Objective: To establish, validate and apply an enzyme-linked immunosorbent assay (ELISA) method for determination of residual human factor Ⅻ(FⅫ) and prekallikrein (PK) in high concentration human intravenous immunoglobulin (10%IVIG). Methods: Microplates were pre-coated with specific antibodies for FⅫ and PK, respectively, and then blocked. The FⅫ and PK in 10%IVIG were allowed to bind with the pre-coated antibodies on the plates. After appropriate washing steps, biotinylated detection antibodies specific for FⅫ and PK were added. Following a second washing, HRP/Streptavidin-Peroxidase conjugates were added and unbound conjugates were washed away with another washing step. After the addition of a chromogenic substrate and a stop solution, the absorbance was measured at 450 nm using a microplate reader. The detection method was validated according to the 2020 edition of Chinese Pharmacopoeia. Then the validated method was used to determine the residual levels of FⅫ and PK in process intermediates and final products 10%IVIG. Results: The dilution linearity results showed a matrix effect and a hook effect in FⅫ and PK detection methods, respectively, which could be solved by diluting 5-20 times and 80-120 times, respectively. The average recoveries in the spiked experiments were 110.3% and 99.1% for FⅫ and PK respectively, with RSD of 3.6% and 2.7%. indicating good accuracy of the method. The RSDs of the repeatability validation were 8.3%、7.7% for both methods, and the RSD of intermediate precision validation were 7.0% and 9.2% for FⅫ and PK, respectively, indicating good precision of the two methods. The limits of quantitation of the two methods were 1.0 ng · mL-1 and 1.25 ng · mL-1 respectively. Both methods exhibited good linearity (r = 0.999 9). The robustness validation, which included incubation time, the effective period of the opened reagent kit, and different batches of reagent kits, showed good robustness of the method. The residues of high concentration human immunoglobulin FⅫ and PK maintained at low levels by using the method, indicating that octanoic acid precipitation combined with two-step anionic exchange chromatography process could effectively remove FⅫ and PK in 10%IVIG. Conclusion: The detection method shows good applicability and can be used for the determination of FⅫ and PK residues in 10%IVIG.
YU Yu-rong
,
LIU Yong
,
YANG Long
,
TANG Liang-yu
,
XIAO Chun-qiao
,
LI Dan
,
JIAN Chang-yong
. Determination of residual human factor Ⅻ and prekallikrein in high concentration human immunoglobulin for intravenous injection by enzyme-linked immunosorbent assay*[J]. Chinese Journal of Pharmaceutical Analysis, 2025
, 45(3)
: 514
-521
.
DOI: 10.16155/j.0254-1793.2024-0235
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