Design and analysis of lyophilized formulation for extending shelf life of Tuberculin PPD

Abstract

Tuberculin test is currently used for diagnosis and epidemiological surveys of tuberculosis. Mantoux method involves the intradermal injection of purified protein derivative (PPD) at the International unit dose. This study aimed to develop a dry PPD preparation to extend the shelf life of tuberculin PPD. Therefore, 5.0% Sucrose (S), 6.5% Mannitol (M), 2.5% Trehalose (T) and 0.3% Haemaccel (H) were added to each formulation. In vivo and in vitro analyses were carried out after lyophilization in order to determine the efficacy of the tuberculin activity. In the in vivo test, the activities of tuberculin shown by Delayed type hypersensitivity (DTH) responses to lyophilized preparations were compared to DTH responses to liquid preparation (CL) after injection into Bacillus Calmette Guerin (BCG) vaccinated guinea pigs. The results from preparations with added H, M, T, and S were 100%, 90%, 89%, and 60% of those from CL respectively. There was no loss of tuberculin activity in H formula. A statistically significant difference was found between S and CL (P <0.05). The cellular test for Interferon-gamma (IFN-γ) secretions was performed by using whole blood of human subjects screened for having DTH responses to tuberculin PPD Mantoux tests. They were assayed for Cell mediated immunity (CMI) responses caused by the stimulation of tuberculin antigens in the formulations. The detection of IFN-γ secretions was done by using Enzyme-Linked Immunosorbent Assay and the efficacy was expressed in terms of percentage of IFN-γ responses to tuberculin antigens. The results of CL, H, M, T and S were 3.28%, 10.40%, 0.84%, 1.52% and 1.29% respectively. The lyophilized H, M and T formulations and the liquid CL were studied for their shelf-life stabilities. Accelerated degradation was induced by storing the samples at 4°C and higher temperatures of 37°C and 56°C for 3, 6, 9 and 12 months. All tuberculin PPD solutions were injected into BCG vaccinated guinea pigs at the end of each storage period and the;"การทดสอบการติดเชื้อวัณโรคด้วยทูเบอร์คูลินในปัจจุบันยังนิยมใช้วิธี Mantoux ที่ใช้ทูเบอร์คูลินชนิด purified protein derivative (PPD) ฉีดเข้าชั้นผิวหนังตามขนาดฉีดมาตรฐาน ซึ่งน้ำยามีความเจือจางสูงของปริมาณโปรตีน ทำให้ไม่มีความคงทน อายุของน้ำยาสั้น งานวิจัยนี้มีวัตถุประสงค์ในการพัฒนาสูตรตำรับทูเบอร์คูลิน PPD เป็นชนิดแห้งเพื่อยืดอายุการใช้งา"