E.coli Host Cell DNA Residue Detection Kit (2G) Validation Report

E.coli Host Cell DNA Residue Detection Kit (2G)

Qualification Summary (Cat No.: 41308ES60)

1. Background

The data summarized in this report are performed by Yeasen Biotechnology for 'E.coli Host Cell DNA Residue Detection Kit (2G) product. The summary data is only for the user's reference, and the user needs to verify the host cell residue DNA method by using their own samples to confirm the method can meet the user's requirements. All laboratories who using this kit are recommended to verify the following parameters: Linearity, Range, Accuracy, Precision, Quantitation Limit, Specificity, and Robustness.

Yeasen Biotechnology can provide the detailed qualification report for this kit. If the user uses this report, then only the suitability (which included Precision, Accuracy and Specificity) should be verified.

2. Experimental materials and methods

2.1 E.coli Host Cell DNA Residue Detection Kit (2G), Vendor: Yeasen, Cat No.: 41308ES60.

2.2 MolPure® Magnetic Residual DNA Sample Preparation Kit, Vendor: Yeasen, Cat No.: 18461ES60.

2.3 National standard for E.coli DNA content: Cat. 270027, Lot No.: 201101, concentration: 96.2 μg/mL, storage condition: -70℃, purchased from: National Institutes for Food and Drug Control.

2.4 Original data: the electronic version were recorded in the 'Experimental Report' the subfile of the parent file 'E.coli Host Cell DNA Residue Detection Kit (2G)'.

2.5 Methods: The materials and operation methods used for the experiment were basically produced or set by Yeasen Biotechnology, which were assessed and verified for a long time, the kit development and manufacture are followed the ISO 13485 system.

3. Qualification contents and results

3.1 Detection range

The linear range of the kit was 30 fg/μL~300 pg/μL with R²=1, and the amplification efficiency was 101.74% with CV<15% for each concentration assay.

Figure 1 E.coli DNA (2G) qPCR Standard Curve

3.2 Accuracy

3.2.1 Deviation from National standard for E.coli DNA content

The E.coli DNA reference material in each batch of the kit was calibrated using the National standard for E.coli DNA content. Yeasen DNA reference material was basically free of deviation from the National standard for E.coli DNA content calibration curve, and the deviation of the assay value was <5%.

3.2.2 Recovery

3.2.2.1 Blank Sample Spiking Recovery Experiment

Samples of E.coli DNA standards at high and low concentrations: 300 pg/μL, 3 pg/μL, and 30 fg/μL were prepared, respectively, and assayed after extraction using the Magnetic Bead Method for Residual DNA Sample Pre-treatment Kit (2 extraction replicates were performed for each sample, and 3 assay replicates were done for each extraction), and the sample recoveries and CVs were analyzed.

For different concentrations of DNA samples, the recoveries ranged from 70% to 130%, and the CVs were all <20%.

Sample type	Theoretical Concentration	Extract 1 mean	Extract 2 mean	Average concentration	CV	Recovery
Blank Sample	/	/	/	/	/	/
Recovery sample 1	300 pg/μL	250.56 pg/μL	262.11 pg/μL	256.33 pg/μL	4.41%	85.44%
Recovery sample 2	3 pg/μL	2.29 pg/μL	2.56 pg/μL	2.42 pg/μL	7.09%	80.77%
Recovery sample 3	30 fg/μL	33.62 fg/μL	32.61 fg/μL	33.12 fg/μL	14.28%	110.39%

Table1 Blank Sample Spiking Recovery

3.2.2.2 Simulated Sample Spiking Recovery Experiment

Samples of the same concentration (3 pg/μL of E.coli DNA) were extracted (2 extraction replicates were performed for each sample and 3 assay replicates were done for each extraction) with 5 different background solutions (high protein, high nucleic acid, high and low pH, high salt) and sample recoveries and CVs were analyzed.

DNA sample recoveries for different background solutions ranged from 70% to 130%, with all CVs <20%.

Sample type	Theoretical Concentration	Extract 1 mean	Extract 2 mean	Average concentration	CV	Recovery
Basic Sample	/	/	/	/	/	/
High Protein	3 pg/μL	2.25	2.23	2.24	1.09%	74.74%
High Nuclear Acid		3.93	3.96	3.95	1.56%	131.52%
High-salt		2.69	2.67	2.68	2.63	89.20%
High pH		2.90	3.67	3.29	13.82%	109.55%
Low pH		2.77	2.87	2.82	3.41%	94.01%

Table2 Basic Sample Spiking Recovery

3.3 Precision

3.3.1 Repeatability

Repeat the assay 10 times for E.coli DNA standards at a concentration of 3 pg/μL with a CV <15%.

Repetitions	1	2	3	4	5	6	7	8	9	10	Mean	CV
Detection Value (fg/μL)	3.16	2.87	2.97	2.86	2.81	2.97	3.14	3.21	3.14	3.1	3.02	4.78%

Table3 Repeatability Results

3.3.2 Intermediate precision

Three experimenters independently tested E.coli DNA standard samples at high and low concentrations: 300 pg/μL, 3 pg/μL, and 30 fg/μL, and three replicates were done for each concentration, and the CVs of all nine data obtained were <15%.

Exp	Actual Concentration Theoretical Concentration	E.coli DNA (2G)
		300 pg/uL	3 pg/uL	30 fg/uL
Exp 1	Determination 1	295.65	3.22	26.90
	Determination 2	287.71	3.24	32.00
	Determination 3	300.23	3.17	27.50
Exp 2	Determination 4	306.06	3.13	37.30
	Determination 5	299.76	3.02	32.70
	Determination 6	299.63	3.02	34.00
Exp 3	Determination 7	266.70	3.01	35.90
	Determination 8	272.89	3.09	40.70
	Determination 9	276.51	3.01	35.20
/	Mean	289.46	3.10	33.60
/	CV	4.89%	3.02%	13.18%

Table4 Intermediate precision Results

3.4 Specificity

The interference of cellular genomic DNA commonly used in the production of biologics was assessed for interference with E.coli DNA detection reagents, and the interference group overlapped with the control group and no interference was seen (the figure below shows, in order, the interference data of HEK293, Vero, and CHO genomic DNAs with E.coli DNA detection reagents).

 

Figure 2 Interference experiment results

3.5 Quantitation Limit

E.coli DNA was detected at 50 fg/μL, 40 fg/μL, 30 fg/μL, 10 fg/μL, and 5 fg/μL, with 10 replicates for each concentration. The results showed that the CV was <20% at concentrations of 30 fg/μL and above. That is, the limit of quantification of the E.coli host cell DNA residue detection kit (2G) was 30 fg/μL.

Repetitions Test Item	E.coli DNA (2G) (fg/μL)
	Quantity	Recalculation Ratio
1	29.43	98.09%
2	29.51	98.35%
3	32.04	106.79%
4	26.07	86.89%
5	34.06	113.53%
6	33.54	111.79%
7	26.95	89.82%
8	27.38	91.26%
9	27.04	90.13%
10	26.10	87.02%
Mean	29.21	/
CV	10.40%	/

 

 

                                 Figure 3 30fg/μL E.coli DNA (2G) qPCR Test Result

3.6 Robustness

This kit has been tested and is suitable for, but not limited to, the following instruments:

Vendor	Instrument Models	Amplification Efficiencies	R2	Limit of quantification (fg/μL)	CV
Thermo	ABI 7500	101.74%	1	30	13.30%
Thermo	ABI QuantStudio5	100.46%	1	30	12.40%
Bio-Rad	CFX96	99.20%	0.999	30	13.76%
Shanghai Hongshi	SLAN	100.40%	0.999	30	11.67%

Table5 Instrument suitability test results

3.7 Stability

3.7.1 Freeze-thaw stability

E.coli Host Cell DNA Residue Detection Kit (2G) was tested by repeated freezing and thawing 10 times, and the performance of the kit was not affected.

Testing Indicators Freeze-thaw Times	Parameter	T0 time	Freeze-thaw 10 times
Standard Curve Parameter	Amplification Efficiencies	98.23%	100.20%
	R2	1	0.999
Limit of quantification (30 fg/μL)	CV	15.07%	9.66%

Table6 Freeze-thaw stability result analysis

3.7.2 Accelerated Stability

E.coli Host Cell DNA Residue Detection Kit (2G) were stored at 2~8°C for 30 days and 37°C for 14 days, respectively. None of the performance of the kit was affected.

Testing Indicators Acceleration Temperature & Time	Parameter	Experiment 1		Experiment 2
		T0 time	2~8℃ 30 Days	T0 time	37℃ 14 Days
Standard Curve Parameter	Amplification Efficiencies	97.77%	99.81%	98.39%	98.65%
	R2	1	1	1	1
Limit of quantification (30 fg/μL)	CV	11.04%	13.79%	9.55%	14.55%

Table7 Accelerated stability result analysis

 

4. 4.Reference

4.1 Chinese Pharmacopoeia Commission (ChPC). Pharmacopoeia of People's Republic of China (Vol Ⅲ) [S]. Beijing：China Medical Science and Technology Press，p.542-543, 2020.

4.2 NMPA, 2007: General Principles for Technical Review of Analytical Method Validation for Quality Control of Biological Products.

4.3 ICH(2022)《Analytical method validation Q2》, draft version.

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