METHOD AND COMPOSITION FOR BIOMOLECULE STABILIZATION

Abstract

The present invention relates to biomolecule stabilization to provide biomolecules, such as sensitive polymerases, in a convenient ready-to-go format. The invention provides a method and composition in which non-ionic surfactant or detergents of the polyoxyethylene cetyl ether family are used, preferably a Brij reagent or a combination of Brij reagents.

Claims

1. A method for stabilizing biomolecules, comprising: treating a biomolecule with a lyophilisation mixture comprising non-ionic surfactant(s) of the polyoxyethylene cetyl ether family; dispensing said mixture into a receptacle; and freeze-drying said mixture.

2. The method according to claim 1, wherein the non-ionic surfactant(s) of the polyoxyethylene cetyl ether family are selected from Brij 52 (Polyoxyethylene-2-cetyl ether; molecular weight: ˜330), Brij 56 (Polyoxyethylene-10-cetyl ether; molecular weight: ˜683) and/or Brij 58 (Polyoxyethylene-20-cetyl ether; molecular weight: ˜112256 & 58.

3. The method according to claim 1 or 2, wherein the mixture also comprises: a collapse temperature modifier selected from dextran, hydroxyethyl starch, Ficoll and gelatin, preferably Ficoll, most preferably Ficoll 70/Ficoll 400; a bulking agent selected from a sugar or sugar alcohol, such as mannitol, lactose, sucrose, trehalose, sorbitol, glucose, raffinose, melezitose, or amino acids, such as arginine, glycine, histidine, leucine; a stabilizing protein, such as bovine serum albumin (BSA); and a buffering agent, preferably Tris HCl.

4. The method according to claim 3, wherein the non-ionic surfactant or detergent is/are Brij 52, Brij 56 and/or Brij 58, preferably Brij 58.

5. The method according to claim 4, for stabilization of protein, such as enzymes.

6. The method according to claim 5, for stabilization of nucleic acid polymerase, such as Taq DNA polymerase.

7. The method according to claim 6, wherein said mixture is formed into beads, cakes, conical, flat or square structure depending on the shape of the receptacle into which the composition is dispensed into.

8. A stabilized biomolecule composition, comprising a biomolecule stabilized in a mixture comprising non-ionic surfactant(s) of the polyoxyethylene cetyl ether family, such as Brij 52, Brij 56 and Brij 58, or any combination thereof.

9. The biomolecule composition according to claim 8, stabilized in a mixture comprising 1-20% Ficoll, 5-25% Melezitose, 0.1-5% Brij 58, 0.1-10 mg/mL BSA; and 5-50 mM Tris-HCl pH 9.0.

10. The biomolecule composition according to claim 9, wherein the stabilized biomolecule is a protein, such as an enzyme.

11. The biomolecule composition according to claim 10, wherein the stabilized biomolecule is in freeze dried or liquid format.

12. Use of the biomolecule composition according to claim 11 in combination with a solid support, preferably a cellulose based matrix or paper, provided with a biological sample, in an assay involving the biological sample and the stabilized biomolecule.

13. Use of the biomolecule composition according to claim 12, wherein all or portions of the solid support is added to said biomolecule composition, and wherein optionally a sequestrant, preferably cyclidextrin, is added to counteract surfactant inhibition of biomolecule (enzyme) activity or activity of a specific binding partner in the biological sample.

14. A method for amplification of nucleic acid comprising the steps: i) contacting a solid support with a biological sample comprising nucleic acid; ii) transferring all or portions of said solid support to a reaction vessel; iii) incubating said nucleic acid on the solid support with a nucleic acid amplification reagent stabilized in a mixture comprising non-ionic surfactant(s) of the polyoxyethylene cetyl ether family, optionally in the presence of a cyclodextrin; iv) amplifying the nucleic acid to produce amplified nucleic acid, preferably by PCR (polymerase chain reaction); and v) detecting said nucleic acid.

15. The method according to claim 14, wherein more than one sample nucleic acid are individually detected simultaneously by detecting a specific amplified nucleic acid sequence associated with each sample nucleic acid, wherein the specific nucleic acid sequences are unique oligonucleotide sequences functioning as labels for each sample nucleic acid.

16. A kit comprising a solid support and a freeze-dried reagent composition comprising a nucleic acid polymerase stabilized in a mixture, comprising non-ionic surfactant(s) of the polyoxyethylene cetyl ether family, for amplifying an oligonucleotide sequence; and a user instruction manual.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1—Genomic DNA Endpoint PCR; Amplification of 1.3 & 3.6 kb amplicons from the human beta-globin gene. The inclusion of Brij 58 into the bulk Taq DNA polymerase Ready-to-Go mix had minimal effect on the amplification efficiency compared to controls consisting of RE-960. This is apparent for both bead and cake formats.

[0036] FIG. 2 Eurkaryotic qPCR using Brij 58 containing Ready-To-Go beads. Controls beads were also tested using the detergent RE-960 as the source of the detergent. Results indicated that the inclusion of Brij 58 in the beads had minimal effect on the amplification efficiency compared to RE-960-containing controls.

[0037] FIG. 3 Eurkaryotic qPCR using Brij 58 containing Ready-To-Go cakes. Controls cakes were also tested using the detergent RE-960 as the source of the detergent. Results indicated that the inclusion of Brij 58 in the cakes had minimal effect on the amplification efficiency compared to RE-960-containing controls.

[0038] FIG. 4 Prokaryotic qPCR using Brij 58 containing Ready-To-Go beads. Controls beads were also tested using the detergent RE-960 as the source of the detergent. Results indicated that the inclusion of Brij 58 in the beads had minimal effect on the amplification efficiency compared to RE-960-containing controls.

[0039] FIG. 5 Prokaryotic qPCR using Brij 58 containing Ready-To-Go cakes. Controls cakes were also tested using the detergent RE-960 as the source of the detergent. Results indicated that the inclusion of Brij 58 in the beads had minimal effect on the amplification efficiency compared to RE-960-containing controls.

[0040] FIG. 6 Amplicon quality assessment based upon DNA Sequencing. Results indicated that equivalent Phred 20 scores were generated using the Taq DNA polymerase Ready-To-Go mixture manufactured in the presence of either Brij 58 or RE-960 and irrespective of bead or cake formats studied.

[0041] FIG. 7 Comparison of the glass transition temperature of Taq DNA polymerase Ready-To-Go mixtures manufactured in the presence of either Brij 58 or RE-960 in bead or cake formats.

[0042] FIG. 8 Comparison of the water content as determined by a Karl Fisher analysis of Taq DNA polymerase Ready-To-Go mixtures manufactured in the presence of either Brij 58 or RE-960 in bead or cake formats.

[0043] FIG. 9 shows Brij 58 cakes stability data physical integrity; glass transition temperature (Tg). All datasets were shown to be normally distributed using the Shapiro-Wilk test and shown to be statistically equivalent with each other using the each pair Student's t test (p>0.05).

DETAILED DESCRIPTION OF THE INVENTION

[0044] Preparation of Taq DNA Polymerase in Ready-to-go Format

[0045] The method of the invention is exemplified by generation of Taq DNA polymerase in Ready-to-Go dry bead and cake formats. Initially, a liquid formulation is generated that is subsequently freeze-dried into the bead and/or cake. Stabilised dry formats facilitate storage and in most instances, easier downstream workflows. The liquid formulation provides for 10 μl aliquot that contains 3.5 U Taq DNA polymerase. The subsequent freeze-dried bead/cake is re-hydrated with 25 μl reaction volume that contains the appropriate oligonucleotide primers and nucleic acid template which can be added either at the time of manufacture or on rehydration by the end user. The final product is a freeze-dried bead or cake reagent that contains all the necessary components to facilitate the PCR amplification of nucleic acids. These reagents are stable at ambient temperatures when stored at low humidity (<20% RH).

Raw Materials

[0046]

TABLE-US-00001 Material Supplier Catalogue Code 10 x Cycle Sequencing Buffer GE Healthcare 30799 Taq DNA Polymerase Enzymatics 29018715 Melezitose Sigma M-5375 1198 DNase I RNase free GE Healthcare 409636 DNA Polymerization Mix GE Healthcare 28406557BS BSA DNase free Calbiochem 28954811 Ficoll 70 GE Healthcare 17-0310 Ficoll 400 GE Healthcare 17-0300 RNase- free DEPC treated water USB 70783 Bacterial Chromosomal DNA Lab 411576 Tris USB 75825 10M Sodium hydroxide Fluka 72068 Potassium chloride Sigma P3911 Magnesium chloride USB 18641 EGTA USB 15703 10% Brij 58 Thermo 28336 1.0M DTT Lab MR014 Ethanol Absolute alcohol Sigma 51976 Calcium chloride Riedel- de Haen 31307 Hydrochloric Acid Riedel- de Haen 30721 0.2 μm cellulose acetate filter Nalgene SFCA 158 Human genomic DNA Promega G152A 1.3 kb beta-globin reverse primer Sigma-Genosys HA05748544 1.3 kb beta-globin forward primer Sigma-Genosys HA05748543 3.6 kb beta-globin reverse primer Sigma-Genosys HA05748546 3.6 kb beta-globin forward primer Sigma-Genosys HA05748545 Molecular Biology grade water Sigma W4502 Exoprostar GE Healthcare US77750V Big Dye 3.1 Sequencing kit Life Technologies 4336917 Beta-actin qPCR reagent ABI 401846 P53 reverse primer Sigma-Genosys HA03008180 P53 forward primer Sigma-Genosys HA03008179 Bacterial chromosomal DNA Promega 411576 Bacterial chromosomal DNA Promega G2212-2014122 reverse primer Bacterial chromosomal DNA Promega G2212-2014123 forward primer

Example of Preferred Biomolecule Stabilisation Mixture

[0047]

TABLE-US-00002 Chemical component Actual conc Range Ficoll 70 9.6% 1-20% Ficoll 400 9.6% 1-20% Melezitose  15% 5-25% Brij 58   1% 0.1-5%  Taq DNA polymerase 3.5 units/bead/cake 1.0-5 units/bead/cake BSA 0.6 mg/ml 0.1-10 mg/ml Tris HCl pH 9.0 20 mM 5-50 mM DNA polymerisation 0.5 mM 0.1-10 mM Mix CaCl.sub.2 0.1 mM 0.01-5 mM MgCl.sub.2 2.5 mM 0.5-5 mM KCl 50 mM 10-100 mM DNase 1 unit/50 units Taq DNA 0.5-5 units polymerase EGTA 10 mM 1-20 mM DTT 0.03 mM 0.01-5 mM

Methods

[0048] Buffer Preparation—

[0049] The following buffers were generated using autoclaved DNAse and RNAse-free water following standard laboratory procedure using commercially available reagents. All manufactured buffers were sterilized using 0.2 μM cellulose acetate filters—1.0 M Tris/HCl pH 9, 3.0 M KCl, 1.0 M MgCl.sub.2 and 1.0 M CaCl.sub.2. Additionally, BSA (10 mg/ml) was prepared and triple filtered using 0.2 μM cellulose acetate filters.

[0050] The above solutions were used to generate the Exchange Buffer (20 mM Tris/HCl pH 9.0, 0.1 mM CaCl.sub.2, 2.5 mM MgCl.sub.2 and 50 mM KCl) supplemented with a concentration range of Brij 58. A control Exchange Buffer was also prepared using the detergent RE-960 as the source of the detergent however in this instance 10 mM Tris/HCl pH 8.0 was used.

[0051] Unexpected experimental results (not shown) indicated that Taq DNA polymerase Ready-to-Go beads and cakes manufactured with Brij 58 worked significantly better during PCR reactions at pH 9.0 compared to pH 8.0. Subsequently the “Brij 58 Exchange Buffer” (as described above) contained 20 mM Tris/HCl pH 9.0.

[0052] Taq DNA polymerase solution (200 KU/ml) was prepared using Exchange Buffer. DNase I was added to the solution at a ratio of 1 unit DNase per 50 units of Taq DNA polymerase. This was incubated at 37° C. for 22 hours. The DNase was heat inactivated at 75° C. for 15 mM in the presence of 0.03 M DTT and 10 mM EGTA pH 8.0

[0053] Preparation of Carbohydrate Excipient Mix—

[0054] Ficoll.sub.70, Ficoll.sub.400, and Melezitose were mixed until dissolved at 2-8° C. to final concentrations of 9.6%, 9.6% and 15% respectively in the presence of 10× Cycle Sequencing Buffer. The resultant solution was sterilized using a 0.2 μM cellulose acetate filter.

[0055] The Taq DNA polymerase Ready-to-Go mix was generated by mixing the following components in a clean sterile polycarbonate bottle to the final concentrations using DNase and RNase-free water—1× Carbohydrate excipient mix, 0.60 mg/ml BSA, 0.5 mM DNA polymerization mix and 0.35 units/μl Taq DNA polymerase. The reagents were stirred for >30 min at 4° C.

[0056] Ready-to-go Bead and Cake Preparation—

[0057] The freeze-drying procedure involves the following; the bead dropping process dispenses the bulk Taq DNA polymerase Ready-To-Go mix in 10 μl aliquots (containing 3.5 U Taq DNA polymerase) that are immediately frozen by submersion in liquid nitrogen. This can be achieved by using either an automated Bead Dropper device or a standard laboratory pipette. The beads are removed from the liquid nitrogen using a sieve that ensures that beads exhibit a spherical shape with consistent diameter size and weight. Sieving is accomplished at low humidity (<15%). The resultant beads are subjected to drying using a Virtis Freeze Drier set at −46° C., under vacuum for 48 hours.

[0058] To generate the Ready-To-Go cakes, bulk Taq DNA polymerase Ready-to-Go mix is treated in a similar way however the mix is dispensed into either the wells of a 96-well PCR or flat bottomed plate etc, immediately frozen and dried under vacuum.

[0059] Test of Functional and Physical Properties—

[0060] Both the functional and physical properties of the Taq DNA polymerase Ready-To-Go bead and cakes containing the detergent Brij 58 were tested. Functional experimental results focussed on the generation of PCR products amplified from the following sources of DNA:—

[0061] [1] Genomic DNA Endpoint PCR: human genomic DNA (1.3 and 3.6 kb amplicons derived from the single copy Beta-globin gene).

[0062] [2] Real-time quantitative PCR; using bacterial chromosomal and human genomic DNA as the PCR templates.

[0063] [3] Amplicon quality assessment based upon DNA Sequencing: Plasmid DNA (910 bp amplicon derived from p53 gene fragment inserted into pUC-19). The quality and integrity of the resultant PCR products were investigated by performing DNA sequencing as a representative downstream application.

[0064] [4] Physical integrity of Ready-To-Go beads and cakes manufactured using either Brij 58 or RE-960.

[0065] This test was based upon a close visual inspection of the structure and appearance of beads and cakes manufactured in the presence of either Brij 58 or RE-960. Inspections indicated that no visible differences were observable between either formats irrespective of the detergent used.

[0066] [5] Physical integrity of bead or cake; glass transition temperature. The Perkin Elmer Model B016-9321 Differential Scanning calorimeter (DSC) measures the energy changes that occur as a sample is heated, cooled or held isothermally, together with the temperature at which these changes occur. It is used for determining melting points, crystallizations, and measurement of glass transitions and other thermal events. The glass transition temperature (Tg) is a fundamental property of all glass-forming materials, and a significant change in the mechanical properties occurs at this temperature. Below Tg an amorphous material is a glass and above Tg it is defined as being in a more flexible state. Room temperature stability requires the Tg of the product to be higher than the storage temperature.

[0067] The DSC was used to measure the Tg of the Taq DNA polymerase Ready-To-Go beads and cakes manufactured using either Brij 58 or RE-960 according to manufacturer's instructions.

[0068] [6] Physical integrity of bead or cake; Water content; Karl Fischer (KF) Coulometry. This method is based on the principle that water reacts quantitatively with I.sub.2 according to the following equation:

H.sub.2O+I.sub.2+[RNH]SO.sub.3CH.sub.3+2RN<---->[RNH]SO.sub.4CH.sub.3+2[RNH]I

[0069] The iodine required for this reaction is generated by electrochemical means in the Coulomat AG Oven. A quantitative relationship between the electric charge and the amount of iodine generated is used for high-precision dispensing of the iodine. The end point of the determination is indicated voltametrically by applying an alternating current to an electrode immersed in the electrolyte. When the free iodine is consumed in the reaction with water the voltage difference across this electrode is drastically reduced and recorded.

[0070] As the material to be tested for residual moisture content is a lyophilised solid i.e Taq DNA polymerase Ready-To-Go beads and cakes manufactured using either Brij 58 or RE-960, the water must be driven out of the cake using an oven that is attached to the KF Coulometer. The water is carried into the titration vessel in a stream of dry Nitrogen.

Results

[0071] An unexpected difference between Brij 58 and RE-960 containing Ready-To-Go beads and cakes was that there appears to be a significant reduction in the amount of electrostatic energy associated with beads and cakes manufactured using Brij 58 compared to those manufactured using RE-960. This is a particular advantage especially for beads dispensed into receptacles such as PCR tubes, Eppendorf centrifuge tube, 96-well PCR and flat-bottomed plates etc as the tendency for the beads to “jump” out of the receptacle due to either electrostatic attractive or repulsive forces is significantly reduced. This represents a major and significant advantage during both the manufacturing process and for the end-user as the bead/cakes exhibit reduced attractive and repulsive forces.

[0072] [1] Genomic DNA Endpoint PCR—Amplification of 1.3 & 3.6 kb amplicons from the human beta-globin gene.

[0073] Ready-To-Go beads—Amplified DNA yield as determined from a visual inspection of the band intensity of PCR products separated on agarose gel electrophoresis indicated that yields derived from Brij 58 containing Ready-To-Go beads were equivalent to that of controls using RE-960. Band width and intensity were considered equivalent (see FIG. 1).

[0074] Ready-To-Go beads cakes—A similar observation was apparent when using Brij 58 containing Ready-To-Go cakes. Band width and intensity were equivalent.

[0075] [2] Real-time quantitative PCR; using human genomic DNA and bacterial chromosomal DNA as the PCR templates.

[0076] Eukaryotic DNA—

[0077] The ability to amplify and quantify a 265 bp amplicon derived from the human Beta Actin gene was used to investigate the efficiency of the Brij 58 containing Taq DNA polymerase Ready-to-Go mixture in both bead and cake formats. Results indicated that the inclusion of Brij 58 had minimal effect on the amplification efficiency compared to RE 960-containing controls (see FIGS. 2 & 3).

[0078] Prokaryotic DNA—

[0079] The ability to amplify and quantify a 100 bp amplicon derived from bacterial chromosomal DNA was used to investigate the efficiency of the Brij 58 containing Taq DNA polymerase Ready-to-Go mixture in both bead and cake formats. Results indicated that the inclusion of Brij 58 had minimal effect on the amplification efficiency compared to RE-960-containing controls (see FIGS. 4 & 5).

[0080] [3] Amplicon quality and integrity assessment based upon DNA Sequencing

[0081] Plasmid-based Endpoint PCR was used to generate a 910 bp amplicon derived from p53 gene fragment inserted into the plasmid pUC-19. The quality and integrity of the resultant PCR products were investigated by performing DNA sequencing as a representative downstream application. The DNA sequencing quality metric Phred 20 score were compared for amplicons generated using Ready-To-Go beads and cakes consisting of either Brij 58 or the equivalent control format containing RE-960.

[0082] Results indicated that equivalent Phred 20 scores were generated in the presence of either Brij 58 or RE-960 and irrespective of bead or cake formats (see FIG. 6).

[0083] [4] Physical integrity of bead or cake; glass transition temperature.

[0084] The glass transition temperature was determined for Taq DNA polymerase Ready-To-Go mixtures manufactured in the presence of either Brij 58 or RE-960 in bead or cake formats. Results indicate (see FIG. 7) that no statistical differences were apparent irrespective of the format i.e. bead versus cake or detergent.

[0085] The stability trails in FIG. 9 were performed by storing Brij 58-containing RTG cakes at both real time i.e. 25° C. with a relative humidity of 60% and accelerated conditions i.e. stored at 40° C. with a relative humidity of 75%. The data presented in FIG. 9 was derived from Brij-58 cakes stored for up to one year. The results demonstrate that the manufactured cakes do not deteriorate as measured by the glass transition temperature (Tg).

[0086] 12 months stability data was performed at accelerated conditions i.e. 40° C. and 75% humidity which equates to 36 months of real time data (25 oc and 60% humidity). This is based upon the Arrenhius equation and an aging factor of (Q10)=2

Reference ASTM F198007

[0087] [00001]$\mathrm{Accelerated}.Math..Math.\mathrm{Aging}.Math..Math.\mathrm{Time}.Math..Math.\left(\mathrm{AAT}\right)=\frac{\mathrm{Desired}.Math..Math.\mathrm{Real}.Math..Math.\mathrm{Time}.Math..Math.\left(\mathrm{RT}\right)}{Q_{10}^{\left[\left(T_{\mathrm{AA}}-T_{\mathrm{RT}}\right)/10\right]}}$

[0088] [5] Physical integrity of bead or cake; Karl Fisher analysis—water content of beads & cakes

[0089] The water content of beads & cakes was determined for Taq DNA polymerase Ready-To-Go mixtures manufactured in the presence of either Brij 58 or RE-960 in bead or cake formats. Results indicate (see FIG. 8) that no statistical differences were apparent irrespective of the format i.e. bead versus cake or detergent.