UNIT-5 B.Pharma 3rd Sem Pharmaceutical Microbiology PDF

# Unit-5 Pharmaceutical Microbiology ## Unit V: 10 Hours - Types of spoilage, factors affecting the microbial spoilage of pharmaceutical products, sources and types of microbial contaminants, assessment of microbial contamination and spoilage. Preservation of pharmaceutical products using antimicr...

# Unit-5 Pharmaceutical Microbiology ## Unit V: 10 Hours - Types of spoilage, factors affecting the microbial spoilage of pharmaceutical products, sources and types of microbial contaminants, assessment of microbial contamination and spoilage. Preservation of pharmaceutical products using antimicrobial agents, evaluation of microbial stability of formulations. - Growth of animal cells in culture, general procedure for cell culture, Primary, established and transformed cell cultures. Application of cell cultures in pharmaceutical industry and research ## Spoilage - Spoilage of pharmaceutical products is the deterioration of their physical, chemical, or biological properties, rendering them unsuitable for use. - This can be caused by a variety of factors, including microbial contamination, chemical degradation, and physical damage. ## Type of Spoilage: ### Microbial spoilage: - This is the most common type of spoilage and is caused by the growth of microorganisms in the product. - Microbes can produce toxins, enzymes, and other metabolites that can degrade the active ingredients, change the physical appearance of the product, and produce off-odors and off-flavors. - **Environment:** Microorganisms can be present in the air, water, and on surfaces in the manufacturing and distribution environment. - **Raw materials:** Microorganisms can be present in raw materials such as water, excipients, and active ingredients. - **Personnel:** Personnel can carry microorganisms on their hands, clothing, and hair. - **Equipment:** Equipment that is not properly cleaned and sanitized can harbor microorganisms. ### Non-Microbial Spoilage: - **Chemical spoilage:** This type of spoilage is caused by chemical reactions that occur within the product or between the product and its packaging. Common causes of chemical spoilage include oxidation, hydrolysis, and photolysis. - **Physical spoilage:** This type of spoilage is caused by physical damage to the product or its packaging, such as breakage, cracking, or crushing. - **Enzymatic spoilage:** Spoilage due to any enzyme like as lipase, protease, maltose etc. - Eg: Thydrolysis of liquid, due to lipase etc. ## Factors Affecting Microbial Spoilage of Pharmaceutical Products: - **Nutritional factors:** Microorganisms require a variety of nutrients to grow and reproduce, including carbon, nitrogen, phosphorus, sulfur, and vitamins. The presence of these nutrients in a pharmaceutical product can make it more susceptible to microbial spoilage. - **Water:** Water is essential for microbial growth. The water activity (aw) of a pharmaceutical product is a measure of the amount of water that is available to microorganisms. Products with a higher aw are more susceptible to microbial spoilage. - **Storage temperature:** The optimal temperature for microbial growth varies depending on the type of microorganism. However, most microorganisms can grow at temperatures between 4°C and 60°C. Pharmaceutical products should be stored at temperatures that are outside of this range to minimize the risk of microbial spoilage. - **pH:** The pH of a pharmaceutical product can also affect microbial growth. Most microorganisms prefer to grow in neutral or slightly acidic conditions. However, some microorganisms can grow at extreme pH values. Pharmaceutical products should be formulated with a pH that is unfavorable for microbial growth. - **Redox potential:** The redox potential of a pharmaceutical product is a measure of its oxidizing or reducing capacity. Microorganisms that require oxygen to grow (aerobes) will prefer products with a high redox potential. Microorganisms that do not require oxygen to grow (anaerobes) will prefer products with a low redox potential. Pharmaceutical products should be formulated with a redox potential that is unfavorable for the types of microorganisms that are most likely to cause spoilage. - **Package design:** The packaging of a pharmaceutical product can also affect microbial spoilage. Packaging materials should be selected to prevent the ingress of microorganisms and to maintain the sterility of the product. ## Source and Types of Microbial Contamination: - The microbial contamination of pharmaceutical products is influenced by the environment in which they are manufactured and by the material used in their formulation. - Source and types are described as follow:- - **Atmosphere:** Microorganism are carried into the atmosphere suspended on particles of dust, skin, clothing, moisture or sputum following coughing or sneezing. The microbial content of the air may be increased during the handling of contaminated material during dispensing, blending and formulation. Microbes commonly isolated from the atmosphere are bacteria and fungi. Eg: Staphytococcus, etc. - **People:** People are the most common source of microbial contamination. Microbes can be found on the skin, hair, clothing, and in the respiratory tract of healthy people. When people handle food, prepare meals, or work in sterile environments, they can easily transfer microbes to surfaces and products. - **Water:** Water can be a major source of microbial contamination, especially if it is not properly treated or disinfected. Microbes can enter water supplies from sewage runoff, agricultural runoff, and animal waste. - **Raw materials:** Raw materials used in food production, such as meat, produce, and dairy products, can be contaminated with microbes. Microbes can also be found on raw materials used in the production of pharmaceuticals and other medical products. - **Packaging:** Packaging materials can also be a source of microbial contamination. Microbes can be introduced into packaging materials during manufacturing, storage, and handling. - **Equipment:** Equipment used in food production, pharmaceutical manufacturing, and other industries can also be a source of microbial contamination. Microbes can become lodged in equipment and grow to form biofilms, which are difficult to remove. ## Assessment of Microbial Contamination and Spoilage: ### Physical & Chemical Changes: - It is the changes of different pharmaceutical formulations indicate microbial contamination and spoilage. - Change in visocosity, pH, emulsion stability and loss of surface activity of formulation indicates microbial spoilage. - Measurement of oxygen consumption of the product can indicate the degree of oxidative attack and microbial growth. ### Sterility test: - Testing which confirms that products are free from the presence of viable microorganism. - Claim to be sterile or free from viable microorganism. - Test is conducted by competent and experienced personnel in an adequately clean room with laminar flow cabinet facilities. - All injectable and ophthalmic preparations are sterile hence, these preparation are tested by the sterility test. ### Non-sterile products: - Non-sterile products are tested for viable microorganism for detection of pathogens and total viable counts. - This can be identified through “Microbial Limit Tests" ## Preservation of pharmaceutical products using antimicrobial agents: - The main purpose of these preservatives is to inhibit growth of m001+6icrobes in the pharmaceutical product. - They can be classified into four major groups as, - Acidic, - Mercuric, - Neutral, - Quaternary Ammonium Compounds. ## Antimicrobial Chemical Preservatives used in Pharm. formulations | Sr No. | Formulation | Preservative | Concentration (% w/v) | |---|---|---|---| | 1 | Tablets | Methyl Paraben | 0.1 | | 2 | Injectables | Phenol | 0.2-0.6 | | | | Cresol | 0.2-0.5 | | | | Benzyl Alcohol | 1.0-2.0 | | | | Thiomersal | 0.01 | | 3 | Eye Drops | Methyl hydroxy benzoate. | 0.1 | | | | Benzalkonium Chloride | 0.01 | | | | Phenyl Mercuric Nitrate | 0.002 | | | | Chlorhexidine acetate | 0.01 | | 4 | Liquids / Mixtures | Bronopol | 0.002 | | | | Alcohol | 15-20 | | | | Methyl Paraben | 0.1 | | | | Chloroform | 0.25 | | | | Benzalkonium Chloride. | 0.005-0.02 | | | | Chlorocresol | 0.1 | | 5 | Semisolids | Chlorocresol | 0.2 | | | | Dichlorobenzyl Alcohol | 0.1-0.2 | | | | Cetyltrimethylammonium Bromide | 0.05-0.1 | ## Characteristics of Preservatives: - Should able to kill all the microbes' contaminants rapidly. - It should be non-toxic. - Not be irritant. - Cost Effective - It should be physically and chemically stable. ## Evaluation of microbial stability of formulations. - Microbial stability of a formulation is dependent on effectiveness of its preservative. - Chemical assay and biological assay may assure effectiveness of preservative but it may lose its activity due to presence of other ingredients in the formulation. - Some formulations do not require preservative because they act as self-preservative. - Some formulation does not require preservative because it contains antimicrobial agents like antibiotics as ingredient. - Some formulation may contain high sugar concentration, salt concentration and may act as self preservative. So, the ability of the formulation to protect itself from microbial growth must ascertain to determine microbial stability of the formulation. It is done by preservative efficacy test. - Basic principle of this test is to inoculate products with different types of specified microorganism with specific quantity. - Little amount of inoculated product is removed at a specific interval. Then viable count of this withdrawn sample is determined. United States Pharmacopoeia, European Pharmacopoeia etc recommends this type of tests. - The concentration of the test organism should be 105 -106 cells per ml or gm. Total microbial count is performed in 0 hr, 6 hrs, 24 hrs, 48 hrs. 7 days, 14 days and 28 days. - British pharmacopeia recommends test even after 28 days. Different bacterial species are used for this purpose. They are Staphylococcus aureus, Pseudomonus aeruginosa and E.coli. Different fungus species are also used. They are Candida albicans, Aspergillus niger etc. - This test allows to add designated micro-organisms if required. After withdrawal of sample and before viable count, sample is mixed with chemicals which can deactivate preservative because presence of even very minute quantity of preservative may hamper microbial viable count. - There are two types of performance criteria. Criteria A is desired and recommended where as criteria B is satisfactory in justified cases. ## Growth of animal cells in culture, general procedure for cell culture, Primary, established and transformed cell cultures. Application of cell cultures in pharmaceutical industry and research ## Animal Cell Culture: - Animal cell culture may be defined as tha cultivation of any animal cells, out from the body (in-vitro) by providing the cells with a suitable environment and required nutrients. ## General Procedure for Cell Culture: - Design of animal cell culture media is more difficult than that of microorganism and plant cultures. - Culture media are used to support the survival as well as growth. - Selection of media is dependent on type of cells and main objective of culture. ## Culture media classified as: 1. Natural 2. Artificial ### 1. Natural media: - This media are obtained from natural sources such as plasma clot or coagulants, biological fluids and tissue extracts. - Blood plasma: Provide a nutritive substrate and supporting structure for many types of cultures. - Blood serum: Blood serum (fibrinogen free plasma) is used in animal tissue culture. It is mixture of plasma proteins, peptides, lipid, carbohydrates, hormones, enzymes and minerals. ### 2. Artificial media: - It is prepared by adding organic and inorganic nutrients, vitamins, salts, serum protein, carbohydrates, O2 and Co2 gases. - Serum containing media: In which serum is added in 5 to 20% amount. - These are developed to overcome the limitations of serum. It has ability to make the medium more selective for a particular cell types. ## Procedure for cell culture: - A piece of tissue from the organism is usually quite complex and it contain connective tissue, variety of blood cells and reticuloendothelial cells. - The first cell suspension is isolated and then inoculated into a new culture vessel along with fresh medium (primary cell culture) ## Stages for cell culture:- 1. Isolation of tissue 2. Disaggregation 3. Seeding the culture into culture vessels. ### 1. Isolation of tissue: - The explant from an excised (cut surgically) postion of the body of an animal is used the culture of animal cells in a suitable nutrient medium. - Animals are mice, rabbits, guinea, pig etc. - Organs from which cells are to be isolated are surface sterilised with 70% alcohol and then removed aseptically. - The tissue isolate from explant is either stored in freeze or used immediately. ### 2. Disaggregation of tissue:- - A primary cell culture obtained by disaggregating the tissue mechanically, enzymatically or by use of chelating agent. (EDTA). ### 3. Seeding the culture into the culture vessels: - The dissociated (primary) cell grow well when seeded on culture plates at high density. ## Types of cell culture: 1. Primary cell Culture 2. Established cell culture 3. Transformed cell culture ### 1. Primary cell culture: - When the cells are surgically removed from an organ and placed into suitable culture environment, they will attach, divide and grow. - It is first step to established an cell culture. - This is called Primary cell culture. ### 2. Established cell culture: - When the primary cell lines continue to grow and divide they are sub cultured in fresh media to maintain their viability and growth. After many sub culturing (about 70 times) these cells are called established cell lines (cell culture). - Primary Cell culture can grow very slowly but established cell line grow faster. - Primary cell maintains the properties of parent cells, but established has some altered properties. ### 3. Transformed cell culture: - In which established cell lines cells become immortal (i.e. cells have the capacity to grown indefinitely). - This capacity is generated due to transformation in genetic material, due to this transformation, cells loose the sensitivity to external stimuli and also sometimes shows changes in chromosomal numbers. ## Organs: - Culturing of single cell in suitable culture media (Primary cell Culture) - Frequent Sub Culturing (70-80 times) (Established cell culture) - indefinitely (Transformed Cell Culture) ## Application of cell cultures in pharmaceutical Industry and research: - **Drug discovery and development:** Cell cultures can be used to screen drug candidates for efficacy and toxicity. They can also be used to study the mechanisms of action of drugs and to identify biomarkers that can be used to predict patient response to treatment. - **Production of biopharmaceuticals:** Many biopharmaceuticals, such as monoclonal antibodies and vaccines, are produced using cell cultures. - **Quality control:** Cell cultures can be used to test raw materials and finished products for the presence of contaminants. - **Model System:** It is used as a model system in research that helps us to study in basic cells biology effects of substances on cells etc. - **Virology:** Cultivation and study of virus is called virology. - **Virus are cultured for vaccine production, genetic engineering or manipulation in genetic material (DNA & RNA).** ## Share with Your Friend