PHA112 Micro 5 (Contamination)2020.pdf

MPharm Programme Sources of Contamination and Sampling Dr Callum Cooper [email protected] Hugo & Russell’s Pharmaceutical Microbiology Useful reference for this section of Mpharm Also useful for future Micro. courses Learning Objectives Microbial contamination of pharmaceutical products...

MPharm Programme Sources of Contamination and Sampling Dr Callum Cooper [email protected] Hugo & Russell’s Pharmaceutical Microbiology Useful reference for this section of Mpharm Also useful for future Micro. courses Learning Objectives Microbial contamination of pharmaceutical products – – – – Origins / sources of contamination Reducing risks of contamination Testing for contamination Results of contamination How did this sample get contaminated? How can we reduce the risk of contamination? Types of contamination in pharmaceuticals Types of Contamination Chemical Biological Bacterial Fungal Viral Physical Sources of biological contamination Utilities Facilities Process Contaminated Product Materials Equipment Personnel Controlling Microbial Contamination Control measures to reduce risk Environmental controls Clean or aseptic preparationareas Clean room Laminar flow cabinets Isolators Air/water controls Personnel controls PPE Hand hygiene (antisepsis) Disinfection/antisepsis Cleaning / disinfection of working environment/personnel Sterilisation Destruction of potential contaminants prior to release Preservation Reduces risk of longer term contamination and spoilage Aseptic Technique Under normal conditions, working areas will be constantly contaminated with microorganisms Free floating or carried on dust particles Personnel Risk of contamination can be reduced using; Bunsen burner Used for heat sterilising metal and glass tools on lab bench Biosafety Cabinets Laminar flow cabinet Pharm. Micro. Glovebox/isolator Aseptic Technique Bunsen Burner Laminar airflow cabinet Biosafety Cabinet Isolator Cabinet Environmental controls Aseptic production areas For the manufacture of sterile medicinal products normally 4 grades can be distinguished: Grades C and D: Clean areas for carrying out less critical stages in the manufacture of sterile products. Less stringent Grade B: In case of aseptic preparation and filling, the background environment for grade A zone. Grade A: The local zone for high risk operations, e.g. filling zone, stopper bowls, open ampoules and vials, making aseptic connections. More stringent Environmental controls Air drawn from outside aseptic area How does this reduce contamination? High efficiency particulate air (HEPA) filtration; Defined in US as removal of at least 99.97% of 0.3 µm diameter airborne particles EU has multiple classifications based on level of filtration Environmental controls Two standards for water in pharmaceutical manufacturing Purified Water for injection Purified water used for non-sterile applications Media preparation Basic preparations e.g cough syrup Water for injection (WFI) is used in sterile applications Stricter quality guidelines than purified water Endotoxin levels Reducing contamination from personnel Humans are disgusting meat bags covered in microbes Facial skin has ~100 million microbes/cm3 Common to find feacal microbes on peoples hands and worksurfaces A single sneeze contains about 40, 000 droplets Saliva has up to 1x108 microbes/ml A sneeze can tracel about 6 meters! Droplets can remain in the air for up to 2 hours Reducing contamination from personnel Hand washing Reduces risk of transmission of contaminants between hands and products, surfaces Reduces risk of transmission to sterile gloves Use of alcohol hand gels to further reduce bacterial numbers Reducing contamination from personnel Protective equipment Includes items for non-sterile manufacturing; Gloves Hairnets Overshoes Different levels of equipment for different levels of production Sterile production often wear oversuit and face mask Microbial sampling Product Sampling / Clinical Samples Filtration Broth / Agar Direct inoculation Environmental Sampling Surface swabbing Contact plates Air sampling Liquid sampling Broth / Agar Counting microbes Serial dilution and plate counts Optical density (OD)/ turbidity Counts everything Inaccurate at high and low OD Direct microscopy Only shows viable cells (colony forming units; CFU) 3 different methods Pour plate Spread plate Drop count Total number of cells in a defined area Flow cytometry Uses fluorescence Microbiological Calculations: Total microbial count Uses a haemocytometer Enables the counting of bacterial cells in a known area Requires multiple fields of viewing VERY time consuming Shows intact bacterial cells Nothing about viability Good for difficult to culture and polymicrobial Microbiological Calculations: Total viable count For bacteria ,yeasts and moulds Can see individual Count colony forming units (CFU) For viruses you don’t see colonies Absence of growth (plaques) Count plaque forming units (PFU) Shows presence of culturable organisms Can get organisms that are difficult to grow Believed to be that only 1-5% of human microbiota can be cultures Microbiological Calculations: Total viable count Calculate CFU/mL of the original inoculum from the plate; Number of colonies: 12 Initial dilution Volume of sample added: 0.1 mL CFU/mL = Number of colonies X (dilution in tubes X no. aliquots in 1mL) CFU/mL = 12 X (1000 X 10) = 12 X 10000 CFU/mL = 120000 = 1.2x105 Regulating Contamination: QC Standards British Pharmacopoeia Official standards for UK medicinal products and pharmaceutical substances (includes Ph. Eur.) Specifies acceptable limits for microbial contamination of non-sterile products AdministrationRoute Max TotalAerobes (CFU / g or /ml) Specified Absences Oral (non-aqueous) 103 E. coli Oral (aqueous) 102 E. coli Rectal 103 - Mucosal / Cutaneous 102 S. aureus; P.aeruginosa Sterile products have to contain no microbial contaminants and also have additional criteria What can microbial contamination do? Spoilage Chemical & physicochemical deterioration Rate of breakdown will depend on; Molecular structure Environmental conditions Type and number of microbial contaminant Inactivation of product e.g. penicillin breakdown by βlactamase Breakdown of thickening/suspending agents e.g. starch breakdown by amylases Synthetic packing materials (e.g nylon) more resistant than naturally derived (e.g. cellophane) Important to understand where and how product is to be used Manipulate formulation to create resistance to spoilage e.g add preservatives What can microbial contamination do? Health hazards to patients Product contamination with pathogens →infections in susceptible patients Response varies; No reaction Local infection GI infection Systemic/bloodstream infection Most serious are from injected products or immunocompromised patients Contamination with toxic microbial metabolites Most serious effects from contaminated injectable products; General bacteraemic shock Death What happens when it all goes wrong? What happens when it all goes wrong? Can lead to; Product recall Litigation https://www.fda.gov/Safety/Recalls/default.htm https://www.gov.uk/drug-device-alerts Summary Looked at different sources of contamination and how they interconnect Methods which can be used to reduce the risk of contamination Introduction to how to test for contamination Introduced regulations around contamination What happens when it goes wrong Extra reading Hugo and Russell's pharmaceutical microbiology; Part 3, Section 16, 21