Collaborative studies on the development of national reference standards for potency determination of H7N9 influenza vaccine

Figures & data

Figure 1. SDS–PAGE analysis of H7N9 whole virus vaccine bulk. The gel was stained by Coomassie blue. The bands were identified by mass spectrometry.

Table 1. Collaborative study to determine the HA content of H7N9 vaccine bulk by SDS-PAGE

Lab. No.	Protein content, (μg/ml, intra CV% )	HA% by SDS-PAGE, (intra CV %)	HA content, (μg/ml)
1	364.00 (4.05)	37.28 (3.00)	135.70
2	349.60 (1.80)	35.88 (0.71)	125.44
3	370.33 (0.95)	36.80 (0.95)	136.28
Average	361.31 (2.94)	36.65 (1.94)	132.47
SD	10.62	0.71	6.10

Note: (1) Three laboratories, including the National Institutes for the Food and Drug Control (NIFDC), Sinovac Biotech Co., Ltd and Hualan Biological Engineering Co., Ltd, took part in the collaborative study. (2) Each laboratory carried out 3 individual tests for protein content; for HA% determined by SDS-PAGE, each laboratory performed 6 separate deglycosylation treatments, analyzed by duplicate gels. SD: standard deviation.

Table 2. HA contents of 18 vials freeze-dried H7N9 antigen reference

Sample	Average μg HA /vial ±SD
1	55.50±0.50
2	58.50±0.87
3	57.50±2.18
4	54.17±0.76
5	57.50±1.80
6	59.50±3.50
7	55.83±3.33
8	57.67±2.52
9	58.50±3.97
10	53.83±1.61
11	55.67±5.01
12	55.17±4.75
13	56.17±1.26
14	56.67±2.08
15	59.50±3.04
16	58.83±1.04
17	57.17±2.25
18	57.50±1.32
Average	56.95±1.70

Table 3. Collaborative study to determine the HA content of freeze-dried H7N9 antigen reference by SRID

HA contents (μg /vial)
Test No.	Lab 1	Lab 2	Lab 3	Lab 4	Lab 5	Average
1	53.38	57.79	52.72	57.67	54.97	\
2	51.04	57.64	53.39	55.85	55.20	\
3	55.55	55.73	51.57	52.76	55.20	\
4	57.08	56.32	52.79	60.63	54.97	\
5	55.35	57.19	53.00	55.83	55.40	\
6	54.11	58.57	52.67	62.32	55.33	\
Average	54.42	57.21	52.69	57.51	55.18	55.40
SD	2.09	1.03	0.61	3.49	0.18	2.00
CV(%)	3.84	1.81	1.16	6.08	0.32	3.62

Note: (1) Five laboratories took part in the collaborative study (3 labs in Table 1 and Shanghai Institute of Biological Products and Beijing Tiantan Biological Products Co., Ltd). (2) H7N9 vaccine bulk tested in Table 1 was used as standard antigen. (3) Each laboratory carried out 6 individual SRID tests for HA content by 2 persons. (4) The “/” denotes not applicable since the values derived from different labs cannot be averaged.

Table 4. Comparison of 2 methods for HA measurement of vaccine bulk derived from strain NRBRG-267

		Inactived vaccine		Split vaccine
		Lot 1	Lot 2	Lot 3	Lot 4
SDS-PAGE	protein conc. (μg/ml)	679.20	753.53	606.62	521.17
	HA% by SDS-PAGE	36.84	37.92	54.45	66.21
	HA content (μg/ml)	250.22	285.74	330.30	345.07
SRID	HA content (μg/ml)	220.13	271.96	347.27	351.82
Recovery (%)*		113.67	105.07	95.11	98.08

Note: The recovery rate refers to the percentage ratio between the HA contents obtained by SDS-PAGE and that determined by the SRID.