Establishment of In Vitro Alternative Methods for Cysteine Pyrogen Testing and Comparison with the Rabbit Method

doi:10.12300/j.issn.1674-5817.2026.029

Abstract

Abstract:

Objective This study attempts to establish a cysteine bacterial endotoxin test as an in vitro alternative method for cysteine pyrogen detection, so as to reduce the use of rabbits in pyrogen detection and ensure the welfare of experimental animals. Methods According to the Chinese Pharmacopoeia (2025 edition) Volume Ⅳ, General Chapters 1142 and 1143, and Guidelines 9251 and 9301, methodological studies on pyrogen and bacterial endotoxin testing were conducted on three batches of cysteine raw material. We used the pyrogen test (rabbit method, RPT) and the in vitro pyrogen test (monocyte activation test, MAT) to detect pyrogens in cysteine and assessed cell viability during the MAT for one batch. Bacterial endotoxin testing was performed using the gel-clot method (limit test) and the bacterial endotoxin photometric assay (microkinetic chromogenic assay, kinetic chromogenic assay, kinetic turbidimetric assay). Results Pyrogen testing of cysteine using the two pyrogen test methods yielded false-negative results. In the RPT, all the test articles spiked with endotoxin did not show a positive pyrogen response. The MAT results showed that cysteine inhibited interleukin-6 (IL-6) secretion by human peripheral blood mononuclear cells (hPBMCs) after incubation, with recovery rates below 50%. Interference was observed in bacterial endotoxin testing using the gel-clot method: all three batches showed interference at dilutions not exceeding the maximum valid dilution (MVD). In the interference test using the microkinetic chromogenic method, when cysteine was prepared at a concentration of 1.05 mg/mL, the recovery rates for all three batches were between 50% and 200%, indicating that the interference was eliminated and the method was feasible. The recovery rates measured by the microkinetic chromogenic assay, kinetic chromogenic assay and kinetic turbidimetric assay fell within the acceptable range of 50%–200% when the cysteine concentration was 1.05 mg/mL. The bacterial endotoxin levels in the three batches met the specified limits. Conclusion This study establishes a bacterial endotoxin assay for cysteine and preliminarily demonstrates that the photometric techniques can replace RPT for the bacterial endotoxin testing of cysteine.

Key words: Cysteine, Bacterial endotoxin test, Micro kinetic chromogenic assay, Pyrogen test, Monocyte activation test, Rabbits

CLC Number:

R917
R927.12

GAI Yating,LIN Lihua. Establishment of In Vitro Alternative Methods for Cysteine Pyrogen Testing and Comparison with the Rabbit Method[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 740-749. DOI: 10.12300/j.issn.1674-5817.2026.029.

Figures/Tables 6

References 25

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组别 Group	体重/kg Weight/kg	正常体温/℃ Normal temperature/℃	给药前最大升温/℃ Maximum temperature rise before drug administration/℃	给药前最大降温/℃ Maximum temperature reduction before drug administration/℃	升温总和/℃ Sum of temperature rise/℃	降温总和/℃ Sum of temperature reduction/℃
热原阳性对照组 Pyrogen positive control group	3.0	38.7	0.8	0.0	2.2	-0.1
	3.0	38.5	1.2	0.0
	3.2	38.9	0.2	-0.1
空白对照组 Blank control group	2.5	38.2	0.4	0.0	0.9	-0.2
	2.2	38.6	0.1	-0.2
	2.4	38.6	0.4	0.0
半胱氨酸（S202302014）组 Cysteine group（S202302014）	2.2	38.6	0.4	0.0	0.8	-0.4
	2.3	38.6	0.2	-0.4
	2.3	38.7	0.2	0.0
半胱氨酸（S202302014）+内毒素对照组 Cysteine （S202302014）+CSE group	2.2	38.6	0.2	0.0	0.7	-0.1
	2.1	38.2	0.5	0.0
	2.2	38.1	0.0	-0.1
半胱氨酸（S202302015）组 Cysteine （S202302015） group	2.8	39.1	0.1	-0.2	0.2	-0.5
	2.6	39.2	0.0	-0.3
	2.6	38.8	0.1	0.0
半胱氨酸（S202302015）+内毒素对照组 Cysteine （S202302015）+CSE group	2.8	39.0	0.0	-0.1	0.6	-0.6
	2.8	38.1	0.5	-0.3
	2.7	38.8	0.1	-0.2
半胱氨酸（S202302016）组 Cysteine （S202302016） group	2.6	38.9	0.0	-0.3	0.0	-0.6
	2.4	38.7	0.0	-0.3
	2.4	38.9	0.0	0.0
半胱氨酸（S202302016）+内毒素对照组 Cysteine （S202302016）+CSE group	3.0	38.7	0.0	-0.5	0.4	-1.0
	2.9	38.0	0.4	0.0
	3.0	38.5	0.0	-0.5

组别 Group	体重/kg Weight/kg	正常体温/℃ Normal temperature/℃	给药前最大升温/℃ Maximum temperature rise before drug administration/℃	给药前最大降温/℃ Maximum temperature reduction before drug administration/℃	升温总和/℃ Sum of temperature rise/℃	降温总和/℃ Sum of temperature reduction/℃
热原阳性对照组 Pyrogen positive control group	3.0	38.7	0.8	0.0	2.2	-0.1
	3.0	38.5	1.2	0.0
	3.2	38.9	0.2	-0.1
空白对照组 Blank control group	2.5	38.2	0.4	0.0	0.9	-0.2
	2.2	38.6	0.1	-0.2
	2.4	38.6	0.4	0.0
半胱氨酸（S202302014）组 Cysteine group（S202302014）	2.2	38.6	0.4	0.0	0.8	-0.4
	2.3	38.6	0.2	-0.4
	2.3	38.7	0.2	0.0
半胱氨酸（S202302014）+内毒素对照组 Cysteine （S202302014）+CSE group	2.2	38.6	0.2	0.0	0.7	-0.1
	2.1	38.2	0.5	0.0
	2.2	38.1	0.0	-0.1
半胱氨酸（S202302015）组 Cysteine （S202302015） group	2.8	39.1	0.1	-0.2	0.2	-0.5
	2.6	39.2	0.0	-0.3
	2.6	38.8	0.1	0.0
半胱氨酸（S202302015）+内毒素对照组 Cysteine （S202302015）+CSE group	2.8	39.0	0.0	-0.1	0.6	-0.6
	2.8	38.1	0.5	-0.3
	2.7	38.8	0.1	-0.2
半胱氨酸（S202302016）组 Cysteine （S202302016） group	2.6	38.9	0.0	-0.3	0.0	-0.6
	2.4	38.7	0.0	-0.3
	2.4	38.9	0.0	0.0
半胱氨酸（S202302016）+内毒素对照组 Cysteine （S202302016）+CSE group	3.0	38.7	0.0	-0.5	0.4	-1.0
	2.9	38.0	0.4	0.0
	3.0	38.5	0.0	-0.5

样品批号 Sample batch number	稀释倍数 Dilution factor	不加内毒素的实测值/（EU·mL^-1） Measured value without endotoxin/(EU·mL^-1)	加入0.06 EU/mL的内毒素 After addition of 0.06 EU/mL endotoxin		回收率/% Recovery rate/%
样品批号 Sample batch number	稀释倍数 Dilution factor		实测值/（EU·mL^-1） Measured value/(EU·mL^-1)	标准差 s	回收率/% Recovery rate/%
S202302014	5	<0.015 0	0.09	0.002	17.0
	10	<0.015 0	0.37	0.006	49.0
	20	<0.015 0	0.64	0.003	40.3
	25	<0.015 0	1.03	0.007	57.1
S202302015	30	<0.007 5	0.60	0.003	17.2
	40	<0.007 5	1.00	0.012	25.6
	45	<0.007 5	0.86	0.002	17.9
	50	<0.007 5	1.05	0.004	19.7
S202302016	30	<0.007 5	0.57	0.004	12.8
	40	<0.007 5	0.76	0.001	14.7
	45	<0.007 5	1.08	0.006	23.6
	50	<0.007 5	0.85	0.002	13.4

样品批号 Sample batch number	稀释倍数 Dilution factor	不加内毒素的实测值/（EU·mL^-1） Measured value without endotoxin/(EU·mL^-1)	加入0.06 EU/mL的内毒素 After addition of 0.06 EU/mL endotoxin		回收率/% Recovery rate/%
样品批号 Sample batch number	稀释倍数 Dilution factor		实测值/（EU·mL^-1） Measured value/(EU·mL^-1)	标准差 s	回收率/% Recovery rate/%
S202302014	5	<0.015 0	0.09	0.002	17.0
	10	<0.015 0	0.37	0.006	49.0
	20	<0.015 0	0.64	0.003	40.3
	25	<0.015 0	1.03	0.007	57.1
S202302015	30	<0.007 5	0.60	0.003	17.2
	40	<0.007 5	1.00	0.012	25.6
	45	<0.007 5	0.86	0.002	17.9
	50	<0.007 5	1.05	0.004	19.7
S202302016	30	<0.007 5	0.57	0.004	12.8
	40	<0.007 5	0.76	0.001	14.7
	45	<0.007 5	1.08	0.006	23.6
	50	<0.007 5	0.85	0.002	13.4

稀释倍数 Dilution factor	外加内毒素/（EU·mL^-1） Endotoxin spiking/（EU·mL^-1）	实测值/（EU·mL^-1） Measured value/（EU·mL^-1）	标准差 s	回收率/% Recovery rate/%	细胞存活率/% Cell survival rate /%
4	/	<0.03	/	/	/
4	0.06	<0.03	/	/	/
8	/	<0.03	/	/	59
8	0.06	<0.03	/	/	68
16	/	<0.03	/	4.67	/
16	0.06	0.53	0.002	4.67	/
30	/	<0.03	/	18.37	94
30	0.06	1.23	0.003	18.37	93
60	/	<0.03	/	18.91	/
60	0.06	2.52	0.003	18.91	/
120	/	<0.03	/	17.37	92
120	0.06	4.92	0.001	17.37	93