DRY COMPOSITIONS FOR USE IN NUCLEIC ACID AMPLIFICATION AND METHODS

Abstract

The invention relates to a single novel dry reagent composition for use in subsequent nucleic acid amplification, such as PCR (Polymerase Chain Reaction) and the isothermal LAMP (loop-mediated isothermal amplification), the composition including a nucleic acid amplification enzyme; a corresponding enzyme binder; a sugar-based stabiliser; deoxyNucleotide TriphosPhates; oligonucleotides; and a zwitterionic agent where a tris-based buffer is specifically excluded. The invention further relates to methods for producing said single dry reagent composition using air drying processes.

Claims

1. A single dry reagent composition prepared by evaporation or air drying for nucleic acid amplification comprising: at least one nucleic acid amplification enzyme; at least one corresponding enzyme binder; a sugar-based stabiliser; deoxyNucleotide TriphosPhates (dNTPs) or Nucleoside Triphosphates (NTPs) or modified versions thereof; labelled or unlabeled oligonucleotides for nucleic acid amplification; and a zwitterionic agent, wherein the composition excludes at least Tris-based buffers and a nucleic acid template to be amplified.

2. The composition of claim 1, wherein the at least one nucleic acid amplification enzyme is provided in a range from 0.01 to 2 units; and/or the at least one enzyme binder is provided in a range from 0.01 μg to 1 μg; and/or the sugar-based stabiliser is provided in a range from 0 to 10% v/v; and/or the NTPs or DNTPs are each provided in a concentration range from 0.1 mM to 0.4 mM; and/or the oligonucleotides are provided in a concentration range from 0.001 mM to 110 mM; and/or the zwitterionic agent is provided in a concentration range from 10 mM to 100 mM.

3. The composition of claim 1, wherein the zwitterionic agent is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and/or 3-(N-morpholino)propanesulfonic acid (MOPS).

4. (canceled)

5. The composition of claim 1, wherein the at least one nucleic acid amplification enzyme is selected from one or more of: polymerase, DNA polymerase, Taq polymerase, transcriptase and reverse transcriptase, alone or in combination.

6. The composition of claim 1, wherein the at least one enzyme binder is adapted to interact with the at least one corresponding amplification enzyme to temporarily block enzyme activity.

7. The composition of claim 1, wherein the at least one enzyme binder is specific for the at least one nucleic acid amplification enzyme.

8. The composition of claim 1, wherein the at least one enzyme binder is selected from one or more of the following: an antibody, antibody fragment, and synthetic molecule, including aptamers, alone or in combination.

9. The composition of claim 1, wherein the NTPs or dNTPs are modified.

10. The composition of claim 1, wherein the zwitterionic agent is an organic chemical buffer and the composition excludes tricine-based buffers: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid (CAPSO).

11. The composition of claim 1, where in the enzyme is DNA Taq polymerase.

12. The composition of claim 1 wherein the composition further comprises one or more salts, optionally in the concentration range of 1 to 70 mM, preferably 1-10 mM.

13. The composition of claim 1, wherein the sugar-based stabiliser is selected from a monosaccharide including: Fructose, Galactose, Glucose, Rhamnose and Xylose, a disaccharides including Lactose, Maltose, Trehalose, Sucrose or a Trisaccharide including Melezitose and Raffinose.

14. The composition of claim 1, wherein the composition remains in a stable form, preferably wherein the composition remains in a stable form in a temperature range 0° C. to about 32° C.

15. The composition of claim 1, wherein the composition of the invention is provided in volumes of 5-20 μg, preferably about 10 μg.

16. A process for producing a dry stable single nucleic acid amplification reagent composition comprising: mixing together non-lyophilised components comprising at least one nucleic acid amplification enzyme, at least one corresponding enzyme binder, a sugar-based stabiliser, Nucleoside Triphosphates or deoxyNucleotideTriphosPhates or modified versions thereof, oligonucleotides and a zwitterionic agent, excluding a Tris-based buffer; and drying the composition by air drying or evaporation, wherein the process excludes lyophilisation of the composition.

17. The process for producing a dry stable single nucleic acid amplification reagent composition according to claim 16, wherein the at least one nucleic acid amplification enzyme is provided in a range from 0.01 to 2 units; and/or the at least one enzyme binder is provided in a range from 0.01 μg to 1 μg; and/or the sugar-based stabiliser is provided in a range from 0 to 10% v/v; and/or the Nucleoside Triphosphates or deoxyNucleotideTriphosPhates are each provided in a concentration range from 0.1 mM to 0.4 mM; and/or the oligonucleotides are provided in a concentration range from 0.001 mM to 101 mM; and/or the zwitterionic agent is provided in a concentration range from 10 mM to 100 mM.

18. The process of claim 16, wherein the process excludes a step of mixing a nucleic acid template component.

19. The process of claim 17, wherein the nucleic acid amplification is DNA amplification.

20. The process of claim 16, wherein the at least one enzyme is selected from one or more of: polymerase, DNA polymerase, Taq polymerase, transcriptase and reverse transcriptase, alone or in combination.

21. The process of claim 16, wherein the at least one enzyme binder is specific for the at least one corresponding enzyme.

22. The process of claim 16, wherein the at least one enzyme binder is selected from one or more of an antibody, antibody fragment, or synthetic molecule, including aptamers, alone or in combination.

23. The process of claim 16, wherein the NTPs or dNTPs are modified.

24. The process of claim 16, wherein the buffering agent is an organic chemical buffer further excluding tricine-based buffers: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), N-Cyclohexylaminoethanesulfonic acid (CHES); and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid (CAPSO).

25. The process of claim 16, wherein the zwitterionic agent is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and optionally the enzyme is DNA Taq polymerase.

26. The process of claim 16, wherein the composition further comprises a salt, preferably a magnesium salt and more preferably Magnesium Chloride.

27. The process of claim 16, wherein the sugar-based stabiliser is selected from a monosaccharide including: Fructose, Galactose, Glucose, Rhamnose and Xylose, a disaccharides including Lactose, Maltose, Trehalose, Sucrose or a Trisaccharide including Melezitose and Raffinose.

28. The process of claim 16, wherein the composition is stable from about −20° C. to about 32° C. and preferably remains stable when stored for 1 to 12 months at that temperature with a desiccant.

29. The process of claim 16, wherein the composition is stable at temperatures of up to 37° C. for at least 3 weeks.

30. The process of claim 16, wherein the drying is by evaporation in a warm environment and/or under vacuum.

31. The process of claim 16, further comprising a step of dividing the composition in to sample volumes of 5-20 μg, preferably volumes of 5-10 μg.

Description

DETAILED DESCRIPTION

[0061] Table 1 exemplifies preferred embodiments of components and their working ranges for use in a working composition of the invention. Such example compositions are stable and therefore useful in nucleic acid amplification processes.

TABLE-US-00001 TABLE 1 Working Example Composition Table 1 provides an example combination of the components of the composition of the invention. Provided the essential components are present these may be available in any combination of the above and in any working concentrations, without being limited there to, provided no tris-based buffer is used. Dry reagent Example Example working component Range Embodiment concentration Example supplier Nucleic acid 0.01 to 2 units Taq Polymerase 0.75 U Qiagen, P7250-5000 enzyme (Enzymatic taq B, 5 U/μl) Enzyme binder 0.01 to 1 ug Anti-taq mAB 0.0825 μg per reaction PCRBio CP004 dNTPs 0.1 to 0.4 mM dNTPs 0.125 mM each Trilink/N-9506 Sugar-based 0 to 10% v/v  D-(+)-Trehalose     8% v/v Sigma T5251 stabiliser dihydrate Zwitterionic agent 10 to 100 mM HEPES, pH 8 70 mM Sigma H0887-100 ml Oligonucleotides 0.001 mM to 10 mM Oligonucleotides 0.1-0.5 uM various Optional components are also included, as indicated in parenthesis below, but are not essential. Colourant (option) <0.001% v/v 0.05% Patent Blue VF 0.00025% v/v 198218 Sigma- solution Aldrich Detergent (option) 0 to 0.5% v/v Triton-X   0.1% v/v Sigma T8787 Salt 2 component 0 to 70 mM Ammonium Sulphate  2 mM Sigma/A4418 (option) Salt 3 component 0 to 70 mM Potassium Chloride 20 mM Sigma/P9333 (option) Salt component 1 to 10 mM MgCl2  6 mM Sigma M1028 (option)

Comparative Testing

Non-Working Examples

[0062] In this example, the effect of tris-based PCR buffers on dry compositions is provided in which six different preparations of dried PCR composition were made with varying concentrations of tris HCL ph8.3 as buffer. The PCR buffer composition is given as given in the table above where variant 1 is 240 mM Tris-HCl pH8.3, variant 2 is 200 mM, variant 3 is 160 mM, variant 4 is 120 mM, variant 5 is 80 mM and variant 6 is 40 mM. The PCR mixtures were completed by combining a 4× concentrated version and in a 4:1 ratio with a 10× concentration of oligonucleotides.

[0063] The resulting combination was dispensed in a volume of 5 μl in a standard white qPCR plate and submitted to air drying for 17 hours at 30° C. The resulting dried mixes were sealed with PCR caps and stored at different stability test conditions until use.

[0064] The oligonucleotides used in this experiment were a multiplexed set designed (reaction identity HPA-2ab.2) to detect the alleles encoding the Human Platelet Antigen 2a/b (HPA-2a & HPA-2b) using a consensus amplification flanking the 2a/2b polymorphism and dual labelled hydrolysis probes specific for the 482C>T nucleotide. The 482C position was detected with a probe labelled on 5′ with CalFluor Orange 560 fluorophore and with BHQ1 on the 3′ end, whereas the 482T position detected by another probe 5′ labelled with FAM and BHQ1 quenched at the 3′ end. Oligonucleotides specific for a consensus region of the human growth hormone (HGH) were included as an ‘internal amplification control’ (IAC). The IAC amplification was detected using a probe labelled with CalFlour Red 610 and quenched with BHQ2 at the 3′ end.

[0065] The dried mixes were subjected to storage at 1 or 2 weeks at both 32° C. and 37° C. and compared to immediate (Time point 0) conditions.

[0066] The dried PCR mixes were tested at each timepoint in duplicate by rehydrating the dried samples with 10ul of 10 ng/μl reference DNA sample (heterozygous for HPA-2a/b). Successful PCR is measured by the final fluorescence measurement (FF) and the crossing point (Cp) when the amplification is deemed to have started.

[0067] PCR was performed at various timepoints using a Bio-Rad CFX Touch qPCR instrument and data was reported, exported and tabulated as below.

TABLE-US-00002 TABLE 2 Stability data on Tris-based air dried compositions in PCR Tris HCL No storage 32° C. storage 37° C. storage Reaction pH8.3 FF Cp FF Cp FF Cp FF Cp FF Cp Id conc TP0 TP1 (1 week) TP2 (2 week) TP1 (1 week) TP2 (2 week) CalFluor Red 610 HPA- 240 mM 855,244 25.99 17,863  0.00 2,321  0.00 5,123 0.00 1,241 0.00 2ab.2 HPA- 240 mM 746,407 26.03 15,773  0.00 6,512  0.00 2,486 0.00 40,727 0.00 2ab.2 HPA- 200 mM 858,225 25.55 43,819  0.00 7,001  0.00 12,561 0.00 3,317 0.00 2ab.2 HPA- 200 mM 842,591 25.70 48,374  0.00 2,409  0.00 2,470 0.00 1,320 0.00 2ab.2 HPA- 160 mM 852,360 25.63 159,211 34.44 3,538  0.00 10,051 0.00 2,210 0.00 2ab.2 HPA- 160 mM 917,672 25.52 155,626 35.17 7,048  0.00 14,267 0.00 1,171 0.00 2ab.2 HPA- 120 mM 874,335 25.73 442,879 28.11 23,658  0.00 21,775 0.00 2,442 0.00 2ab.2 HPA- 120 mM 840,110 25.53 362,657 28.84 19,570  0.00 22,672 0.00 510 0.00 2ab.2 HPA-  80 mM 652,546 25.46 404,273 26.49 183,206 33.04 195,822 30.82 6,435 0.00 2ab.2 HPA-  80 mM 780,706 25.74 471,135 26.46 449,766 28.00 321,308 28.44 33,658 0.00 2ab.2 HPA-  40 mM 484,665 26.36 396,065 27.26 116,940 38.98 331,813 26.18 1,519 0.00 2ab.2 HPA-  40 mM 507,995 26.17 440,107 26.91 167,117 30.66 333,124 26.17 13,638 0.00 2ab.2 CalFluor Orange 560 HPA- 240 mM 574,018 28.49 285,632 34.42 8,485  0.00 5,700 0.00 2,416 0.00 2ab.2 HPA- 240 mM 497,642 28.39 280,499 35.88 25,880  0.00 2,982 0.00 6,450 0.00 2ab.2 HPA- 200 mM 521,620 27.78 325,336 32.11 35,537  0.00 3,735 0.00 −228 0.00 2ab.2 HPA- 200 mM 558,661 27.98 354,844 31.46 2,251  0.00 3,319 0.00 767 0.00 2ab.2 HPA- 160 mM 510,763 27.92 357,024 28.94 14,236  0.00 123,103 40.17 1,482 0.00 2ab.2 HPA- 160 mM 498,380 27.83 388,371 29.08 49,351  0.00 126,058 38.99 207 0.00 2ab.2 HPA- 120 mM 428,867 27.90 362,364 27.54 164,961 34.91 234,497 31.79 3,336 0.00 2ab.2 HPA- 120 mM 443,102 27.35 362,101 27.93 165,665 34.35 249,253 31.05 4,111 0.00 2ab.2 HPA-  80 mM 314,716 27.40 333,146 27.82 312,552 29.03 364,106 26.78 62,106 44.09 2ab.2 HPA-  80 mM 359,741 27.87 378,057 27.83 326,262 28.78 359,394 27.25 174,458 34.40 2ab.2 HPA-  40 mM 177,927 28.98 240,426 29.60 124,410 31.25 289,342 27.99 60,020 44.04 2ab.2 HPA-  40 mM 192,608 28.61 289,458 28.96 153,513 30.83 306,511 27.73 89,670 35.98 2ab.2 FAM HPA- 240 mM 1,343,596 28.52 1,011,692 33.61 225,478 39.33 47,507 0.00 112,079 0.00 2ab.2 HPA- 240 mM 1,103,278 29.02 1,371,969 34.30 59,805  0.00 19,816 0.00 106,666 0.00 2ab.2 HPA- 200 mM 1,318,780 27.90 1,154,808 31.28 236,601 38.54 19,842 0.00 9,873 0.00 2ab.2 HPA- 200 mM 1,229,919 28.05 1,128,279 31.44 13,896  0.00 16,314 0.00 12,769 0.00 2ab.2 HPA- 160 mM 1,272,638 27.75 1,209,810 28.67 163,749 47.63 982,272 36.27 17,774 0.00 2ab.2 HPA- 160 mM 1,334,130 27.99 1,313,123 28.77 443,204 41.08 942,343 36.02 12,002 0.00 2ab.2 HPA- 120 mM 1,243,945 27.71 1,055,108 27.58 931,887 32.64 1,090,081 30.70 17,671 0.00 2ab.2 HPA- 120 mM 1,206,441 27.85 1,106,790 28.24 946,335 32.67 1,128,047 30.17 54,342 0.00 2ab.2 HPA-  80 mM 1,164,727 26.30 1,239,714 27.49 1,154,421 28.95 1,168,007 27.35 625,679 34.67 2ab.2 HPA-  80 mM 1,040,246 27.73 1,272,979 28.03 891,815 29.30 1,060,398 27.56 903,965 33.06 2ab.2 HPA-  40 mM 638,441 29.02 883,271 30.00 700,234 29.22 1,055,898 27.95 541,851 29.27 2ab.2 HPA-  40 mM 665,639 28.50 1,038,919 29.47 852,584 28.95 1,061,526 28.18 664,476 29.05 2ab.2

[0068] The data in Table 2 shows that the concentration of tris at time point zero (just after drying) does not have any influence on amplification as all three fluorescent channels show similar Cp values although lower levels of tris are detrimental to the fluorescence. It can be seen that at 32° C. storage of 1 week the Cp values are starting to drift to larger numbers showing a loss of activity and the fluorescence values are diminishing showing less efficacy in amplification.

[0069] After two weeks at 32° C. the reactions at higher concentrations are non-functional.

[0070] The pattern is repeated but exaggerated in the 37° C. tests where it can be seen that at 2 weeks at 37° C. the dried reaction containing only tris has completely failed in the red fluorescence, is very poor in the orange fluorescence and just about working only at low concentrations in the FAM channel.

Working Examples

[0071] The experiment (with the same conditions as above) was then repeated, this time replacing the tris buffers with HEPES pH 8.0 at varying concentrations, in accordance with an embodiment of the invention.

[0072] The HEPES containing buffers were dried as per the methods previously described and the results shown in Table 3

TABLE-US-00003 TABLE 3 data on 3-week stability using HEPES in air dried composition HEPES TP0 TP1 28 c TP1 37 c TP2 28 c TP2 37 c TP3 28 c TP3 37 c pH8.0 FF CP FF CP FF CP FF CP FF CP FF CP FF CP CalFlour RED610 20   892,091 25.79   982,917 25.72   729,868 26.37   746,778 26.08 5,411 #DIV/0!   681,876 26.73 7,698 #DIV/0! mM 30 1,008,673 25.73 1,108,360 25.53   971,687 26.03   903,910 25.96 533,877 27.49   796,482 26.27 225,308 32.85 mM 40 1,074,817 25.63 1,008,663 25.70   918,775 25.89 1,010,065 25.76 668,233 27.01   880,033 26.09 400,279 27.20 mM 50 1,008,634 25.65 1,030,270 25.72   900,183 25.96   938,229 25.96 648,531 26.42   784,684 26.21 381,381 26.97 mM 60 1,212,936 25.24 1,212,725 25.49   871,449 25.85 1,086,757 25.39 830,128 25.75   914,503 25.82 152,109 28.77 mM 70 1,278,079 25.31 1,170,598 25.65 1,064,040 25.70 1,126,608 25.44 985,246 25.72   938,929 26.05 701,556 26.32 mM 80 1,219,190 25.40 1,070,810 25.72 1,061,808 25.86 1,042,284 25.50 935,677 26.55 1,056,785 26.07 618,266 27.44 mM 90 1,167,274 25.44 1,097,234 26.03 1,004,293 26.06 1,098,709 25.47 679,583 26.46   972,910 26.16 325,961 28.52 mM Caflour Orange 560 20   447,228 26.94   530,149 26.86   439,731 27.68   478,255 26.72 21,888 #DIV/0!   455,122 27.22 25,099 #DIV/0! mM 30   550,317 26.80   568,917 27.10   525,460 27.70   549,970 26.55 407,173 28.03   482,825 27.15 239,941 29.84 mM 40   604,078 27.19   555,716 27.20   516,609 27.58   601,661 26.94 444,838 27.85   528,893 27.47 356,236 27.90 mM 50   589,227 27.11   557,454 27.85   515,372 27.31   562,933 27.36 422,780 27.70   490,287 27.32 346,233 27.24 mM 60   636,969 26.51   689,121 26.72   539,079 27.06   637,489 26.32 569,438 26.85   529,404 26.60 204,088 30.11 mM 70   738,288 26.51   667,991 27.27   636,669 27.23   667,699 26.41 635,250 27.14   574,773 27.18 515,480 26.83 mM 80   711,484 27.30   594,445 27.74   614,528 27.68   619,347 26.89 552,562 28.12   614,056 27.46 524,090 26.64 mM 90   696,240 27.31   664,984 26.86   579,623 27.68   633,991 27.31 410,055 28.10   614,821 26.61 318,171 28.03 mM FAM 20 1,374,242 26.54 1,391,435 26.95 1,330,491 28.02 1,379,889 26.11 30,857 #DIV/0! 1,363,067 27.07 312,227 34.89 mM 30 1,550,810 26.01 1,479,267 27.30 1,330,151 28.37 1,555,453 25.38 1,251,725 28.20 1,389,686 27.14 1,192,865 29.26 mM 40 1,447,142 27.84 1,371,329 27.61 1,306,909 27.76 1,502,855 26.77 1,131,914 28.66 1,293,229 28.02 1,420,533 28.24 mM 50 1,486,067 27.03 1,441,244 26.63 1,331,521 27.59 1,439,182 26.72 1,194,653 28.03 1,296,000 26.65 1,573,202 26.67 mM 60 1,656,613 25.92 1,597,425 27.10 1,437,913 27.02 1,559,157 25.92 1,429,090 27.30 1,416,478 27.05 1,123,832 29.32 mM 70 1,605,305 27.40 1,392,401 28.25 1,376,084 28.12 1,566,966 25.89 1,453,600 27.94 1,285,866 28.03 1,818,891 26.21 mM 80 1,428,624 28.42 1,296,857 28.35 1,341,319 28.41 1,404,135 27.15 1,312,113 28.78 1,369,914 28.40 1,577,736 27.94 mM 90 1,481,640 27.22 1,350,244 27.85 1,291,958 28.59 1,400,952 27.84 1,055,228 28.77 1,374,016 27.22 1,273,325 28.29 nM

[0073] At compositions above 40 mM both final fluorescence values (FF) and Cp values were stable at 37° C. for at least 3 weeks compared to failure at two weeks for the Tris-based versions shown in Table 2.

Long-Term Stability Study

[0074] A composition as per Table 1 in accordance with embodiments of the invention was formulated for long term stability studies at three different temperatures: −20° C., 4° C. and 32° C., shown in Tables 4a, 4b and 4c, respectively below.

[0075] Two different real time PCR primer and probe sets were utilised and given the reaction identities HPA-1ab and BG65. Reactions were prepared as previously described and the PCR plate strips were sealed with PCR caps and sealed in a foil pouch with a desiccant pouch to prevent light and moisture ingress. The real time 3-plex PCR reactions were both utilising the same HGH IAC oligos using CalFluor Red610 fluorescence as previously described, and the two allele channels utilised were CalFluor Orange 560 and FAM.

[0076] A ‘day zero test’ was used to establish the baseline for the two real time PCR reactions with the two DNA samples utilised throughout the experiment.

[0077] The reactions were considered positive if the following conditions were met:

TABLE-US-00004 Red channel +ve if FF > 1,000,000 RFU, & Cp > 25 < 34 Orange channel +ve if FF > 800,000 RFU, & Cp > 25 < 33 FAM channel +ve if FF > 1,000,000 RFU, & Cp > 25 < 34

[0078] The study illustrates that those example embodiments, using dry reaction components as disclosed herein in accordance with the invention remained active after 1 year at the elevated temperature of 32° C. This data compares favourably to tris-based reaction examples that are not stable after 2 weeks at 32° C.

TABLE-US-00005 TABLE 4 1 year stability data Table 4a Stored −20° C. in foil pouch with desiccant −20° C., 1 −20° C., 2 −20° C., 4 −20° C., 8 −20° C., 12 day zero month months months months months Plate 1 Plate 61 Plate 70 Plate 79 Plate 88 Plate 97 Reac- R610 R610 R610 R610 R610 R610 tion D Ex- Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA pected FF CP FF CP FF CP FF CP FF CP FF CP HPA- DN + 4,274,163 27.50 4,465,486 27.35 4,277,193 27.25 4,635,319 26.76 4,307,561 26.19 4,083,307 27.46 1ab A1 HPA- DN + 3,920,229 27.93 4,136,895 27.66 4,072,936 27.33 4,516,147 27.66 4,108,928 26.22 3,792,774 27.56 1ab A2 BG65 DN + 3,806,896 27.67 4,158,943 27.36 4,019,397 27.25 4,291,091 27.09 3,997,458 26.21 4,041,178 27.59 A1 BG65 DN + 4,047,448 27.89 4,702,533 27.26 4,225,402 27.33 4,423,207 27.93 4,417,546 25.99 4,200,765 27.48 A2 Table 4a Plate 1 Plate 61 Plate 70 Plate 79 Plate 88 Plate 97 Reac- O560 O560 O560 O560 O560 O560 tion D Ex- Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA pected FF CP FF CP FF CP FF CP FF CP FF CP HPA- DN + 1,855,897 30.75 1,947,722 30.58 1,860,973 30.46 2,052,017 30.03 1,927,184 29.91 1,834,431 30.69 1ab A1 BG65 DN + 5,960,056 30.11 6,697,150 29.68 6,256,776 29.36 6,700,824 30.00 6,504,509 27.54 6,414,525 29.71 A2 HPA- DN −   198,758  0.00   215,571  0.00   285,987  0.00   299,193  0.00   190,562  0.00   179,967  0.00 1ab A1 BG65 DN − 5,232,042 36.27 5,994,140 35.19 5,669,089 35.50 5,811,295 35.98 5,967,236 33.72 5,849,151 35.76 A2 Table 4a Plate 1 Plate 61 Plate 70 Plate 79 Plate 88 Plate 97 Reac- FAM FAM FAM FAM FAM FAM tion D Ex- Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA pected FF CP FF CP FF CP FF CP FF CP FF CP HPA- DN + 11,330,278 27.98 12,087,051 27.83 11,846,478 27.66 12,471,076 27.33 11,348,168 27.15 10,947,773 27.96 1ab A1 HPA- DN + 14,193,985 27.59 15,431,130 27.47 15,079,926 27.10 16,712,622 27.56 14,716,029 26.57 14,049,945 27.30 1ab A2 BG65 DN −   −342,350  0.00   −392,106  0.00   −302,871  0.00   −400,031  0.00   −441,206  0.00   −476,491  0.00 A1 BG65 DN −   −362,061  0.00   −388,242  0.00   −388,176  0.00   −401,190  0.00   −465,862  0.00   −467,381  0.00 A2 Table 4b Stored 4° C. in foil pouch with desiccant 4° C., 1 month 4° C., 2 months 4° C., 4 months 4° C., 8 months 4° C., 12 months Plate 62 Plate 71 Plate 80 Plate 89 Plate 98 Reac- Ex- R610 R610 R610 R610 R610 tion D pected Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA result FF CP FF CP FF CP FF CP FF CP HPA- DN + 4,655,499 27.20 4,513,128 26.14 4,039,185 27.22 4,082,121 27.39 3,835,899 27.53 1ab A1 HPA- DN + 4,262,928 27.49 4,113,765 26.27 3,830,204 27.36 3,912,511 27.93 3,681,906 27.53 1ab A2 BG65 DN + 4,100,826 27.68 4,292,196 25.91 3,817,734 27.26 3,890,431 28.15 3,795,536 27.62 A1 BG65 DN + 4,579,944 27.53 4,528,343 25.89 3,905,266 27.46 3,863,082 28.29 3,954,394 27.53 A2 Table 4b Plate 62 Plate 71 Plate 80 Plate 89 Plate 98 Reac- Ex- O560 O560 O560 O560 O560 tion D pected Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA result FF CP FF CP FF CP FF CP FF CP HPA- DN + 2,027,601 30.40 1,955,508 29.64 1,743,712 30.63 1,835,866 30.68 1,679,630 30.94 1ab A1 BG65 DN + 6,729,158 29.55 6,426,464 28.24 5,907,745 29.31 6,193,300 29.66 6,271,353 28.87 A2 HPA- DN −   223,489  0.00   256,879  0.00   250,575  0.00   153,702  0.00   163,367  0.00 1ab A1 BG65 DN − 6,000,782 35.32 5,872,005 34.63 5,263,808 35.55 5,691,067 35.57 5,806,485 34.52 A2 Table 4b Plate 62 Plate 71 Plate 80 Plate 89 Plate 98 Reac- Ex- FAM FAM FAM FAM FAM tion D pected Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA result FF CP FF CP FF CP FF CP FF CP HPA- DN + 12,565,316 27.68 11,903,427 27.03 10,735,200 27.77 10,998,578 27.86 10,431,208 28.01 1ab A1 HPA- DN + 15,774,975 27.32 14,809,750 26.58 14,135,356 27.15 14,226,396 27.76 13,684,465 27.41 1ab A2 BG65 DN −   −390,599  0.00   −355,787  0.00   −361,781  0.00   −429,394  0.00   −467,773  0.00 A1 BG65 DN −   −412,632  0.00   −361,592  0.00   −344,490  0.00   −441,525  0.00   −493,004  0.00 A2 Table 4c Stored 32° C. in foil pouch with desiccant 32° C., 1 month 32° C., 2 months 32° C., 4 months 32° C., 8 months 32° C., 12 months Plate 63 Plate 72 Plate 81 Plate 90 Plate 99 Reac- Ex- R610 R610 R610 R610 R610 tion D pected Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA result FF CP FF CP FF CP FF CP FF CP HPA- DN + 4,425,588 27.24 4,579,700 27.09 4,035,520 26.18 3,989,009 26.25 3,453,396 27.11 1ab A1 HPA- DN + 4,422,687 27.21 3,558,889 27.45 3,867,236 36.38 3,936,280 26.29 3,554,085 27.62 1ab A2 BG65 DN + 4,145,554 27.29 3,738,079 27.39 3,898,030 26.17 4,009,788 26.15 3,742,340 27.44 A1 BG65 DN + 4,504,940 27.23 4,289,849 27.48 4,067,059 26.27 4,759,142 25.48 3,830,815 27.55 A2 Table 4c Plate 63 Plate 72 Plate 81 Plate 90 Plate 99 Reac- Ex- O560 O560 O560 O560 O560 tion D pected Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA result FF CP FF CP FF CP FF CP FF CP HPA- DN + 1,906,248 30.53 1,932,223 30.52 1,657,414 30.17 1,703,615 30.19 1,356,415 31.18 1ab A1 BG65 DN + 6,652,584 29.48 6,148,614 29.78 5,835,810 28.29 6,034,462 27.98 5,587,700 30.62 A2 HPA- DN −   210,699  0.00   213,110  0.00   200,827  0.00   162,424  0.00   126,578  0.00 1ab A1 BG65 DN − 5,916,969 35.79 3,944,496 36.38 5,469,641 34.81 5,836,179 34.73 4,984,385 36.99 A2 Table 4c Plate 63 Plate 72 Plate 81 Plate 90 Plate 99 Reac- Ex- FAM FAM FAM FAM FAM tion D pected Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean ID NA result FF CP FF CP FF CP FF CP FF CP HPA- DN + 11,992,451 27.69 11,407,593 27.66 10,455,389 27.15 10,341,138 27.18  8,698,892 27.75 1ab A1 HPA- DN + 15,922,432 27.12 12,571,504 27.26 13,320,371 26.75 13,912,265 26.57 12,028,268 27.39 1ab A2 BG65 DN −   −402,292  0.00   −263,831  0.00   −323,044  0.00   −434,705  0.00   −416,573  0.00 A1 BG65 DN −   −383,518  0.00   −377,673  0.00   −321,353  0.00   −439,087  0.00   −414,986  0.00 A2