Freeze-Dried Composition

Abstract

The invention relates to the use of a polysaccharide having at least four saccharide units, such as stachyose, as a glass-forming agent for the freeze-drying of a reaction mixture comprising an enzyme. In particular, the enzyme is a polymerase useful in a nucleic acid amplification reaction such as a Polymerase Chain Reaction.

Compositions comprising such polysaccharides as well as methods for preparing them, kits containing them and methods for using them form further aspects of the invention.

Claims

1. A dried composition haying a cake three-dimensional structure which is resistant to collapse in an environment of 75% humidity, the composition comprising: (i) a polymerase that is useful in a nucleic acid amplification reaction; and (ii) a glass-forming agent comprising a polysaccharide having at least 4 saccharide units, wherein the dried composition is free of gelatin and is in a freeze-dried form, and the polymerase is an isothermal polymerase or a thermostable polymerase.

2. The composition of claim 1, wherein the composition is free of stabilizer polysaccharide.

3. The composition of claim 1, wherein the composition is free of cross-linked polysaccharide.

4. The composition of claim 1, wherein the composition further comprises a buffer selected from the group consisting of Iris, Trizma, HEPES, tricine, and bicine.

5. The composition according to claim 1, wherein the polysaccharide has 4-6 saccharide units.

6. The composition according to claim 1, wherein the polysaccharide is selected from stachyose, verbascose and lycopose.

7. The dried composition of claim 1, which further comprises at least one of the following: (i) a metal salt; (ii) nucleotides which may be fluorescently labelled; (iii) a blocking compound; (iv) an anti-oxidant and/or anti-maillard reagent; (v) a primer useful in an amplification reaction which is optionally labelled with a fluorescent label; (vi) a probe useful in detection of an amplification reaction which is optionally labelled with one or more fluorescent labels; (vii) a fluorescent dye; (viii) an anti-Taq antibody that binds to the active site of the polymerase and inactivates it at ambient temperature, but denatures and dissociates from the enzyme at elevated temperatures; (ix) a pyrophosphate salt and a pyrophosphatase enzyme; (x) a nucleic acid hat is able to act as an internal control for an amplification reaction; or (xi) a primer that is able to amplify the nucleic acid of item (x).

8. The dried composition of claim 7, which is in the form of a freeze-dried cake which comprises two or more distinct layers, and wherein at least one of said further reagents is comprised within one distinct layer within the freeze-dried cake.

9. The dried composition of claim 1, which further comprises a polymeric compound that is able to stabilize a freeze-dried cake, wherein the polymeric compound is selected from the group consisting of polyethylene glycol, polyvinylpyrrolidine, and polysaccharides.

10. A kit comprising the dried composition of claim 1, and a second composition comprising a reagent for use in a reaction with said polymerase.

11. The kit of claim 10, wherein the second composition is in freeze-dried form.

12. The kit of claim 10, wherein said second composition comprises stachyose and a reagent mixture for use in a reaction with said polymerase.

13. A method for preparing the dried composition of claim 1, wherein the method comprises forming a mixture by combining the polymerase and the glass-forming agent and optionally one or more further components, and freeze-drying the mixture to form the dried composition.

14. The method of claim 13, wherein the polymerase is a polymerase solution free of glycerol.

15. The method of claim 14, wherein the isothermal polymerase solution further comprises at least one of a detergent, an antioxidant and an anti-reducing agent.

16. The method of claim 15, wherein the freeze-drying further comprises: (i) freezing a first portion of the mixture to form a first frozen layer; (ii) freezing an additional portion of the mixture to form an additional frozen layer; (iii) optionally repeating step (ii) until all components of the composition are present in distinct frozen layers; and (iv) freeze-drying the frozen layers to form the dried composition.

17. The method of claim 13, further comprising dividing the mixture amongst multiple reaction vessels so that each vessel contains sufficient reagents to carry out a single reaction using the polymerase, and wherein the freeze-drying further comprises freeze-drying the divided mixture within said reaction vessels.

18. A method for carrying out a chemical or biochemical reaction using a polymerase, the method comprising forming a reaction mixture by adding a liquid and optionally one or more additional components for carrying out the reaction to the dried composition of claim 1, and subjecting the reaction mixture to suitable reaction conditions.

19. The method of claim 18, wherein the chemical or biochemical reaction is a polymerase chain reaction.

20. A method for enhancing the stability of a composition comprising a polymerase, said method comprising freeze-drying a mixture comprising the polymerase with a glass forming agent, wherein the mixture is free of gelatine.

21. A composition of claim 1, further comprising an anti-maillard reagent, wherein the anti-maillard reagent optionally comprises threonine.

22. The composition of claim 1, wherein the isothermal polymerase is selected from M-MuLV reverse transcriptase, AMV reverse transcriptase, and Tth reverse transcriptase.

23. The composition of claim 1, wherein the isothermal polymerase is an isothermal polymerase for an isothermal amplification reaction selected from transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA (LAMP), isothermal multiple displacement amplification, helicase-dependent amplification, single primer isothermal amplification, circular helicase-dependent amplification, and whole genome amplification.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The invention will now be particularly described by way of example with reference to the accompanying drawings, in which:

[0079] FIG. 1 shows the results of a stability trial carried out on a variety of freeze-dried sugar solutions;

[0080] FIG. 2 shows the results of PCR reactions carried out using reaction mixtures in accordance with the invention and fresh undried reaction mixture; and

[0081] FIGS. 3-5 show the results of amplification reactions carried out using various PCR enzymes which had been incorporated into compositions in accordance with the invention.

EXAMPLES

Example 1

Preparation of Compositions of the Invention

[0082] Freeze dried compositions of the invention are prepared by mixing together the components required for the reaction including enzymes and optionally other components discussed above, with a sugar as defined above such as stachyose in a glass-forming amount and a small quantity (less than 1% M/v and preferably less than 0.5% M/v) of a structural stabiliser such as PEG, also as described above. Any enzyme used should be substantially free of glycerol. At this stage, the mixture will contain a solvent and in particular water. The amount of water present is selected to give the desired cake volume after freeze drying. Specifically, during a freeze drying process, the mixture is first frozen, which effectively determines the volume of the cake. During subsequent sublimation of the solvent, the frozen material may undergo some shrinkage, but will generally retain a substantial proportion of the volume of the frozen material. This is useful in that it allows a cake of a suitable volume to be produced. Thus the amount of solvent such as water present in the mixture at this time will vary depending upon factors such as the amount required for dissolution of the components and the desired cake volume. Typically, any solution suitably contains less than 50% water as solvent compared to the final reconstituted reaction composition.

[0083] The solution is then freeze-dried to remove substantially all of the water in a freeze dryer used in accordance with the manufacturer's instructions. The freeze-dried composition forms a cohesive and malleable cake.

[0084] In particular, the or each container holding the freeze-dried composition is sealed under an inert atmosphere such as a nitrogen atmosphere.

[0085] Just prior to use, the freeze-dried compositions are reconstituted by addition of a suitable volume of water which may include a sample under test. The reaction mixture is then processed in the usual way.

[0086] It has been found that the freeze-dried compositions of the invention are more stable for prolonged periods, for example in high humidity prior to use, when compared to published methods. They also reconstitute more easily after exposure to form active reaction mixtures which are extremely reliable in use.

Example 2

Comparative Environmental Exposure Properties of Freeze-Dried Cakes of Various Sugars

[0087] The following solutions were prepared: [0088] A) 2.5% (m/v) Raffinose , 0.5% (m/v) PEG, 500 uL cake [0089] B) 2.5% (m/v) Stachyose , 0.5% (m/v) PEG, 500 uL cake [0090] C) 2.5% (m/v) Trehalose, 0.5% (m/v) PEG, 500 uL cake
These were each freeze dried to form a similar 500 μL cake. The cakes were stored in a desiccator at about 75% humidity in the presence of a saturated solution of sodium chloride at ambient temperature. The stability of the cakes over time was monitored and the results showed that the stachyose composition (B) retained stability better than both compositions (A) and (C).
The experiment was repeated using slightly larger (525 μL) cakes. The materials were placed in glass lyovials, and then dried using a Virtis Advantage+ freeze drier, operating a lyophilisation cycle comprising a thermal treatment step, a primary drying step and a Post hold step as summarised in the following tables:

TABLE-US-00001 Thermal Treatment Step No Temp° C. Time (minutes) Ramp/Hold 1 10 15 Hold 2 −45 110 Ramp 3 −45 120 Hold

TABLE-US-00002 Primary Drying Step No Temp° C. Time (minutes) Vacuum Ramp/Hold 1 −45 900 100 Hold 2 0 120 100 Ramp 3 0 60 100 Hold 4 20 50 100 Ramp 5 20 120 100 Hold

TABLE-US-00003 Post hold Temp° C. Time (minutes) Vacuum 20 1000 100
Condenser temperature: −55° C.

Secondary Set Point: 45° C.

[0091] A bell jar was filled with a saturated sodium chloride solution at ambient temperature to produce an approximate relative humidity environment of ˜75%. The caps from the three lyovials were removed immediately before placement within, and closure of, the bell jar. The hydration of the cakes was filmed using time-lapse photography.

[0092] The results are shown in FIG. 1. These show that the stachyose cake (B) was resistant to collapse showing marginal alteration in cake structure within the period of the experiment (˜80 time minutes). The raffinose cake (A) showed almost complete collapse after ˜65 minutes. The trehalose cake (C) showed approximately 50% collapse at the completion point of the experiment (˜80 time minutes) minutes.

[0093] As a result, stachyose appears to provide a more stable freeze-dried composition than either raffinose or trehalose.

Example 3

Generation and Testing of a Lyophilised PCR Mastermix

[0094] This example illustrates the generation of a lyophilised core master mix preparation according to the invention. This includes the use of a chemically modified hot start polymerase Taq DNA dependent polymerase in a suitable buffer composition dried in a glass lyovial.

[0095] Example of freeze-dried master mix composition:

TABLE-US-00004 Reagent Cake Concentration (2x) Final Concentration (1x) Tris 20 mM 10 mM MgCl2 6 mM 3 mM BSA 500 ng/μl 250 ng/μl dNTPs with dUTPS 0.4 mM 0.2 mM Stachyose 2.5% 1.25% PEG 0.5% 0.25% TAQ polymerase 0.08 U/μl 0.04 U/μl TAQ additive* 0.08 U/μl 0.04 U/μl *TAQ additive is a detergent mix obtainable from Fluorogenics Ltd.

[0096] A cake comprising the above components was prepared and was freeze dried in a Virtis Advantage+ freeze drier using the program described in Example 2 to create active lyophilised product having a final concentration as listed above.

[0097] The resulting freeze dried mixture was reconstituted by addition of various concentrations of water as detailed below and used to amplify a DNA target (derived from bacteriophage λ) using custom primers in combination with SYBR®Green-1 DNA binding dye to a final concentration of 1:10,000 of reference solution. The mixture was amplified on the Genie thermal cycler using:

PCR parameters

TABLE-US-00005 Denature 95° C. 900 seconds Amplification 95° C. 20 seconds 55° C. 20 seconds 74° C. 20 seconds Optical Read X5 45 Cycles Melt 50° C. 20 seconds 95° C. 10 seconds Continuous read −1 Rate = 0.1° C./second

[0098] The resulting mixture was used to amplify a DNA target (derived from bacteriophage λ) using custom primers in combination with SYBR®Green-1 DNA binding dye to a final concentration of 1:10,000 of reference solution. The data (FIG. 2) shows three 10-fold dilutions (labelled 10{circumflex over ( )}5, 10{circumflex over ( )}4, & 10{circumflex over ( )}3) of template and a no template control in duplicate amplifications. The solid lines are reactions prepared using lyophilised master mix. The dotted line shows the same formulation prepared as a non-lyophilised control.

[0099] The results are substantially equivalent, showing that the stachyose has protected the activity of the active components through the freeze-drying process.

Example 4

Preparation and Use of a Two-part Composition Kit

[0100] A lyophilised core PCR master mix was prepared according to the invention, and a second composition comprising a lyophilised primer and probe mixture was also prepared. The Taq enzyme used in this example was an anti-Taq antibody mediated hot start Taq DNA dependent polymerase in a suitable buffer composition, available from Promega (USA).

Primer Probe Recipe (2X cake formulation), dried to 12.5 μL volume final.

TABLE-US-00006 REAGENT CAKE CONCENTRATION (2x) Units HSV 1 F 1 uM HSV 1 R 1 uM HSV 1 P 0.4 uM HSV 2 F 1.8 uM HSV 2 R 1.8 uM HSV 2 P 0.8 uM Trizma 2 mM STACHYOSE 2.5 % M/V PEG 0.5 % M/V
Master Mix Recipe (2.5X cake formulation) dried to 5 μL volume final (1x).

TABLE-US-00007 REAGENT CAKE CONCENTRATION (2.5 x) MgCl2 10 mMols/L BSA 625 mg/L dUTPS(dUTP/dNTP 0.5 mMols/L mixture) Taq 100000 UNITS/L Trizma 25 mMols/L STACHYOSE 2.5 % (M/v) PEG 0.5 % (M/v)

[0101] Both compositions were freeze dried as described in Example 2, but in separate micro-amp style vessels.

[0102] The resulting compositions were used to amplify a DNA target (derived from HSV 1 and 2) using custom primers in combination with dual labelled fluorogenic probes for each target using the 5′ nuclease assay process.

[0103] The primer probe pots were resuspended in 12.5 μL of water, to which 12.5 μL of either template or water (for no amplification controls) was added and mixed. 12.5 μL was transferred to the core master mix pot and mixed before transfer to an ECO plate. The mixture was amplified on the Illumina ECO thermal cycler using the following parameters:

PCR parameters

TABLE-US-00008 Denature 98° C. 600 seconds Amplification 95° C. 5 seconds 45 Cycles 62° C. 35 seconds, Optical Read in FAM and HEX channels
The results are shown in FIG. 3. They illustrate efficient amplification for both templates tested.

Example 5

Generation and Testing of Other Lyophilised PCR Reaction Mixtures

[0104] This example illustrates the generation and use of a lyophilised core master mix preparations according to the invention.

[0105] A first mastermix included as the enzyme a high fidelity recombinant polymerase Pfu DNA dependent DNA polymerase in a suitable buffer composition.

TABLE-US-00009 Reagent Cake Concentration (2X) Final Concentration Trizma 20 mM 10 mM BSA 500 ng/μl 250 ng/μl dUTP/dNTP 0.4 mM 0.2 mM Pfu enzyme 0.04 U/μl 0.02 U/μl Stachyose 2.5% 1.25% PEG 0.5% 0.25%

[0106] The composition was freeze dried as described in Example 2 in a glass lyovial. The resulting mixture was resuspended in resuspension buffer that was able to provide dissolution and an active complete PCR reaction mixture. This mixture was then used to amplify a DNA target (derived from bacteriophage λ) using custom primers in combination with SYBR®Green-1 DNA binding dye to a final concentration of 1:10,000 of reference solution. The mixture was amplified on the Genie thermal cycler using:

PCR parameters

TABLE-US-00010 Denature 98° C. 30 seconds Amplification 98° C. 10 seconds 55° C. 30 seconds 72° C. 30 seconds Optical Read X5 45 Cycles Melt 50° C. 20 seconds 95° C. 10 seconds Continuous read −1 Rate = 0.1° C./second

[0107] The data shows three 10-fold dilutions (labelled 10{circumflex over ( )}5, 10{circumflex over ( )}4, & 10{circumflex over ( )}3) of template and a no-template control in duplicate amplifications. The results illustrated in FIG. 4 show efficient amplification across the tested target template concentrations.

[0108] In a second experiment, a chemically modified hot start polymerase Taq DNA dependent polymerase in combination with a thermo-stable MMuLV RNA dependant DNA RNase H-polymerase was used as the enzyme. This was combined with other reagents including salts and appropriate concentrations of stachyose to form a PCR mastermix. This was dried in a glass lyovial using a method as described in Example 2.

[0109] The resulting mixture was used to amplify a RNA target (derived from rat 18s RNA) using custom primers in combination with dual labelled fluorogenic probes for each target using the 5′ nuclease assay process. The mixture was amplified on the Genie thermal cycler using:

PCR parameters

TABLE-US-00011 RT-Step 48° C. 600 seconds Denature 95° C. 900 seconds Amplification 95° C. 20 seconds 45 Cycles 60° C. 60 seconds Optical Read X10

[0110] The amplifications were carried out at three 10-fold dilutions (labelled 4.5 ng (per reaction), 0.45 ng (per reaction), 0.045 ng (per reaction) of template and a no-template control in duplicate amplifications. The results are shown in FIG. 5 solid lines are reactions prepared using lyophilised master mix. The dotted line shows a commercial 2x master mix (non-lyophilised) control. The results are substantially equivalent.

Example 6

Alternative Separate Compositions

[0111] As described above, one embodiment of the invention includes the use of a core lyophilised PCR master with a lyophilised resuspension buffer, which forms a second composition. This resuspension buffer may also be lyophilised using the same freeze drying procedure as the PCR master, but in a separate vial (although alternative drying techniques may be used as described above). This means that salts required for the buffer composition in the final reaction, but which could be inhibitory, for example, to the stability of the polymerases during and post drying to be kept away from the enzymes until required. Likewise, some of the enzyme cofactor, which in this case is magnesium ions in the form of magnesium chloride, may be dried in this buffer.

[0112] This is illustrated here by an example of the use of an anti-Taq antibody mediated hot start Taq DNA dependent polymerase lyophilised master mix for use in combination with the reconstitution buffer. The reconstitution buffer mixture and lyophilised PCR master is dried using the same process but in separate vessels.

Reconstitution buffer cake recipe Recipe (2X cake formulation), dried to 12.5 μL volume final.

TABLE-US-00012 REAGENT CAKE CONCENTRATION (2x) Magnesium Chloride 4 mMols/L Ammonium Sulphate 20 mMols/L Potassium Chloride 20 mMols/L STACHYOSE 2.5 % M/V PEG 0.5 % M/V
PCR Master Mix Recipe (2 X cake formulation) dried to 12.5 μL volume final (2x). The final PCR reaction would be 25 μL.

TABLE-US-00013 REAGENT CAKE CONCENTRATION (2 x) MgCl2 6 mMols/L BSA 500 mg/L dUTPS (dUTP/dNTP 0.4 mMols/L mixture) Taq 80000 UNITS/L Trizma 20 mMols/L STACHYOSE 2.5 % (M/v) PEG 0.5 % (M/v)

[0113] The two separate dried compositions as set out above may be collected together in a kit.

[0114] A number of reconstitution regimes and solute permutations are possible. For example, in a first embodiment, the reconstitution buffer cake is re-suspended using only water to a 2x composition. The resulting mixture is used to effect dissolution of the lyophilised master mix to a 2x solution. The complete solution will form a 2x mixture that may be further diluted with primers, probes and template to carry out a PCR.

[0115] Alternatively, the reconstitution buffer cake is re-suspended using a primer-probe cocktail to a 2x composition. The resulting mixture is used to effect dissolution of the lyophilised master mix to a 2x solution. The complete solution will form a 2x mixture that may be further diluted with template to carry out a PCR.