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Quantification of the percentage of lymphocyte-binding components in polyclonal anti-thymocyte/lymphocyte globulin
Wenqu Yin1, Haoyong Zou1, Yujuan Xu1
1YuJin Bio-pharma Basic Research Department, Wuhan Institute of Biological Products Co., Ltd., Wuhan 430207, China.
The immunomodulatory and immunosuppressive properties of anti-thymocyte globulin (ATG)/anti-lymphocyte globulin (ALG) are primarily facilitated through the interaction between the lymphocyte-binding components of ATG/ALG (active ATG) and the antigens expressed on various immune cell populations. Nevertheless, the precise concentration and proportion of active ATG present in ATG/ALG preparations have yet to be definitively ascertained. In this study, we employed a methodology integrating flow cytometry and ELISA to quantify the percentage of active ATG in ATG/ALG preparations. The method was replicated six times to assess active ATG in ATG-Fresenius utilizing Jurkat cells, resulting in a coefficient of variation (CV) of 11.05 %. Analysis of 4 porcine immune plasma batches showed significant inter-batch variability in active ATG concentrations and total IgG concentrations. Analysis of 12 batches of porcine anti-thymocyte globulin (pATG) stock solutions revealed that the mean percentages and CV value for active ATG were 45.08 % and 23.58 %, respectively. Moreover, the biological activity assay demonstrated a strong correlation between the biological activity of pATG and the percentage of active ATG in the pATG preparation (r = 0.9283). In conclusions, we have developed an absolute quantification method for active ATG, which will facilitate precise pharmacokinetic analysis of active ATG and contribute to improving the potency testing and dosing regimens of ATG.
The immunomodulatory and immunosuppressive properties of anti-thymocyte globulin (ATG)/anti-lymphocyte globulin (ALG) are primarily facilitated through the interaction between the lymphocyte-binding components of ATG/ALG (active ATG) and the antigens expressed on various immune cell populations. Nevertheless, the precise concentration and proportion of active ATG present in ATG/ALG preparations have yet to be definitively ascertained. In this study, we employed a methodology integrating flow cytometry and ELISA to quantify the percentage of active ATG in ATG/ALG preparations. The method was replicated six times to assess active ATG in ATG-Fresenius utilizing Jurkat cells, resulting in a coefficient of variation (CV) of 11.05 %. Analysis of 4 porcine immune plasma batches showed significant inter-batch variability in active ATG concentrations and total IgG concentrations. Analysis of 12 batches of porcine anti-thymocyte globulin (pATG) stock solutions revealed that the mean percentages and CV value for active ATG were 45.08 % and 23.58 %, respectively. Moreover, the biological activity assay demonstrated a strong correlation between the biological activity of pATG and the percentage of active ATG in the pATG preparation (r = 0.9283). In conclusions, we have developed an absolute quantification method for active ATG, which will facilitate precise pharmacokinetic analysis of active ATG and contribute to improving the potency testing and dosing regimens of ATG.
