Evaluation Report Of Stability Was Officially Released!

Evaluation of stability is the basis for setting the expiration date of the product, and the stability of in vitro diagnostic reagents is also an important indicator of the effectiveness of the product during use.

In December 2021, the“Announcement on In Vitro Diagnostic Reagent Registration Application Materials Requirements and Approval Document Format (No. 122 of 2021)”issued by the National Medical Products Administration clearly required that the evaluation of stability in vitro diagnostic reagents should submit the reports, including: real-time stability evaluation (shelf life), In-use stability and stability during transport.

Materials and Methods

The NEXome Core Panel and NadPrep NanoBlockers (for MGI, DI) were selected for stability testing

Freeze-thaw stability testing

Three independent batches of reagents were repeatedly frozen and thawed for several times. After repeated freezing and thawing for 0, 10, 20, 30, 40 and 50 times, the standard pre-libraries were used for hybridization capture and sequencing to verify the performance of two products through a number of basic quality control indicators, such as mappability, on-target, target covered and so on

Accelerated stability testing

Take three independent batches of reagents and divide them into several parts, one part is normally stored at -20°C±5°C, and the other six parts are stored at room temperature for 1 day, 3 days, 8 days, 12 days, 19 days and 28 days respectively, the standard pre-libraries were used for hybridization capture and sequencing to verify the performance of two products through a number of basic quality control indicators, such as mappability, on-target, target covered and so on.

Results of Freeze-thaw stability testing

NEXome Core Panel: After repeated frozen and thawed for 0, 10, 20, 30, 40 and 50 times, compared with the starting point (C0), there was no significant difference in the basic quality control performance of mappability and on-target rate (Fig. 1. A).

Likewise, the coverage uniformity under different freeze-thaw times, such as 0.2x mean depth of coverage, 0.5x mean depth of coverage, and Fold 80 base penalty, were also consistent with the C0 (Fig. 1. B & C). Among the three batches , there were only differences in GC bais (Fig. 1. D).

Fig. 1: Freeze-thaw stability testing performance of NEXome Core Panel.

A. Mappability and On-target rate; B. Target covered; C. Fold 80 Base Penalty; D.GC bias.

NadPrep NanoBlockers (for MGI, DI): After repeated frozen and thawed for 0, 10, 20, 30, 40 and 50 times, Compared with the C0, therewas no significant difference in basic quality control performance such as mappability and on-target rate, coverage uniformity and old 80 base penalty.

Among them, the average on-target rate at freezon-thawed for 10 times was slightly lower, which was due to the manual operation within the group (Fig. 2. A & B & C). The GC bias were also largely consistent in the three batches (Fig. 2. D).

Fig. 2: Freeze-thaw stability testing performance of NadPrep NanoBlockers (for MGI, DI).

A. Mappability and On-target rate; B. Target covered; C. Fold 80 Base Penalty; D.GC bias.

Results - Accelerated stability testing

NEXome Core Panel: stored at room temperature for 28 days can still maintain the stability of performance. Compared with the C0, there was no significant difference in the basic quality control performance of mappability and on-target rate (Fig. 3. A). In terms of coverage uniformity, the 0.2x mean depth of coverage, 0.5x mean depth of coverage, and Fold 80 base penalty were also consistent with the C0 (Fig. 3. B & C), with only differences in GC bias (Fig. 3. D).

Fig. 3: Accelerated stability testing performance of NEXome Core Panel.

A. Mappability and On-target rate; B. Target covered; C. Fold 80 Base Penalty; D.GC bias.

NadPrep NanoBlockers (for MGI, DI): stored at room temperature for 28 days can still maintain the stability of performance. Compared with the C0, there was basically no difference in the performance of mappability, but there was a certain fluctuation in the on-target rate in each group(Fig. 4. A). Among the 3-batch replicates within the group 2 and group 5 (stored at room temperature for 1 day and 12 days, respectively), the target rate of at least two batches was higher than 89%, which was met with expectations.

In terms of coverage uniformity, the 0.2x average coverage depth, 0.5x average coverage depth and Fold 80 base penalty are consistent with the C0 for each treatment group (Fig. 4. B & C), with only slight differences in high GC regions (Fig. 4.D).

Fig. 4: Accelerated stability testing performance of NadPrep NanoBlockers (for MGI, DI).

A. Mappability and On-target rate; B. Target covered; C. Fold 80 Base Penalty; D. GC bias

Conclusions

Whole exome panel: NEXome Core Panel and blockers: NadPrep NanoBlockers (for MGI, DI) are typical reagents in the targeted capture sequencing. When they were frozen and thawed for 50 times and stored at room temperature for 28 days, the comparison results of different dimensions show that the overall performance are relatively stable. We speculate that the possible reason is that since the oligonucleotide fragments, the main components of the reagents, are shorter, higher in concentration, and kept in a stable solvent, they are more resilient to environmental damage[1]. In order to ensure the long-term stability of the reagent performance, they should be used and stored in strict accordance with the protocol.

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