MB_6_T/F

MPharm Programme: Purification & Sterilization

• The presentation covers purification and sterilization processes within the MPharm program.
• Dr Callum Cooper is the lecturer.
• A recommended resource is "Principles and Practice of Disinfection, Preservation and Sterilization" (5th edition) by Russell, Hugo & Ayliffe.

Learning Objectives

• Introduction to downstream processing
• Purification
• Sterilization
• Various sterilization processes
• Sterility checking
• Sterility testing

Recap (MAb Upstream Process)

• The process starts with thawing and expanding cells.
• Cells are further expanded in seed bioreactors.
• The antibody is expressed into the medium in a production bioreactor.
• Filtration and centrifugation processes follow.
• Cell culture broth is harvested.

Recap (Monoclonal Antibody Downstream Process)

• The process begins with protein A chromatography.
• Polishing chromatography steps remove impurities.
• Orthogonal steps remove viruses (low pH viral inactivation, viral filtration).
• The final step is ultrafiltration/diafiltration to formulate and concentrate the product.

Purification

• Separation of products from production mixtures.
• Removal of unwanted components or contaminants.
• Methods include: sedimentation, precipitation (e.g., heat, pH, organics), centrifugation, adsorption (e.g., ion exchange, immuno-affinity), microfiltration (specified MWCO).
• Purification minimizes side effects while maintaining yield.
• Yield depends on the number of steps and product loss in each step.

Downstream Processing: Sedimentation and Precipitation

• Sedimentation speed depends on cell size, density, and mixing speed.
• Precipitation lowers solute (media) solubility, causing product to precipitate out of solution.
• Methods can be chemical or based on temperature/pH changes.
• Precipitation is used in recombinant DNA polymerase production.

Downstream Processing: Centrifugation

• Application of centrifugal force to separate products based on density differences.
• Denser particles move outward first.
• Requires consideration of component density relative to the medium.
• Factors affecting separation include particle size, density, and relative rotational speed (rpm).

Downstream Processing: Adsorption

• Chromatography-based approach.
• Liquid phase passes through a semi-solid phase.
• Ion exchange binds proteins based on their charge.
• Can be used for capturing or allowing passage of specific proteins.
• Immuno-affinity uses antigenic regions to bind contaminants.
• Common use: removal of bacterial endotoxins (LPS).

Sterilization

• Process that removes or kills all forms of life (bacteria & fungi).
• Can also disable/destroy/remove infectious proteins (e.g., prions).
• Used when a medical product will interact with bodily defenses against infection (e.g., IV administration, broken skin contact, or mucosal surfaces).

Microbial Sensitivity to Sterilization

• Different microbes have varying sensitivities.
• Sensitivity isn't tied to a specific sterilization method but influences method choice.
• Higher resistance is seen in prions, bacterial spores, and gram-negative bacteria, with progressively lower resistance exhibited by other organisms. Prions are notoriously hard to sterilise.

Selection of Sterilization Method

• All methods have some product damage risk; harsh methods impact stability and efficiency.
• The level of microbial reduction must be chosen to minimize product damage.
• 5 common methods recognized by the European Pharmacopoeia (2002): steam, gas, filtration, dry heat, and ionizing radiation.

Downstream Processing: Filtration

• Removes, rather than destroys, microorganisms.
• Filtration efficacy assessed by bacterial count reduction.
• Pore size affects contaminant retention.
• 0.22µm pore size is a widely used standard.
• Other factors: membrane composition, MWCO (molecular weight cut-off).
• Major uses include heat-sensitive solutions, air, gases, and biological products.

Filtration (Methods)

• Membrane filters: particles retained on the filter surface (sieving).
• Depth filters: particles trapped within the filter structure.
• Combinations of depth filters and membranes are possible.

Heat-Based Sterilization Methods

• Moist heat (e.g., autoclave): uses hydrolytic action, typically at >120°C and 1 atm pressure.
• Dry heat (e.g., oven): utilizes oxidative action, with temperatures typically at >150°C.

Gas-Based Sterilization Methods

• Ethylene oxide (EtO): chemically reactive gas.
• Formaldehyde: chemically reactive gas.
• Packaging materials must be EtO permeable.
• Less reliable than heat-based methods, but utilized for temperature-sensitive equipment - (surgical instruments).

Sterilization Using Radiation

• Ionizing (y-rays, accelerated electrons, x-rays) and non-ionizing (UV light).
• Ionizing radiation methods require shielding to prevent damage to surrounding material.
• UV light is best for surface disinfection and certain air/shallow water applications (not ideal for deep water).

Sterilization

• Sterilization process success depends on numerous factors, including equipment maintenance and steam circulation.

Checking Sterilization

• Sterilization success can be verified by physical indicators, chemical indicators, and biological indicators.

Physical/Chemical Sterilization Indicators

• Temperature/pressure record charts.
• Thermometer probes (thermocouples).
• Chemical indicators (e.g., autoclave tape).

Biological Sterilization Indicators

• Standardized bacterial spore preparations (non-pathogenic).
• Spores (e.g., Geobacillus stearothermophilus) are placed in test packs around the sterilizer.
• Post-sterilization, spores grow in nutrient medium; visible changes (like color changes) confirm sterilization process success.

Sterility Assurance

• Microbial populations decrease exponentially with sterilization exposure, independent of initial numbers; sterile means no detectable survivors.

Sterility Testing

• Assesses the absence of microbial contamination after sterilization.
• Membrane filtration is a common method (liquid products).
• Sterile filters collect microorganisms from liquid products.
• Product transferred to media for incubation (long incubation times).
• Turbidity visually confirms contamination.

Issues with Sterilization (Failures)

• Can lead to product recall, product failure, regulatory issues, and economic losses for businesses.

Summary (Presentation Material)

• Subsequent manufacturing stages (purification/sterilization).
• Different purification methods.
• Risks and benefits of purification.
• Sterilization concept and methods.
• Suitable method selection for various product types.

Extra Reading

• "Russell, Hugo & Ayliffe's principles and practice of disinfection, preservation and sterilization".
• "Hugo and Russell's pharmaceutical microbiology".