Parentrals , preparation and evaluation

preparation and evaluation of parenterals

1. PARENTERALS RINCY RAJAN 1 st YEAR M.PHARM DEPT. OF PHARMACEUTICS St. JOSEPH’S COLLEGE OF PHARMACY.

2. Processing of parenteral preparation Cleaning equipment and containers Sterilization of equipment Compounding the product Filtration Filling procedures Sealing Test for Quality Control Sterilization Packaging Labelling

3. 1. Cleaning equipment and containers Equipment and containers are thoroughly cleaned with detergent and washing → tap water →clean distilled water →WFI Debris more difficult to remove →hot detergent Equipments are mostly disassembled and each parts are thoroughly scrubbed and cleaned Two types of rinsers are used Rotary type rinser Convey type rinser

4. CLEANING RUBBER AND GLASS BOTTLES: Rubber closures and glass bottles are cleaned in mechanical equipment which is heated electrically. Detergent are mostly used for cleaning the rubber closures. This rubber closures are washed first with water → autoclave → rinse second time with preservatives → autoclaved Glass containers are cleaned by mechanical equipment ,which is rotating and the glass bottles are washed and cleaned.

5. 2. Sterilization of equipment Mostly in parenteral equipments are sterilized by using autoclave before starting of manufacturing or filling process to remove micro-organism or pyrogen . All the equipment including filling equipment, manufacturing equipments are sterilized before use. Methods of Sterilization Steam sterilization Dry heat sterilization Filtration sterilization Gas sterilization Ionizing radiation sterilization

6. 1. Steam sterilization : Method : Applying steam under high pressure Equipment : Autoclave (steam sterilizer) Mechanism of microbial destruction : denaturation and coagulation of some of organism's essential protein. In presence of moisture, MO are destroyed at lower temp. than dry heat. Temperature- 121-124°C for 15min.

7. 2.Dry Heat Sterilization: Method : using heated air Equipment : Oven Mechanism of microbial destruction : dehydration of microbial cell after oxidation. Usually conducted at 150 to 170°C for not less than 2 hour. Application : Generally employed for substances that are not effectively sterilized by moist heat such as fixed oil, glycerol, petrolatum, heat stable powder.

8. Advantages Easy to install Non toxic and does not harm the environment Non-corrosive for metal Disadvantages Time consuming method - Slow heat penetration High temp. are not suitable for most material

9. 3.Sterilization by filtration: Method : filtration allows for the exclusion of organisms based upon size. Membrane filtration are mainly used method for system sterilization. Membrane filtration traps contamination larger than the pore size on the surface of the membrane.

10. Example : Millipore filter which is a thin plastic membrane of cellulose ester containing uniform pores constituting up to 80% of membrane's volume. Pore size range from 14 to 0.025μm Advantages : Rapid, inexpensive, effective, large volume Disadvantages : 1. cannot use for oily preparation 2. cannot use for moisture sensitive preparations 3. cannot kill spores

11. 4. Gaseous sterilization Method : use of gas, such as Ethylene Oxide Equipment : special oven, for admission of gas and humidity Application : Thermolabile powder, plastic (e.g. syringes), polymers, ophthalmic prep., tubing sets.

12. Ethylene Oxide (EO) Used to sterilize heat- sensitive materials, MO and spores. Used in combination with CO2 to avoid explosion, >3% EO in air is explosive. Mechanism of action of EO is alkylation of hydroxyl, carbonyl and amino groups of bacterial enzyme. Disadvantages 1. Explosive hazards 2. Toxic 3. Not appropriate for solutions

13. 5.Radiation sterilization Equipment : Ultraviolet lamp; for surface sterilization -Ionization (Beta rays, Gamma rays, X-rays) Application : Thermolabile drugs (powder) Disadvantages : 1. Highly specialized equipment required 2. Effect of irradiation on product and their containers

14. 3. Compounding the product Compounding of product should be performed under clean environmental condition. It involves two steps Collection of material Preparation of parental product

15. 4. Filtration It is defined as the process in which particles are separated from a liquid by passing the liquid through a semi permeable membrane. Filtration is frequently the method of choice for sterilization of solution. For filtration of aqueous solutions, semi permeable 0.2 to 2 micron filter papers are used. 2 micron filter paper is used as a pre filter, while 0.2 micron paper is used as a last paper before filling. For oily solution, cartridge filter paper is used. After filtration , the solution must be protected from environmental contamination until it is sealed in the final container.

16. 5.Filling Procedures FILLING EQUIPMENT FOR LIQUIDS: Here, the measured amount of liquids deliver from the small orifice into the container. The size of the delivery tube is governed by opening in the container to be used, the viscosity and density of the liquid and the speed of delivery desired. The tube must free enter the neck of the container and deliver the liquid deep enough to permit air to escape without sweeping the entering liquid into the neck or out of the container. Filling machine parts should be constructed of nonreactive materials such as borosilicate glass or stainless steel.

17. The solution are usually filled in the bottle by gravity, pressure or vacuum filling device. Emulsion and suspension required specially designed filling equipment because of their high viscosity. Powders such as antibiotics, are more difficult to subdivide accurately and precisely into individual dose containers than are liquid.

18. 6.Sealing Container should be sealed in the aseptic area in immediately adjacent to the filling machine. It is obvious that a sterile container that has been opened can no longer be considered to be sterile. Therefore, temperature proof sealing is essential.

19. SEALING AMPOULES: Ampoules may be closed by melting a portion of the glass of neck to form either tip-seals or pull seals. Tip-seals are made by melting sufficient glass at the tip of the ampoule neck to form a bead of glass and close the opening. Pull-seals are made by heating the neck a rotating ampoule below the tip, then pulling the tip away to form a small, twisted capillary just prior to being melted closed. Excessive heating of air and gasses in the neck causes expansion against the soft glass with the formation of fragile bubbles at the point of seal.

20. SEALING OF VIALS,PLASTIC BOTTLE: When closures are to be inserted by machines, the surface of the closures is usually halogenated or coated with silicone to reduce the friction. Aluminum caps are used to hold rubber closures in place

21. 7 . Quality control test Quality control involves operational techniques and activities that are used to assure product compliance to specifications . There are 3 general areas of QC. They are - incoming stock - manufacturing & - the finished product.

22. Quality control are classified into:- Sterility Test Clarity Test/ Particulate matter monitoring Leakage Test Pyrogen Test

23. 1 . STERILITY TEST: Most reliable step of QC. To detect the presence of viable forms of microbes in pharmacopeial final preparations. All the final preparations must confirm to the test for sterility as prescribed in pharmacopoeia. The test for sterility is usually carried out under aseptic conditions.

24. PRINCIPLE: If viable microbes are placed in a nutritive medium, kept at favourable temperature, the organisms will grow & their presence can be identified by turbidity in the clear medium. Sterility test is mainly carried out by 2 methods. Membrane filtration method Direct inoculation method To carry out these tests, a sterility test media is essential.

25. Sterility test media: It should be such that it supports the growth of aerobes, anaerobes and fungi. If the final product is clear, then fluid thioglycollate medium can be employed. If the product is turbid & viscid, fluid thioglycollate medium devoid of agar is used. Fluid thioglycollate medium Anaerobes & aerobes Soya bean-casein digest medium Fungi & aerobic bacteria

26. Membrane filtration method: This method is employed in the following cases: 1. oil & oily preparations 2. alcoholic preparations 3. for preparations miscible with or soluble in aqueous or oily solvents. This methods can be explained through various simple steps.

27. Step 1: Selection of membrane: Membrane selected should have a nominal pore size not greater than 0.45µm and diameter approximately 47mm. Should have effectiveness to retain micro-organisms to be established. Eg : cellulose nitrate filters cellulose acetate filters, etc. For products containing antibiotics, specially adapted membrane filters are used.

28. Step 2: Washing the membrane Before the start of sterility test, the membrane is rinsed with diluting or rinsing fluid. Fluid A- Sterile solution- 1gm peptic digest of animal tissue in 1lt of water. Fluid D- Contains suitable emulsifying agent like polysorbate 80 at a conc of 10gm/lt. Fluid A Aqueous solutions or soluble solids Fluid D (3 TIMES) Oil, oily solutions, w/o emulsions

29. Step 3:Transfer of product to the membrane After rinsing, the contents in the product container is transferred to the rinsed membrane. Aqueous & oily solutions of low viscosity may be filtered without prior dilution. Viscous oils, w/o emulsions, etc may be suitably diluted with sterile diluents like isopropyl myristate before passing through the membrane. Pre-filled syringes(without attached needles), the contents of syringes are expelled into separate pooling vessels prior to transfer whereas if separate needle is attached, the contents are directly expelled.

30. Step 4: Culturing in broth After filtration of contents, the membrane is aseptically cut into 2 halves. Each half transferred to both the medium. Both incubated for not less than 14 days. Fluid thioglycollate Incubated at 32.5±2.5 ◦c Soya bean-casein Incubated at 22.52±1◦c

31. Step 5: Interpretation of result The media is examined for microbial growth at intervals during the incubation period. The test may become invalid if The data of microbiological monitoring or testing facility or the testing procedure used reveals a fault. or 2. Negative control that shows microbial growth. When a test is declared to be invalid, it is repeated same as original If no evidence of microbial growth Product complies the test for sterility If evidence of microbial growth (bacterial colonies) Product does not complies the test for sterility

32. Direct inoculation method Implemented when membrane filtration method appears to be difficult. In here, the quantity of preparation to be tested is directly transferred directly into the culture medium or vice versa, so that the volume of product is not more than 10% of the volume of the medium. If the product to be tested contain any anti-microbial agent, using suitable reagent it should be neutralised before the test. For large volume products, we use conc. culture medium.

33. The inoculated medium is then incubated for not less than 14 days. Observe the culture during incubation period. If the medium render to be turbid, result interpretation will be impossible. In such cases, after 14 days of incubation, transfer the contents to a fresh vessel of the same medium. Then incubate both the fresh and the original for 4 more days. At last, the result interpretation is done as same as for membrane filtration method.

34. If no evidence of microbial growth - Product complies the test for sterility If evidence of microbial growth (bacterial colonies) -Product does not complies the test for sterility The test may become invalid if The data of microbiological monitoring or testing facility or the testing procedure used reveals a fault. or 2. Negative control that shows microbial growth. When a test is declared to be invalid, it is repeated same as original

35. Scheme for sterility test by direct inoculation method

36. 2. CLARITY TEST This test is also referred as particulate matter monitoring. Particulate matter refers to unwanted mobile insoluble foreign matter other than gas bubbles present in the product. It is to be noted that there occur a very dangerous situation when the particle size is greater than RBC because they may block the blood vessels. The QC department detect and discard individual containers of a product that is considered to be unclean.

37. For particulate matter, the pharmacopoeia has established certain limits such as 50 particles of 10µm & larger 5 particles of 25µm & larger Mainly we discuss here 2 methods Visual method Coulter counter method

38. Visual method: It is the most used and relevant method for clarity testing. It is an old method. According to this method, the filled containers are examined against a strong illuminating background with a swirling motion since moving particles are easier to identify than stationary particles. Inversion of containers can be done to view heavier particles. Dark background- Light particles Light background- Dark particles

39. This is a model for visual inspection machine.

40. Coulter counter method: This is the test that is based on the principle that there will be an increase in the resistance as a particle approaches and passes through the orifice (2 electrodes). This method require destruction of the product unit since an electrolyte is added to the preparation before its evaluation.

41. Some other methods of clarity testing can be listed as Filtration method, Light scattering method, Light absorption, Light blockage methods, etc... Once the particles are detected, then they are identified by various methods like microscopy, X-ray powder diffraction, mass microscopy, micro-chemical tests, polarised light microscopy and scanning electron microscopy .

42. 3. Leakage test Ampoules are intended to provide hermetically sealed container for a single dose. If any capillary pore tiny cracks are present microbes or any other contaminants may enter the ampoule or the contents may leak out and spoil the product. The leak test is intended to detect any incompletely sealed ampoules, presence of cracks, openings, etc so that they may be discarded.

43. Leak test is usually done by producing a negative pressure within an incompletely sealed ampoule, usually in a vacuum chamber, while the ampoule is entirely submerged in a deeply coloured dye solution. The atmospheric pressure then causes the dye to penetrate if any opening is present. Only a tiny drop of dye may penetrate a small opening. Dye solution- 0.5-1.0% methylene blue. The presence of dye colour confirms the leakage & hence the product is rejected.

44. Representation of leakage test machine

45. Vials and bottles are subjected to such leak tests because the rubber closures are not rigid. For such cases, a spark tester probe is applied to the outside of the bottle moving from the liquid layer to the air space. A blue spark discharge occurs if the air space is identified. In case of bottles, a vacuum remains during its shelf-life. The presence of vacuum is detected by striking the base of bottle with the heel of hand to produce a typical water hammer sound.

46. 4. Pyrogen test PYROGENS: These are the metabolic products of microbes. Most bacteria, moulds and viruses produce Pyrogen . Most potent pyrogenic substance called endotoxins are produced by gram negative bacteria . Pyrogens when injected into a human, shows marked rise in the temperature , chills, body aches, cutaneous vasoconstriction and increased arterial blood pressure. The most likely source of pyrogens are water, contaminated solutes and containers.

47. Rabbit test

48. Rabbit Pyrogen Test Rabbits must be healthy and mature New Zealand or Belgian Whites used Either sex may be used Must be individually housed between 20 and 23°C Rabbit test Selection of animals - healthy, adult, not less than 1.5kg. Housing of animals - environmental problems: presence of strangers (unknown place), noise. Equipment and material used in test - glassware, syringes, needles. Retaining boxes - comfortable for rabbits as possible. Thermometers - standardized position in rectum, precision of 0.1°C.

49. Preliminary test ( ShamTest ) Intravenous injection of sterile pyrogen -free saline solution To exclude any animal showing an unusual response to the trauma (shock) of injection Any animal showing a temperature variation greater than 0.5 C is not used in them a in test All glassware, syringes and needles must be pyrogen free by heating at 250 C for not less than 30min.

50. Main test: Group of 3 rabbits Preparation and injection of the product: Warming the product to 37±2°C Dissolving or dilution Injection site: ear vein The injected volume: about 10ml per kg of body weight over 10min. duration Record temperature at 30min. Interval for 3 hours

51. Interpretation of the results: The test is carried out on the first group of 3 rabbits; if necessary on further groups of 3 rabbits to a total of 4 groups, depending on the results obtained. Intervals of passing or failing of products are on the basis of summed temperature response.

52. If the difference is negative, the result is counted as zero response. Result interpretation : Later with the 2 nd case, if not more than 3 of 8 rabbits show individual response of 0.6 or more and if the sum of the response of 8 rabbits does not exceed 3.7 , the preparation passes the test for pyrogen . Sum of response of 3 rabbits Individual response of a rabbit Result Does not exceed 1.4 ◦ c Less than 0.6 ◦c Product pass the test Is or Exceed 1.4 ◦c Is or is m ore than 0.6 ◦c Continue the test with 5 more rabbits

53. Limulus Amebocyte Lysate {LAL} Test Recently added method. Invitro test. Rapid. 5 to 10 min more sensitive than rabbit test. Semi-quantitative. PRINCIPLE: The endotoxins of gram negative bacteria forms a firm gel within 60 mins in the presence of lysate of amebocytes of limulus polyphemus {horseshoe crab} when incubated at 37◦c. Hence the test is only effective with gram negative bacteria which constitute the majority and the most potent of the pyrogens .

54. The test also has been automated. This test can also be called as Bacterial Endotoxins Test {BET}. To provide standardization for the test, the USP has established a reference endotoxins against which lots of lysate are standardized. Therefore, the sensitivity of the lysate is given in terms of Endotoxin Unit {EU}. HORSESHOE CRAB

55. Enzyme + protein Endotoxins Coagulation - Formation of gel ~ 30 mins Extract from blood cells ( Amoebocyte lysate ) Horseshoe crab (Limulus polyphemus )

56. 10. Packaging and labeling : The packaging should be neat ,clean and attractive in appearance if it is to convey to the user . The quality, purity and reliability that should be conformed from the carton or label. The package must be accurately and completely provide the user information necessary for its use. The labeling should be identified and shows the required things on label. It should be stored as per its necessary.

57. Reference : Pharmaceutical dosage forms: parenteral medications of industrial pharmacy , Lachmann / Lieberman's, 4 th edition, pg.no . 864-871. Dispensing pharmacy by R M Mehta, pg.no . 303-311. The science & practice of pharmacy, Remington, 21 st edition, pg.no . 1367-1374.