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Presented by Kunche Satya ASHOK M.Pharm 1 st year PHARMACEUTICAL STERILIZATION


INTRODUCTION DEFINTIONS RELATED TO STERILIZATION CLASSIFICATION STERILIZATION PARAMETERS METHODS OF CONTROL:- Control of Microorganisms by Physical Methods Control of Microorganisms by Chemical Methods FDA Guidance Conclusion References CONTENTS


The Egyptians used fire to sterilize infectious materials and disinfectants to embalm bodies, and the Greeks burned sulfur to fumigate sick rooms (a gas or vapor that’s smells strongly or is dangerous to breath). To day the ability to destroy microorganisms is No less important; it makes possible the aseptic techniques used in microbiological research , the preservation of food and the preservation of disease. These techniques are essential to personal safety in both the laboratory and hospital. The goal is two fold 1)To destroy pathogens and prevents their transmission . 2)To reduce or eliminate microorganisms responsible for the contamination of water, food and other substances. These two points are together referred as CONTROL INTRODUCTION

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Sterilization is an essential concept in the preparation of sterile pharmaceutical products. It’s main aim is to provide a product that is safe and eliminates the possibility of introducing infection. Definition :- sterilization is a process used to destroy or eliminate viable microorganisms that may be present in or on a particular product or package. (or) Is the process by which a product is rendered sterile i.e. the destruction or removal of microorganisms. The term sterile indicates a probable condition of complete freedom from viable microorganisms with the limitations. The currently accepted performance target for a sterilization process is that it provides for a probability of finding a non sterile unit less than 1 in 1 million


STERILIZATION:- Sterilization is the process of killing all forms of microbial life in or on the given object or preparation. Microbiologically, sterile material is one that contains No living organisms at all and the term sterile is therefore an absolute one. Accordingly, an object is sterile or non sterile but it can never be semi sterile or almost sterile. ANTISEPTIC:- A substance that arrests sepsis i.e. prevents the growth or action of microorganisms by inhibiting their activity without necessarily destroying them. These can be applied on human body. DEFINTIONS

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VIABLE:- Live and growing bacteria or microorganisms + spores. DISINFECTION:- A process that removes infection potential by destroying microorganisms but not ordinarily bacterial spores. DISINFECTANTS:- These are generally meant for application on inanimate objects. GERMICIDES:- A substance that kills disease microorganisms i.e. pathogens/germs but not necessarily spores. STERILITY:- The absence of viable organisms. DEATH:- The term death as used in microbiology is defined as the irreversible loss of the ability to reproduce


Sterilization process can be basically separated as Terminal and Non Terminal process based on stage at which the preparation is subjected to the process of sterilization. Terminal Sterilization: Different types of terminal sterilization techniques employed are physical sterilization and chemical sterilization. Non Terminal Sterilization: it includes the filtration procedure. Based on principle of mechanism involved in the process are further classified as Physical sterilization: this class includes heat sterilization and radiation sterilization as well. CLASSIFICATION


Heat sterilization procedure is one in which the destruction of microorganisms mainly occurs due to the high temperatures employed includes a) Moist heat sterilization b) Dry heat sterilization Radiation sterilization: this process is accomplished by exposure to UV light or high-energy ionizing radiation such as gamma rays and accelerated electrons i.e. particulate radiation as well.. Chemical sterilization: the procedures which involve treatment of the preparations to be sterilized with certain chemicals in either gaseous form or in liquid form are categorized under this class. Contd ….


Definitions of terms Bioburden :- The population or number of living microorganisms per defined unit. D value :- It is the parameter calculated as the time taken for one log 90% reduction (Decimal reduction) in the number of microorganisms. Z value :- It calculates the temperature or dose of radiation sterilization required to produce a one log 90% reduction in D value for a particular organism. F value :- This value is used to compare the lethality of different heat sterilization procedures. Survival curves :- They are plots of the logarithm of the fraction of survivals (microorganisms which retain viability following a sterilization process) against the exposure time or dose. STERILIZATION PARAMETERS


Sterility Assurance Level :- An estimation of the effectiveness of a sterilization process. It usually expressed in terms of negative power of 10 (i.e. 1 in 1 million = 10-6). Validation :- The act of verifying that a procedure is capable of producing the intended result under prescribed circumstances and challenges to predefined specifications. Contd ….

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Source:- REMINGTON Page No -778 Source:-Encyclopedia of pharmaceutical Technology Page No -3513

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Source:- Encyclopedia of Pharmaceutical Technology, James Sarbwick , Page No 3530

Control of M.O's by Physical Methods:

Includes :- Thermal methods (Heat) Radiation method Filtration method Thermal methods or Heat sterilization :- Heat sterilization is most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents. This method of sterilization can be applied only to the thermo stable products, but it can be used for Moisture – Sensitive materials – Dry heat sterilization Moisture – Resistance materials – Moist heat Control of M.O's by Physical Methods


The efficiency with which heat is able to inactivate microorganisms is depends up on Degree of heat The exposure time & The presence of water This process is of two types Dry heat sterilization Moist heat sterilization Dry heat sterilization :- Examples of dry heat sterilization are 1) Incineration 2) Red heat 3) flamming Contd …

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Hot air oven There are various temperatures and periods of treatment for dry heat depending on the pharmacopeia. The U.S Pharmacopeia states that the dry heat sterilization process for containers for sterile pharmaceutical products should be at a temperature of 160-170 0C for a period of 2-4 hr. The British Pharmacopeia states that items sterilized by dry heat should be kept at a temperature not less than 160 0C for at least 1 hr. The benefit of dry heat includes good penetrability and non corrosive nature which makes it applicable for sterilizing glass ware and metal surgical instruments also non aqueous thermo stable liquids and thermo stable powders.

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Hot Air Oven :- Dry heat sterilization is usually carried out in a hot air oven , which consists of the following.. 1) An insulated chamber surrounded by an outer case containing electric heater. 2) A fan. 3) Thermocouples. 4) Temperature Sensor. 5) Door locking controls. Operation :- Articles to be sterilized are first wrapped or enclosed in a container of card board paper or aluminium. Then the material are arranged to ensure uninterrupted air flow. Hot Air Oven

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Moist heat sterilization :- Moist heat sterilization is otherwise referred as Steam Sterilization Under Pressure. Moist heat may be used in three forms to achieve microbial inactivation. Dry saturated steam – Autoclaving Boiling water / steam at atmospheric pressure Hot water below boiling point Conditions to be followed for the moist heat sterilization According to USP XXI and BP 1988 are given as Pressure : 15lb / square inch (psi) Temperature : 121 0C Time : 15 minutes.

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Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable systems available for the decontamination of laboratory glass ware, media and reagents. For efficient heat transfer, steam must flush the air out of the autoclave chamber. Before using the autoclave, check the drain screen at the bottom of the chamber and clean if blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the autoclave , preventing efficient operation. Autoclaves should be tested periodically with Biological Indicators like of Bacillus sterothermophilus to ensure proper function.

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Autoclaves or steam sterilizers essentially consists of :- 1) A cylindrical or rectangular chamber, with capacities ranging from 400-800 lit. 2) Water heating systems or steam generating systems. 3) Steam outlet and inlet valves. 4) Single or double doors with locking mechanism. 5) Thermometer or temperature gauge. 6) Pressure gauge. Operation : For porous loads (dressings) sterilizers are generally operated at a minimum temperature of 134 oC For bottled fluid, sterilizers employing a minimum temperature of 121 oC are used

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Ensure that there should be sufficient water in the autoclave to produce the steam. The stages of operation of autoclaves includes air removal, steam admission and sterilization cycle (including heating up, holding / exposure & cooling stages). INTERNAL STRUCTURE OF AUTOCLAVE

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It sterilizes containers such as 1) LVPs in glass bottles 2) LVPs and SVPs in plastic containers 3) Prefilled glass or plastic syringes 4) Jars and similar containers with press on or screw caps 5) Blisters containing various materials, for example Disposable contact lenses. AUTOCLAVE

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Agent Mechanisms of Action Comments Moist Heat, boiling Denatures proteins Kills vegetative bacterial cells and viruses Endospores survive Moist Heat, Autoclaving Denatures proteins 121°C at 15 p.s.i. for 30 min k ills everything Moist Heat, Pasteurization Denatures proteins Kills pathogens in food products Dry Heat, Flaming Incineration of contaminants Used for inoculating loop Dry Heat, Hot air oven Oxidation & Denatures proteins 170°C for 2 hours ; Used for glassware & instrument sterilization Filtration Separation of bacteria from liquid (HEPA: from air) Used for heat sensitive liquids Cold, Lyophilization (also desiccation) Desiccation and low temperature Used for food & drug preservation; Does not necessarily kill so used for Long-term storage of bacterial cultures Cold, Refrigeration Decreased chemical reaction rate Bacteriostatic Osmotic Pressure, Addition of salt or sugar Plasmolysis of contaminants Used in food preservation (less effective against fungi) Radiation, UV DNA damage (thymine dimers) Limited penetration Radiation, X-rays DNA damage Used for sterilizing medical supplies Strong vis. Light Line-drying laundry

Control of M.O's by chemical agents:

Also called as BIOCIDES Factors influencing on microbial chemical agent. The kinds of microorganisms potentially present The concentration and nature of the disinfectant to be used The disinfectant should be stable upon storage , odorless or with odor, soluble in water and lipids for permeation into microorganisms, and a low surface tension so that it can enter cracks in surfaces The main factor is chemical must be toxic for infectious agents, it should not be toxic to people or corrosive for common materials in practice, this balance between effectiveness and low toxicity for animals is hard to achieve. Control of M.O's by chemical agents


Phenol is the first widely used antiseptic and disinfectant. In 1867 Joseph leister employed it to reduce the risk of infection during operations. Today phenol and phenolics(phenol derivatives) such as cresols,xylenols and ortho phenyl phenol are used as disinfectants in laboratories and hospitals. Phenolics act by denaturing proteins and disrupting cell membranes they have some advantages as disinfectants; phenolics are tuberculocidal , effective in the presence of organic material and remain active on surface long after application. Remarkable success has been achieved in modifying the phenol molecule by the introduction of chlorine and methyl groups , as in chlorocresol and chloroxylenol. PHENOLS

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Chlorocresol is used as a bactericide in injections and to preserve oil-in-water creams, whereas chloroxylenol is employed as a house hold and hospital antiseptic. Phenol may it self be rendered less caustic by dilution to 1% w/v or less for lotions and gargles or by dissolving in glycerol for use as ear drops. Bisphenols such as hexachlorophane useful in skin antiseptic property but it restricted in UK because of brain damage. Chloro cresol Chloroxylenol


Alcohols are among the most widely used disinfectant and antiseptics they are bactericidal and fungicidal but not sporicidal; some lipid containing viruses can also destroyed. The two most popular alcohols germicides are ethanol and isopropanol usually used in about 70-80% concentration. They act by denaturing proteins and possibly by dissolving membrane lipids. A 10-15 min soaking in sufficient to disinfectant thermometers and small instruments. The effect of aromatic substitution is to produce a range of compounds which are less volatile and less rapidly active and final use as preservation. ALCOHOLS

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Example :- phenyl ethanol for eye drops and contact lens solution, benzyl alcohol in injections. PHENYL ETHANOL BENZYL ALOHOL


The halogens Iodine and Chlorine are important antimicrobial agents. Iodine is used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins. At high conc it may kill some spores . Iodine often has been applied as tincture of iodine(BP 1973), 2% or more iodine in water – ethanol soln of potassium iodide( lugol’s iodine BP 1973) Although it is an effective antiseptic the skin may be damaged a stain is left and iodine allergies can result. The staining and irritant properties of iodine have resulted in the development of IODOPHORES, mixtures of iodine with surface active agents which hold the iodine in micellar combination from which it released slowly. HALOGENS

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Some popular brands are Wescodyne for skin and Betadine (polyvinyl pyrrolidone iodine formulated as 10% povidone iodine) used as a non staining, non irritant antiseptic for wounds.

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Chlorine is usual disinfectant for municipal water supplies. One potential problem is that chorine reacts with organic compounds to form carcinogenic trihalomethanes which must be monitored in drinking water. Ozone some times has been used successfully as a alternative to chlorination in Europe and Canada. Traditional chlorine containing pharmaceutical formulations are Eusol- chlorinated lime and boric acid solution and Dakin’s solution (surgical chlorinated soda solution BPC 1973) both of which are designed to provide slow release of chlorine. Sometimes alternatively Chloramine T used for prolonged release of chlorine. Halazone slowly released chloride when added to water and disinfects.


Many metallic ions are toxic to essential enzyme systems particularly those utilizing thiol (-SH) groups, but those used medically are restricted to mercury, silver and aluminium. Phenyl mercuric nitrate(and acetate) as a bactericide in eye drops and injections and Thomersal as a preservative in biological products and certain eye drops. A 1% solution of silver nitrate is used into eyes of infants to prevent ophthalmic gonorrhea Silver sulfadiazine is used on burns. Heavy metals combine with proteins often with their sulfhydryl groups and inactivate them they may also precipitate cell proteins. HEAVY METALS


The most popular of these disinfectant are quaternary ammonium compounds characterized by a positively charged quaternary nitrogen and a long hydrophobic aliphatic chain. They disrupt microbial membranes and may also denature proteins. Cationic detergents like benzalkonium chloride and cetylpyridinium chloride kill most bacteria but not M.Tuberculosis or Endospores. Used as food utensils and skin antiseptics. Brands are Zephiran – Benzalkonium chloride Ceepryn - Cetylpyridinium chloride QUATERNARY AMMONIUM COMPOUNDS


Commonly used aldehydes formaldehyde and glutaraldehyde are highly reactive molecules that combine with nucleic acids and proteins and inactivate them , probably by cross linking and alkylating molecules. Formaldehyde is usually dissolved in water or alcohol before use. A 2% buffered solution of glutaraldehyde in an effective disinfectant. It is less irritating than formaldehyde and is used to disinfect hospital and laboratory equipment. Glutaraldehyde usually disinfectant objects within about 10 min but may require as long as 12 hrs to destroy all spores. ALDEHYDES


Benzoic acid , sorbic acid mechanism is these prevents the uptake of substrates requiring a proton-motive force to enter the cell. Preservation in oral products. Organic acid esters (parabens) – methyl, ethyl, butyl, propyl, benzyl parabens and their salts. Preservation used principally in topical and oral products and in some injections . Good activity against fungi. ORGANIC ACIDS


Chlorhexidine is a widely used biocide which has activity against gram +ve and gram –ve bacteria but little activity against endospores or viruses. It is widely used in general surgery , both alone and in combination with cetrimide and can also be used as a preservative in eye drops. Biguanides act on the cytoplasmic membrane, causing leakage of intracellular constituents. The aromatic diamidines , propamidines & di bromo propamidine are non-toxic antiseptics mainly active against gram +ve bacteria and fungi. BIGUANIDES AND AMIDINES


The chemically reactive gases such as formaldehyde and ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a liquid at temperature below 10.8 0C above this temperature it vaporizes rapidly. Ethylene oxide is a colorless, odorless, & flammable gas. So this flammable nature overcome by preparing mixtures of ethylene oxide in carbon dioxide which is now available commercially. The CO2 – Ethylene oxide are non flammable and there is NO alteration of the microbial activity of the ethylene oxide. The mechanism of Anti microbial action of these gases assumed to be ALKYLATION of sulfhydryl , amino, hydroxyl and carboxylic groups on proteins and amino groups of nucleic acids. GESEOUS STERILIZATION

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Both of these gases being Alkylating agents are potentially Mutagenic & Carcinogenic. They also produce acute toxicity including irradiation of the skin , conjunctiva & nasal mucosa. It is used for sterilizing heat or moisture sensitive materials in hospitals, industry & laboratories has become Universal.

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Agent Mechanisms of Action Comments Surfactants Membrane Disruption; increased penetration Soaps; detergents Quats (cationic detergent) Denature proteins; Disrupts lipids Antiseptic - benzalconium chloride, Cepacol ; Disinfectant Organic acids and bases High/low pH Mold and Fungi inhibitors; e.g., benzoate of soda Heavy Metals Denature protein Antiseptic & Disinfectant; Silver Nitrate Halogens Oxidizing agent Disrupts cell membrane Antiseptic - Iodine (Betadine) Disinfectant - Chlorine ( Chlorox ) Alcohol s Denatures proteins ; Disrupts lipids Antiseptic & Disinfectant Ethanol and isopropyl Phenolics Disrupts cell membrane Disinfectant Irritating odor Aldehydes Denature proteins Gluteraldehyde - disinfectant (Cidex) ; Formaldehyde - disinfectant Ethylene Oxide Denaturing proteins Used in a closed chamber to sterilize Oxidizing agents Denature proteins Hydrogen peroxide – antiseptic ; Hydrogen peroxide – disinfectan ; Benzoyl peroxide – antiseptic

Guidances as per FDA:

Guidance for the submission of documentation for sterilization process validation in applications for Human and Veterinary drug products . Additionally organizations like International Society For Pharmaceutical Engineering (ISPE) and Parenteral Drug Association have issued various documents which include all facts of the International Regulatory Requirements. Guide to inspections of Lyophilization of parenterals. Guide to inspections of High Purity Water Systems. Guide to inspections of Microbiological Pharmaceutical Quality Control Laboratories. Bacterial Endotoxins / Pyrogens; (Inspection Technical Guide). Guidances as per FDA


Heat Exchangers to Avoid Contamination; (Inspection Technical Guide). ICH Q5A, Guidance on Viral Safety Evaluation of Biotechnology products derives from cell lines of Human or Animal Origin. C ontd …


Sterilization is a special process because it’s efficacy cannot be verified by simple inspection and testing on the final product….. For this reason , sterilization process have to be validated before use, the performance of the process monitored routinely and equipment regularly maintained. CONCLUSION


Prescott – Harley – Klein; Microbiology, Fifth Edition, Section II , Microbial nutrition growth and control chapter 7; Control of Microorganisms By Physical and Chemical agents, The MEGRAW – HILL companies 2002 , Page. No ; 136 – 149. Microbiology by Michael J Pelczar , JR ; E.C.S Chan; Noel R . Krieg, Fifth Edition , TATA MEGRAW – HILL Edition – 1993 ; Page. No 469-507. Remington’s Theory and Practice of Pharmacy ; Sterilization Vol I, chapter 40 ; 21 st Edition, Page. No 776-801. Aulton’s Pharmaceutics ; The Design and Manufacturing of Medicines ; Edited by ; Michael E Aulton ; Third edition; Chrchill Livingston Elesevir ; Page. No 235-241. Pharmaceutical Microbiology and Biotechnology ; sterilization methods and principles ; Dr Yashmin Sultana lecturer. Dept of pharmaceutics, Jamia Hamdard New Delhi , Date 11-07-2007. References

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Encyclopedia of PHARMACEUTICAL TECHNOLOGY Third Edition VOLUME I Edited by James Swarbrick ; Sterilization Dry heat, Page. No 3512 – 3517 and Sterilization Moist heat ,Page. No 3529 – 3539. Sterilization; The Theory and Practice of Industrial Pharmacy by Leon Lachman , Herbert A Lieberman, special Indian edition 2009; CBS Publishers Page. No 619-622. Sterilization (Microbiology)

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