl at room temperature in order to synthesize the Biotin-C10-NH<NUM> with terminal amino group. The synthesis route is described in <FIG>. The compound Biotin-C10-NHz was fully characterized by H-NMR, C-NMR and LC-MS. Formula weight is <NUM>, measured mass is found to be <NUM>.

The synthesis of Biotin-C17-NH<NUM> (<FIG>) followed the same peptide coupling reactions by HATU/DIPEA, Boc deprotection with <NUM> HCl, Fmoc-deprotection with piperidine and biotinylation with Biotin-NHS as described for Biotin-C10-NH<NUM>.

The Boc protecting group of Fmoc-EDA-MeAla-β-Ala-NHBoc (C-<NUM> linker) was removed by <NUM> HCl treatment to form Fmoc-EDA-MeAla-β-Ala-NH2. The terminal amino group was then coupled to Boc-MeAla by HATU/DIPEA in acetonitrile. The terminal Boc protecting group was removed by <NUM> HCl and then coupled to β-Ala-NHBoc by HATU/DIPEA coupling. Then the Fmoc group was removed with <NUM>% piperidine in acetonitrile, Biotin was coupled to the terminal NH2 group as NHS active ester and finally the terminal Boc group removed with <NUM> HCl to form the Biotin-C17-NH<NUM> affinity linker molecule. Finally, after the deprotection of Biotin-C17-NHBoc, <NUM> of product was obtained with an overall yield of <NUM> % (<NUM> steps). The compound was fully characterized by H-NMR, C-NMR and LC-MS. Formula weight is <NUM>, measured mass is found to be <NUM>.

The synthesis of Biotin-C24-NH<NUM> (<FIG>) followed the same peptide coupling reactions by HATU/DIPEA, Boc deprotection with <NUM> HCl, Fmoc-deprotection with piperidine and biotinylation with Biotin-NHS as described for Biotin-C10-NH<NUM> and Biotin-C17-NH<NUM>.

The Boc protecting group of Fmoc-EDA-MeAla-β-Ala-MeAla- β-Ala-NHBoc (C-<NUM> linker) was firstly removed by <NUM> HCl treatment to form Fmoc-EDA-MeAla-β-Ala-MeAla- β-Ala-NH<NUM>. The terminal amino group was then again coupled to Boc-MeAla by HATU/DIPEA in acetonitrile. The terminal Boc protecting group was again removed by <NUM> HCl and then coupled to β-Ala-NHBoc by HATU/DIPEA coupling. Then the Fmoc group was removed with <NUM>% piperidine in acetonitrile, Biotin was coupled to the terminal NH2 group as NHS active ester and finally the terminal Boc group removed with <NUM> HCl to form the Biotin-C24-NH2 affinity linker molecule. With the experience from the synthesis of Biotin-C10-NH2 and Biotin-C17-NH<NUM>, this synthesis of Biotin-C24-NH<NUM> resulted in <NUM> of material were obtained with an overall yield of <NUM> % (<NUM> steps). The compound was fully characterized by H-NMR, C-NMR and LC-MS. Formula weight is <NUM>, measured mass is found to be <NUM>.

To a solution of Biotin-EDA-NH<NUM>, Biotin-C10-NH<NUM> or Biotin-C17-NH<NUM> in water was added a <NUM> solution of sodium bicarbonate in water until pH <NUM>. To that solution was added VDMA. The mixture was concentrated and subjected to silica chromatography (liquid injection in methanol, DCM <NUM> CV, DCM/MeOH <NUM> to <NUM> % in <NUM> CV, <NUM> to <NUM> % in <NUM> CV, <NUM> to <NUM> % in <NUM> CV and <NUM> % for <NUM> CV). The pooled fractions were concentrated. The products were dissolved in methanol, concentrated and dried under vacuum to yield Biotin-C10-ene, Biotin-C17-ene and Biotin-C24-ene, respectively.

Biotin-C10-ene (<FIG>) with terminal vinyl group was synthesized by addition of <NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone VDMA to the terminal amino group of Biotin-EDA-NH2 in water. Biotin-C10-ene was finally purified by silica chromatography and fully characterized by H-NMR, C-NMR and LC-MS. Formula weight is <NUM>, measured mass is found to be <NUM>.

Biotin-C17-ene (<FIG>) with terminal vinyl group was accordingly synthesized by addition of <NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone VDMA to the terminal amino group of Biotin-C10-NH2 in water. Biotin-C17-ene was finally purified by silica chromatography and fully characterized by H-NMR, C-NMR and LC-MS. Formula weight is <NUM>, measured mass is found to be <NUM>.

Biotin-C24-ene (<FIG>) with terminal vinyl group could be synthesized following the same strategy: Addition of <NUM>-Vinyl-<NUM>,<NUM>-dimethyl azlactone VDMA to the terminal amino group of Biotin-C17-NH<NUM> in water resulted in the formation of the C7 elongated Biotin-C24-ene affinity ligand almost quantitatively. Biotin-C24-ene was finally purified by silica chromatography and fully characterized by H-NMR, C-NMR and LC-MS. Formula weight is <NUM>, measured mass is found to be <NUM>.

WorkBeads <NUM>/<NUM><NUM> ACT (Bio-Works AB) containing reactive bromohydrin groups were treated with <NUM>,<NUM>-diamino ethane (<NUM>% in water) at room temperature overnight. The beads were washed with water and a triethyl amine buffer (<NUM>). The resulting primary amine functionalized beads were used for coupling of the medium and the long length linkers with biotin.

The aminated substrate (example <NUM>) was mixed with the Biotin-C10-ene or the Biotin-C17-ene synthesized as describes in example <NUM> and <NUM> respectively (<NUM> nmol/ml aminated support) in <NUM> triethylamine (<NUM>/ml aminated support) for <NUM> hours. Reaction stopped by washed with water extensively to avoid any leakage.

The resulting two types of beads with Biotin-C10 or Biotin-C17, may be used for purification of e.g. an antibody by capturing a Dual Affinity Polypeptide (DAP) or Target Binding Polypeptide (TBP) molecules according to previously described antibody purification methods, such as the purification processes disclosed in <CIT> or in <CIT>.

These biotinylated agarose resins with Biotin-C10-NH-, Biotin-C17-NH- and Biotin-C24-NH linker are tested for dynamic binding capacity and binding rate studies with DAP.

To determine the DBC of DAP or TBP to biotin linker coupled to the solid support surface the Langmuir adsorption isotherm is applied. This is based on the assumptions listed below:.

The Langmuir adsorption isotherm is based on the equilibrium between the DAP and the biotin linked to the solid support. Therefore, it is mandatory that DAP is in excess of the maximum binding capacity for each analysis to be able to reach equilibrium
Using the Langmuir isotherm, the binding of DAP to the biotin surface can be described as: <MAT>.

Where q* is the mass of bound DAP expressed as mg DAP bound per ml of biotin resin beads or membrane, c* is the equilibrium concentration of DAP in solution, DBC is the maximum binding capacity and k is the adsorption equilibrium constant. (ref: <NPL>).

The DBC can be determined by plotting C* against C*/q*. The slope is equal to the DBC. Functionalized solid substrates prepared as described in the Examples above were analysed for dynamic binding capacity by incubating a known amount of solid substrate with four different amounts of DAP in solution.

The solid substrate (<NUM>) was placed in an Omnifit column (<NUM>/<NUM>) (Diba Industries Inc. , Danbury, CT, USA) and equilibrated with <NUM> Citrate pH <NUM>, <NUM>% tween <NUM>. An Äkta Pure (GE Healthcare, Chicago, IL, USA) was used to load <NUM> of DAP solution at concentrations of <NUM>, <NUM>, <NUM> and <NUM>/mL respectively. to the Omnifit column containing the functionalized solid substrate. The pump flow speed was set at <NUM> per minute, which corresponds to a residence time I the column of <NUM> seconds. After washing out unbound DAP, and collecting the flow through fraction, it was analysed for DAP content using a Nanodrop spectrometer (ThermoFisher, Waltham, MA, USA). Extinction coefficient for DAP: εDAP = <NUM><NUM>/mg and MW=<NUM>,<NUM> kDa.

The equilibrium concentration in bulk liquid (C*, mg/mL) was calculated by determining the total DAP content in solution after incubation with the solid substrate. The equilibrium concentration (q*, mg/mL) on the solid substrate was calculated by subtracting the DAP content in flow through solution from the starting DAP content in solution. The data collected from the experiments were used by plotting C* (y) as a linear function of C*/q* (x) as shown in <FIG>. The slope of the linear trendline gives the maximum binding capacity for the amount of solid substrate used in the binding experiment. In this analysis <NUM> of solid substrate was tested, to the dynamic binding capacity (DBC) in mg/mL is obtained by multiplying the slope by four.

In Table <NUM> are the DBC results summarised from analysing the Biotin-C10, Biotin-C17 and Biotin-C24 functionalized solid substrates. From the table it is clear that the Biotin-C17 functionalized solid substrate leads to the highest DBC.

Functionalized solid substrates prepared as described in the Examples above were analysed further to understand if residence time will affect the DBC. For each pump speed was the DBC determined as above in example <NUM>. Pump flow speed was varied to achieve different residence times of the DAP in contact with the solid substrate, i.e., <NUM>, <NUM> and <NUM> per minute, which corresponds to residence times of <NUM>, <NUM> and <NUM> seconds, respectively. The dynamic binding capacity was determined as described in EXAMPLE 9above for the Biotin-C10, Biotin-C17 and Biotin-C24 functionalized solid substrates and results are summarized in Table <NUM>.

The Biotin-C17 functionalized solid substrate provide DBC maximum compared to the other Biotin Linkers. It is also observed that the DBC for Biotin-C17 is nearly constant (less than <NUM>% reduction), which means the binding rate is much faster for Biotin-C17 compared to the other linker tested. Biotin-C10 and Biotin-C24 have DBC reduction of <NUM>% and <NUM>% respectively over the measured range of Residence times.

Table <NUM>. Summary af measured dynamic binding capacity ("DBC", mg/mL) of the Biotin-C10, Biotin-C17 and Biotin-C24 functionalized solid substrate at flow speeds of <NUM>, <NUM> and <NUM>/minute, corresponding to a residence time of <NUM>, <NUM> and <NUM> seconds, respectively. The Biotin-C17 functionalized solid substrate leads to a higher DBC at all residence times measured and is affected only to a minor extent by the decrease in residence time.

Radical initiated co-grafting of three different lengths linkers: Biotin-C10-ene, Biotin-C17-ene or Biotin-C24-ene, respectively, with N,N-dimethyl acryl amide to polysulfone ether membrane sheets (<NUM>).

The membrane was cut into round pieces (<NUM> in diameter) and washed with water, followed by dioxane. The membrane was soaked in <NUM> % N,N-dimethyl acryl amide (Fluka) in dry and oxygen free dioxane and <NUM>% of the Biotin-C10-ene, Biotin-C17-ene and Biotin-C24-ene, respectively, and with benzoequinone (Fluka, <NUM> ppm) as radical initiator. The membranes were irradiated with an UV lamp for <NUM> minutes under neutral atmosphere (dry nitrogen). The membranes were washed with dioxane.

Washed out homopolymer was collected and used to approximate the length of the grafted polymer by SEC. The homopolymer distribution was broad at approx. <NUM> to <NUM> Da in molecular weight, as compared to a MW standard.

The resulting three different membranes with drafted poly acrylamide and Biotin-C10, Biotin-C17 and Biotin-C24 were washed extensively with hot water (<NUM>) to remove any homopolymer and avoid later leakage.

The resulting membranes were physical intact, though slightly yellowish, and had graft copolymer consisting of a polyacrylamide backbone and the different lengths semi-stiff branches. The branches (Comb) different from the backbone.

The different membranes with polyacrylamide grafts with different lengths linkers from branch points (Comb) were tested in a set-up similar to example <NUM>. The membranes was cut into small pieces (approx. 2x2 mm) and packed in the Omnifit column.

Claim 1:
A use of a ligand-functionalized substrate for purification of a target biomolecule,
wherein the ligand-functionalized substrate comprises:
a. A substrate having carboxylic (-COOH), hydroxy (-OH), thio (-SH), amino groups (-NH2), C-C double bonds (-ene) or C-C triple bonds (-yne) at the surface thereof;
b. Covalently bound to said carboxylic (-COOH), hydroxy (-OH), thio (-SH), amino groups (-NH2), C-C double bonds (-ene) or C-C triple bonds (-yne) of said substrate a linker having a linker length of <NUM>-<NUM>, such as <NUM>-<NUM> bonds, wherein bonds are C-C, C-N, C-N(H), C-C(O) and/or C-O; and
c. A ligand-functional group bound to the surface of the substrate via said linker;
the purification comprising the steps:
(a) contacting (i) a target biomolecule, (ii) a dual affinity polypeptide, and (iii) the ligand-functionalized substrate, and
(b) recovering the target biomolecule by elution, wherein the target biomolecule and the dual affinity polypeptide are contacted in solution before the mixture is contacting the ligand-functionalized substrate, and optionally
(c) washing away impurities and/or removing the dual affinity polypeptide,
wherein said ligand-functional group and said linker are selected from the group consisting of:
<CHM>
<CHM>
<CHM>