1. E COLOGY OF MICROORGANISMS MIC 306 1

2. P HARMACEUTICAL MICROBIOLOGY Pharmaceutical microbiology is the part of industrial microbiology that is responsible for ensuring medication do not contain harmful levels of microbes- such as bacteria, yeast & molds. 2

3. The microbiological quality of pharmaceutical products is influenced by the environment in which they are manufactured and the materials used in their formulation 3

4. S PECIFICATIONS FOR M ICROBIOLOGICAL Q UALITY CategoryType of PreparationSuggested Limit 1 Products for Topical Application (including those for use in body cavities) 1aFor use on broken* and unbroken skin (other than antiseptics and corticosteroids) TAMC** not more than 10 2 per mL or per gram amongst which there should be no pseudomonads no Staphylococcus aureus 1bAntiseptics and corticosteroids TAMC** not more than 10 per mL or per gram amongst which there should be no pseudomonads no Staphylococcus aureus 4

5. S PECIFICATIONS FOR M ICROBIOLOGICAL Q UALITY CategoryType of PreparationSuggested Limit 2 Products for Oral Use 2aProducts other than those containing raw material of vegetable or animal origin TAMC** not more than 10 3 per mL or per gram amongst which there should be not more than 10 2 yeast and mould not more than 10 2 enterobacteria no E coli in 1mL or 1g no salmonellae in 10mL or 10g 2bProducts containing raw materials of vegetable or animal origin TAMC** not more than 10 4 per mL or per gram amongst which there should be not more than 10 2 yeast and mould not more than 10 2 enterobacteria no E coli in 1mL or 1g no salmonellae in 10mL or 10g 2cHerbal teasTAMC** not more than 10 5 per gram amongst which there should be not more than 10 2 yeast and mould not more than 10 2 enterobacteria no E coli in 1mL or 1g no salmonellae in 10mL or 10g * "broken skin" refers to minor cuts and abrasions; products intended for use on large open wounds or severely damaged skin should be sterile. ** Total Aerobic Microbial Count 5

6. S OURCE OF MICROFLORA The microflora of the final product may represent 1. the contaminants from the raw materials 2. the equipment with which it was made 3. the atmosphere 4. the person operating the process or 5. the final container into which it was packed 6

7. 7

8. H OW TO M INIMIZE ? With improvements in manufacturing technology there have been developments in Good Manufacturing Practices (GMP) to minimize contamination By a study of the ecology of microorganisms The hazards posed by them and Any points in the process which are critical to their control. 8

9. G OOD M ANUFACTURING P RACTICE (GMP) T HE MAIN CONCEPT HERE IS THAT PRODUCTION TAKES PLACE ACCORDING TO COMPREHENSIVE, APPROVED STANDARDS ( AND NOT ACCORDING TO INDIVIDUAL PERSONAL EXPERIENCE AND KNOWLEDGE ) WITH PROPER MONITORING AND DOCUMENTATION. Good Analytical Practice (GAP) This is equivalent to GMP, as applied to analytical procedures. It is also called Good Laboratory Practice (GLP). 9

10. I MPLEMENTATION OF THESE APPROACHES This approach has been distilled into the concept of Hazard Analysis of Critical Control Points (HACCP ), with the objective of Improving the microbiological safety of the product in a cost-effective manner, which has been assisted by the development of rapid methods for the detection of microorganisms 10

11. I MPROVING GUIDELINE BY HACCP 11

12. A TMOSPHERE Air is not a natural environment for the growth and reproduction of microorganisms, as it does not contain the necessary amount of moisture and nutrients Any sample of untreated air contains suspended bacteria, moulds and yeasts, but to survive they must be able to tolerate desiccation and the continuing dry state. 12

13. A VAILABILITY OF MICROORGANISMS Microorganisms commonly isolated from air are the spore-forming bacteria Bacillus spp. and Clostridium spp., the non-sporing bacteria Staphylococcus spp., Streptococcus spp. and Corynebacterium spp., the moulds Penicillium spp., Cladosporium spp., Aspergillus spp. and Mucor spp., as well as The yeast Rhodotorula spp. 13

14. The number of organisms in the atmosphere depends on the activity in the environment and the amount of dust that is disturbed An area containing working machinery and active personnel will have a higher microbial count than one with a still atmosphere the air count of a dirty, untidy room will be greater than that of a clean room. The microbial air count is also influenced by humidity. 14

15. H OW THE LOAD IS INCREASED The microbial content of the air may be increased during the handling of contaminated materials during dispensing, blending and their addition to formulations. the use of starches and some sugars in the dry state may increase the mould count. Some packaging components, e.g. card and paperboard, have a microflora of both moulds and bacteria, and high counts around packaging machines 15

16. C OMMON METHODS FOR CHECKING THE MICROBIOLOGICAL QUALITY OF AIR INCLUDE THE FOLLOWING The exposure of Petri dishes containing a nutrient agar to the atmosphere for a given length of time. This relies upon microorganisms or dust particles bearing them settling on the surface. The 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 a Petri dish, a plastic strip or a membrane filter which may then be incubated with a nutrient medium. 16

17. P HARMACEUTICAL POINT OF VIEW In areas where products for injection and ophthalmic use which cannot be terminally sterilized by moist heat are being manufactured, the air count should be very low and regarded as a critical control point in the process The manufacture of liquid or semi-solid preparations for either oral or topical use requires a clean environment for both the production and filling stages. 17

18. While many formulations are adequately protected by chemical preservatives or a pH unfavorable to airborne bacteria that may settle in them, preservation against mould spores is more difficult to achieve 18

19. R EDUCTION OF MICROBIAL COUNT The microbial count of air may be reduced by filtration, chemical disinfection and to a limited extent by ultraviolet (UV) light Filtration Is the most commonly used method and filters may be made of a variety of materials such as cellulose, glass, wool, fibreglass mixtures or polytetrafluorethylene (PTFE) with resin or acrylic binders. 19

20. Chemical disinfectants Are limited in their use as air sterilants because of their irritant properties when sprayed. atomized propylene glycol at a concentration concentration of 0.05–0.5mg/L and quaternary ammonium compounds (QACs) at 0.075% may be used. For areas that can be effectively sealed off for fumigation purposes, formaldehyde gas at a concentration of 1–2 mg/L of air at a relative humidity of 80–90% is effective 20

21. UV irradiation At wavelengths between 240 and 280 nm (2400 and 2800 Å) is used to reduce bacterial contamination of air, but it is only active at a relatively short distance from the source. Bacteria and mould spores, particularly those with heavily pigmented spore coats, are often resistant to such treatment. It is however, useful if used in combination with air filtration. 21

22. W ATER The microbial ecology of water is of great importance in the pharmaceutical industry owing to its multiple uses as a constituent of many products as well as for various washing and cooling processes. Two main aspects are involved: the quality of the raw water and any processing it receives and the distribution system. 22

23. G ENERAL FLORA PRESENT IN WATER Microorganisms indigenous to fresh water in clude Pseudomonas spp., Alcaligenes spp., Flavobacterium spp., Chromobacter spp. and Serratia spp. Such bacteria are nutritionally undemanding and often have a relatively low optimum growth temperature. Bacteria which are introduced as a result of soil erosion, heavy rainfall and decaying plant matter include Bacillus subtilis, B. megaterium, Enterobacter aerogenes and Enterobacter cloacae. 23

24. Contamination by sewage results in the presence of Proteus spp., Escherichia coli and other enterobacteria, Streptococcus faecalis and Clostridium spp. Bacteria which are introduced as a result of animal or plant debris usually die as a result of the unfavorable condition 24

25. R AW OR MAINS WATER When the supply is derived from surface water the flora is usually more abundant and faster- growing than that of supplies from a deep-water source such as a well or spring. This is due to 1. surface waters receiving both microorganisms and 2. nutrients from soil and sewage while water from deep sources has its microflora filtered out 25

26. D OMESTIV VS I NDUSTRIAL STORAGE On prolonged storage in a reservoir, in domestic storage waterborne organisms tend to settle out, but in industrial storage tanks the intermittent (IRREGULAR ) throughput ensures that, unless treated, the contents of the tank serve as a source of infection. The bacterial count may rise rapidly in such tanks during summer months and reach 10 5 –10 6 per ml. 26

27. S OFTENED WATER This is usually prepared by either a base- exchange method using sodium zeolite, by a lime- soda ash process, or by the addition of sodium hexametaphosphate. Softened water is often used for washing containers before filling with liquid or semi-solid preparations and for cooling systems. 27

28. D EIONIZED OR DEMINERALIZED WATER is prepared by passing mains 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 beds which are not regenerated frequently with strong acid or alkali are often heavily contaminated and add to the bacterial content of the water Deionized water is used in pharmaceutical formulations, for washing containers and plant, and for the preparation of disinfectant solutions 28

29. D ISTILLED WATER Distilled water 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. A level of organisms up to 106 per ml has been recorded Distilled water is often used in the formulation of oral and topical pharmaceutical preparations and a low bacterial count is desirable 29

30. Water produced by reverse osmosis Water produced by reverse osmosis (RO) is forced by an osmotic pressure through a semi-permeable membrane which acts as a molecular filter 30

31. D ISINFECTION OF WATER Three methods are used for treating water, namely chemicals, filtration or light. 1. Chemical treatment Chemical treatment is applicable usually to raw, mains and softened water, but is also used to treat the storage and distribution systems of distilled and deionized water and of water produced by reverse osmosis 31

32. Treatment process Sodium hypochlorite and chlorine gas are the most common agents for treating the water supply Level required For most purposes a free residual chlorine level of 0.5–5 ppm is adequate. For storage vessels, pipelines, pumps and outlets a higher level of 50–100 ppm may be necessary 32

33. 2. F ILTRATION Membrane filtration is useful where the usage is moderate and a continuous circulation of water can be maintained As many waterborne bacteria are small, it is usual to install a 0.22-μm pore-size membrane as the terminal filter 33

34. 3. L IGHT UV light at a wavelength of 254 nm is useful for the disinfection of water of good optical clarity. Such treatment has an advantage over chemical disinfection as there is no odor or flavor problem and, unlike membrane filters, it is not subject to microbial colonization. 34

35. R AW M ATERIALS Raw materials account for a high proportion of the microorganisms introduced during the manufacture of pharmaceuticals The selection of materials of a good microbiological quality aids in the control of contamination levels in both products and the environment 35

36. G ENERAL CONSIDERATION Common to have to accept raw materials which have some nonpathogenic microorganisms present An assessment must be made 1. to the risk of their survival to spoil the finished product by growing in the presence of a preservative system or 2. the efficacy of an in-process treatment stage to destroy or remove them 36

37. S OURCE Untreated raw materials that are derived from a natural source usually support an extensive and varied microflora. 1. Products from animal sources such as gelatine, desiccated thyroid, pancreas may be contaminated with animal-borne pathogens - Escherichia coli and Salmonella spp 2. The materials of plant origin such as gum acacia agar, powdered rhubarb and starches may arise from that indigenous to plants and may include bacteria such as Erwinia spp., Pseudomonas spp., Lactobacillus spp., Bacillus spp. 3. streptococci, moulds such as Cladosporium spp., Alternaria spp. and Fusarium spp and non-mycelated yeasts, or those introduced during cultivation. 37

38. C ONTROL OF RAW MATERIAL CONTAMINATION 1. Some refining processes modify the microflora of raw materials, for example drying may concentrate the level of spore-forming bacteria 2. Synthetic raw materials are usually free from all but incidental microbial contamination 3. The storage condition of raw materials, particularly hygroscopic substances, is important, and as a minimum water activity is required for contamination Eg: 0.70 for osmophilic yeasts, 0.80 for most spoilage moulds and 0.91 for most spoilage bacteria 38

39. 4. Some packaging used such as unlined paper sacks, may absorb moisture and may itself be subject to microbial deterioration. For this reason polythene-lined sacks are preferable 5. Some liquid or semi-solid raw materials contain preservatives, but others such as syrups, it is important that they are held at a constant temperature, as any variation may result in evaporation of some of the water content followed by condensation and dilution of the surface layers to give an Aw value which may permit the growth of osmophiles and spoil the syrup 39

40. P ACKAGING M ATERIAL Packaging material has a dual role and acts both 1. to contain the product and to prevent the entry of microorganisms or 2. moisture which may result in spoilage The microflora of packaging materials is dependent upon both its composition and storage conditions 40

41. P URPOSE OF PACKAGING 1. To keep the product safe 2. To make it look more attractive 3. To retain and maintain the product’s quality for longer duration 4. To keep the product dust free and germ free 5. To give it a good brand appeal 6. To give the product a distinct identity amongst the other products 41

42. P ACKAGING MATERIALS CRITERIA They must protect the product from environmental conditions They must not be reactive with the product. They must not impart to the product taste or odor. They must be nontoxic They must be FDA approved They must meet applicable tamper resistance requirements. They must be adaptable to high speed packaging equipments

43. T YPES OF MATERIALS Glass Plastics Metals

44. G LASS CONTAINERS commonly used in packaging because it has superior protective properties. Economical Readily available in a variety of sizes and shapes. Chemically inert. Impermeable Rigid. Strong FDA approved. Does not deteriorate with age. Excellent barrier from all environmental conditions except light. Colored glass, amber, gives protection from light.

45. G LASS CONTAINERS Disadvantages of glass: fragility. Weight.

46. C OLORED GLASS, LIGHT PROTECTION Clear Amber Blue Emerald Green Only Amber and green glass can protect products from sunlight by screening out UV light

47. P LASTIC CONTAINERS Usefulness: Ease of formation. High quality. Freedom of design. Extremely resistant to breakage.

48. P LASTIC CONTAINERS Polymers usually used in the making of plastic containers: Polyethylene. Polypropylene PVC Polystyrene.

49. P OLYETHYLENE Most widely used for containers by the pharmaceutical industry. Good barrier against moisture. Unaffected by strong acids and alkalies. Low cost Permeable to oxygen, odors, and flavors. Limits its use for some pharmaceutical preparations.

50. P OLYPROPYLENE Good resistance to almost all types of chemicals such as strong acids alkalies, etc. Has high boiling point, good for sterilizable products. Excellent gas and vapor barrier. Slightly better than high density polyethylene.

51. P OLYPROPYLENE Good resistance to almost all types of chemicals such as strong acids alkalies, etc. Has high boiling point, good for sterilizable products. Excellent gas and vapor barrier. Slightly better than high density polyethylene.

52. P OLYSTYRENE Rigid, crystal clear. Low cost. High water vapor transmission as well as high oxygen permeability. Not usefull for packaging liquid products. Resistant to strong acids but not to strong alkalies or oxidizing agents.

53. B UILDINGS - W ALLS AND C EILINGS Moulds are the most common flora of walls and ceilings and the species usually found are Cladospo rium spp., Aspergillus spp., particularly A. niger and A. flavus, Penicillium spp. and Aureobasidium ( Pullularia) spp The organisms derive most of their nutrients from the plaster onto which the paint has been applied The addition of up to 1% of a fungistat such as pentachlorophenol, 8-hydroxyquinoline or salicylanilide is an advantage To reduce microbial growth, all walls and ceilings should be smooth, impervious and washable 53

54. B UILDINGS - F LOORS AND D RAINS To minimize microbial contamination, all floors should be 1. easy to clean, 2. impervious to water and 3. laid on a flat surface Any joints in the floor, necessary for expansion, should be adequately sealed. The floor-to-wall junction should be coved In areas where acid or alkaline chemicals or cleaning fluids are applied, a resistant sealing and jointing material must be used otherwise the surface becomes pitted and porous and readily harbours microorganisms 54

55. Connections to drains should be outside areas where sensitive products are being manufactured drains should be avoided in areas where aseptic operations are being carried out. 55

56. D OORS, WINDOWS AND FITTINGS To prevent dust from collecting, all ledges, doors and windows should fit flush with walls. Doors should be well fitting to reduce the entry of microorganisms all windows in manufacturing areas should serve only to permit light entry and should not be used for ventilation Overhead pipes in all manufacturing areas should be sited away from equipment to prevent condensation and possible contaminants from falling into the product 56

57. E QUIPMENT The type and extent of growth in equipment will depend on 1. the source of the contamination, 2. the nutrients available and 3. The environmental conditions, in particular the temperature and pH 57

58. H OW TO C ONTROL All equipment should be easy to dismantle and clean. All surfaces that are in contact with the product should be smooth, continuous and free from pits all sharp corners eliminated and junctions rounded or coved There should be no inside screw threads and all outside threads should be readily accessible for cleaning. Coupling nuts on all pipework and valves should be capable of being taken apart and cleaned 58

59. Valves should be plug type instead of diaphragm type All pipelines should slope away from the product source and all process and storage vessels should be self-draining If a vacuum exhaust system is used to remove the air or steam from a vessel, it is necessary to clean and disinfect all fittings regularly If any filters or straining bags made from canvas, muslin or paper are used,they are cleaned and sterilized regularly to prevent the growth of moulds such as Cladosporium spp., Stachybotrys spp. and Aureobasidium (Pullularia) pullulans, which utilize cellulose 59

60. 60

61. 61

62. 62

63. The agent selected must fulfill the following criteria. 1. It must suit the surface to be cleaned and not cause corrosion. 2. It must remove the product without leaving a residue. 3. It must be compatible with the water supply. 63

64. 64

65. 65

66. 66