Sansure Biotech Inc.
Novel Coronavirus 2019-nCoVnucelci acid diagnostic kit (PCR-fluorescence
probing)
Study on stability
Contents
1. Purpose of stability study...................................................................................................................... 1
2. Basis for the determination of stability research methods.................................................................... 1
3. The specific method and process of accelerated destabilization research on reagent...........................1
3.1 Reagent and its source and proposed storage condition..............................................................1
3.2 Standards for determining whether the reagents are qualified.................................................... 1
3.3 Quality control of the test............................................................................................................2
3.4 Specific method and process of accelerated destabilization test.................................................2
3.5 Summary of the results of the accelerated destabilization experiment of the finished kit at
37 °C..................................................................................................................................................2
3.6 Conclusion...................................................................................................................................3
4. Study purpose of sample stability......................................................................................................... 3
5. Specific method and process of sample stability study.........................................................................3
5.1 Reagent and sample source......................................................................................................... 3
5.2 Technical indicator...................................................................................................................... 4
5.3 Quality control of the experiment............................................................................................... 4
5.4 Specific method and process of stability study........................................................................... 4
5.5 Summary of stability test results of samples stored at -20 °C.....................................................4
5.6 Conclusion on sample stability study..........................................................................................5
Sansure Biotech Inc.
1. Purpose of stability study
Stability is the basic attribute that in vitro diagnostic reagents must have, and an important
indicator to ensure that the product is safe and effective during use. The expiration date of the kit is a
common concern of manufacturers and users, and it is also a vital indicator for evaluating the quality
of the kit.
2. Basis for the determination of stability research methods
Due to the limited time and that it is less than one month from the production date of this kit, the
routine stability study of the kit has not been carried out, instead, the accelerated destructive stability
study has been carried out in advance to initially evaluate or predict the stability of this kit.
According to the relevant content stipulated in the Guidelines for Stability Testing of Bulk Drugs
and Pharmaceutical Preparations in Appendix XIXC, Part II of the Chinese Pharmacopoeia (2010
Edition) and the related literature (Zhang Li. Research on the stability of in vitro diagnostic reagents.
Chinese Journal of New Drugs, 2006, 15(22): 1899-1900), the accelerated destabilization studies were
designed.
3. The specific method and process of accelerated destabilization research on
reagent
3.1 Reagent and its source and proposed storage condition
The reagents used to carry out the accelerated destabilization study are the 2020001ZC batch,
2020002ZC batch, and 2020003ZC batch of finished kits that are continuously produced by Production
Department of Sansure Biotech Inc. and passed the inspection of the Quality Control Department. And
the production dates are 22th January 2020. The proposed storage temperature of this kit is -20 ± 5 °C.
3.2 Standards for determining whether the reagents are qualified
Fully considering the actual situation of this kit in clinical application, the testing items are in
accordance with the verification items of finished product, including the accuracy and specificity
(positive and negative coincidence rate), limit of detection (analytical sensitivity) and repeatability
(precision) of this kit.
3.2.1 Visual inspection
The outer packing box is intact, undamaged, and clean; The instructions are correct, clear and
complete; The appearance of each tube of reagent should be complete and the label should be clear and
undamaged, and the product name, batch number and expiration date should be clear and easy to
distinguish; After the reagent has melted, the solution is clear with no obvious precipitation or
suspension.
3.2.2 Technical indicator
2019-nCoV-PCR-Positive control, 2019-nCoV-PCR-negative control and enterprise references
are used ad the test samples.
1)Accuracy (Positive coincidence rate): 10 positive reference samples of enterprise references for
2019-nCoV virus were tested by using the diagnostic kit, and the results are all positive.
2)Specificity (Negative coincidence rate): 13 negative reference samples of enterprise references
for 2019-nCoV virus were tested by using the diagnostic kit, and the results are all negative.
3)Precision: Precision references R1 and R2 were repeatedly tested 10 times, respectively, and
CV of their Ct value is less than 5%。
4)Limit of detection: Detection limit reference sample S was tested, and the test result is negative.
1
Sansure Biotech Inc.
3.3 Quality control of the test
1) 2019-nCoV-PCR-negative control: FAM, ROX channels and internal standard (HEX) channel
have no Ct value or Ct > 40;
2) 2019-nCoV-PCR-Positive control: Ct value of FAM, ROX channels and internal standard (HEX)
channel are ≤ 35;
3) The above requirements must be met at the same experiment, otherwise, this experiment is invalid
and needs to be repeated.
3.4 Specific method and process of accelerated destabilization test
Three batches of kits were placed in the incubator at 37 °C, and was taken out after 48 h and 72
h, respectively. The enterprise references were used as the test samples and were then tested by using
the aforementioned kits according to the operation manual, to test if each indicator meets the
requirements of Section 3.2 and Section 3.3.
3.5 Summary of the results of the accelerated destabilization experiment of the
finished kit at 37 °C
Table 1 Test results of the accelerated destabilization experiment of the 2020001 batch of reagents at
37 °C
Storage time (accelerated
48 h 72 h
at 37 °C)
Outer appearance Qualified Qualified
CV of precision Ct value (ORF R1 (%) 0.15 0.12
1ab) R2 (%) 0.70 0.92
R1 (%) 0.12 0.14
CV of precision Ct value (N)
R2 (%) 0.66 0.76
Positive reference 10/10 10/10
Coincidence rate Negative reference 13/13 13/13
Detection limit reference Positive Positive
FAM 27.65 27.39
Positive control HEX 24.76 24.65
ROX 24.52 24.40
FAM NoCt NoCt
Negative control
HEX NoCt NoCt
ROX NoCt NoCt
Conclusion Qualified Qualified
Table 2 Test results of the accelerated destabilization experiment of the 2020002 batch of reagents at
37 °C
Storage time
48 h 72 h
(accelerated at 37 °C)
Outer appearance Qualified Qualified
CV of precision Ct value (ORF R1 (%) 0.14 0.11
1ab) R2 (%) 0.69 0.91
R1 (%) 0.10 0.14
CV of precision Ct value (N)
R2 (%) 0.79 0.81
Positive reference 10/10 10/10
Negative reference 13/13 13/13
Coincidence rate
Detection limit
Positive Positive
reference
FAM 27.28 26.79
Positive control HEX 24.31 24.41
ROX 24.18 24.53
Negative control FAM NoCt NoCt
2
Sansure Biotech Inc.
HEX NoCt NoCt
ROX NoCt NoCt
Conclusion Qualified Qualified
Table 3 Test results of the accelerated destabilization experiment of the 2020003 batch of reagents at
37 °C
Storage time
48 h 72 h
(accelerated at 37 °C)
Outer appearance Qualified Qualified
R1 (%) 0.10 0.14
CV of precision Ct value (ORF 1ab)
R2 (%) 0.50 0.62
R1 (%) 0.10 0.13
CV of precision Ct value (N)
R2 (%) 0.58 0.66
Positive reference 10/10 10/10
Negative reference 13/13 13/13
Coincidence rate
Detection limit
Positive Positive
reference
FAM 28.65 28.31
Positive control HEX 25.19 25.07
ROX 24.50 24.72
FAM NoCt NoCt
Negative control HEX NoCt NoCt
ROX NoCt NoCt
Conclusion Qualified Qualified
This kit is qualified after being accelerated at 37 °C for 48 h and 72 h, and the acceleration
stability is stable.
3.6 Conclusion
The performance of this kit is still stable after accelerating at 37 °C for 72 h. Referring to the
expiration date of the similar registered products of our company using the same technology platform,
such as Hepatitis C virus nucleic acid diagnostic kit (PCR-fluorescence probing), Coxsackie virus A16
nucleic acid detection kit (PCR-fluorescence probing) Enterovirus universal nucleic acid detection kit
(PCR-fluorescence probing), Enterovirus 71 nucleic acid detection kit (PCR-fluorescence probing) and
Universal nucleic acid detection kit for influenza A virus (PCR-fluorescence probing), the tentative
storage condition and expiration date of this kit are as follows: the kit can be stored at -20 ± 5 °C, and
its expiration date is 12 months.
4. Study purpose of sample stability
Stability is the basic attribute that in vitro diagnostic reagents must have, and an important
indicator to ensure that the product is safe and effective during use. At the same time, the study of
sample stability is crucial to the success of the experiment.
By carrying out sample stability studies under different conditions, it provides a basis for
determining the storage time of applicable samples at -20 °C for the new coronavirus 2019-nCoV
nucleic acid diagnostic kit (fluorescent PCR method).
According to the clinical use, the storage conditions and storage time of the samples were studied.
5. Specific method and process of sample stability study
5.1 Reagent and sample source
The reagents used for the sample stability study are the company's 2020001ZC batch of kits and
its production date is 22th January 2020.
The samples used for the stability study are the clinical samples of 2019-nCoV coronavirus
3
Sansure Biotech Inc.
collected from the hospital. Nucleic acid sample was collected from Wuhan Jinyintan Hospital. The
clinical positive sample was collected from Sanway medical laboratory center, and the patient was
confirmed to be positive for the new coronavirus by Hunan Provincial Center for Disease Control and
Prevention on 21th January 2020.
5.2 Technical indicator
Clinical nucleic acid and clinical sample of 2019-nCoV virus at the high, medium and low
concentration are used as the test samples, and each sample was repeatedly tested 10 times. The results
should meet the following requirements:
1) All 10 tests for both samples were positive (Ct ≤ 40)
2) The coefficient of variation (CV,%) of the two samples for 10 tests is not more than 5%;
3) The difference between the average Ct value of 10 times of the current test and the value of 10
times of the reference control test is not more than 1.
5.3 Quality control of the experiment
1) 2019-nCoV-PCR-negative control: FAM, ROX channels and internal standard (HEX)
channel have no Ct value or Ct > 40;
2) 2019-nCoV-PCR-Positive control: Ct value of FAM, ROX channels and internal standard
(HEX) channel are ≤ 35;
3) The above requirements must be met at the same experiment, otherwise, this experiment is
invalid and needs to be repeated.
5.4 Specific method and process of stability study
5.4.1 Sample detection
2019-nCoV nucleic acid samples and clinical samples at high, medium and low concentration
were chosen and repeatedly tested 10 times to calculate the average Ct value, so as to provide
reference for the sample stability study below.
5.4.2 Stability test of samples stored at -20 °C
1) Nucleic acid samples of 2019-nCoV virus at high, medium and low concentration were placed
in refrigerator at -20 °C and were taken out after 15 days, and then were tested by using the qualified
kit of our company and operating according to the manual, to test whether each indicator meet the
requirements of Section 2.2 and Section 2.3.
2) Clinical samples of 2019-nCoV virus at high, medium and low concentration were placed in
refrigerator at -20 °C and were taken out after 10 days, and then were tested by using the qualified kit
of our company and operating according to the manual, to test whether each indicator meet the
requirements of Section 2.2 and Section 2.3.
5.5 Summary of stability test results of samples stored at -20 °C
Table 1 Ct value of stability test results of nucleic acid sample stored at -20 °C for 15 days
Storage condition Control (Orf 1ab) -20 °C (Orf 1ab) Control (N) -20 °C (N)
Medi Medi Medi Medi
Storage time High Low High Low High Low High Low
um um um um
1 18.62 26.64 32.13 18.92 27.08 32.11 17.14 23.66 31.18 17.09 23.79 31.92
2 18.64 26.58 32.31 18.91 26.99 32.01 17.20 23.63 31.39 17.07 23.77 31.42
3 18.72 26.58 32.39 18.93 25.59 32.34 17.25 23.61 31.63 17.06 23.76 32.11
4 18.60 26.59 32.36 19.02 25.61 32.51 17.24 23.87 31.08 17.07 23.78 31.69
4
Sansure Biotech Inc.
5 18.59 26.24 32.29 18.99 26.99 32.18 16.99 23.55 30.99 17.06 23.64 31.17
6 18.66 26.68 32.18 18.98 27.01 32.38 17.16 23.65 31.36 17.22 23.52 31.02
7 18.67 25.42 32.22 19.00 26.42 32.22 17.03 24.09 31.44 17.13 23.18 31.04
8 18.69 25.41 32.28 18.94 26.90 32.61 17.11 24.05 31.33 17.28 23.41 31.32
9 18.64 26.28 32.31 18.96 26.90 32.35 17.10 23.42 31.43 17.02 23.71 31.49
10 18.63 26.66 32.44 18.87 27.00 32.22 17.20 23.75 31.51 16.99 23.52 31.22
Average 18.65 26.31 32.29 18.95 26.65 32.29 17.14 23.73 31.33 17.10 23.61 31.44
0.21 1.88 0.29 0.24 2.18 0.56 0.50 0.91 0.63 0.52 0.85 1.17
CV, %
% % % % % % % % % % % %
The difference from the
average Ct value of the 0.3 0.34 0 -0.04 -0.12 0.11
control
Quali Quali Quali Quali Quali Quali Quali Quali
Conclusion
fied fied fied fied fied fied fied fied
Table 2 Ct value of stability test results of clinical sample stored at -20 °C for 10 days
Storage condition Control (Orf 1ab) -20 °C (Orf 1ab) Control (N) -20 °C (N)
Medi Medi Medi Medi
Storage time High Low High Low High Low High Low
um um um um
1 18.63 26.63 32.06 18.93 26.61 32.39 17.22 23.69 31.39 17.13 23.52 31.6
2 18.63 26.63 32.1 20.01 26.95 32.38 17.00 23.71 31.62 17.71 23.52 31.5
3 18.60 26.57 32.74 18.92 25.51 32.15 17.13 23.59 31.54 16.99 23.76 31.27
4 18.64 26.69 32.13 19.03 25.71 32.31 17.12 23.69 31.24 17.12 24.01 31.76
5 18.62 26.48 32.31 18.79 26.84 31.96 17.10 23.60 31.05 16.91 23.39 31.09
6 18.57 26.51 32.24 18.91 26.76 32.14 17.11 23.60 31.27 17.2 23.34 31.63
7 18.96 25.43 32.36 18.86 26.81 32.57 17.21 24.02 31.24 17.09 23.43 31.29
8 18.67 25.43 32.22 18.9 25.69 31.96 17.22 24.09 31.31 17.04 24.00 31.55
9 18.66 26.49 32.33 18.98 25.61 32.45 17.17 23.61 31.52 17.05 24.05 31.22
10 18.53 26.36 32.34 19.03 25.70 32.04 17.12 23.56 31.24 17.15 23.79 31.47
Average 18.65 26.32 32.28 19.04 26.22 32.24 17.14 23.72 31.34 17.14 23.68 31.44
0.62 1.82 0.60 1.84 2.34 0.67 0.40 0.79 0.56 1.27 1.16 0.67
CV, %
% % % % % % % % % % % %
The difference from the
average Ct value of the 0.39 -0.1 -0.04 0 -0.04 0.1
control
Quali Quali Quali Quali Quali Quali Quali Quali Quali Quali Quali Quali
Conclusion
fied fied fied fied fied fied fied fied fied fied fied fied
5.6 Conclusion on sample stability study
From the test results of above-mentioned nucleic acid samples and clinical samples stored at
-20 °C, the following conclusions can be drawn:
1) The nucleic acid samples were valid after being stored at -20 °C for 15 days. The difference
between the average Ct value of 10 times of the current test and the value of 10 times of the reference
control test is not more than 1. The test result is qualified.
2) The clinical sample were valid after being stored at -20 °C for 10 days. The difference
between the average Ct value of 10 times of the current test and the value of 10 times of the reference
control test is not more than 1. The test result is qualified.
5
Sansure Biotech Inc.