New sterilization techniques

Recent advances in sterilization

1. STERILIZATION TECHNIQUES ‘NEW STERILIZATION METHODS’ Presented by: NAIR RAHUL RAGHAVAN 1 st yr M.Pharm (Pharmaceutics), SRIPMS 1

2. Contents What is sterilization? Methods of sterilizations(traditional methods) New sterilization methods Conclusion 2 2

3. What is sterilization? Sterilization can be defined as any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses and prions ) from a surface, equipment, foods, medications, or biological culture medium. DISINFECTION: Disinfection describes a process that eliminates many or all pathogenic microorganisms, except bacterial endospores 3 3

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5. PHYSICAL METHODS HEAT STERILIZATION : -Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents i ) Dry Heat (160-180˚C): Oxidative changes occur. Sterilization for thermostable products, moisture sensitive materials ii) Moist heat (121-134˚C): Hydrolysis & denaturation occurs Sterilization is used for moisture-resistant materials 5 5

6. The efficiency with which heat is able to inactivate microorganisms is dependent upon > the degree of heat, the exposure time and the presence of water . The action of heat will be due to induction of lethal chemical events mediated through the action of water and oxygen. In the presence of water much lower temperature time exposures are required to kill microbe than in the absence of water. 6 6

7. Thermal methods includes: i ) Dry Heat Sterilization Ex:1. Incineration 2. Red heat 3. Flaming 4. Hot air oven ii) Moist Heat Sterilization Ex:1.Dry saturated steam – Autoclaving 2. Boiling water/ steam at atmospheric pressure 3. Hot water below boiling point 7 7

8. Dry heat sterilization Temp range of 160-180˚C & exposure time up to 2 hrs Good penetrability & non-corrosive nature: useful for sterilizing glass wares & metal surgical instruments. Also used for sterilizing non-aqueous thermostable liquids and thermostable powders . Dry heat destroys bacterial endotoxins (or pyrogens): applicable for sterilizing glass bottles which are to be filled aseptically Application: Sterilization of dry powdered drugs, Suspensions of drug in non aq. solvents, Oils, fats, waxes, soft & hard paraffin, Oily injections, implants, ophthalmic ointments & ointment bases etc 8 8 HOT AIR OVEN

9. Moist heat sterilization Moist heat sterilization involves the use of steam in the range of 121-134˚C . Steam under pressure( upto 15lbs) is used to generate high temperature needed for sterilization. Saturated steam acts as an effective sterilizing agent Application : Sterilization of surgical dressings & instruments, containers, closures, medical devices, culture media, etc AUTOCLAVE 9 9

10. Radiation sterilization EMR e.g. Gamma rays and UV light & Particulate radiation (e.g. Accelerated electrons ). Major target for these radiation is Microbial DNA . Use: Sterilization of heat & moisture sensitive products . Gamma-rays ( from Cobalt 60) are used to sterilize antibiotic , hormones , sutures , plastics and catheters etc. UV light : Has Lower energy & poor penetrability Surface sterilization of aseptic work areas ; for treatment of manufacturing grade water ; but is not suitable for sterilization of pharmaceutical dosage forms. 10 10

11. Filtration sterilization Does not destroy but removes the microorganisms Used for clarification and sterilization of liquids and gases The major mechanisms of filtration are Sieving, Adsorption and Trapping within the matrix of the filter material Ex: HEPA FILTERS Used in the treatment of heat sensitive injections & ophthalmic solutions , biological pdts & air & other gases for supply to aseptic areas . Used in industry as part of the venting systems on fermentors , centrifuges, autoclaves and freeze driers . Membrane filters are used for sterility testing 11 11

12. 2 types of filters used: (a) Depth filters : fibrous or granular materials so packed as to form twisted channels of minute dimensions. Made of diatomite, porcelain, sintered glass or asbestos . (b) Membrane filters : Porous membrane 0.1 mm thick, made of cellulose acetate , cellulose nitrate, polycarbonate , etc. Membranes are supported on a frame and held in special holders. Fluids are made to transverse membranes by positive or negative pressure or by centrifugation. 12 12

13. CHEMICAL METHODS GASEOUS STERILIZATION -Gases like formaldehyde & ethylene oxide have biocidal activity – MOA: A lkylations of sulphydryl , amino, hydroxyl and carboxyl groups on proteins & amino gps of nucleic acids . -Conc. Range 800- 1200 mg/L for ethylene oxide & 15-100 mg/L for formaldehyde with operating temperatures of 45-63°C & 70-75°C respectively. - Sterilizing hormones, proteins, various heat sensitive drugs etc. - Demerits: These gases are potentially mutagenic & carcinogenic. They also produce acute toxicity including irritation of the skin, conjunctiva and nasal mucosa 13 13

14. LIQUID STERILIZATION Peracetic acid is sporicidal at low concentrations. Merits: Water soluble & leaves no residue after rinsing. Has no harmful health or environmental effects. MOA: Disrupts bonds in proteins and enzymes and may also interfere with cell membrane transportation through the rupture of cell walls and may oxidize essential enzymes and impair vital biochemical pathways . 14 14

15. RECENT ADVANCES IN STERILIZATION Sterilization, as a specific discipline, has been with us for approximately 120 years, since the invention of the steam autoclave by Charles Chamberland in 1879 We have seen progressive refinement in steam sterilizers: from the early, manually operated equipment to modern microprocessor-controlled, automatic machines. Although the efficiency, reliability, and performance monitoring of modern equipment is continually improving, the fundamental process remains essentially the same. 15 15

16. NEW STERILISATION METHODS Chemical methods Physical methods Physicochemical methods Synergistic methods - Surfacine -Pulsed light sterilization -Gas plasma sterilization - Psoralen and UVA - Superoxidized water -Ultra high pressure sterilization -Ultrasound and bactericide -Chlorine dioxide - Hydroclave - Glutaraldehye -Ortho phthalaldehyde - Endoclens 16

17. SURFACINE Surfacine is a surface coating that kills microorganism on contact, by selectively delivering Silver . Used on animate or inanimate surfaces. Despite the fact that silver ions possess antimicrobial efficacy equal to or greater than other heavy metals ions, but is almost non toxic to mammals. Silver has a broad spectrum of activity (bacteria, yeast and fungi) CHEMICAL METHODS OF STERILIZATION 17 17

18. Mechanism of SURFACINE It incorporates silver iodide in a surface-immobilized coating (a modified polyhexamethylenebiguanide ) that is capable of chemical recognition and interaction with the lipid bilayer of the bacterial cell membrane by electrostatic attraction . The intimate microbial contact with the surface results in transfer of silver directly from the coating to the organism. Microorganisms contacting the coating accumulate silver until the toxicity threshold is exceeded; dead microorganisms eventually lyse and detach from the surface. 18 18

19. A 3-D polymeric network is immobilized onto the substrate surface The immobilized surface network is impregnated with sub-micron particles of silver iodide that result in the formation of AgCl-polymer complex Solubility characteristics of this complex prevent ionic silver from leaching into solutions contacting the surface coating Silver is available, however, to react with bacterial cells contacting the coating 19 19

20. Silver is preferentially transferred directly to the microorganism causing a toxic accumulation that results in cell death. The silver halide reservoir within the polymeric network replenishes the coating surface with silver, allowing the coating to maintain high surface anti microbial activity to microorganisms that contact it for further challenges 20 20

21. Advantages Very low leaching of silver into solutions Duration of activity: Long term Reservoir capacity: High Non toxic to humans Disadvantage Not active against viruses and very less activity against spores. Applications: Can be used in: Medical devices: prostheses, catheters, endotracheal tubes etc Dental care products Food preparation & packaging Water storage, treatment and delivery systems 21 21

22. SUPEROXIDIZED WATER (STERILOX/MEDILOX) Broad spectrum disinfectant, introduced recently. Prepared by electrolyzing saline solution with titanium-coated electrodes at 9 amp The main products are Hypochlorous acid[ HClO ] as well as free chlorine radicals & superoxide radicals The product generated has a pH of 5.0-6.5 and an redox potential of >950 mV. MOA is not clear but probably relates to a mixture of oxidizing species. 22 22

23. Applications: Freshly generated superoxidized water is rapidly effective (<2 minutes) against microorganisms ( Mycobacterium tuberculosis ,  M. chelonae , poliovirus, HIV, MRSA ,  E.coli ,   Candida albicans ,  Enterococcus faecalis ,  Pseudomonas aeruginosa ) Advantages: Basic materials i.e saline and electricity, are cheap & the end product (water) is not damaging to the environment. Nontoxic to Humans Noncorrosive and nondamaging to endoscopes Disadvantage: Equipment used to produce the product may be expensive because parameters such as pH, current, and redox potential must be closely monitored. 23 23

24. CHLORINE DIOXIDE(ClO 2 ) Prepared by reacting Hypochlorus acid and sodium or potassium chlorate. MOA It is a molecular free radical and disinfects by oxidation Organic materials in bacterial cells react with chlorine dioxide, causing several cellular processes to be interrupted Chlorine dioxide reacts directly with amino acids and the RNA in the cell -> proteins synthesis is blocked-> cell death Application: -Drinking water disinfection -Can be used against anthrax (ClO2 is effective against spore forming bacteria ) 24 24

25. Advantage s: Alternative to chlorine- Better disinfectant activity than chlorine. Deactivates chlorine resistant Giardia and Cryptosporidium No odour nuisance, unlike chlorine. Unlike chlorine, prevents formation of harmful halogenated disinfection by-products. Low contact time required . Disadvantages: Explosive Safety equipment must be used while handling as it causes irritation, watery eyes Highly unstable when in contact with sunlight. More expensive than chlorine . 25 25

26. GLUTARALDEHYDE Trade names : Cidex , Sonacide , Sporicidin , Hospex . Cold sterilant - used to sterilize a variety of heat sensitive instruments viz. endoscopes, bronchoscopes, dialysis equipment, anesthesia & respiratory equipment, transducers & spirometry apparatus Concentration: 2-3% -is active against a wide range of microorganisms: Gram positive & negative, mycobacteria & spores MOA: Causes Alkylation of sulfhydryl, hydroxyl, carboxyl, and amino groups of microorganisms -> alters RNA, DNA, and protein synthesis . Aqueous solution of glutaraldehyde is not sporicidal . Only when it is activated(made alkaline ) using alkylating agents ( pH 7.5-8.5 ) does the solution become sporicidal . 26 26

27. Spectrum of activity: Active against MRSA (methicillin resistant Staphylococcus aureus), VRE (vancomycin resistant enterococci), Influenza A virus, E.coli , Salmonella typhi , P.aeruginosa , Klebsiella , Avian rotavirus. Inactive against Mycobacterium chelonae , M.avium-intracelulare , M.xenopi , fungal ascospores, Cryptosporidium Engineering controls: The goal of engineering controls is to keep the vapours from entering the work room & the employee’s breathing zone by containing & removing at the source of release. General room ventilation Local exhaust hoods Transfer procedures 27 27

28. Use and handling: While transferring, pour the liquids carefully and minimize splashing and agitation Rinse soaked instruments under running water. Use adequate ventilation Use protective equipments : Gloves, Glasses, gas masks, etc Transportation and storage: Should be done in closed containers wih tight fitting lids to minimize potential for spills Store in a cool, secured and properly labelled area Dispose off outdated solutions properly 28 28

29. Advantages: Biodegradable Non carcinogenic (unlike formaldehyde) Non corrosive to metals, rubbers, plastics Relatively inexpensive Excellent material compatibility Disadvantages: Side effects due to glutaraldehyde vapours: Respiratory and dermal irritant, occupational asthma, itching of eyes, rhinitis. Pungent & irritating odour Slower mycobacterial activity as compared to OPA Exposure to vapours should be monitored Protective equipment to be used during handling and transfer of gas . 29 29

30. ORTHO-PHTHALDEHYDE(OPA) OPA received clearance by FDA in Oct. 1999. MOA – similar to glutaraldehyde Advantages: Potential advantages compared with glutaraldehyde : Active against glutaraldehyde resistant Mycobacterium Non-irritant to the eyes and nasal passages Has excellent stability over a wide range of pH (pH 3-9) Does not require exposure monitoring, and has a barely perceptible odour. Like glutaraldehyde, OPA has excellent material compatibility.   30 30

31. Disadvantages: OPA stains proteins gray (including unprotected skin) and thus must be handled with caution (i.e., use of gloves, eye protection, fluid-resistant gowns when handling contaminated instruments, contaminated equipment, and chemicals). Limited clinical studies of OPA are available. Disposal: If OPA disposal in the sanitary sewer is restricted, glycine (25 g/gallon) can be used to neutralize the OPA and make it safe for disposal.   31 31

32. ENDOCLENS Used for sterilization of flexible endoscopes Consists of a computer-controlled endoscope- reprocessing machine that uses performic acid as a sterilant . The sterilant is produced by automatic mixing of the two component solutions of hydrogen peroxide and formic acid The system's major features are: an automatic cleaning process capability to process two flexible scopes asynchronously filter water rinsing and scope drying after sterilization hard-copy documentation of key process parameters user-friendly machine interface total cycle time (scope testing, washing, sterilization, and drying) is less than 30 minutes. 32 32

33. PHYSICAL METHODS OF STERILIZATION PULSED LIGHT STERILIZATION It is a nonthermal method for sterilization that involves the use of intense, short duration pulses of a broad spectrum to ensure microbial decontamination. MOA: It appears that both the visible and infrared regions, combined with the high peak power of pulsed light , contribute to killing microorganisms. Various mechanisms have been proposed to explain the lethal effect of pulsed light, all of them related to the UV part of the spectrum and its photochemical and(or) photo-thermal effect. 33 33

34. Photo chemical mechanism Primary target of pulsed light is nucleic acids because DNA is a target molecule for these UV wavelengths. The germicidal effect of UV light has been attributed primarily to a photo-chemical transformation of pyrimidine bases in the DNA of bacteria ,viruses ,and other pathogens to form dimers . Formation of such bonds prevents DNA unzipping for replication & the organism becomes incapable of reproduction. Without sufficient repair mechanisms , such damage results in mutations , impaired replication and genetranscription , and ultimately the death of the organism. 34 34

35. Photo thermal mechanism : The lethal action of pulsed light also can be due to a photothermal effect. Disinfection is achieved through bacterial disruption during their temporary over heating resulting from the absorption of all UV light from a flashlamp . This over heating can be attributed to a difference in UV light absorption by bacteria and that of a surrounding medium. The water content of bacteria is vaporized , that induces membrane disruption. Types of damage induced by pulsed UV light are: Photolysis Loss of colony-forming ability Inability to support phage growth (enzyme inactivation) Destruction of nucleic acid. 35 35

36. CLARANOR STERILIZATION EQUIPMENT: 36 36

37. Operation: The pulse is produced in 2 steps: 1. A 20 kV pulse lasting a few nanoseconds makes the xenon-filled lamp conductive while creating an electric arc in the lamp 2. The capacitor charged at 3000 V discharges in this arc during a 300 microsecond pulse, which ionizes the gas in the lamp and generates a plasma emitting a very high intensity white light (20,000 times greater than sunlight on the earth's surface) The energy of the lamp, which is delivered over a very short period (0.3 ms ), produces a power of 1 MW , half of which is dissipated in heat and the other half in optical energy. 37 37

38. Advantages : Total DNA destruction Quick process No chemicals used. Worker-friendly (safe and easy to use) Minimum space requirements. Water circulated for cooling of lamps can be recycled. Disadvantages : Pulsed light  is a  surface treatment . The decontaminated areas are those which receive the light pulse Applications :  Sterilization of caps ,  cups ,  lids and other packaging materials Sterilization of food packaging 38 38

39. HYDROCLAVE The Hydroclave is essentially a double-walled (jacketed) cylindrical, pressurized vessel, horizontally mounted , with one or more side or top loading doors, and a smaller unloading door at the bottom. CHARACTERISTICS: Sterilizes the waste utilizing steam , similar to an autoclave , but with much faster & much more even heat penetration Hydrolyses the organic components of the waste such as pathological material. Removes the water content ( dehydrates ) the waste. Breaks up the waste into small pieces of fragmented material. Reduces the waste substantially in weight and volume 39 39

40. STEPS INVOLVED IN THE TREATMENT CYCLE a) Loading b) Heat-up and fragmentation c) Sterilization period d) De-pressurization and De-hydration e) Unloading Loading In smaller units, materials to be sterilized are packed & loaded manually In large sized units, a combination of conveyors, hoppers and tippers are available to load the waste into large top loading doors. 40 40

41. b) Heat-up and fragmentation After loading, the vessel doors are closed, and the outer jacket of the vessel is filled with high temperature steam , which acts as an indirect heating medium for heating the waste During heat-up, the shaft and mixing arms rotate , causing the waste to be fragmented & continuously tumbled against the hot vessel walls. The moisture content of the waste will turn to steam & the vessel will start to pressurize.  At the end of this period, the correct sterilization temperature and pressure are reached, and the sterilization period is initiated automatically 41 41

42. c) Sterilization period By computer, the temperature & pressure are maintained for the desired time to achieve sterilization. The mixing/fragmenting arms continue to rotate during the entire sterilization period, to ensure thorough heat penetration into each waste particle. Sterilization time of 15 minutes at 132 C, or 30 minutes at 121 C d) De-pressurization and De-hydration After sterilization period ends, the vessel is de-pressurized via a steam condenser, which causes initial waste dehydration due to depressurization.  The steam to the jacket will remain on, agitation continues, and the waste loses its remaining water content through a combination of heat input from the jacket and continued agitation. 42 42

43. e) Unloading Due to the unique construction of the mixing arms, the opposite rotation causes the fragmented waste to be pushed out of the vessel discharge door, into a waste container, or onto a conveyor. The vessel is now ready for another treatment cycle, having retained most of its heat for the treatment of the next batch. Uses: The Hydroclave can sterilize: Bagged waste, in ordinary bags Sharps containers and needles Liquid containers Cardboard containers Metal objects Plastics 43 43

44. Advantages: Performance Guaranteed high level of sterilization, including wet waste, metals, liquids and sharps. Automatic operation, and not dependent on operator skill for sterility. No infectious or harmful emissions. Economic Low operating cost with low energy consumption. Low maintenance costs. No costly bags, filters or chemicals in the process. Very large weight and volume reduction of the waste.   Dry waste, regardless of its original water content. Low odour , due to the dryness. 44 44

45. Hydroclave Autoclave Low operating cost by recycling steam. No special bags required Treats wet or liquid loads easily Strong weight reduction Strong volume reduction Consistent high sterility Higher operating cost, no steam recycling High temp. bags required Cannot treat wet or liquid loads Weight increase No volume reduction Spotty sterility 45 45

46. Also called High hydrostatic pressure sterilization(HHP)/ Pascalization . Mainly used in food sterilization technology It is a cold sterilization technique by which products, already sealed in its final package , are introduced into a vessel and subjected to a high level of isostatic pressure ( 200–600MPa/43,500-87,000psi ) transmitted by water , for few seconds to few minutes. Pressures > 400 MPa at cold (+ 4ºC to 10ºC)inactivate vegetative flora (bacteria, virus, yeasts, moulds & parasites ) present in food, extending its shelf life ULRA HIGH PRESSURE (UHP) STERILIZATION 46 46

47. MOA: Under UHP the volume of the pdt is compressed , results in the deeper penetration of proteins & other macromolecules into the pdt , resulting in the destruction of its 3-D structure Thus the food's proteins are denatured, hydrogen bonds are fortified, & noncovalent bonds  in the food are disrupted , while the product's main structure remains intact -> causes starch and protein denaturation and enzyme inactivation , all of which may have an effect on the texture of the product. Pressure is instantly and evenly applied to the product, regardless of its size, shape and volume. Because UHP sterilization is not heat-based ,  covalent bonds  are not affected , causing no change in the food's taste 47 47

48. Advantages: Being a cold sterilization the characteristics of the fresh product are retained, nutritional properties, flavor and taste remain almost intact. Destroys pathogens  ( Listeria, Salmonella, Vibrio, Norovirus , etc.) Extends product shelf life : improved customer satisfaction. Avoids or reduces the need for food preservatives : Clean label foods (Natural/Additive Free). Only needs water (which is recycled) & lower electricity consumption: Environment friendly . Disadvantages: For the inactivation of bacterial endospores require synergistic action of very high pressure(>600MPa) & temperature(>60*C) 48 48

49. PHYSICOCHEMICAL METHODS OF STERILIZATION GAS PLASMA STERILIZATION Discovered by Sir William Crookes in 1879. This technology was patented in 1987 and marketed in U.S in 1993 Plasma is defined as an ionized gas with an equal no. of + ve & - ve ions. 4 th state of matter. Its properties are similar to those of both gases and liquids. Gas plasma is generated in an enclosed chamber under deep vacuum (low pressure) using radiowaves or microwaves to excite gas molecules (hydrogen peroxide) to produce ionized gas particles 49 49

50. MOA : It operates synergistically via three mechanisms: Free radicals interactions UV/VUV radiative effects Volatilization UV/VUV radiation causes -Formation of thymine dimers in DNA, inhibiting bacterial replication. -Base damage -Strand breaks Volatilization: It is able to vaporize microbiological matter, causing physical destruction of spores. Application: STERRAD System enable sterilization of surgical instruments, rigid and flexible endoscopes, cameras, catheters,etc . 50 50

51. Steps involved in plasma steriliztion : 1. The Vacuum Phase: Internal pressure is reduced by evacuating the chamber 2. The Injection Phase: Liquid peroxide is injected into the chamber, evaporating the aqueous hydrogen peroxide solution & dispersing it into the chamber, where it kills bacteria on any surface it can reach 3. Diffusion phase: The H202 vapor permeates the chamber, exposing all load surfaces to the Sterilant and rapidly sterilizes devices and materials without leaving any toxic residues 4. The Plasma Phase An electromagnetic field is created in which the H2O2 vapour breaks apart, producing a low-temperature plasma cloud that contains ultraviolet light and free radicals. 5. The Vent Phase The chamber is vented to equalize the pressure enabling the chamber door to be opened. There is no need for aeration or cool-down. Devices are ready for immediate use. 51 51

52. Advantages : The process is usually at room temperature and hence poses no dangers associated with high temperatures (unlike autoclaves) Byproducts are generally water and oxygen-harmless to the environment Time of treatment is fast(1 min or less) Disadvantages : Weak penetrating power of the plasma. Complications arise in: -Presence of organic residue -Packaging material -Complex geometries -Bulk sterilization of many devices High power consumption Can corrode certain materials 52 52

53. SYNERGISTIC STERILIZATION METHODS PUVA ( Psoralen and UVA) Psoralen is a coumarin derivative which occurs naturally in the seeds of  Psoralea corylifolia , as well as in the common fig, celery, parsley and in all citrus fruits MOA : Psoralen intercalates into DNA and on exposure to ultraviolet (UVA) radiation can form monoadducts and covalent interstrand cross-links (ICL) with thymine, resulting in apoptosis . Uses: -Sterilization of Blood plasma and platelets -It is also active against viruses ex. HIV, hepatits , etc -Other uses of PUVA: Treatment of psoriasis, eczema, vitiligo 53 53

54. ULTRASOUND AND BACTERICIDE: Low(20kHz) or High(250kHz) frequency ultrasound energy is synergistic with low concentration Glutaraldehyde This technique is effective in killing bacterial endospores . Advantages: - Heat sensitive materials can be sterilized(plastics and rubber equipments) -Inexpensive and quick sterilization is possible. 54 45

55. CONCLUSION We have seen the new methods used in sterilization, which have been developed in the past few decades and currently in use in the industries for sterilizing medical instruments, foods and pharmaceuticals. Most of these methods are automated and efficient, environment friendly, have low running cost, can sterilize on a large scale, highly effective against certain resistant microorganisms and ensure complete sterility of products. 55 55

56. REFERENCE The theory and practice of industrial pharmacy by Lachman and Lieberman Gas Plasma Sterilization in Microbiology: Theory, Applications, Pitfalls and New Perspectives by Hideharu Shintani , Akikazu Sakudo Disinfection, sterilization and preservation by Seymour Stanton Hospital sterilization by Anand Nagaraja Prem www.Medscape.com www.mddionline.com Thank You…. 56 56