1. Snake and scorpion bites represent a serious public health problem around the world. Antitoxic sera preparations are the only treatment options. These products are composed of antibodies from immunized animals, especially equines (horses). The purification of antibodies (immunoglobulin fragments) should aim at obtaining products of consistent quality, safety, efficacy and adhering to the GMP principles. Endotoxins are an integral component of the outer cell surface of Gram-negative bacteria. Endotoxins are shed from living bacteria and are also released into the environment where bacteria die and decompose. Due to the existence of bacteria in air, water, and soil, bacterial endotoxins are common contaminates of the raw materials and processing equipment used in the manufacturing of antivenoms. in this work, we establish an environmental monitoring program for identification of potential sources of endotoxin contamination, due to air or personnel, throughout the whole process of antivenom preparation. In addition, we attempt to optimize the biochemical test used for endotoxin detection during preparation and purification of the product. The limulus amebocyte lysate or (LAL) test is the standard pharmacopeial test for endotoxin detection. Carrying out the test on the samples collected at various stages of preparation of the antivenom proved to be a challenging task due to the presence of different sources of interference that needed to be eliminated for proper testing procedures. Removal strategies for endotoxin are under evaluation to complete the process aiming at the production of endotoxin free product that can pass quality control testing both in Egypt and outside. Determination of Possible Sources of Endotoxin Contamination during Different Steps of Antisera Production Norhan. S. Sheraba 1, Aymen. S. Yassin 2, Mohamed. R. Diab 1, Magdy. A. Amin 2 and Hamdallah. H. Zeden 2 1 VACSERA, the Holding Company for Biological Products and Vaccines, Giza, 22311, Egypt. 2 Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt. Determination of sources of antisera contamination and identification of contaminants. Detection and quantitative determination of endotoxins at different steps during the preparation of antisera. Depyrogenation of pyrogenic antisera by various techniques and validation of the depyrogenation methods. MIB-3 Immunization of horses (or sheep) Selection and collection of venom Quality control for endotoxin Final product Fractionation and purification of F(ab') 2 or purification Production of Animal Antivenom Bleeding and separation of plasma/serum Poor Physicochemical Characteristics Of Antisera Protein aggregates Poor stability of proteins Residual non-IgG proteins and fragment Presence of bacterial products (LPS) F(ab')2 fragments were prepared by pepsin digestion after adjusting the pH of plasma to 3.3 by 1.76N acetic acid followed by the addition of 3.5g pepsin/liter plasma. Digestion was performed at 22-25°C for 1 hour, and the pH was elevated to 3.6 using 1N NaOH for 30 min. Afterward plasma pH was readjusted to 5.8 and the mixture was incubated for 15 min. at 56°C followed by centrifugation for 10 min. at 900 x g to remove fibrinogen. Caprylic acid was added dropwise to the undiluted plasma to attain a final concentration of 5% (v/v). The mixture was stirred vigorously for 24 h, Afterwards, NaCl and tricresole were added to a final concentration of 0.15M and 0.35%, respectively. The preparation was sterilized by filtration through 0.22-μm membranes. NaCl Saline Diluted plasma Digested solution Pepsin14% NHSO4 first precipitation mixture Horse plasma Supernatant Precipitate Second precipitation mixture 32% NHSO4 Precipitate Supernatant Resuspended solution NaCl Saline Purified serum Acetic acid + caprylic acid Diafiltration Conc. plasma AL(OH)3 Bulk Final product Final product Sterile filtration Mixing of different species of serum Stream samples were taken at different stages during the production of polyvalent snake & Antiscorpion venom antiserum and analyzed for endotoxin content by LAL Gel Clot method. Also, we have measured the microbial air contamination by passive sampling and the microbial monitoring of surfaces and personnel by means of Rodac plates using MacConkey as a selective media for gram negative bacteria from specified areas of manufacturing of antisera. The environmental monitoring tests were carried out in accordance with European and US pharmacopeia guidelines. The micro- organisms were identified by biochemical API20NE & API20E.The setting, the layout, and the strategy of the sampling points for endotoxin assay in these processes are illustrated in the above Figure. Is to detect presence of endotoxin in a given sample to which the test is applied using lysate derived from the animal Limulus polyphemus, also called as Horseshoe crab. This animal is a living fossil whose blood is blue in colour due to the presence of Hemocyanin instead of Hemoglobin. The unique property of the blood is to react with Lipopolysaccharides (LPS) present in the membrane of Gram- negative bacteria. Bacterial endotoxin test by using Semi-Quantitative Gel Clot Prepare Standards or Standard Series and Add to Reaction Tubes Prepare Samples at Correct Dilution and Pipette into Reaction Tubes Prepare Positive Product Control Reconstitute Lysate with LRW Add Lysate to Reaction Tubes Incubate for 60 minutes (+/- 2 mins) Gel Clot (Positive) Results & Discussions Environmental isolates were collected from different stages of antivenom production where the number of overall isolates with 63 (31 air, 24 surface & 8 personnel). The most frequent isolates were gram negative cocci & short bacilli; The detected gram negative bacteria can be attributed to poor HVAC, non effective cleaning and sanitization procedures at predetermined locations and inadequate education and training in aseptic preparation technique. Aeromonas hydrophilia/caviae Orchrobactrum anthropi Pseudomonas aeruginosa Burkholderia cepaciaStenotrophomonas maltophilia The Validation study of Gel Clot LAL Method consists of : Phase I Preliminary Screening / interference Study : In this two identical series of product dilution, one spiked with 2 λ, and one left unspiked. The result of Phase I will tell you the noninterfering dilution (NID) of the product, which is used for the actual validation (Phase II). The non-interfering dilution (NID) is the first set of PPC that shows a gel. Phase I Preliminary Screening / interference Study 1:1001:801:401:20Sample dilution ++ Spiked ++ Unspiked MVD = ENDOTOXIN LIMIT/ λ = 3.58/0.03125 = 1:114 OUR FUTURE WORK IS RESOLVING INTERFERENCE OF ENDOTOXIN IN ANTISERA