Ecology of Microorganisms as it affects the pharmaceutical industry - PowerPoint Presentation

1. Ecology of Microorganisms as it affects the pharmaceutical industry

2. Microbiological quality of pharmaceutical products is influenced by: Environment Materials

3. Good Manufacturing Practices (GMP)It is developed to minimize contamination by a study:Ecology of microorganismsHazards posed by them Control the critical points in the process

4. The source of pharmaceutical products contamination:AtmosphereWaterPersons (Skin & respiratory tract flora)Raw materialsPackagingBuildingsEquipmentCleaning equipment & utensils

5. 1. AtmosphereMicroorganisms commonly isolated from air are: * Spore- forming bacteria: Bacillus spp.; Clostridium spp. * Non- sporing bacteria: Staphylococcus spp.; Streptococcus spp.; Corynebacterium spp. * Moulds: Penicillium spp.; Aspergillus spp.; Cladosporium spp.; Mucor spp. * Yeasts: Rodotorulla spp.

6. Methods for checking the microbiological quality of air:Simple methods: including, exposure of petridishes containing a nutrient agar to the atmosphere for a given length of time (not more than 7 hours). Quantity methods: including, use of an air- sampling machine which draws a measured volume of air from the environment and impinges it on a nutrient agar surface on either petridishes, plastic strip or a membrane filter, then incubated with a nutrient medium.

7. environmental monitoring

8. How can reduced the microbial count of air:It may be reduced by:Filtration: It is the most commonly used method, using laminar flow, which sterilizing air by permits to passage through a High Efficiency Particulate Air (HEPA) filter, to remove all particles in excess of 0.1-0.3 um in size.Chemical disinfection: Chemical disinfectants are limited in their use as air sterilants because of their irritant properties when sprayed. Like propylene glycol; quaternary ammonium compounds. UV irradiation at wave lengths 240-280 nm, it used to reduce bacterial contamination of air. *Note: The combination of UV light & air filtration gives useful results in air sterilization. Compressed air: It has many applications in the manufacture of pharmaceutical products, it is used in the conveyance of powders & suspension. Compressed air is sterilized by heat & filtration.

9. The number of organisms in the air depends on: * Activity in the environment. * Amount of dust that is disturbed. usually content of air is increased in the summer more than winter.The movement of microorganisms in the atmosphere: Microorganisms are carried into the air suspended on: * Dust * Skin or clothing * Droplets of moisture * Sputum following talking, coughing, sneezing.

10. 2. WaterThe microbial ecology of water is of great importance in the pharmaceutical industry owing to its multiple uses as constituent of many products as well as for various washing & cooling processes.We can divided the water which used in pharmaceutical industry into many types:Raw water or mains water: The quality of the water from the mains supply varies , when the supply is derived from surface water the flora is usually more abundant(microorganisms & nutrients introduced from soil& sewage) than that of supplies from a deep- water (its microflora filtered out).

11. Microorganisms indigenous to fresh (mains) water: Pseudomonas/ Alcaligenes/ Chromobacter/ Sertia.On prolonged storage in the industrial storage tanks, the bacterial count may rise rapidly during summer months and reach 105 – 10 6 per ml. * Uses of raw water: Washing & cleaning. * Note: The microbial count of the mains water will reflected in both softened & deionized water which may prepared from it.

12. B. Softened water:It is prepared by either : Base- exchange method, using sodium zeolite or, by addition of sodium hexametaphosohate. In addition to the bacteria derived from the mains water, additional flora of Bacillus & Staphylococcus aureus may be introduced into system Uses of softened water: - Washing of containers. - For cooling systems.*Unless precaution are taken, the microbial count in cooling system or jacketed vessel will rise rapidly & if faults develop in cooling pates or vessel wall, contamination of the product may occur.

13. C. Deionized or demineralized water:It is prepared by passing raw water through anion and cation exchange resin beds to remove the ions. Thus, any bacteria present in the mains water will also be present in the deionized water, and beds which are not regenerated frequently with strong acid or alkaline are often heavily contaminated & add to the bacterial content of the water. * Uses of deionized water: Its used in: - pharmaceutical formulations - washing containers - preparation of disinfectant solutions.

14. D. Distilled water:It is free from microorganisms, and contamination occurs as a result of a fault in the cooling system, the storage vessel or the distribution system.The flora of contaminated distilled water is usually Gram- negative bacteria.Uses : It is used in: -Formulation of oral & topical pharmaceutical preparations, and low bacterial count is desirable. -Manufacture of parenteral preparations and a post- distillation heat sterilization stage is commonly included in the process. * Water for such preparation is stored at 80C to prevent bacterial growth & the production of pyrogenic substances.

15. E. Water produced by reverse osmosis(RO): It is forced by an osmotic pressure through a semi- permeable membrane which acts as a molecular filter, which prevents molecular in excess of 250 Dalton from passing . The process, which is the reverse of the natural process of osmosis, thus removes microorganisms and their pyrogens.Post- RO contamination may occur if the storage vessel or the distribution system is not kept free from microorganisms.

16. F. Distribution System:An optimum distribution system for reducing growth of microbial flora is:Ensures a constant recirculation of water at a positive pressure through a ring- main without” dead- legs”.Only very short branches to the take- off points.There should be a system to re-sterilize the water, usually by membrane filtration or UV light treatment, just before return to the main storage tank.

17. Disinfection of water:There are chemical& physical methods used for treating water.Chemical treatment: * Sodium hypochlorite & chlorine gas are common agents for treating water supply itself in concentration 0.5 – 5 ppm for most purposes 50 – 100 ppm for storage vessels, pipelines, pumps. 1% formaldehyde solution for treated RO systems, distilled, deionized water.Physical methods:Filtration: membrane filtration is useful where the usage is moderate & continuous circulation of water .UV light : 254 nm is useful for the disinfection of water of good optical clarity.

18. 3. Persons:Microorganisms may be transferred to pharmaceutical preparations from the process operator. Natural skin flora may contaminated the products: Staphylococcus aureus: it is common on hands& face. Saprophytic bacteria: from ear secretions. Dermatophytic fungi: from different parts of the body. * Bacteria other than skin flora may transferred from operator as a result of poor personal hygiene is E.coli .

19. For the manufacture of products intended for injection & eye or ear preparations, it is necessary for the operators to: - Wear sterilized clothing, trousers, boots, face masks, gloves,…….etc. - Washing their hands before entering the production area.

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21. 4. Raw materials:Raw materials account for a high proportion of the microorganisms introduced during the manufacture of pharmaceuticals, and the selection of materials of a good microbiological quality aids in the control of contamination levels in both products & the environment.It is accept to raw materials, have some non- pathogenic microorganisms.Microflora of untreated raw materials, various depending upon natural sources, which derived from it.

22. Sources of raw materials:a. Natural source b. Synthetic sourcea. The natural sources divided into: 1. Animal source2. Plant sourceProducts from animal source: gelatin, desiccated thyroid , pancreas.Contaminated with animal- borne pathogens like: E. coli, Salmonella.b. Synthetic source: Usually raw materials from this source free from contaminants, but incidental microbial contamination.Products from plant origin: gum, agar, starches.Contaminated with: Bacteria: Erwinia, Pseudomonas, Bacillus, Lactobacillus, Streptococci. Moulds: Cladosporium, Alternaria, Fusarium.

23. Note: Raw material should be stored at(Aw) under 0.65. How can reduce the microbial count of raw materials:By sterilization procedures, include: - Heat treatment - Filtration - Irradiation - Recryatallization from bactericidal solvent such as alcohol, or for dry products ethylene oxide gas.

24. 5.Packaging:Packaging material has acts to contain the product & to prevent the entry of microorganisms or moisture which may result in spoilage, and it is therefore important that the source of contamination is not the packaging itself.The microflora of packaging materials is dependent upon : * Its composition. * Storage conditions.

25. Containers used in packaging: There are tow types of containers:a. Glass containers: Are: b. Plastic bottles: That:Sterile when leaving furnace.Stored in dusty conditions & packed in cardboard boxes.Contaminate during packed & transport. Thus,Wash glass containers to remove dust which present.Include a disinfection stage if the product being filled is liquid or semi- solid preparation.In the case of injectables & ophthalmic preparations which are manufactured aseptically but do not receive a sterilization treatment in their final container, the packaging has to be sterilized.Dry heat used for vials & ampoules.Have low microbial count.Contaminate during transport. Thus, - Used a moist heat or ethylene oxide gas or formaldehyde to sterilized the plastic bottles.

26. Closure liners of pulpboard or cork considers a source of microbial contamination, thus, must be treated with preservatives, foil, wax coating in liquid or semi- solid preparations. A closure with a plastic flowed-in liner is better than one stuck in with an adhesive, especially on a natural product (like casein).Note: Containers & closures must be sterilized by chemicals & irradiation to reduced the microbial contamination of the final products.

27. 6. Buildings:There are include, walls, ceilings, floors, drains, doors, windows, & fittings.Common flora of walls & ceilings: Moulds are the most common flora like: Cladosporium/ Aspergillus/ penicillium/ AureobasidiumTo reduce microbial contamination of buildings: - All walls & ceilings should be smooth, impervious & washable . - Painted with hard gloss finish. - Addition of up to 1% of fungistate. - In areas where aseptic filling operations are carried out it is useful to have a false ceiling with the services or lighting & ventilation sited above it. - The joint between the false ceiling & the room below is well sealed. - All electric cables & ducting for other services should be installed deep in cavity walls. - All floors should be easy to clean & to slope towards a drain, no pools of water form.

28. - In areas where acid & alkaline chemicals or cleaning fluids are applied, a resistant sealing & jointing material must be used to prevent pitted & porous formed. - Floor drainage channels should be open, shallow & easy to clean. - Drains should be outside areas ,where sensitive products are manufactured. - All doors & windows should fit flush with walls to prevent dust from collecting. - All windows in manufacture should serve only to permit light entry & not be used for ventilation. - Stainless steel piper support little microbial growth, but lagged pipes present a problem and must be treated with disinfectant.

29. 7. Equipment:Each piece of equipment used to manufacture or pack pharmaceuticals has its own peculiar area where microbial growth may be supported. * To reduce the risk of microbial colonization in equipments:All equipment should be easy dismantle & clean.All surfaces that are in contact with the product should be smooth, continuous, free from pits & eliminate sharp corners and junctions must be rounded.All internal welding should polished out & there is no dead ends.There should be no inside screw threads & all outside threads readily accessible for cleaning.Coupling nuts on all pipework & valves should be capable of being taken a part and cleaned.Mechanical seals are preferable to packing boxes as packing material & must be far away from product lines.Valves should be of a sanitary design, and all contact parts must be treated during cleaning & sanitation.

30. 8. All pipelines should slope away from the product source and all process and storage vessels should be self- draining. 9. If a vacuum exhaust system is used to remove the air or steam from a vessel, it is necessary to clean & disinfect all fittings regularly.Cleaned & sterilized regularly filters or straining bags made from natural materials, to prevent the growth of moulds. * Sterilization and disinfection of equipment: Equipment may be sterilized or disinfected by heat, chemical disinfection or by combination of both. * Microbial cheeks: The efficacy of cleansing- in- place(CIP) system can be checked by plating out a sample of the final rinse water with nutrient agar, or by swab test.

31. 9. Cleaning equipment & utensils:The misuse of brooms & mops can substantially increase the microbial count of the atmosphere by raising dust or splashing with waterborne contaminants. To prevent this, either a correctly designed vacuum cleaner or a broom made of synthetic materials, which is washed regularly. Use of a neglected dry mop may redistributes microorganisms which it has picked up, but a neglected wet mop redistributes many times the number of organisms it picked up originally.In order to maintain mops and similar non- disposable cleaning equipment in a good hygienic state, at first wash & then to boil or autoclave the items, and finally to store them in a dry state. Disinfectant solutions were found to be inadequate.

32. Many chemical disinfectants like halogens & QACs are inactivated in the presence of organic matter, thus, all cleaning materials( buckets & fogging sprays) are kept clean. The bulk of the disinfectant must be store in a concentrated form and diluted to use concentration as required.

33. 4th Lecture:

34. Disinfectants are chemical agents which are used to destroy microorganisms on inanimate (dead) objects.Antiseptics are chemical agents which are used to destroy or inhibition of microorganisms on living tissues. Disinfectant does not necessarily kill all microorganisms, but it reduces them to a level which is acceptable for a define purpose.The term disinfectant can be used to include antiseptics, in a wider sense ( but British Standards Institution consider antiseptic is not a synonym for disinfectant).

35. Antiseptics have more selectivity in their action toward the bacteria than the living tissues, at the recommended concentration ,therefore antiseptics usually exert a bacteriostatic or low bactericidal effect at the recommended concentration. Antiseptics must not be toxic or irritating to skin, and they are often lower concentrations of the agents used for disinfection.Disinfectants on the other hand are non selective, irritant, corrosive or toxic to be applied to the skin or to tissues.

36. Preservatives: These are included in pharmaceutical preparations to prevent microbial spoilage of the product and to minimize the risk of the consumer acquiring an infection when the preparation is administered. Preservatives must be able to limit proliferation of microorganisms that may be introduced unavoidably into non- sterile products( oral& topical medications) during their manufacture and use, while in sterile products(eye drops and multi- dose injections) preservatives should kill any microbial contaminants introduced inadvertently during use.

37. Preservative is not toxic, and employed at lower concentrations, and levels of antimicrobial action lower order than for disinfectants or antiseptics(European Pharmacopoeia ).There are around 250 chemicals that have been identified as active components of microbiocidal products in the European Union.

38. Factors affecting choice of antimicrobial agent:(P:286)Properties of the chemical agent.Microbiological challenge.Intended application.Environmental factors.Toxicity of the agent.

39. Factors affecting the activity of antimicrobial agents:1- Temperature: In general, as the temperature increased in arithmetical progression, the rate of kill increase geometrically. The effect of temperature increase on the rate of bactericidal activity at a fixed concentration and inoculum size is expressed quantitatively as a temperature coefficient, usually in θ10 value: which is the change in activity per 100C rise in temperature, e.g. rising the temperature of phenol from 200C to 300C increased the killing activity by a factor of 4. θ10 value may be calculated by determining the extinction time at two temperature differing exactly by 100C. θ10 =Time require to kill at T0 Time require to kill at(T+10)0 * This value is constant for each compound. Compounds with high θ10 are more effective with increases in temperature than those with low θ10.

40. θ10 Compound 30- 50Aliphatic alcohol 3- 5Phenols 1.5Formaldehyde

41. 2- Concentration:The rate of kill of bacterial population is directly affects with concentration or dilution, therefore slightly increase or decrease in the concentration of certain agents can increase or decrease their bactericidal effect.The graph plotting the log 10 of a death time(i.e. the time required to kill a standard inoculums) against the log 10of the concentration is usually a straight line, the slope of which is the concentration exponent(η)η= concentration exponent (dilution coefficient),which is measure the effect of changes in concentration(or dilution) on cell death rate η=log t2 – log t1 log c1 – log c2It is constant for each compound, and the dilution does not affect the cidal attributes of all disinfectants in a similar manner. Therefore compounds with low η are less affected by dilution, but those with high η will be readily inactivated.

42. Reduce the activity(the time required)No. of dilution ηCompound 21 double the time 2 1Mercuric chloride 31 three times 3 1Formaldehyde 26 64 (the time) 36 726 (the time) ½ 1/3 6Phenol

43. :3- PHChanges in PH of the medium affect both bacterial cell and disinfectant activity. Bacterial growth is optimums at PH 6- 8, any change in this PH will decrease the bacterial growth as well as change the physicochemical state of their surface. The degree of ionization of acidic or basic disinfectant will obviously depend on the PH.

44. 4- Formulation:Formulation is greatly affect the activity of the disinfectant and the recommendation of the manufacturer for dilution should strictly be followed. This is because that penetrating power and hence the effectiveness of some agents such as chlorhexidine and QAC may be greater in 70% alcohol than in aqueous solutions.Phenols have low solubility in water, therefore concentrated solutions are prepared used soap, hydrocarbon carriers.Iodine have low solubility in water, therefore alcohol, potassium iodide solution or surface active agents are used to dissolved it.

45. 5- Physicochemical factors:Although the action of disinfectant depend largely upon its adsorption & penetration through bacterial cell wall and cytoplasmic membrane and or reaction with cellular constituents yet these processes depend largely upon a- molecular size b- configuration c- partitioning of germicide. The presence of other substance in solution can greatly effect the previous phenomenon of these substances.Nature of solvents: the nature of solvent is important in relation to the partition coefficient of the germicide. Water is the best media for the action of germicides, since bacteria can grow therefore only in this medium, therefore the addition or the presence of any other material which attracts the germicide will effect its concentration in water, thereby, it will reduce its concentration in the effective site. Oils, fats and glycols depress the activity of phenol in this way. On the other hand compounds with high partition coefficient might be more effective against microorganisms of high lipid content e.g. Mycobacterium tuberculosis, and other acid- fast bacilli.

46. B. Soaps: soaps increase the activity of phenol because it reduce the surface tension and aids the contact of the disinfectant with bacterial cell and perhaps it increases the permeability of the cell membrane. QAC like benzalkonium chloride may act by this way as well as many sanitizer( which are used in cleansing utensils and equipments) which contain detergents. 6. Interfering substance in the environments: Organic materials such as blood, pus, saliva, mucous, milk, food, feces(stool) and proteinous material, highly influence the effectiveness of disinfectant. This is because of adsorption or chemical interaction or shielding of microorganisms from adequate contact with the germicide.

47. Off the compounds which are largely affected, are biguanides; chlorhexidine, QAC, chlorine compounds & some phenolic disinfectants. Organic materials such as fabrics, rubber, cork and plastics are also capable of adsorbing certain disinfectant.Soaps and anionic detergents can inactivate QAC.Thiol groups can react with mercurial's.Diluted phenolic compounds such as chloroxylenols lose activity on standing due to change in the physical state of the emulsion also hard water may break emulsions & precipitate soaps.

48. 7. Nature, number and location of microorganisms:The kind & the number of microorganism greatly affect the power of disinfection, in general, vegetative bacteria are the most sensitive sort of microorganisms followed by fungi, viruses and the most resistance forms are the bacterial spores. Off the vegetative bacteria the G+ve are more sensitive than the G-ve .High inocula of microorganisms needs higher concentration of disinfectant due to inactivate of these agents with organic depress.The presence of water( aqueous solution) is essential for chemical disinfection. Dried blood film, mucous, feces, sputum may prevent the penetration of disinfectant, therefore, the cleansing with tap water is required before disinfection.

49. 5th Lecture:

50. 1. High Germicidal: (p:286)Killed all microorganisms unless extreme challenge or resistance exhibited. M. surviving ( challenge of resistant bacterial spore, prions) Bactericidal, Sporocidal, Fungicidal & Virucidal e.g. Ethylene oxide, Formaldehyde, Glutaraldehyde.2. Intermediate Germicidal:Killed most vegetative bacteria including M. tuberculosis, most viruses including hepatitis B virus (HBV), most fungi. M. surviving (bacterial spores, prions) Bactericidal, Non- sporocidal, Fungicidal &Virucidal e.g. Alcohol, Chlorine compounds & Iodine solution.3. Low Germicidal:Killed most vegetative bacteria, some viruses, some fungi. M. surviving (M. tuberculosis, bacterial spore, some viruses, prions) Bactericidal, Fungicidal & kill lipid viruses only e.g. Chloroxylenoles, Thiomersal, QACs, Chlorhexidine, Iodophors.

51. Evaluation of liquid disinfectants:1. Suspension Tests: Essentially are tests of sterility a. Phenol Coefficient Test: * Rideal – Walker (RW) test * Chick – Martin (CM) test b. Capacity use – dilution test Kelsey – Sykes (SK) test2. Quantitative suspension test

52. The Rideal- Walker Test: 1903Briefly, dilutions of the disinfectant are compared with standard dilution of phenol( from 1 in 95 to 1 in 115) for their lethal activity against Salmonella typhi NCTC786To each 5 ml volume of disinfectant or phenol solution in distilled water held at 17-18oC is added 0.2 ml of 24 hour culture. At intervals of 2.5,5,7.5 and 10 minutes, subcultures using a standard loop are made into 5 ml volume of broth; these are than incubated for 48- 72 hours at 37oC after which presence or absence of growth in each broth is recorded.

53. Contact time(min.) of culture and disinfectantDilutionDisinfectant 10 7.5 5 2.5 _ _ _ + 1 in 250 X _ _ + + 1 in 300 X _ + + + 1 in 350 X + + + + 1 in 400 X _ _ + + 1 in 100 Phenol

54. The phenol coefficient of the disinfectant(X) is calculated by dividing the dilution of X which allows survival of test organism at 2.5 and 5 but not at 7.5 and 10 minutes by dilution 0f phenol giving the same response. The Rideal- Walker phenol coefficient(RW) of disinfectant X is: RW of X =300 = 3 100

55. The Chick- Martin Test: 1908Chick & Martin, realizing that disinfectants usually had to act in the presence of organic material, suggested the inclusion of 3% dried human feces in the test. They also considered the 10 minutes disinfection time allowed in the Rideal- Walker test was too short and they introduced a 30 minutes contact time with subcultures in duplicate at the end of this. A different strain of Salmonella typhi from that of (RW) test was used. The human fecal suspension was later replaced by dead yeast cells and the Chick-Martin test become the subject of British Standard Specification BS 808: 1938.

56. Series of dilutions of the unknown disinfectant and of phenol are made in distilled water in regular diminishing stages of 10%. To 2.5 ml volume of these held at 200C are added 2.5 ml of the culture- yeast suspension(2 ml of a 24 hours Salmonella typhi culture + 48 ml of 5% dry weight yeast suspension). After a contact time of 30 minutes, a standard loopful of disinfectant- culture- yeast mixture is transferred in duplicate to 10 ml of broth. These broth tubes are incubated at 370C for 48 hours, then the presence or absence of growth is recorded.The Chick- Martin Coefficient (CM) is calculated by dividing the mean of the highest concentration of phenol permitting growth in both subcultures and the lowest concentration showing absence of growth in both subcultures by the corresponding mean concentration of the unknown disinfectant.

57. Subculture tubeDisinfectant X(%)Subculture tubePhenol(%) 2 1 2 1 _ _ 1.00 _ _ 2.00 _ _ 0.90 _ _ 1.80 + + 0.81 + + 1. 62 + + 0.73 + + 1.46

58. Mean of phenol concentration is: ½ ( 1.80+ 1.62)=1.71Mean of disinfectant X concentration is: ½ (0.90+ 0.81)=0.85 Chick- Martin Coefficient of X= 1.71 = 2.0 0.85If growth occurs in one, but not in the other, of the a pair of subculture tubes the concentration value corresponding to that pair is used. Chick- Martin Coefficient of X = 1.62 = 2.0 0.81

59. Subculture tubeDisinfectant X(%)Subculture tubePhenol(%) 2 1 2 1 _ _ 1.00 _ _ 2.oo _ _ 0.90 _ _ 1.80 _ + 0.81 _ + 1.62 + + o.73 + + 1.46

60. Capacity use- dilution tests:There are four test organisms, Pseudomonas aeroginosa, Proteus vulgaris, E.coli and Staphylococcus aureus; the bacteria are suspended in standard hard water for the test under clean conditions, and in a yeast suspension for the test under dirty conditions, the disinfectant is diluted in hard water. To 3 ml of each disinfectant dilution add 1 ml of the bacterial suspension prepared in broth, yeast, or serum as required, and shake gently. After 8 minutes, remove the sample of the mixture with a dropper, pipette and transfer one drop to each of five tubes of the liquid recovery medium.

61. Alternatively, five drops may be placed separately on a nutrient agar plate. Two minutes later, i.e. 10 min. after the first inoculation, re-inoculate the disinfectant mixture with a further 1 ml of bacterial suspension, and 8 min. later subculture as before. A further 2 min. later ,i.e. 20 min. after the first inoculation, repeat the process again. The initial test is carried out at 20 to 22o C, and all subcultures are incubated at about 32o C for 48 hours. Record the number of tubes showing growth in the liquid medium or the number of colonies growing on the surface plate cultures. A capacity test (the Kelsey- Sykes): use- dilution 1.o%

62. Number of subculture broth ( out of five) showing growth afterDisinfectant Concentration (%) 28 min. 18 min. 8 min. 3 0 0 2.0 5 2 0 1.0 5 4 1 0.5 5 5 5 o.25

63. 5thLecture:

64. Quantitative suspension tests:The number of survivors expression as the percentage remaining viable at the end of a giving time may be determined by viable counts & this parameter is often used in assessing bactericidal. After exposure of bacterial cells to the disinfectant, surviving organisms can be counted by two techniques , either by direct culture or by membrane filtration.The basic principle of the quantitative suspension tests using direct culture is as follows: after contact with the disinfectant, a sample of the reaction mixture is inoculated on a solid nutrient medium; after incubation the number of survivors is counted and compared with initial inoculums size.

65. The pour- plate technique as well as surface plates may also be used for subculturing.The decimal reduction rate, or Microbial Effect(ME) can be calculated using the formula: ME= log NC- log ND ( NC: CFU in control, ND: CFU after exposure to disinfectant).ME= ( 4+ 2.04) – (1+ 1.94) = 3.10 ( after 5 min.)

66. No. of CFUDilution of subculture in disinfectant in control _ tntc _ tntc 100 1.94 88 _ tntc 10-1 0.78 6 _ tntc 10-2 _ o _ tntc 10-3 _ 0 2.04 110 10-4

67. Methods which measure only growth inhibition( bacteriostasis) are:1. Serial dilution: Graded doses of the test substance are incorporated into broth and the tubes inoculated with the test organisms and incubated. The point at which no growth occurs is taken as the bacteriostatic concentration( Minimum Inhibitory Concentration, MIC). It is essential when performing these tests to determine the size of the inoculum at the position of the endpoint varies considerably with inoculum size, which should always be defined in any description of results.

68. The test is carried out in practice by mixing the appropriate volume of the solution under test with double- strength broth and making it up to volume with water as illustrated in table below:Tube contents for determining the MIC of phenol Final volume555555Double-strength broth5432100.5% phenol solution012345Sterile distilled water0.250.20.150.10.050Final con. phenol (%w/v)

69. 2. Ditch- plate technique:The test solution is placed in a ditch cut in nutrient agar contained in a petridish, or it may be mixed with a little agar before pouring into the ditch. The test organisms( as many as six may be tested) are streaked up to the ditch. The plate is then incubated.3. Cup- plate technique:The solution is placed in contact with agar, which is already inoculated with the test organism and after incubation, zones of inhibition observed. A method used widely in antibiotic assays. The solution may be placed in a small amount in a well cut from agar with a sterile cork- borer.

70. 4. Disc tests:These are modification of the earlier cup or ditch- plate procedures where filter-paper discs impregnated with the antimicrobial agents. For disc tests, standard suspensions are prepared and inoculated onto the surface of appropriate agar plates. Commercially available filter-paper discs containing known concentrations of antimicrobial agent.5. E-tests:The most presently accepted method of determining bacterial MICs, however, is the E(Epsilometer)-test. Basically this is performed a similar manner to the disc test except that nylon strips that have a linear gradient of antimicrobial lyophilized on one side and the on the other side of the nylon strip are a series of lines and figures denoted MIC values.

71. Cup- plateDisc- plate

72. E- Test:

73. 6. Solid dilution method:In this method the dilutions of the substance under test are made in agar instead of broth. The agar containing the substance under test is subsequently poured onto a petridish. It has the advantage that for any one concentration of the test substance, several organisms, may be tested.7. Gradient- plate technique:In this technique the concentration of a drug in an agar plate may be varied infinitely between zero and a given maximum. To perform the test, nutrient agar is molted, the solution under test added, and the mixture poured into a sterile petridish and allowed to set in the form of a wedge(A). A second amount of agar is then poured onto the wedge and allowed to set with the petridish flat on the bench.

74. 8. NCCLS:Regularly updated guidelines have been provided by the National Committee for Clinical Laboratory Standards (NCCLS) and are widely used in many countries.

75. 8th Lecture :

76. Products made in the pharmaceutical industry today must meet high microbiological specification, i.e. if not sterile, they are expected to have no more than a minimal microbial population at the time of product release. Nevertheless, from time to time a few rogue products with an unacceptable level and type of contamination will occasionally escape the quality assurance net.

77. Spoilage-chemical and physicochemical deterioration of pharmaceuticals.Mixed natural communities are often far more effective co-operative biodeteriogens than the individual species alone, and sequences of attack of complex substrates occur where initial attack by one group of microorganisms renders them susceptible to further deterioration by secondary, and subsequent, microorganisms. However, the rates of degradation of materials released into the environment can vary greatly, from half-lives of hours (phenol) to months (hard detergents) to years(halogenated pesticides).

78. The overall rate of deterioration of a chemical will depend upon:Its molecular structure.The physicochemical properties of a particular environment.The type and quantity of microbes present.Whether the metabolites produced can serve as sources of usable energy and precursors for the biosynthesis of cellular components, and hence the creation of more microorganisms.

79. Pharmaceutical ingredients susceptible to microbial attack:Therapeutic agents: Through spoilage, active drug constituents may be metabolized to less potent or chemically inactive forms. Materials as diverse as alkaloids(morphine, strychnine, atropine), analgesics ( aspirin, paracetamol), thalidomide, barbiturates, steroids esters and mandelic acid can be metabolized and serve as substrates for growth.The metabolism of atropine in eye-drops by contaminating fungi; inactivation of penicillin injections by β-lactamase -producing bacteria; steroid metabolism in damp tablets and creams by fungi; microbial hydrolysis of aspirin in suspension by esterase producing bacteria; and chloramphenicol deactivation in an oral medicine by chloramphenicol acetylase- producing contaminant.

80. Organic polymers: many of thickening and suspending agents used in pharmaceutical formulations are subject to microbial depolymerization by specific of extracellular enzymes, yielding nutritive fragments and monomers. Examples of such enzymes, with their substrates in parentheses are: amylases ( starches) pectinases ( pectins) cellulases (carboxmethylcelluloses but not alkylcelluloses) uronidases (polyuronides such as in tragacanth and acacia) dextranases ( dextrans) proteases ( proteins) .* Agar is an example of a relatively inert polymer and, as such, is used as a support for solidifying microbiological culture media. The lower molecular weight polyethylene glycols are readily degraded by sequential oxidation of the hydrocarbon chain, but the larger congeners are rather more recalcitrant. Nylon, polystyrene and polyester(packaging polymers) are extremely resistant to attack.

81. Fats and oils: These hydrophobic materials are usually attacked extensively when dispersed in aqueous formulations such as oil-in- water emulsions. Fungal attack has been reported in condensed moisture films on the surface of oils in bulk, or where water droplets have contaminated the bulk oil phase.While the microbial metabolism of pharmaceutical hydrocarbon oils is rarely reported, this is a problem in engineering and fuel technology when water droplets have accumulated in oil storage tanks & subsequent fungal colonization has catalyzed serious corrosion. Sweetening, Flavouring and colouring agents:Many of sugars and other sweetening agents used in pharmacy are ready substrates for microbial growth. However, some are used in very high concentrations to reduce water activity(Aw) in aqueous products and inhibit microbial attack.

82. At one time, a variety of colouring agents( such as peppermint water)were kept as stock solutions, but they frequently supported the growth of Pseudomonas aeruginosa. Such stock solutions should now be preserved or freshly made as required by dilution of alcoholic solutions which are much less susceptible to microbial attack.Humectants: Low molecular weight materials such as glycerol and sorbitol are included in some products to reduce water loss and may be readily metabolized unless present in high concentrations.

83. Preservatives and disinfectants: Many preservatives and disinfectants can be metabolized by a wide variety of Gram-negative bacteria. Growth of Pseudomonas in stock solutions of quaternary ammonium antiseptics & chlorhexidine has resulted in infection of patients. Pseudomonas spp. Have metabolized 4-hydroxy-benzoate ester preservatives contained in eye-drops and caused serious eye infection, and have also metabolized the preservatives in oral suspensions and solutions.Surface-active agents: * Anionic surfactants, such as amine soaps of fatty acids, are generally stable due to the slightly alkaline PH of the formulations. Alkyl and alkylbenzene sulphonates and sulphate esters are metabolized by α-oxidation of their terminal methyl groups followed by sequential β-oxidation of the alkyl chains and fission of the aromatic rings. The presence of chain branching involves additional α-oxidative process. Generally, degradation decreases with increasing chain length and complexity of branching of the alkyl chain.

84. *The cationic surfactants, used as antiseptics and preservatives in pharmaceutical applications are usually only slowly degraded at high dilution in sewage.*Non-ionic surfactants, such as alkylpolyxyethylene alcohol emulsifiers, are metabolized by a wide variety of microorganisms. Increasing chain lengths and branching again decrease ease of attack. Lipolytic cleavage of the fatty acids from sorbitan esters, polysorbates and sucrose esters is often followed by degradation of the cyclic nuclei, producing numerous small molecules readily utilizable for microbial growth.*Ampholytic surfactants, based on phosphatides, alkylamino-substituted amino acid, are an increasingly important group of surfactants and are generally reported to be reasonably biodegradable.

85. Observable effects of microbial attack on pharmaceutical products:To observe the microbial attack, the microorganisms must replicate to levels where obvious spoilage becomes apparent(i.e. the number of M.O. more than 106 . Early indications of spoilage are often organoleptic, with the release of unpleasant smelling and tasting metabolites such as "sour” fatty acid, “fishy” amines, “bad eggs” bitter, “earthy”. Products may become unappealingly discoloured by microbial pigments of various shades. Thickening and suspending agents such as tragacanth, acacia or caroxymethylcellulose can be depolymerized resulting in loss of viscosity, and sedimentation of suspended ingredients.

86. Alternatively, microbial polymerization of sugars and surfactant molecules can produce slimy, viscous masses in syrups, shampoos and creams, and fungal growth in creams has produced (gritty) textures. Changes in product PH can occur depending on whether acidic or basic metabolites are released, and become so modified as to permit secondary attack by microbes previously inhibited by the initial product PH. Gaseous metabolites may be seen as trapped bubbles within viscous formulation.When a complex formulation such as an oil-in-water emulsion is attached, a gross and progressive spoilage sequence may be observed.

87. Lecture 7th :

88. Factors affecting microbial spoilage of pharmaceutical products:Types & size of contaminant inoculum.Nutritional factors.Moisture content: water activity(Aw).Redox potential.Storage temperature.PH.Packaging design.Protection of microorganisms within pharmaceutical products.

89. Types& size of contaminant inoculum:Bioburden: The term is used to describe the concentration of microorganisms in a material; this may be either a total number of organisms per milliliter or per gram, regardless of type. Or the maximum permitted concentrations of contaminants may be those specified in various pharmacopoeias or the levels established by the manufacturer during product development.Low levels of contaminants may not cause appreciable spoilage, if unable to replicate in a product; however, an unexpected surge in the contaminant bioburden may present an unacceptable challenge to the designed formulation.This could arise if,1. raw materials were unusually contaminated; 2. there was a lapse in the plant-cleaning protocol;3. a biofilm detached itself from within supplying pipework;4. the product had been grossly misused during administration(saliva contain 109 M.O. /1mm3 ).Low levels of aggressive pseudomonas in a weakly preserved solution may suggest a greater risk than tablets containing fairly high numbers of fungal and bacterial spores.

90. When an aggressive microorganism contaminates a medicine, there may be an appreciable lag period before significant spoilage begins, there is delay between manufacture and administration of factory- made medicines, growth and attack could ensue during this period unless additional steps were taken to prevent it.The isolation of a particular M.O. from a markedly spoiled product does not necessarily mean that it was the initiator of the attack. It could be a secondary opportunist contaminant which had overgrown the primary spoilage organism once the physicochemical properties had been favourably modified by the primary spoiler.

91. Nutritional factors:The simple nutritional requirements and metabolic adaptability of many common spoilage m.o. enable them to utilize many formulation components as substrates for biosynthesis and growth. The use of crude vegetable or animal products in formulation provides an additionally nutritious environment. Even demineralized water prepared by good ion-exchange methods will normally contain sufficient nutrients to allow growth of many waterborne G-ve bacteria such as Pseudomonas.

92. Moisture content: water activity(Aw )Microorganisms require readily accessible water in appreciable quantities for growth to occur. By measuring a products Aw , it is possible to obtain an estimate of the proportion of uncomplexed water that is available in the formulation to support microbial growth, using the formula: Aw = vapour pressure of formulation/vapour pressure of water under similar conditions.Limiting Aw values are of the order of: G-ve rods, 0.95; staphylococci,micrococci and lactobacilli, o.90; and most yeasts, 0.88. Syrup-fermenting osmotolerant yeasts have spoiled products with Aw levels as low as 0.73, while some filamentous fungi such as Aspergillus glaucus can grow at 0.61.

93. The Aw of aqueous formulation can be lowered to increase resistance to microbial attack by the addition of high concentrations of sugars or polyethylene glycols. However, even syrup(67% sucrose; Aw =.86) has failed to inhibit osmotolerant yeasts and additional preservation may be necessary.Aw can also be reduced by drying, although the dry, often hygroscopic medicines(tablets, capsules, powders) will require suitable packaging to prevent resorption of water and consequent microbial growth.Condensed water films can accumulate on the surface of otherwise dry products such as tablets or bulk oils following storage in damp atmospheres, resulting in sufficiently high localized Aw to initiate fungal growth. Condensation similarly formed on the surface of viscous products such as syrups and creams, may well permit surface yeast and fungal spoilage.

94. Redox potential:The ability of microbes to grow in an environment is influenced by its oxidation-reduction balance (redox potential), as they will require compatible terminal electron acceptors to permit their respiratory pathways to function. The redox potential even fairly viscous emulsions may be quite high due to the appreciable solubility of oxygen in most fats and oils.

95. Storage temperature:Spoilage of pharmaceuticals could occur in range of about (-20 to 60)0C. A deep freeze at -200C or lower is used for long-term storage of some pharmaceutical raw materials. Reconstituted syrups and multi-dose eye-drop packs are sometimes dispensed with the instruction to store in cool place such as domestic fridge(8-12) 0C, partly to reduce the risk of growth of contaminants introduced during use. Conversely water for injection(EP) should be held at 800C or above after distillation and before packing and sterilization to prevent possible re-growth of G-ve bacteria and the release of endotoxins.

96. PH:Extremes of PH prevent microbial attack. Around neutrality bacteria spoilage is more likely, with reports of pseudomonas and related G-ve bacteria growing in antacid mixtures, flavoured mouthwashes and in distilled or demineralized water. Above pH spoilage is rare. In products with low pH levels(fruit juice-flavoured syrups with a pH 3-4), mould or yeast attack is more likely. Yeasts can metabolize organic acids and raise the pH to levels where secondary bacterial growth can occur.

97. Packaging design:Packaging can have a major influence on microbial stability of some formulations in controlling the entry of contaminants during both storage and use.Self-sealing rubber wads must be used to prevent microbial entry into multi-dose injection containers.Wide-mouthed cream jars have now been replaced by narrow nozzles and flexible screw-capped tubes, thereby removing the likelihood of operator-introduced contamination during use of product.Where medicines rely on their low Aw to prevent spoilage, packaging such as strip foils must be of water vapour-proof materials with fully efficient seals.

98. Protection of microorganisms within pharmaceutical products: The survival of microorganisms in particular environments is sometimes influenced by the presence of relatively inert materials. Thus, microbes can be more resistant to heat or desiccation in the presence of polymers such as starch, acacia or gelatin.There is a limited hard evidence, that the presence of suspended particles such as kaolin, magnesium trisilicate or aluminium hydroxide gel may influence contaminant longevity in those products containing them, and that the presence of some surfactants, suspending agents and proteins can increase the resistance of microorganisms to preservatives.

99. Lecture 8th:

100. * Hazard to health:Pseudomonads contaminating antiseptic solution have infected the skin of badly burnt patients, resulting in the failure of skin grafts and subsequent death from G-ve septicaemia. Infection of eczematous skin and respiratory infections in neonates have been traced to ointments and creams contaminated with G-ve bacteriaSee table 16.1 in 270pp.Microbial toxins: G-ve bacteria contain lipopolysaccharides (endotoxins) in their outer cell membranes; these can remain in an active condition in products even after cell death and some can survive moist heat sterilization. Endotoxin can induce a number of physiological effects if they enter the blood- stream via contaminated infusion fluids, even in nanogram quantities, or via diffusion across membranes from contaminated haemodialysis solutions.

101. Such effects may include fever, inactivation of the cytokine system, endothelial cell damage, all leading to septic and often fatal febrile shock. The acute bacterial toxins associated with food poisoning episodes are not commonly reported in pharmaceutical products, although aflatoxin producing aspergilli have been detected in some vegetable ingredients.However, many of the metabolites of microbial deterioration have quite unpleasant tastes and smell even at low levels, and would deter most patients from using such a medicine.Sources of contamination: see p: 271-275, particularly fig:16.3

102. Although impossible to quantify, the use of contaminated medicines has undoubtedly contributed to the spread of cross- infection in hospitals. Clinical reactions may not be evident in one patient, yet in another, this reactions may range from inconvenient local infections of wounds or broken skin, caused possibly from contact with a contaminated cream, to gastrointestinal infections from the ingestion of contaminated oral products, to serious widespread infections such as a bacteraemia or septicaemia, possibly resulting in death, as caused by the administration of contaminated infusion fluids.The outcome of any episode is determined by a combination of several factors:Type and degree of microbial contaminationThe route of administration The patients resistance.

103. 1.Type & degree of microbial contamination M.O. that contaminate medicines and cause disease in patients may be classified as true pathogens or opportunist pathogens. Pathogenic organisms like Clostridium tetani and Salmonella ssp.rarely occur in products, but when present cause serious problems. Wound infections and several cases of neonatal death have resulted from use of talcum powder containing Cl. tetani . Outbreaks of salmonellosis have followed the inadvertent ingestion of contaminated thyroid and pancreatic powders. On the other hand, opportunist pathogens like Ps. aeruginosa, Klebsiella, Serratia and other free living organisms are isolated from medicinal products and, as their name suggests, may be pathogenic if given the opportunity.

104. These organisms needs simple nutritional requirements to enable them to survive in a wide range of pharmaceuticals, and thus they tend to be present in high numbers( in excess of 106 -107 CFU/g or CFU/ml.Compromised hospital patients, i.e. the elderly, burned, traumatized or immunosuppressed, are considered to be at risk from infection with these organisms.The critical dose of microorganisms that will initiate an infection is largely unknown and varies not only between species but also within species.

105. 2. The route of administration:Contaminated products injected directly into the bloodstream or instilled into the eye cause the most serious problems. Injectable and ophthalmic solutions are often simple solutions and provide Gram- negative opportunist pathogens with sufficient nutrients to multiply during storage; if contaminated, a bioburden of 106 CFU as well as the production of endotoxins should be expected. Total parenteral nutrition fluids, formulated for individual patients’ nutritional requirements, can also provide nutritional support for invading contaminants.Ps. aeruginosa, contaminant of eye- drops, has caused serious ophthalmic infections, including the loss of sight in some cases. The problem is compounded when the eye is damaged through the improper use contact lenses or scratched by fingernails or cosmetic applicators.

106. The fate of contaminants ingested orally in medicines may be determine by several factors: The acidity of the stomach may provide a successful barrier, depending on whether the medicine is taken on an empty or full stomach and also on gastric empting time.Contaminants in topical products may cause little harm when deposited on intact skin. Not only dose the skin itself provide an excellent mechanical barrier, but few contaminants normally survive in competition with its resident microbial flora. Skin damaged during surgery or trauma or in patients with burns or pressure sores may, however, be rapidly colonized and subsequently infected by opportunist pathogens.

107. 3. Resistance of the patient A patient’s resistance is crucial in determining the outcome of a medicament- borne infection. Hospital patients are more exposed and susceptible to infection than those treated in the general community. Neonates, the elderly, diabetics and patients traumatized by surgery or accident may have impaired defence mechanisms. People suffering from leukaemia and those treated with immunosuppressants are most vulnerable to infection; there is an undeniable case for providing all medicines in a sterile form for these patients.

108. Preservation of medicines using antimicrobial agents:1- basic principlesAn antimicrobial preservative may be included in a formulation to minimize the risk of spoilage and preferably to kill low levels of contaminants introduced during storage or repeated use of a multi- dose container. However, where there is a low risk of contamination, as with tablets, capsules and dry powders, the inclusion of a preservative may be unnecessary. Preservatives should never be added to mask poor manufacturing processes.The properties of an ideal preservative are well recognized:1. Broad spectrum of activity .2. Rapid rate of kill.3. Selectivity in reacting with the contaminants and not the formulation ingredients.4. Non- irritant and non- toxic to patient.5. Stable and effective throughout the life of the product.

109. Unfortunately, the most active antimicrobial agents are often non-selective in action, inter-reacting significantly with formulation ingredients as well as with patients & microorganisms. Having excluded the more toxic, irritant and reactive agents, those remaining generally have only modest antimicrobial efficacy, and there are now no preservatives considered sufficiently non-toxic for use in highly sensitive areas, e.g. for injection into central nervous system tissues or for use within the eye. Most effective preservatives used in cosmetics have caused a significant number of cases of contact dermatitis.Although a rapid rate of kill may be preferable, this may only be possible for relatively simple aqueous solutions such as eye-drops or injections. For physicochemically complex systems such as emulsions and creams, inhibition of growth and a slow rate of killing may be all that can be realistically achieved.

110. 2. Effect of preservative concentration, temperature and size of inoculum: Changes in the efficacy of preservatives vary exponentially with changes in concentration. The effect of changes in concentration(concentration exponent,η) varies with the type of agent. For example, halving the concentration of phenol(η=6)gives a 64-fold(26) reduction in killing activity. Changes in preservative activity are also seen with changes in product temperature, according to the temperature coefficient,θ10 . Thus, a reduction in temperature from 30 to 200C could result in a significantly reduced rate of kill for Escherichia coli, fivefold in the case of phenol(θ10 =5).Preservative molecules are used up as they inactivate microorganisms and as they interact non-specifically with significant quantities of contaminant introduced during use. This will result in a progressive and exponential decline in the efficiency of remaining preservative. Preservative capacity is a term used to describe the cumulative level of contamination that a preserved formulation can tolerate before becoming so depleted as to become ineffective. This will vary with preservative type and complexity of formulation.

111. 3. Factors affecting the availability of preservativesEffect of product pH: In the weakly acidic preservatives, activity resides primarily in the unionized molecules and they only have significant efficacy at pHs where ionization is low. Thus, benzoic and sorbic acids(pKa =4.2 and 4.75,respectively)have limited preservative usefulness above pH 5. The activity of quaternary ammonium preservatives and chlorhexidine probably resides with their cations; they are effective in products of neutral pH. Formulation pH can also directly influence the sensitivity of microorganisms to preservatives.Efficiency in multiphase systems: In a multiphase formulation, such as an oil-in-water emulsion, preservative molecules will distribute themselves in an unstable equilibrium between the bulk aqueous phase and :

112. (i) the oil phase by partition, (ii) the surfactant micelles by solubilization, (iii) polymeric suspending agents and other solutes by competitive displacement of water of solvation, (iv) particulate and container surfaces by adsorption and (v) any microorganisms present. Generally, the overall preservative efficiency can be related to the small proportion of preservative molecules remaining unbound in the bulk aqueous phase. In view of these major potential reduction in preservative efficacy, considerable effort has been directed to devise equations in which one might substitute variously derived system parameters( such as partition coefficients, surfactant and polymer binding constants and oil: water ratios) to obtain estimates of residual preservative levels in aqueous phases.

113. C. Effect of container or packaging: Preservative availability may be appreciably reduced by interaction with packaging materials. Phenolics, for example, will permeate the rubber wads and teats of multi-dose injection or eye-drop containers and also interact with flexible nylon tubes for creams. Quaternary ammonium preservative levels in formulations have been significantly reduced by adsorption onto the surfaces of plastic and glass containers. Volatile preservatives such as chloroform are so readily lost by the routine opening and closing of containers.

114. Lecture 9th:

115. Quality assurance(QA)This term refers to the sum total of the arrangements made to ensure that the final product is of the quality required for its intended purpose. It consists of good manufacturing practice plus factors such as original product design and development.Good manufacturing practice(GMP): GMP comprises that part of quality assurance that is aimed at ensuring the product is consistently manufactured to a quality appropriate for its intended use. GMP requires that:1. The manufacturing process is fully defined before it is commenced.

116. 2. The necessary facilities are provided. In practice, this means that:Personnel must be adequately trained.Suitable premises and equipments employed.Correct materials used.Approved procedures adopted.Suitable storage and transport facilities available.Appropriate records made.

117. Quality control (QC):Quality control refers to the part of GMP that ensures that: 1. at each stage of manufacture the necessary tests are conducted. 2. the product is not released until it has passed these tests. An example is the test for pyrogens applied to sterile pharmaceutical products. In-process control: This comprises any test on a product, the environment or the equipment that is made during the manufacturing process. An example of this is testing that an autoclave is functioning correctly.

118. Control of microbial contamination during manufacture: general aspectsA pharmaceutical product may become contaminated by a number of means and at several points during manufacture. There are several ways in which this risk can be minimized. Any such measures require an understanding of the risks involved.While there is general agreement on the need to control total microbial levels in non-sterile medicines and to exclude certain species that have previously proved troublesome, the precision and accuracy of current methods for counting(or even detecting) some microbes in complex products are poor.Pathogens, present in low numbers, and often damaged by processing, can be very difficult to isolate.

119. Products showing active spoilage can yield surprisingly low viable counts on testing; although present in high numbers, ungerminated spores or a secondary contaminant which has outgrown the initiating spoilerThe type of culture medium and conditions of recovery and incubation may greatly influence any viable counts obtained from products.The British Pharmacopoeia has now included both quantitative and qualitative tests, but suggests that they should be used to assist in validating GPMP processing procedures and not as conformance standards for routine end-product testing. Thus, for a medicine to be administered orally, there should not be more than 103 aerobic bacteria or 102 fungi per gram or cm3 of product, and there should be an absence of Escherichia coli.

120. Hazard analysis of critical control points(HACCP):HACCP has been widely used in the food industry and is becoming more commonly used in the pharmaceutical industry. HACCP is a tool for evaluating steps in a manufacturing process. It provides a structured through process for GMP. The seven steps involved are:Analysis and identification of the potential risk (hazard)represented by each step in the process.Determination of the critical control points(CCP) where it is necessary to control the hazards.Definition of the limits within which each critical parameter should be controlled.Establishment(and validation) of in-process control methods and tests to be used to determine, for each critical control point, whether or not the potential hazard is maintained within the defined limits.

121. 5. Establishment of corrective measures designed to correct any out of control situation at each and every CCP. 6. Confirmation that the HACCP regime(and hence the manufacturing process adopted) is functioning as intended.7. Documentation of the entire system, in terms of both HACCP steps to be followed, and of the results obtained.

122. Manufacture of sterile products:For production purposes an important distinction exists between sterile products which have been terminally sterilized and those which are not. Terminal sterilization involves the product being sealed in its container and then sterilized, usually by heat, but ionizing radiation or, less commonly, ethylene oxide may be employed. Such product must be produced in a clean area. A product cannot be terminally sterilized is prepared aseptically from previously sterilized materials or by sterile filtration and then filled into sterile containers. Strict aseptic conditions are required.Vaccines, consisting of dead microorganisms, microbial extracts or inactivated viruses may be filled in the same premises as other sterile medicinal products.Non-sterile products and sterile products must not be processed in the same area.

123. * Clean & aseptic area : general requirementsDesign of premises: Sterile production should be carried out in a purpose- built unit separated from other manufacturing areas and should ensure a safe and organized workflow.2. Internal surfaces, fittings and floor: All surfaces must have smooth, impervious surfaces which will: a. prevent accumulation of dust or other particulate matter; b. permit easy, repeated cleaning and disinfection. Smooth rounded coving should be used where the wall meets the floor and the ceiling. * Suitable flooring may be provided by welded sheets of polyvinyl chloride(PVC). * The preferred surfaces for walls are plastic, epoxy-coated plaster. False ceilings should be adequately sealed to prevent contamination from the space above . * Doors & windows should be flush with the walls. Windows should not be openable.

124. 3. Services:* Clean and aseptic areas must be adequately illuminated; lights are best housed in translucent panels set in a false ceiling. Electrical switches and sockets must be flush with the wall. * Gases should pumped in from outside the unit. * Pipes and ducts, if they must be brought into the clean area, must be sealed through the walls, and must be boxed in or readily cleanable(to prevent dust accumulation). Alternatively they may be sited above false ceilings. * Sinks should be of stainless steel with no over-flow, and water must be of at least potable quality. * Drains should be avoided. * Any floor channels should be open, shallow and cleanable and connected to drains outside the area.

125. 4. Air supply: Areas for sterile manufacture are classified according to the required characteristics of the environment. Each operation an appropriate level of microbial and particulate cleanliness; four grades (see table 21.1 in 382pp.). * The air supplied to the manufacturing environment substantially influences environmental quality. Filtered air is used to achieve the necessary standards; this should be maintained at positive pressure throughout a clean or aseptic area, with the highest pressure in the most critical rooms(aseptic or clean filling rooms)and a progressive reduction through the preparation and changing rooms; a minimum pressure differential of 10-15 Pa is normally required between each class of room. A minimum of 20 changes of air per hour is usual in clean and aseptic rooms. * See fig.21.4 in 382pp; and table 21.2 in 383pp.

126. 5. Clothing: Clothing worn in the clean area must be of non-shedding fibres; terylene is a suitable fabric. * Air contamination, both microbial and particulate, is reduced when trouser suits, close-fitting at the neck, wrists and ankles, are worn. Clean suits should be provided once a day, but fresh headwear, overshoes and power-free gloves are necessary for each working session. 6. Changing facilities: Entry to clean or aseptic area should be through a changing room fitted with interlocking doors; this acts as an airlock to prevent influx of air from outside. This rout not for the transfer of materials and equipment. For entry into a clean area, passage through the changing room should be from a “black” to a “grey” area, via a dividing step-over sill. Movement trough these areas and finally into the clean room is permitted only when observing a strict protocol, whereby outer garments are removed in the black area and clean room trouser-suits donned in the grey area. After hand-washing in a sink fitted with elbow-or foot-operated taps the operator may enter the clean room.

127. 7. Cleaning and disinfection: Cleaning agents used include alkaline detergents and ionic and non-ionic surfactants. A wide range of chemical disinfectants is available Commonly usedDisinfectantsInterior services & fittingsClear soluble phenolsWorking surfacesAlcohols(70% ethanol or isopropanol)Skin , gloves hands.Cationic detergents such as cetrimide or chlorhexidine usually formulated with 70& alcohol

128. 8. Operation: The number of persons involved in sterile manufacture should be kept to minimum . * Containers made from fibrous materials such as paper, cardboard and sacking are generally heavily contaminated(especially with bacterial spores & moulds) and should not be taken into clean areas. * Containers and closures for use in aseptic manufacture must, in addition, be sterilized after washing and rinsing in preparation for aseptic filling.