CHO Host Cell DNA Residue Detection Kit (3G) Validation Report

CHO Host Cell DNA Residue Detection Kit (3G)

Qualification Summary (Cat No.: 41332ES60)

1. Background

The data summarized in this report are performed by Yeasen Biotechnology for CHO Host Cell DNA Residue Detection Kit (3G) 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 CHO Host Cell DNA Residue Detection Kit (3G), Vendor: Yeasen, Cat No.: 41332ES60.

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

2.3 Original data: the electronic version were recorded in the 'Experiment Report' the subfile of the parent file CHO Host Cell DNA Residue Detection Kit (3G).

2.4 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 3 fg/μL~300 pg/μL with R²=1, and the amplification efficiency was 99.09% with CV<15% for each concentration assay.

 

Figure 1 CHO DNA (3G) qPCR Standard Curve

3.2 Accuracy

3.2.1 Recovery

3.2.1.1 Blank Sample Spiking Recovery Experiment

Samples of CHO DNA standards at high and low concentrations: 30 pg/μL, 300 fg/μL, and 3 fg/μL were prepared, respectively, and assayed after extraction using the Magnetic Bead Method for Residual DNA Sample Pre-treatment Kit (3 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	Extract 3 mean	Average concentration	CV	Recovery
Blank Sample	/	/	/		/	/	/
Recovery sample 1	30 pg/μL	27.07	27.51	28.27	27.62	2.20%	92.06%
Recovery sample 2	300 fg/μL	285.53	301.25	295.71	294.16	2.71%	98.05%
Recovery sample 3	3 fg/μL	3.50	3.54	3.31	3.45	3.56%	115.00%

Table1 Blank Sample Spiking Recovery

3.2.1.2 Simulated Sample Spiking Recovery Experiment

Samples of the same concentration (300 fg/μL of CHO DNA) were extracted (3 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	Extract 3 mean	Average concentration	CV	Recovery
Basic Sample	/	/	/		/	/	/
High Protein	300 fg/μL	302.29	332.86	342.76	325.97	6.47%	108.66%
High Nuclear Acid		284.47	290.66	308.76	294.63	4.28%	98.21%
High-salt		272.96	286.25	312.91	290.71	7.00%	96.90%
High pH		296.04	320.58	324.56	313.72	4.92%	104.57%
Low pH		314.77	327.89	340.26	327.64	3.89%	109.21%

Table2 Basic Sample Spiking Recovery

3.3 Precision

3.3.1 Repeatability

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

Repetitions	1	2	3	4	5	6	7	8	9	10	Mean	CV%
Detection Value (fg/μL)	3.13	3.26	2.89	3.16	2.95	2.87	2.88	2.75	2.87	3.15	2.99	5.65%

Table3 Repeatability Results

3.3.2 Intermediate precision

Three experimenters independently tested CHO DNA standard samples at high and low concentrations: 300 pg/μL, 3 pg/μL, and 3 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	CHO DNA (3G)
		300 pg/uL	3 pg/uL	3 fg/uL
Exp 1	Determination 1	302.63	2.97	3.40
	Determination 2	294.54	3.01	3.14
	Determination 3	287.67	2.99	3.01
Exp2	Determination 4	292.27	2.92	3.47
	Determination 5	299.61	2.90	2.82
	Determination 6	305.73	2.93	2.90
Exp 3	Determination 7	301.06	3.17	3.15
	Determination 8	299.17	3.00	3.21
	Determination 9	290.66	2.90	3.05
/	Mean	297.04	2.98	3.13
/	CV%	2.03	2.80	6.85

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 CHO 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 Mouse, MDCK, HEK293 and E.coli genomic DNAs with CHO DNA detection reagents).

Figure 2 Interference experiment results

3.5 Quantitation Limit

CHO DNA was detected at 3 fg/μL, 1 fg/μL, 0.5 fg/μL, 0.3fg/uL and 0.1 fg/μL, with 10 replicates for each concentration. The results showed that the CV was <20% at concentrations of 0.3 fg/μL and above. That is, the limit of quantification of the CHO host cell DNA residue detection kit (3G) was 0.3 fg/μL.

Repetitions Test Item	CHO DNA (3G) (fg/μL)
	Quantity	Recalculation Ratio%
1	0.28	93.33%
2	0.26	86.67%
3	0.32	106.67%
4	0.30	100.00%
5	0.33	110.00%
6	0.23	76.67%
7	0.29	96.67%
8	0.37	123.33%
9	0.31	103.33%
10	0.28	93.33%
Mean	0.30	/
CV%	13.09%	/

 

                                 Figure 3.  CHO DNA (3G) qPCR Test Result

3.6 Detection Limit

CHO DNA was detected at 0.3 fg/μL, 0.1 fg/μL, 0.05 fg/μL and 0.01 fg/μL, with 20 replicates for each concentration. The results showed that the detection rate was ≥19 (i.e., detection rate ≥95%) in 20 replicate wells at concentrations of 0.05 fg/μL and above. That is, the limit of detection of the CHO host cell DNA residue detection kit (3G) was 0.05 fg/μL.

Figure 4. CHO DNA (3G) qPCR Test Result

3.7 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	CV	Limit of Detection	Detection Rate
Thermo	ABI 7500	99.69%	1	0.3 fg/μL	12.40%	0.05 fg/μL	95.65%
Thermo	ABI QuantStudio5	99.26%	1	0.3 fg/μL	15.30%	0.05 fg/μL	100.00%
Roche	LightCycler480	2.05%	0.978	0.3 fg/μL	/	0.05 fg/μL	/
Shanghai Hongshi	SLAN	101.14%	0.999	0.3 fg/μL	14.41%	0.05 fg/μL	95.65%

Table5 Instrument suitability test results

3.8 Stability

3.8.1 Freeze-thaw stability

CHO Host Cell DNA Residue Detection Kit (3G) 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	95.70%	98.62%
	R2	1	1
Limit of Quantification (0.3 fg/μL)	CV	15.31%	17.16%
Limit of Detection (0.05 fg/μL)	Detection Rate	95.65%	100%

Table6 Freeze-thaw stability result analysis

3.7.2 Accelerated Stability

CHO Host Cell DNA Residue Detection Kit (3G) 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	100.53%	102.61%	101.76%	101.84%
	R2	1	1	1	1
Limit of Quantification (0.3 fg/μL)	CV	17.16%	12.63%	16.27%	12.49
Limit of Detection (0.05 fg/μL)	Detection Rate	95.65%	100%	95.57%	100%

Table7 Accelerated stability result analysis

3.9 Limit of Blank

3.9.1 No Template Control (NTC)

No template control (NTC) was the DNA dilution with 96 repetitions to do qPCR quantitative assay, all CT values above the 96 replicate wells tested were >40.

Figure 5 NTC experiment results

3.9.2 Negative Extraction Control Sample (NCS)

Negative Extraction Control Sample (NCS) was the DNA dilution, and it was first extracted by sample pre-treatment, then qPCR was done, all CT values above the 48 replicate wells tested were >40.

 

Figure 6 NCS experiment results

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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