QC Testing of Sterile Products

1. QC Testing of Sterile Products Dr.M.Shakil.S.Siddiqui

2. Introduction • Pyrogens:- • Pyrogens are the heat stable, filterable and soluble substances of 0.05-1.0 micrometer size and arise from microbial contamination. Chemically these are lipopolysaccharides from the outer cell wall of the bacteria. • The term endotoxins is also used interchangeably • Both G+ and G- bacteria produce pyrogens, however, the pyrogens of G- bacteria are more potent. The pyrogens are heat stable up to some extent, thus, withstand normal sterilization temperatures.

3. Depyrogenation • Depyrogenation is the removal of pyrogen. This is achieved by the following methods. • Inactivation - Application of very high dry heat (in excess of 250 degrees) for not less than 30 minutes is the desired method for rendering material pyrogen free.

4. Detection and quantification of Pyrogens • In-vivo pyrogen test involves the evaluation of the presence of pyrogens in parenteral sample by quantitative fever response produced in rabbits. The principle is based on the fact that the human and rabbits are equally responsive to pyrogen injected intravenously on a dose per weight basis. • This test requires the following. • Test animals: healthy adult rabbits (of either sex) weighing not less than 1500 gm(1.5kg). The animals have been properly maintained in terms of environment and diet prior to the performance of test. The animals are screened for their temperature.

5. Pyrogen Test • Their control temperature must not differ more than 1°C from each other. Any individual animal having temperature 39.8°C or less than 38.0°C is excluded from the test

6. LAL Testing • 2) Limulus Amebocyte Lysate Test(LAL)- • The limulus amebocyte lysate test is also called as in-vitro pyrogen test (USP )Specified new test). Officially it is termed as bacterial endotoxin test (BET). • The test principle is based on the clotting of lysate of amebocyte (an enzyme obtained from the horse shoe crab) in the presence of pyrogens

7. LAL Testing ( contd ) • The extract from the blood cells of horse shoe crab, Limulus Polyphemus contains an enzyme and protein system called "Limulus- Amebocyte Lysate" (LAL) which reacts with pyrogens so that an assay mixture increases in viscosity and opacity until an opaque gel is formed. • Amebocyte + Pyrogen ~ Opaque gel • The reaction accomplishes within 15-60 minutes, depending on concentration of pyrogens after mixing. The concentrated pyrogens make the gel more turbid and thick.

8. Advantages of LAL Test • Advantage of LAL test • 1. It is in-vitro and does not require animal handling, thus is more convenient • 2. It is 10 times more sensitive than that of the in-vivo rabbit test • 3. It is economical • 4. It consume less time, i.e., 1 vs 3 hours required by rabbits test • 5. It requires less laboratory facilities and minimum equipments • 6. It requires less test volume • 7. It is more accurate

9. Particulate Matter • Particulate matter in injections and parenteral infusions consists of mobile undissolved particles, other than gas bubbles, unintentionally present in the solutions.

10. CLARITY TESTING (DETECTION OF PARTICULATE MATTER) • Particulate matter can be detected in parenteral product by two methods, including visual inspection and electronic particulate counting. • A) Visual methods • B) Automated visual inspection • 1) Visual inspection by naked eye • In visual inspection, each injectable is inspected visually against white and black backgrounds. The white background aids in diction of dark colored particles. The light or reflective particles will appear against the black back ground. Some visual-enhancing aids can increase the efficiency. A magnifying lens at 2.5 × magnification set at the eye level facilitates the inspection.

11. Automated Visual inspection • Automated visual inspection • The automatic systems are also called as the electron particles counter. The electronic particles counter evaluates the particles in injectables automatically. However, this method requires destruction of the ampoule/container for the particle examination. • Electronic particles counting may be based on any one of the following principles: a) change in electrical resistance, b) light blockages principle and c) light scattering. Some of the automated systems for visual particle inspection include Autoskan, Eisai Ampoule inspection machine, Schering PDS/A-V system, etc. Autoskan System