BACKGROUNDReagents used for molecular diagnostics are typically stored frozen or at least refrigerated to maintain viability. The proprietary NeuDry process developed by NeuMoDx offers a simple, low cost, and rapid process using a conventional conveyer oven to produce ready to use, ambient condition stable reagents capable of providing long open-shelf life for active reagents used in molecular diagnostic testing. The performance of reagents produced using the NeuDry process across wide variety of molecular diagnostic applications is presented here.

RESULTSNeuDry extraction reagents demonstrated >24 months shelf life and 63 days in-use stability in real time stability studies, with excellent sensitivity in a variety of matrices, such as plasma, serum, whole blood, urine and swab specimens. The NeuDry PCR/RT-PCR reagents, for both qualitative and quantitative assays, provided excellent amplification relative to liquid reagents as measured by Ct shift (<1 cycle) and relative fluorescence increase (>80%). The PCR Master Mix formulated for the detection of GBS provided 25 months shelf life when stored at 18-28°C and up to 63 days in-use shelf life in real time stability studies. For both DNA and RNA viral quantitative assays, formulations for detecting HBV, CMV and HCV, have demonstrated over 12 months stability and up to 63 days in-use shelf life with no loss of activity in real time and/or accelerated stability studies. The NeuDry process has also proven to be easily applicable to master mixes using different Taq polymerases without substantial re-formulation. The entire drying process takes less than 30 min resulting in over 10,000 tests produced in a single 8-hour shift with two operators and one conveyor oven. In addition, the continuous workflow enables significantly easier automation (& reduced cost) compared to batch processes such as lyophilization process.

CONCLUSIONNeuDry reagents demonstrated excellent nucleic acid isolation from several clinical matrices as well as efficient and robust PCR/RT-PCR amplification using the eluted DNA/RNA directly. In addition, the NeuDry reagents have demonstrated a long shelf life of 25 months with an in-use life of 63 days.

ACKNOWLEDGMENTSThe authors gratefully acknowledge the help and support provided by all members of the NeuMoDx team. We would also like to thank our collaborators for their invaluable assistance in providing access to clinical specimens for testing and evaluation.NeuMoDx GBS Test and NeuMoDx Extraction Plates on NeuMoDx 288 is For In Vitro Diagnostics only. NeuMoDx HBV, CMV and HCV tests are For Research Use Only. Not for use in diagnostic procedures.

METHODSThree types of reagents formulated and optimized for both long term storage and in-use shelf life were produced using the NeuDry process - 1) Nucleic acid isolation reagents including NeuMag™ affinity particles, lytic enzyme, and encapsulated DNA/RNA process controls, 2) PCR Master mix, and 3) RT-PCR Master mix. Performance of the NeuDry reagents was initially evaluated against the liquid version of these reagents. Efficacy of the reagents was further evaluated by testing across a variety of clinical matrices encompassing both DNA and RNA targets. Long-term stability and in-use shelf life was determined via accelerated and real time stability studies on fully automated NeuMoDx Molecular System.

PCR OR RT-PCR FORMULATIONNUCLEIC ACID EXTRACTION FORMULATION

DRY, 15-20 MIN

PACKAGE

DRYING PROCESS

DISPENSE

READY TO USE MOLECULAR DIAGNOSTIC REAGENTS WITH LONG TERM AMBIENT AND ON-BOARD STORAGE FOR FULLY AUTOMATED SYSTEMSBetty Wu, Elizabeth Craig, Crystal Nguyen, Hui Lin Lee, Lijie Gong, Will Marshall, Mark Olson, and S Brahmasandra, NeuMoDx Molecular, Inc, Ann Arbor, MI

NEUMODX GBS TEST STRIP: REAL TIME STABILITY STUDY, 25 MONTHS

NEUMODX EXTRACTION PLATE STABILITY – REAL TIME, 25 MONTHS

NEUMODX HBV QUANT ASSAY TEST STRIP: REAL TIME STABILITY - 13 MONTHS

NEUMODX CMV QUANT ASSAY TEST STRIP: ACCELERATED STUDY

NEUMODX HCV QUANT ASSAY TEST STRIP: ACCELERATED STUDY

NEUMODX HBV QUANT ASSAY TEST STRIP: IN-USE STABILITY – 63 DAYSNEUMODX EXTRACTION PLATE STABILITY – IN-USE, 63 DAYS AT 25 MONTH

• Two lots NeuMoDx GBS Test Strips were pre-aged as part of real time stability study for 12 months.

• The test strips were removed from the packaging, and stored at “in-use” conditions - average temperature at 28°C & average of 40-45% humidity - for up to 63 days

• Activity of the strips accessed by testing of the aged strips on a NeuMoDx System using GBS spiked into Lim Broth at concentration of 1000 CFU/mL.

• 31 replicates of GBS positive samples and 3 negatives tested for each lot of GBS Test Strip per pre-storage condition.

• The test strips aged 63 days at “in-use” conditions performed equivalently to day 0 based on the positive detection rate and ΔCt from time 0.

• NeuMoDx Group B Strep (GBS) Assay is a qualitative in vitro diagnostic test designed to detect Group B Streptococcus (GBS) DNA.

• Three lots of GBS Test Strips were stored at ambient temperature (~ 23°C) and 28°C at an average 40-45% humidity for 25 months.

• Stability of the NeuMoDx GBS Test Strips by testing aged strips on a NeuMoDx System using GBS spiked into Lim Broth at concentration of 1000 CFU/mL.

• 27 replicates of GBS positive samples and 3 negatives tested for each lot of NeuMoDx GBS Test Strip per storage condition.

• Aged group performed equally well as unaged Control group based on positive detection rate of 100% demonstrated for both storage conditions.

• NeuMoDx Extraction Plate contains all the reagents required for DNA and RNA extraction, such as lytic enzymes and Neu magnetic beads, as well as DNA and RNA Sample Process Controls.

• Three lots of Extraction Plates stored at ambient temperature (~ 23°C) and 28°C with average 40-45% humidity for 25 months

• Stability of the Extraction Plates was evaluated by testing of the aged Extraction Plates on the NeuMoDx System using NeuMoDx HCV Assay (GBS Assay Data previously presented in the figures on the left.)

• Stability of the Extraction Plates for RNA assays was evaluated with a model RNA assay, NeuMoDx HCV Assay. HCV viral particles were spiked into human plasma at concentration of 60 IU/mL (27 replicates) and processed on a NeuMoDx Molecular System using aged and unaged extraction plates.

• Aged group performed equally well based on the 100% amplification of both HCV target and the RNA Sample Process Control with low SD of Ct values.

• Three lots of NeuMoDx HBV Quant Assay Test Strips were stored at ambient (~ 23°C) and 28°C with average 40-45% humidity for 13 months.

• Activity of the test strips was evaluated by testing the aged test strips on a NeuMoDx System using human plasma spiked with HBV at concentrations of 5.5, 3.5 (10 replicates at each level) and 1.4 log10 IU/mL (20 replicates) per lot for each storage condition

• Linear regression analysis demonstrated minimal drift after 13 months - ≤ 1%, in quantification of HBV after storage for 13 months.

• NeuMoDx CMV Assay is an in vitro diagnostic test for quantitative detection of Human Cytomegalovirus (CMV) DNA in human plasma.

• Three lots of CMV Test Strips were stored at 50°C up to 61 days, equivalent to 13 months at 23°C.

• The activity of the strips were accessed by testing the aged strips on the NeuMoDx System using human plasma spiked with CMV at concentrations of 5, 3 and 2 log10 IU/mL (6 replicates per level) per lot for each storage condition.

• There is minimum drift, ≤ 0.25%, in quantification of CMV after storage for 61 days at 50°C (equivalent to 13 month when stored at 23°C) with 100% detection rate at all the levels.

• Three lots of test strips performed reproducibly with SD < 0.07 Log10 IU/mL across all the testing points and concentrations.

• Representative PCR amplification curves from one of the three lots of HCV Test Strips are shown above.

• NeuMoDx HCV Assay is a in vitro diagnostic test for quantitative detection of Human Hepatitis C Virus (HCV) RNA in human plasma and serum.

• Three lots of HCV Test Strips were stored at 40°C up to 149 days, equivalent to 17 months at 23°C.

• The activity of the strips was evaluated by testing the aged strips on the NeuMoDx System using human plasma spiked with HCV at concentrations of 5, 3 and 1.88 log10 IU/mL (6 replicates per level) per lot for each storage condition.

• After storage up to 149 days at 40°C, the test strips quantified HCV targets with minimum bias to the expected concentrations. The difference between D149 and D0 is < 0.17 Log10 IU/mL and almost no drift, ≤ 0.07%, was observed when analyzed with linear regression.

• Three lots of test strips performed reproducibly with SD < 0.17 Log10 IU/mL across all the testing points and concentrations.

• Representative PCR amplification curves from one of the three lots of HCV Test Strips are shown above.

• In-Use stability study of NeuMoDx HBV Test Strips. HBV Test Strips were first aged at ambient or 28°C as part of the Real Time Stability study. Two lots of HBV Test Strips were removed from their primary storage condition and stored at In-Use condition (31°C with average 40-45% humidity) up to 61 days.

• The activity of the strips was evaluated by testing the aged strips on the NeuMoDx System using human plasma spiked with HBV at concentrations of 5.5, 3.5 (10 replicates at each level) and 1.4 log10 IU/mL (20 replicates) per lot for each storage condition.

• There is minimum drift, ≤ 0.07%, in quantification of HBV up to 61 days at the In-Use condition for both pre-aging groups, ambient or 28°C. The data from the 28 °C pre-aging group are presented here.

• Two lots NeuMoDx Extraction Plates were pre-aged as part of real time stability study for 25 months.

• The Extraction Plates were removed from the packaging, and stored at “in-use” conditions - average temperature at 31°C & average of 40-45% humidity - for up to 63 days

• Stability of the Extraction Plates was evaluated by testing of the aged Extraction Plates on the NeuMoDx System using NeuMoDx HCV Assay (GBS Assay Data previously presented in the figures on the left.)

• HCV viral particles were spiked into human plasma at concentration of 60 IU/mL and processed on a NeuMoDx Molecular System.

• Aged group performed equally well based on the 100% amplification of both HCV target and RNA sample process control with low SD of Ct values.

Pre-Aging  Ambient (N=62) 28°C (N=62)

In-Use, Days Avg SD % Pos Avg Ct SD % Pos

0 32.9 0.59 100% 32.8 0.55 100%

15 32.6 0.13 100% 33.1 0.60 100%

32 32.5 0.11 100% 32.6 0.36 100%

43 33.6 0.13 100% 32.7 0.37 100%

63 32.8 0.52 100% 33.2 0.53 100%

ΔCt -0.1   0.4    

25

27

29

31

33

35

37

Lot 1 Lot 2 Lot 3

Cycl

e

NeuMoDx GBS Test Strip Real Time Stability, 25 Months, GBS

Controls Ambient (23°C) 28°C

25

26

27

28

29

30

31

32

33

34

Lot 1 Lot 2 Lot 3

Cycl

e

NeuMoDx GBS Test Strip Real Time Stability, 25 Months, Sample Process

Control

Controls Ambient (23°C) 28°C

28

29

30

31

32

33

34

35

1 2 3 4 5

Cycl

e

NeuMoDx GBS Test Strip, In-Use Stability Study

Ambient (N=62) 28°C (N=62)

-37

4963

9963

14963

19963

24963

29963

1 3 5 7 9 11131517192123252729313335373941

Fluo

resc

ence

Cycle

Lot 1, 63 Days, (28°C Pre-aging Group)

28

30

32

34

36

38

Lot 1 Lot 2 Lot 3

Cycl

e

N=27

NeuMoDx Extraction Plates, Real Time, RNA (HCV), 25 months

Control Ambient 28°C

-722

9278

19278

29278

39278

49278

1 6 11 16 21 26 31 36 41 46 51

Fluo

resc

ence

Cycles

HCV Amplification

-18

982

1982

2982

3982

4982

5982

6982

7982

8982

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49

Fluo

resc

ence

Cycle

SPC2 PCR Amplification

27

28

29

30

31

32

Lot 1 Lot 2 Lot 3

Cycl

e

N=27

NeuMoDx Extraction Plates, Real Time, RNA Sample Process Control, 25 months

Control Ambient 28°C

30

31

32

33

34

35

36

37

Lot 1 Lot 2

Cycl

e

Extraction Plate, In-Use 63 Days, RNA (HCV)

Control (N=27) HCV, Ambient (N=31) 28°C (N=31)

2728282929303031313232

Lot 1 Lot 2

Cycl

e

Extraction Plate, In-Use 63 Days, RNA (Sample Process Control)

Control (N=27) HCV, Ambient (N=31) 28°C (N=31)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 2 4 6 8 10 12 14

HBV

Conc

. Log

10 IU

/mL

Storage Time in Months

HBV Test Strip Stability, Real Time, 13 Months

5.5 Log10 IU/mL, 28°C 3.3 Log10 IU/mL, 28°C1.4 Log10 IU/mL, 28°C 5.5 Log10 IU/mL, Ambient3.3 Log10 IU/mL, Ambient 1.4 Log10 IU/mL, Ambient

NeuMoDx Test Strips Shelf Life, 13 Months

Storage Condition

Time 0 3 Months 7 Months 13 MonthsSlope %

DriftΔTime 0 at 13 MAvg SD Avg SD Avg SD Avg SD

Ambient 5.48 0.01 5.49 0.02 5.53 0.07 5.53 0.04 0.0038 0.38% -0.02

28°C 5.48 0.01 5.46 0.05 5.57 0.03 5.58 0.01 0.0094 0.94% 0.10

Ambient 3.41 0.05 3.42 0.05 3.52 0.03 3.52 0.06 0.0100 1.00% -0.05

28°C 3.41 0.05 3.42 0.05 3.52 0.02 3.52 0.04 0.0098 0.98% 0.11

Ambient 1.22 0.02 1.28 0.05 1.28 0.04 1.31 0.07 0.0065 0.65% -0.01

28°C 1.22 0.02 1.28 0.03 1.28 0.04 1.31 0.13 0.0015 0.15% 0.09

Expected HBV Conc,

[Log10 IU/mL]

D0 D15 D32 D43 D63 Δ fr D0 [Log10 IU/

mL]%

DriftAvg SD Avg SD Avg SD Avg SD Avg SD

5.63 5.58 0.02 5.56 0.01 5.59 0.01 5.59 0.09 5.60 0.03 0.02 0.03%

3.63 3.51 0.01 3.47 0.01 3.54 0.00 3.52 0.05 3.55 0.05 0.03 0.07%

1.40 1.28 0.04 1.27 0.13 1.41 0.04 1.33 0.11 1.28 0.03 0.00 0.06%

-112.00

4888.00

9888.00

14888.00

19888.00

24888.00

1 5 9 13 17 21 25 29 33 37 41 45

Fluo

resc

ence

Cycle

HBV Test Strip, Ambient Storage

-329.00

4671.00

9671.00

14671.00

19671.00

24671.00

29671.00

1 4 7 10131619222528313437404346

Fluo

resc

ence

Cycle

HBV Test Strip, 28°C Storage

HB

V C

onc

[L

og10

IU

/mL]

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 20 40 60 80

HBV

Conc

entr

atio

n, L

og10

IU/m

L

Days in the In-Use Condition

HBV Test Strip, In-Use Stability Study, 63 Days

5.5 Log10 IU/mL 3.5 Log10 IU/mL 1.4 Log10 IU/mL

-37

4963

9963

14963

19963

24963

29963

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41

Fluo

resc

ence

Cycle

Lot 1, 63 Days, (28°C Pre-aging Group)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 10 20 30 40 50 60 70

CMV

Conc

. Log

10 IU

/mL

Storage Time in Days

CMV Test Strip, Accelerated Study, 50°C

5 log10 IU/mL 3 log10 IU/mL 2 log10 IU/mL-262

4738

9738

14738

19738

24738

29738

34738

39738

44738

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46

Fluo

resc

ence

Cycle

CMV Test Strip, 61 Days at 50°C

D0 33 Days (7 Months Equivalent) 61 Days (13 Months Equivalent)%

DriftAvg Δ fr D0CMV

Conc Lot 1 Lot 2 Lot 33

Lots Avg

SD Lot 1 Lot 2 Lot 33

Lots Avg

SD Lot 1 Lot 2 Lot 33

Lots Avg

SD

5.00 log10 IU/mL

4.87 4.93 4.84 4.88 0.05 4.89 5.03 4.95 4.96 0.07 4.92 5.05 4.93 4.97 0.07 0.14 0.09

3.00 log10 IU/mL

2.90 2.86 2.89 2.88 0.02 2.99 3.00 2.95 2.98 0.03 3.01 3.04 3.05 3.03 0.02 0.25 0.15

2.00 log10 IU/mL

2.00 1.88 1.88 1.92 0.07 2.00 1.99 1.85 1.95 0.08 2.04 2.12 2.05 2.07 0.04 0.24 0.15

0

1

2

3

4

5

6

0 50 100 150

HCV

Conc

, Log

10 IU

/mL

Storage Time in Days

HCV Test Strip, Accelerated Stability Study at 40°C

-254.00

9746.00

19746.00

29746.00

39746.00

49746.00

59746.00

1 4 7 1013161922252831343740434649

Fluo

resc

ence

Cycle

HCV – 149 days at 40°C

HCV Expected Conc.

[Log10 IU/mL]

D0 D44 (Eq 5 months)

D70 (Eq 8 months)

D96 (Eq 11 months)

D149 (Eq 17 months) Δ fr

D0%

DriftAvg SD Avg SD Avg SD Avg SD Avg SD

5.00 5.16 0.03 4.82 0.07 4.89 0.02 4.90 0.15 5.00 0.17 -0.16 0.07%3.00 3.11 0.05 2.76 0.11 2.81 0.02 2.86 0.09 2.94 0.08 -0.17 0.07%1.88 1.86 0.10 1.61 0.06 1.65 0.07 1.69 0.12 1.76 0.17 -0.11 0.04%