PHA112 Micro 6 (Sterilization)2020.pdf

MPharm Programme Purification & sterilization Dr Callum Cooper [email protected] Russell, Hugo & Ayliffe Useful reference for this section of Mpharm Also useful for future Micro. courses Learning Objectives Introduction to Downstream processing Purification Sterilisation Different proce...

MPharm Programme Purification & sterilization Dr Callum Cooper [email protected] Russell, Hugo & Ayliffe Useful reference for this section of Mpharm Also useful for future Micro. courses Learning Objectives Introduction to Downstream processing Purification Sterilisation Different processes of sterilisation Sterility checking Sterility testing Recap Recap Purification Separation of products from production mixtures/removal of unwanted components/contaminants – Sedimentation and precipitation e.g. heat, pH, organics – Centrifugation – Adsorption e.g. ion exchange, immuno-affinity – Micro-filtration with specified molecular weight cut-off (MWCO) Why purify? Yield at different recovery % Reduces risk of side effects while maintaining yield 1. 2. How many steps are used Loss of product at each step % Yield Yield depends on: 120.0 100.0 95 80.0 90 60.0 85 40.0 80 20.0 0.0 1 2 3 4 5 6 Step 7 8 9 10 Downstream processing: Sedimentation and precipitation Sedimentation: Speed depends on cell size, density and mixing speed * * * * * * * * * ** * * * * * * * * * ** * * * * * * * * * ** * * * * * * * * * ** ******************** ******************** *************** * * * * * * * ** Precipitation: Lowers solute (media) solubility and causes product to fall out of solution Can be done through various routes Chemical, temperature, pH etc Used in production of recombinant DNA polymerases Downstream processing: Centrifugation Application of centrifugal force to separate out products Denser particles move to outside first Requires components to have a different density from the medium Sedimentation speed depends on cell size, density and rpm By Zuzanna K. Filutowska Own work, CC BY-SA 3.0, https://commons.wikimedia. org/w/index.php?curid=2976 4058 Downstream processing: Adsorption Principle is based on that of chromatography Passage of a liquid phase through a semi-solid phase Ion exchange: Binds proteins based on protein charge Can be used to capture or allow passage of proteins of interest Charged + - + - proteins + + + + + + + + ++ + + + + + + + + + + + + Immuno-affinity: Uses antigenic regions to bind unwanted components Usually targeting specific contaminants Industrial removal of bacterial endotoxins (LPS) + -+ + -+ + + + + + + + + + + + - + + + + + + + -- Sterilization Process that removes / kills everything – Normally refers to bacteria & fungi Viruses must be removed from biologically- derived therapeutics – e.g. monoclonal antibodies, plasma components Modern usage may include disabling/destruction/removal of infectious proteins e.g. Prions (TSE) When? Any medical product where use will breach normal bodily defences against infection: 1. 2. 3. Parenteral (IV) administration Contact with broken skin (e.g. wounddressings) Contact with mucosalsurfaces or internal organs Microbial Sensitivity to sterilisation Different microbes have different levels of sensitivity to sterilisation Generally independent of sterilising method used but will influence choice of method Prions More resistant Spores Gram negative bacteria Small non-enveloped viruses Fungi Large non-enveloped viruses Gram positive bacteria Lipid enveloped viruses Less resistant Prions exhibit exceptional resistance to all known sterilizing agents – may even survive 18 minutes @ 134-138 °C Selection of Sterilization Method All sterilization methods involve some risk of product damage Especially harsh ones Product damage can reduce therapeutic efficiency, stability, patient acceptability Limit to level of microbial reduction Important to minimise microbial contamination Product Damage Sterilization Failure 5 methods recognised by European Pharmacopoeia(2002): Gas sterilization Steam (autoclave) Filtration Dry heat (oven) Ionising radiation Downstream processing & Sterilisation: Filtration Removes rather than destroys microorganisms Filter grades vary by size and ability to remove microbes Filtration efficacy assessed by reduction in bacterial count Pore size plays a role in retaining contaminants Composition of the membrane etc will also play a role 0.22µm pore size generally used MWCO filters can remove on basis of atomic mass (Da) Major uses; Heat sensitive solutions Biological products Air and other gases Water Filtration Membrane filters: particles retained on filter surface (sieving) Depth filters: particles trapped within the filter Depth (prefilter) and membrane (sterilizing) filters can be combined Heat-Based Sterilization Methods Moist Heat Methods Uses hydrolytic action Steam at >120oC > 1 atmosphere pressure Dry Heat Methods Uses oxidative action Temperature >150oC Broad spectrum antimicrobial Standard method for inactivating bio hazardous waste Compatibility Issues Gas-Based Sterilization Methods Chemically reactive gases – Ethylene oxide (CH2)2O – Formaldehyde H.CHO Packaging materials must be permeable Not as reliable as heat-based methods Generally reserved for temperature sensitive items: – Reusable surgical instruments, medicaldiagnostic, electrical equipment, powders Broad spectrum antimicrobials Mechanism of action assumed to be alkylation of various protein functional groups Ethylene oxide is flammable, toxic and carcinogenic Sterilization Using Radiation Two types of radiation used; Ionising: γ-rays, accelerated electrons, X rays Non-Ionising: UV light (optimum  = 260 nm) Ionising radiation Facility must be heavily shielded Can damage some materials (e.g. radiolysis of water) UV light only used for air/surface/ shallow water sterilisation Lower energy than ionising Both primarily target microbial DNA Sterilisation Should we automatically assume that sterilisation is successful? NO Various factors which can influence outcome of these processes; Poor circulation of steam Poor equipment maintenance/cleaning What do we do? Sterilisation can be checked in one of three ways; Physical indicators Chemical indicators Biological indicators Physical / Chemical Sterilisation Indicators Temperature/pressure record chart of each heat sterilization cycle Thermometer probe (thermocouple) located at coolest part of loaded sterilizer or inserted into test packs Chemical indicators based on ability to visibly alter chemical characteristics e.g. autoclave tape Also available for gaseous and radiation based sterilisation Biological Sterilization Indicators Standardised bacterial spore preparations; Non-pathogenic Possess good thermal resistance Geobacillus stearothermophilus Placed in dummy packs located around sterilizer After processing spores are grown in nutrient medium Delay from incubation time can be reduced by using visible indicator pH decrease causes purple →yellow change (steam) sterile nonsterile Sterility Assurance On exposure to sterilization process, microbial populations loose viability exponentially, independent of initial numbers REMEMBER: Sterile means no survivors 1 Surviving Fraction 10-1 10 Achieving true sterility could 10 take forever Microbial safety index: 10 chance of a single surviving 10 organism ExposureTime The probability of a non-sterile unit is no more than 1 in 1 million (10-6) -2 -3 -4 -5 The nature of the contaminant will have a direct impact on the success of sterilisation Sterility Testing Assesses whether a sterilized product is free from microbial contamination by incubation of a sample in nutrient medium Membrane Filtration Method of choice for pharmaceutical products Microorganisms from liquid product collected on sterile filter Filter transferred to appropriate media Long incubation times e.g. 14 days Visual inspection for turbidity What happens when it all goes wrong? Can lead to; Product Recall Litigation https://www.fda.gov/Safety/Recalls/default.htm Summary Looked at latter stages of manufacturing Purification Sterilisation Different purification methods Risks Vs Benefits of purification Introduced concept of Sterilisation and Sterility Different methods used Suitability of different methods for different types of product Extra reading Russell, Hugo & Ayliffe's principles and practice of disinfection, preservation and sterilization: Section 2 Chapter 15 Hugo and Russell's pharmaceutical microbiology; Part 3, Section 17,19, 20, 21