GB2636805A - Cation exchange paper - Google Patents

Abstract

method of phosphorylating cellulose filter paper in an aqueous solution comprising phosphate and urea comprising: a. determining the number of anyhydroglucose units in the cellulose (AGU) of the filter paper by weighing the filter paper and calculating the AGU using the formula: AGU in cellulose (mol) = {weight of cellulose (g)}× 6.2×10-3(mol/g) b. incubating the filter paper in an aqueous solution comprising a phosphate source and urea; c. swelling the cellulose of the filter paper; Optionally the paper may be dried, cured, washed and dried further. The phosphate source may be a salt, optionally ammonium phosphate monobasic or dibasic. Incubation may be in the aqueous solution for 10-60 minutes and a temperature of 15-105 °C, swelling of the paper may be at a temperature of 30-200 °C. The paper is suitable for use as cation exchange filter paper for radiometric protein kinase assays and included is a claim for the paper obtains by the method.

Description

CATION EXCHANGE PAPER

Field

This disclosure relates to a method of preparing cation exchange paper, in particular cation exchange paper for radiolabelled ATP kinase assays.

Background

The human kinome encodes approximately 520 protein kinases and approximately 20 lipid kinases, which play a central role in health and diseases such as cancer. Protein kinases constitute the largest proportion of targets in drug development programs in the biotechnology and pharmaceutical industry. To date, 121 protein kinase inhibitors are currently approved for clinical use. For example, Imatinib/Gleevec was a game changer for patients with chronic myelogenous leukemia and heralded the age of targeted therapies and precision oncology.

There is an ongoing requirement for reliable protein kinase assays for the identification and profiling of protein kinase inhibitors.

Radiometric assays are considered the "gold standard" for protein kinase assays because of their direct readout, high sensitivity, reproducibility, reliability, and very low background signals.' There are numerous companies around the world that deliver kinase assays as a commercial service.

Non-radiometric high throughput screening assays include luciferase-based and fluorescence-based assays. These assays are indirect and typically measure a physical or chemical phenomenon that is a downstream effect of phosphorylation. Therefore, non-radiometric assays are affected by factors that interfere with these physical or chemical effects, such as background fluorescence and interference with coupling enzymes, and are consequently inferior to radiometric assays discussed above.

Radiometric ATP kinase assays rely on Whatman® P81 grade phosphocellulose cation exchange filter paper (P81 paper) for capturing peptide and protein analytes on the phosphorylated substrate of the paper, after which unreacted material is washed off prior to analysis via scintillation counting. The global production of Whatman® P81 phosphocellulose cation exchange filter paper has been discontinued, and stockpiled supplies are running out.

Although alternatives have been reported, such as Macherey-Nagel® LSA-50 paper, they do not match the performance of P81 paper. LSA-50 filter paper features a strong acidic cation exchange resin. The matrix is polystyrene cross-linked with 8.5% divinyl benzene and the active groups are SO3H. A common issue experienced in practice with alternatives to P81 paper is that they do not bind both peptide and protein substrates under varied concentrations of radiolabelled ATP.

The gold standard performance of Whatman® P81 paper has resulted in several small-scale producers of phosphocellulose paper, but the supply does not currently meet the demand and prices of these alternative phosphocellulose papers are high.

Methods to phosphorylate cellulose were initially developed to flameproof cloth.9,10,11 The phosphate ester favours dehydration of cellulose and therefore the formation of char, which is responsible for flame retardance (Scheme 1).11 Ot_. _HO H OH OH OH 4.( OH H2O (ct HO H OH 0 HO OH 0 HO

O

OH OH 0

+ H3PO4 -H2O 0.0 OH OH

OH

Scheme 1: Proposed mechanism for thermally stable char formation of phosphorylated cellulose.11 These initial reports used phosphorous oxychloride and pyridine or phosphoric acid and urea as reagents,"° however the use of phosphorylating agents including POCI3, H3PO4, P205, (NH4)2HPO4, NH4H2PO4, and organophosphates, and amines including N, N-dimethylformamide, pyridine, and urea have been reported to date.11*12 However, the use of these protocols is potentially risky as they involve dangerous reagents and therefore can only be performed by trained personnel under strict safety conditions.

Recent advances in the functionalisation of nanocellulose have renewed interest in the aqueous preparation of phosphocellulose using ammonium phosphates and urea since this approach is safer and more environmentally friendly.6'7 However, these current approaches are suitable for the phosphorylation of bulk cellulose. There remains a need for simple, safe and economical methods for producing phosphocellulose cation exchange filter paper.

The present disclosure provides methods that address at least some of these problems. 25 Summary Described herein is a safe, cost effective, aqueous method of preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays. The paper produced by this method is suitable for use in radiometric protein kinase assays with comparable results to those achieved with the Whatman® P81 paper. Advantageously, the method employs user-friendly, safe reagents and can easily be performed with minimal skill and basic equipment.

In a first aspect there is provided a method of phosphorylating a cellulose filter paper in an aqueous solution comprising phosphate and urea for preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays.

Phosphorylation of cellulose takes place by reacting the anhydroglucose units (AGU) of cellulose with a phosphate source in the presence of a base (Scheme 2).

Scheme 2: Phosphorylation of cellulose. The method may comprise the steps of: a) Determining the number of anyhydroglucose units in the cellulose (AGU) of a filter paper; b) Incubating the filter paper in an aqueous solution comprising a phosphate source and a base; c) Swelling the cellulose of the filter paper; d) Optionally drying the filter paper; e) Curing the filter paper; f) Washing the filter paper; and g) Drying the filter paper.

The cellulose filter paper may be any filter paper or cellulose based material, such as wood composite. The cellulose filter paper may be any suitable grade, such as grade 1, grade 2, grade 3, grade 4, Grade 5, Grade 50 Hardened Ashless, Grade 541 Hardened Ashless, 1 Chr, 3mm Chr, filter paper cellulose filter paper. Most preferably, the cellulose filter paper may be grade 1 filter paper such as Whatman® 1Chr filter paper, Appleton® grade 1 filter paper, Supertek® grade 1 filter paper, smith filters® grade 1 filter paper, and the like.

The cellulose filter paper may have any shape and dimensions. For example, the paper may be compatible with 96-well plates. The cellulose filter paper may be round. The cellulose filter paper may have a diameter from about 5 mm to about 7 mm.

The base may be any suitable base such as an amine, a hydroxide salt, a carbonate salt, etc. Preferably, the base may not be pyridine. Preferably, the base may be urea. Without wishing to be bound by theory, using urea may be advantageous because due to its melting point, urea is liquid at the reaction temperatures and acts as a solvent for the phosphate source.

OH

HO

HO OH HO

OH

OH

Anydrounglucose unit (AGU)

OH

HO OH

HO -OH0

OH

OH

OH

OH HO',

OH

OH

o-&52--\

O

HO H

OH

The phosphate may be provided by any suitable water soluble phosphate source, such as a phosphate salt or phosphoric acid. The phosphate salt may be mono basic, dibasic or tribasic. The phosphate may be H3PO4, [H2P041, [HPO4]2-, [PO4]3-, an organophosphate (e.g. fertiliser grade organophosphate). Preferably, the phosphate may be selected from [H2PO4]-or [HPO4]2-. The phosphate source may be selected from calcium a phosphate salt, a potassium phosphate salt, a sodium phosphate salt, an aluminium phosphate salt, an ammonium phosphate salt. The phosphate may be provided by an ammonium phosphate salt, such as ammonium phosphate monobasic (NH4H2PO4), or ammonium phosphate dibasic ((NH4)2HPO4). Without wishing to be bound by theory, the inventor has discovered that employing ammonium phosphate salts leads to faster phosphorylation rates than other phosphate sources.

The aqueous solution may further comprise a buffer. The buffer may be an acidic buffer or a basic buffer. In some cases, the buffer may be selected from phosphate buffered saline (PBS) based buffer or tris-buffered saline (TBS).

The amount of reagents required for the method will depend on the number of anyhydroglucose units (AGU) of the cellulose and the degree of phosphorylation of cellulose required. Therefore, the method may comprise an initial step (i.e. prior to the phosphorylation step) of determining the number of anhydroglucose units (AGU) in the cellulose.

Method for determining the AGU of cellulose The concentration of anyhydroglucose units in cellulose is based on 162 g mol-1 for AGU, or 6.2 mmol AGU per gram of cellulose.

For determining the AGU of the cellulose (e.g. cellulose filter paper), the cellulose filter paper may be weighed and the AGU may be calculated using the formula: AGU in cellulose (mol) = [weight of cellulose (g)]-6.2 10-3(mo/ g-1) The AGU: phosphate source: base molar ratio and the wt% cellulose may be varied to alter the degree of phosphorylation. The ratio of AGU: phosphate: urea may be 1:0.6:3.2. The ratio of AGU: phosphate: urea may be 1:2.5:10. The weight % of cellulose may be calculated by taking into account the weight of the filter paper and the weight of water in the solution. For example, the wt% of a filter paper weighing 1 g would be 1% when the filter paper is placed in a solution containing 100 ml (100g) of water.

The molar ratio of AGU in the cellulose filter paper to phosphate source (AGU: phosphate source molar ratio) may be selected from about 1:0.3 to about 1:5, or from about 1:0.3 to about 1:4, from about 1:0.3 to about 1:3, from about 1:0.3 to about 1:2.5, from about 1:0.3 to about 1:2, or from about 1:0.3 to about 1:1, or from about 1:0.5 to about 1:3, from about 1:0.6 to about 1:2.5, or from about 1:0.5 to about 1:1.5.

The molar ratio of AGU in the cellulose filter paper to base (AGU: urea molar ratio) may be selected from about 1:2.5 to about 1:12, or from about 1:2. to about 1:11, from about 1:2.5 to about 1:10, from about 1:3 to about 1:12, from about 1:4 to about 1:12, or from about 1:5 to about 1:12, or from about 1:5 to about 1:10, from about 1:3.2 to about 1:10, or from about 1:3 to about 1:10.5.

In some embodiments, the phosphate source may be ammonium phosphate monobasic, the base may be urea, and the ratio of AGU: ammonium phosphate monobasic: urea may be 1:0.6:3.2. The pH in those embodiments may be at least 2.5, or at least 3, for example 4.

In some embodiments, the phosphate source may be ammonium phosphate dibasic, the base may be urea, and the ratio of AGU: ammonium phosphate dibasic: urea may be 1:2.5:10. The pH in those embodiments may be at least 7, or at least 8, for example 8.6.

The method may comprise incubating the cellulose filter paper in the aqueous solution.

Incubating cellulose in an aqueous solution may comprise maintaining the cellulose inside the aqueous solution for a period of time sufficient to allow the cellulose to be impregnated with the reagents. The cellulose filter paper may be maintained in the aqueous solution from about 10 minutes to about 60 minutes, or from about 10 minutes to about 45 minutes, or from about 10 minutes to about 30 minutes, or from about 15 minutes to about 30 minutes, or about 20 minutes to about 35 minutes, or about 20 minutes to about 30 minutes, or about 25 minutes to about 35 minutes. The cellulose may be maintained in the aqueous solution for about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes. Preferably, the cellulose may be maintained in the aqueous solution for about 25 minutes.

Incubating the cellulose filter paper in an aqueous solution may comprise immersing filter paper in the aqueous solution, covering the system, and maintaining the closed system for a period of time as discussed above. The incubation step may be performed while the system is stationary or while it is agitated (e.g. in a rocker, shaker, via stirring (e.g. with a glass rod or magnetic stirrer) or the like). Incubating cellulose in an aqueous solution may be performed at any desired temperature, e.g. from about 15 °C to about 105 °C, or from about 15 °C to about °C, or from about 15 °C to about 60 °C, or from about 15 °C to about 40 °C, or from about 15 °C to about 30 °C, or from about 15 °C to about 25 °C, or from about 18 °C to about 25 °C, or from about 18 °C to about 30 °C, or from about 30 °C to about 60 °C, or from about 60 °C to about 80 °C, or from about 80 °C to about 105 °C. Incubating cellulose in an aqueous solution may be preferably performed at room temperature (i.e. from about 18 °C to about °C). In some embodiments, the incubation step may be carried out at about 80 °C for about 25 minutes.

The method may further comprise the step of swelling the cellulose of the filter paper. Swelling the cellulose of the filter paper in the aqueous solution may be performed at any suitable temperature. For example, it may be performed at room temperature (i.e. from about 18 °C to about 25 °C), or it may be performed at a raised temperature, for example from about 30 °C to about 200 °C, or from about 70 °C to about 180 °C, or from about 80 °C to about 160 °C, or from about 70 °C to about 100 °C, or from about 100 °C to about 160 °C, or from about 120 °C to about 150 °C, or from about 30 °C to about 90 °C, or from about from about 30 °C to about 90 °C. Swelling cellulose in an aqueous solution may be performed at about 70 °C, or at about 80 °C, or at about 100 °C, or at about 120 °C, or at about 150 °C. In some embodiments, swelling cellulose in an aqueous solution may be performed from about 80 °C to about 150 °C, for example at 80 °C, or at 150 °C.

Urea is liquid between 140-160 °C, at which temperature it swells cellulose and acts as a solvent for phosphoric acid all whilst buffering cellulose from phosphoric acid to reduce degradation." Where phosphoric acid is used with urea, the reaction mixture typically needs to be refluxed for several hours. If ammonium phosphates are used as the phosphorylating agent instead, the sample only needs to be cured at high temperatures for less than an hour, which makes this approach practical for functionalising sheets of filter paper whilst maintaining the characteristics of the filter paper.13 The swelling and incubation steps may be combined into a single step.

The method may further comprise the step of drying the filter paper (after the incubation step -i. e. drying the incubated cellulose paper). Prior to drying, the filter paper may be removed from the aqueous solution. Advantageously, removing the filter paper from the aqueous solution may not require filtering the filter paper. For example, the filter paper may be lifted out from the aqueous solution by any suitable means such as with tweezers, spatula, by hand using gloves, or the like. The aqueous solution may be poured out of the container. The aqueous solution may be allowed to evaporate (e.g. at room temperature or at a raised temperature). The aqueous solution may be decanted out of the system comprising the aqueous solution and filter paper.

In some embodiments, the drying step is not required. For example, in embodiments in which the wt% of cellulose is high (e.g.: about 10-20 wt.%), the filter paper may undergo the curing step directly after incubation step. However, in embodiments in which the wt% of cellulose is low (e.g. from about 0.1 wt% to about 1.5 wt%), the filter paper may undergo a drying step between the swelling and curing steps. Without wishing to be bound by theory, drying the filter paper between the swelling and curing steps may increase the degree of phosphorylation achieved by the method. Suitability of the paper to perform in kinase assays may be determined by reviewing the charge density of the phosphorylated filter paper and comparing it to a control (e.g. VVhatman® P81 paper).

Drying may be performed at room temperature (i.e. from about 18 °C to about 25 °C) or at a raised temperature (e.g. from about 50 °C to about 120 °C, for example at about 100 °C). The drying step may be performed in an oven. The drying step may be performed under vacuum. The drying step may be performed in ambient conditions (e.g. on a plate, airer, dish or the like). Drying may be assisted (e.g. to speed up the drying process) by an air current such as compressed air, or by placing the filter paper on a funnel and applying vacuum. Drying may be assessed by weighing the filter paper at time intervals until the weight of the paper has stabilised (i.e. until the weight of the paper does not change over time).

The drying step may be performed for as long as it is required until the filter paper is dry (i.e. until the weight of the filter paper is stable).

The method may further comprise the step of curing the filter paper. Without wishing to be bound by theory, phosphorylation may take place during the curing step. Curing the paper may be performed at a raised temperature (e.g. in an oven). The curing the paper may be performed from about 130 °C to about 200 °C, or from about 130 °C to about 160 °C, or from about 130 °C to about 160 °C, or from about 130 °C to about 150 °C, or at about or at about 130 °C, or at about 140 °C, or at about 150 °C, or at about 160 °C.

The curing step may be performed for any length of time required for phosphorylation to take place. The curing step may be performed for about 10 minutes to about 45 minutes, or for about 15 minutes to about 40 minutes, or for about 20 minutes to about 35 minutes, or for about 20 minutes to about 30 minutes. The curing step may be performed for about 10 minutes to about 180 minutes, or from about 20 minutes to about 60 minutes, or about 10 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes, or from about 15 minutes to about 45 minutes, or for about 30 minutes to about 60 minutes, or from about 45 minutes to about 180 minutes, or for about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes.

The curing step may be performed at about 150 °C for about 20-30 minutes.

In some embodiments, the phosphate source may be ammonium phosphate monobasic and the ratio of AGU: ammonium phosphate monobasic: urea may be 1:0.6:3.2. The pH in those embodiments may be at least 2.5, or at least 3, for example 4. The curing time required for these embodiments may vary, but in some examples the curing step may be maintained for about 20 minutes.

In some embodiments, the phosphate source may be ammonium phosphate dibasic and the ratio of AGU: ammonium phosphate dibasic: urea may be 1:2.5:10. The pH in those embodiments may be at least 7, or at least 8, for example 8.6. The curing time required for these embodiments may vary, but in some examples the curing step may be maintained for about 30 minutes.

Without wishing to be bound by theory, increasing the temperature at which any of the steps is carried out may decrease the time required for completing said step(s).

The method may comprise the step of washing the (functionalised) filter paper with any suitable solvent. Deionised water may be preferred. Washing the filter paper may remove unreacted reagents from the filter paper, for example to prevent contamination or cross-reaction with excess reagents during assays. The washing step may be performed at any required temperature, but room temperature may be preferred (e.g. about 18 °C to about 22 °C). The washing step may be performed by rinsing the filter paper with the solvent or by immersing the filter paper in the solvent and draining the solvent.

The method may comprise drying the (functionalised) filter paper. The drying step may be performed at room temperature or at a raised temperature. For example the drying step may be performed (e.g. in an oven) at a temperature between about 30 °C and about 105 °C, for example at about 100 °C. Additionally or alternatively, the drying step may be assisted, for example by washing with an organic solvent (e.g. acetone). The drying step may be performed under vacuum. The drying step may be performed in ambient conditions (e.g. on a plate, airer, dish or the like). Drying may be assisted (e.g. to speed up the drying process) by an air current such as compressed air, or by placing the filter paper on a funnel and applying vacuum.

Advantageously, the method may not require filtration of the cellulose filter paper as opposed to methods of phosphorylation of cellulose pulp. The present method allows simpler and easier handling of the starting material (filter paper) compared to loose cellulose pulp or fibres. The reagents and reaction conditions employed in the present method are safer than other methods of cellulose phosphorylation in the art. Furthermore, the method does not require a complex and cumbersome step of forming paper sheets as it employs off the shelf filter paper, therefore simplifying the procedure, for example enabling any lab to perform it in house. Therefore, the present method provides a fast and convenient method for phosphorylating cellulose filter paper which can be subsequently used in radiometric protein kinase assays.

The degree of substitution and grafting mode (i.e.: mono, disubstituted, or crosslinked phosphoester) depends on variables including the: 1) molar ratio between the phosphorylation agent and the cellulose monomer, 2) reaction time, and 3) reaction temperature. The degree of phosphorylation not only influences the charge density but also the tensile strength and appearance of the filter paper product. The inventor has found that the proposed reaction conditions provide suitable phosphorylation levels with minimal reaction times and temperatures, therefore providing a simple, safe cost-effective method for preparing functionalised phosphocellulose cation exchange filter paper which can be used for radiolabelled ATP kinase assays.

In a second aspect there is provided a phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays. The phosphocellulose cation exchange filter paper may be achieved directly by the method of phosphorylating a cellulose filter paper described above. The phosphocellulose cation exchange filter paper may have comparable performance to Whatman® P81 filter paper in radiometric protein kinase assays.

Brief Description of the Figures

Figure 1: Shows an image of functionalised filter paper and control filter paper incubated in aqueous CuSO4 and washed with boiling water (Left: functionalised and washed with NaCI; Middle: functionalised; Right: untreated control).

Figure 2 is a graph displaying the quantitation of radiation on cation exchange papers via scintillation for the kinase assay of EP975 using PKA (count time = 1 minute).

Figure 3 shows a graph displaying the quantitation of radiation on cation exchange papers via scintillation for the kinase assay of: a) EP4562 using Aurora A, b) POLY using BRK, and c) c-kit peptide using c-kit (count time = 1 minute).

Figure 4 shows the quantitation of radiation on cation exchange papers via scintillation for a kinase assay and IC50 curve of Sapk2b with inhibitor SB203580 using 20 pM 33P ATP (count time = 1 minute). Results for VVhatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

Figure 5 shows quantitation of radiation on cation exchange papers via scintillation for the kinase assay of PKBa using 5 pM 33P ATP (count time = 1 minute). Results for VVhatman® P81 paper and a functionalised paper obtained directly from a method according to an

embodiment of the disclosure are compared.

Figure 6 shows quantitation of radiation on cation exchange papers via scintillation for the kinase assay of Sapk2b using 20 pM 33P ATP (count time = 1 minute). Results for VVhatman® P81 paper and a functionalised paper obtained directly from a method according to an embodiment of the disclosure are compared.

Figure 7 shows quantitation of radiation on cation exchange papers via scintillation for the kinase assay of SRPK1 using 50 pM 33P ATP (count time = 1 minute). Results for Whatman® P81 paper and a functionalised paper obtained directly from a method according to an

embodiment of the disclosure are compared.

Detailed Description

Disclosed herein is a safe, cost effective, aqueous method to functionalise cellulose filter paper to prepare phosphocellulose cation exchange filter paper for radiolabelled ATP kinase assays.

The method may comprise the steps of: a) Determining the number of anyhydroglucose units in the cellulose (AGU) of a filter paper; b) Incubating the filter paper in an aqueous solution comprising a phosphate source and a base (e.g. urea); c) Swelling the cellulose of the filter paper; d) Optionally drying the filter paper; e) Curing the filter paper; f) Washing the filter paper; and g) Drying the filter paper.

The conditions and reagents of the method are discussed in the summary section of this application.

Examples

The following method protocol was followed to prepare two phosphocellulose cation exchange filter papers.

Protocol 1. Weigh Whatman® 1Chr paper (or another grade of cellulose filter paper) and calculate the number of anhydroglucose units (AGU).

2. Incubate the filter paper in a glass dish containing an aqueous solution of ammonium phosphate monobasic or dibasic and urea on a rocker at room temperature. The AGU: phosphate source: urea molar ratio and wt% cellulose (i.e.: the amount of water) can be varied within the ranges discussed above to alter the degree of phosphorylation (incubation step).

3. Heat the covered mixture in an oven at 80 °C for 25 minutes to swell the paper (swelling step).

4. Remove the solution from the dish or remove the filter paper from the solution and place it in a fresh glass dish. Alternatively, where the wt% cellulose is high (i.e.: 10-20 wt.% compared to 1 or 0.1 wt.%, for example), the paper can be dried from solution to increase the degree of phosphorylation.

5. Dry the paper in an oven at 100 °C until the weight has stabilised (i.e.: the paper is dry) (drying step).

6. Cure the paper in an oven at 150 °C for 20-30 minutes. Note: This is the step during which cellulose is functionalised, and varying the cure time and temperature impacts the charge density (curing step).

7. Wash the paper extensively in deionised water at room temperature (washing step).

8. Dry the paper at ambient temperature. Alternatively, drying can be accelerated by using an oven, vacuum or organic solvent such as acetone (drying step).

Example 1

Reagent ratios: AGU: ammonium phosphate monobasic: urea = 1:0.6:3.2 (the unadjusted pH is acidic, pH = 4).

Curing conditions in step 6: cured at 150 °C for 30 minutes. Example 2 Reagent ratios: AGU ammonium phosphate dibasic: urea = 1:2.5:10 (the unadjusted pH is basic, pH = 8.6).

Curing conditions in step 6: cured at 150 °C for 20 minutes. Characterisation of examples and comparison with VVhatman® P81 The cation binding properties of the functionalised filter paper was first confirmed by incubating the paper in aqueous CuSO4 and washing it with boiling water to yield green, copper-bound paper (Figure 1).

Thereafter, the functionalised paper was tested in a radiolabelled 32P ATP kinase assay against VVhatman® P81 cation exchange filter paper in triplicate (Scheme 3). Comparable results were obtained (Figure 2).

Samples obtained by the methods of Example 1 (using ammonium phosphate monobasic) and Example 2 (using ammonium phosphate dibasic) (namely Inventive paper 1 and Inventive paper 2, respectively) were tested using a range of different protein and peptide substrates for suitability in radiolabelled ATP kinase assays.

[y-32P]ATP (500 xM), MgCl2 (10 mM), PKA (1 ocg) EP975 (biotinylated peptide -residues around LKB1 Ser431) (5 ocg) ^ pH 7.5, 30 PC, 30 mins.

TRIS (50 mM) EGTA (1 mM) p-Glycerophosphate (20 mM) NaF (20 mM) DTT (2 xM) PhosSTOP Scheme 3: Radiolabelled 32P ATP kinase assay of EP975 using PKA.

In one assay, both paper samples were compared against Whatman® P81 paper in a 33P ATP kinase assay using 3 very different substrates, both of which performed similarly to Whatman® P81 paper for all substrates tested in duplicate (Figure 3).

The paper samples were also compared against Whatman® P81 paper in the following 33P ATP kinase assays, and comparable results were obtained in each assay: 1. IC50 curve, 7 datapoints, single shot (Figure 4) 2. Low [33P ATP], peptide substrate, performed in triplicate (Figure 5) 3. Medium [33P ATP], protein substrate, performed in triplicate (Figure 6) 4. High [33P ATP], peptide substrate, performed in triplicate (Figure 7) These experiments show that the same trends in data are observed for the papers obtained with the novel method and for Whatman® P81 paper.

Conclusion

The inventor has herein proven that methods according to the present disclosure yield phosphocellulose cation exchange filter paper which can be used in radiometric protein kinase assays with comparable results to Whatman® P81 paper. Therefore, the methods described herein address the urgent need to provide stocks of phosphocellulose cation exchange filter paper that can replace the discontinued Whatman® P81 product in order to enable the diagnostic and research communities to continue using the gold-standard radiolabelled ATP kinase assays employed to date.

References: 1. Hastie, C. J.; McLauchlan, H. J.; Cohen, P. Assay of protein kinases using radiolabeled ATP: a protocol. Nat. Protoc., 2006, 1(2), 968-971. DOI: ()Fr:// 2. Applemans, 0.; Kashyap, R. S.; Gilles, P.; De Borggraeve, W. M.; Voet, A.; Van Lint, J. LSA-50 paper: an alternative to p81 phosphocellulose paper for radiometric protein kinase assays. Anal. Biochem., 2021, 630, 114313. DOI: ht 3. Macherey-Nagel. htt (Accessed 10 January 2023) 'a 50-aaao le-srhoo 4. St Vincent's Institute of Medical Research.

Str1hn-r1b' paparl (Accessed 10 January 2023).

5. Lab Alley. htt.s Gist:A/ha man-q hariae-aendose -togastv:paogr. (Accessed 10 January 2023).

6. Ghanadpour, M.; Carosio, F.; Larsson, P. T.; Wagberg, L. Phosphorylated cellulose nanofibrils: a renewable nanomaterial for the preparation of intrinsically flame-retardant materials. Biomacromolecules, 2015, 16, 3399-3410. DOI: hz4.

7. Chen, S.; Ren, N.; Cui, M.; Huang, R.; Qi, W.; He, Z.; Su, R. Heat soaking pretreatment for greener production of phosphorylated cellulose nanofibrils with higher charge density. ACS Sustain. Chem. Eng., 2022, 10, 8876-8884. DOI: ieou2;..0)4 8. Rol, F.; Sillard, C.; Bardet, M.; Yarava, J. R.; Emsley, L.; Gablin, C.; Leonard, D.; Belgacem, N.; Bras, J. Cellulose phosphorylation comparison and analysis of phosphorate position on cellulose fibers. Carbohydr. Polym., 2020, 229, 115294. DOI: or-0 0, 1-15294 9. Reid, J. D.; Mazzeno, L. W.; Buras, E. M. Composition of two types of cellulose phosphates. Ind. Eng. Chem., 1949, 41(12), 2831-2834. DOI: s 1141n - 10. Reid, J. D.; Mazzeno, L. W. Preparation and properties of cellulose phosphates. Ind. Eng. Chem., 1949, 41(12), 2828-2831. DOI: hitussildni.or211 0.10211:350480s039 11. Thomas, B.; Raj, M. C.; Athira, K. B.; Rubiyah, M. H.; Jithin, J.; Moores, A.; Drisko, G. L.; Sanchez, C. Nanocellulose, a versatile green platform: from biosources to materials and their applications. Chem. Rev., 2018, 118(24), 11575-11625. DOI: 12. Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagenknecht, W.; Eds. Comprehensive cellulose chemistry: fundamentals and analytical methods, Vol. 1. Wiley-VCH, 1998.

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Claims (1)

1. Claims 1-A method of phosphorylating cellulose filter paper in an aqueous solution comprising phosphate and urea for preparing phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays, the method comprising the steps of: a. determining the number of anyhydroglucose units in the cellulose (AGU) of the filter paper by weighing the filter paper and calculating the AGU using the formula: AGU in cellulose (moo = [weight of cellulose (g)] * 6.2 * 10-3(moi * g-') b. incubating the filter paper in an aqueous solution comprising a phosphate source and a base; c. swelling the cellulose of the filter paper; d. optionally drying the filter paper; e. curing the filter paper; f. washing the filter paper; and g. drying the filter paper.

   The method of claim 1 wherein the base is selected from an amine, a hydroxide salt, and a carbonate salt, optionally wherein the base is an amine.

   The method of claim 1 or 2, wherein the base is urea.

   The method of any preceding claim, wherein the phosphate source is a phosphate salt, optionally wherein the phosphate salt is selected from ammonium phosphate monobasic (NH4H2PO4), or ammonium phosphate dibasic ((\1H4)2HPO4).

   The method of any preceding claim, wherein the molar ratio of AGU in the cellulose filter paper to phosphate source (AGU: phosphate source molar ratio) is selected from about 1:0.3 to about 1:5.

   The method of claim 5, wherein the molar ratio of AGU in the cellulose filter paper to phosphate source (AGU: phosphate source molar ratio) is selected from about 1:0.6 to about 1:2.5.

   7-The method of any preceding claim wherein the molar ratio of AGU in the cellulose filter paper to urea (AGU: urea molar ratio) is selected from about 1:2.5 to about 1:12.

   8-The method of claim 7 wherein the molar ratio of AGU in the cellulose filter paper to base (AGU: base molar ratio) is selected from about 1:3.2 to about 1:10.

   9-The method of claim 1 or 2, wherein the phosphate source is ammonium phosphate monobasic and the ratio of AGU: ammonium phosphate monobasic: urea is 1:0.6:3.2, optionally wherein the pH in those is at least 2.5, or at least 3, for example 4.

   10-The method of claims 1 to 4, wherein the phosphate source is ammonium phosphate dibasic and the ratio of AGU: ammonium phosphate dibasic: urea is 1:2.5:10, optionally wherein the pH in those embodiments may be at least 7, or at least 8, for example 8.6.

   11-The method of any one of claims 1 to 8 wherein the phosphate source is ammonium phosphate monobasic and the ratio of AGU: ammonium phosphate monobasic: urea is 1:0.6:3.2, optionally wherein at least one of: the pH is at least 3 and/or wherein the curing time is about 20 minutes.

   12-The method of any one of claims 1 to 8 wherein the phosphate source is ammonium phosphate dibasic and the ratio of AGU: ammonium phosphate dibasic: urea is 1:2.5:10, optionally wherein at least one of: the pH is at least 7 and/or wherein the curing time is about 30 minutes.

   13-The method of any preceding claim, wherein the cellulose filter paper is incubated in the aqueous solution from about 10 minutes to about 60 minutes.

   14-The method of any preceding claim, wherein the cellulose filter paper is incubated at a temperature from about 15 °C to about 105 °C.

   15-The method of any preceding claim, wherein swelling the cellulose filter paper is performed at a temperature from about 30 °C to about 200 °C.

   16-The method of any preceding claim, wherein the drying steps d and / or g are performed by at least one of: evaporation of solvent at room temperature, evaporation of solvent at a raised temperature (e.g. in an oven at about 100 °C), under vacuum, washing with organic solvent and evaporating the solvent at room temperature.

   17-The method of any preceding claim, wherein curing the paper is performed from about 130 °C to about 200 °C, optionally at about 150 °C.

   18-The method of any preceding claim, wherein the curing step is performed for about minutes to about 45 minutes, optionally for about 20-30 minutes.

   19-The method of any preceding claim, wherein the cellulose filter paper is compatible with 96-well plates.

   20-A phosphocellulose cation exchange filter paper suitable for use in radiometric protein kinase assays obtained directly by the method of any preceding claim.