Pharmaceutics semester 1

• Dissolution is a three step process: disintegration into granules or aggregates, deaggregation into finer particles and then dissolution occurs. Dissolution meaning the drug is now in solution.
• Diffusion layer of a particle is based on the noyes-whitney equation. Simply describes: when a solid dissolves, the components pass down a diffusion gradient, until finally the particles completely dissolve and hence enter the bulk solution. The diffusion layer can be thought of as a thin stagnant layer around the particle.
• Simplified diffusion controlled process: this is dissolution rate limited i.e. the drug [ ] decreases as more drug diffuses and is absorbed. The drug molecule is then immediately replaced in the diffusion layer, and becomes more saturated as more drug moves into the bulk solution. 
• One of the factors which affects the diffusion layer of the drug, is the pH of the unstirred layer: E.g.: the dissolution rate of a weak acid in the stomach would be low because the drug wouldnt be ionised. However, by forming an alkaline salt from this drug will improve solubility of the drug. E.g.: Na and K salts, which will thus dissolve more rapidly. 
• Permeation: how it gets through the GI wall. Basically, this has been broadly oversimplified i.e. small hydrophylic drugs pass through channels paracellularly (there are a few drugs such as these), lipophylic drugs permeate transcellularly. Membrane and drug efflux transporters are increasingly becoming more relevant. Transport through epithelial and endothelial layer i.e. BBB need more advanced mechanisms. 
• Passive diffusion: when the drug permeates via the membrane i.e. lipophilic drug, and partitions through the lipid, follows the simple fick's law. This can be simplified to first order kinetics, where we assume the [ ] on the outside is higher than the [ ] on the inside. i.e. when blood stream is higher than in cells. 
• pka is the pH at which the [ ionised ] = [unionised ] of the drug. i.e. where the drug is 50 % ionised or unionised. Log P: is a pH independent term, therefore it cant be used as most drugs look at ionisation. It is always used by looking at log P at various pH's. Log P is the measure of how lipophilic a drug molecule is: it is the partition coefficient of a drug between the lipophilic and aqueous phase.
• Distribution coefficient: this is known as the effective partition coefficient (i.e. instead of using water like log P, the aqueous phase is adjusted to a certain pH), as it takes into account the degree of ionisation. i.e. looks at the pka and the pH. There are many limitations to using D i.e. not stirred- convective flow, ionised drug may also be absorbed, different (lower) pH at membrane surfaces, secretions, disruptions of lipid membranes e.g. surfactants. 
• Not permeating into lipid layer: carrier mediated transport: via facilitated or active transport driven by energy. The difference here is that saturation can occur. Involves the affinity of the carrier binding to the drug and the maximum rate of transport or saturation of the carrier. 

• Weakly basic drug generalisations: in the stomach, the drug is ionised, therefore dissolution and solubility increases, whereas permeation decreases. However, not a downside as the stomach has a limited surface area. Food in the stomach would delay gastric emptying, which will increase solubility. In the small intestine, drug changes states towards being non-ionised: this increases permeation and reduces solubility. Reduced solubility shouldnt matter as much, because the solubilised drug is moved into the SI from the stomach (and that diffusion layer is constantly being replaced) Furthermore, another factor is the counter flow of blood, which is affected by the patient. e.g. a sitting patient, would decrease blood flow, and thus reduce absorption of the drug. Blood flow also rises, on stimulation of gastric secretions. NB If the drug however is poorly soluble regardless of pH, the drug would precipitate, but if the drug is taken up so fast in the SI, it should be okay.
• Weakly acid drug generalisations: in the stomach, the drug is unionised due to it being protonated, more lipophylic, but absorption is limited by the stomach's small surface area. In the small intestine, a higher proportion of the drug will be ionised, increases solubility, but a lower proportion of the drug is absorbed. However, if there is rapid uptake of those molecules due to an increased blood flow then its okay. Taking a weak acid with food, increases solubility but delays absorption. if the weak acid has both good solubility and absorption, taking it with food, will lead to toxicity, as high drug peak levels are achieved. 
• Overall, pka of the drug, gastric emptying, blood flow will all affect the solubility and permeation of the drug. 
• Strong acids and strong bases are rarely used in drug formation. 
• Very Weak acids and bases: i.e. pka >8, pka <7 are both usually unionised at most pH's. 
• Moderate acids and bases: are what we covered above. 
• SINK CONDITIONS: dissolution media with or without a solubiliser which is enough to solublise a product. 

• Biopharmaceutics classification system: 
• Class 1: high solubility and high permeability. 
• Class 2: high permeability: only eligible for a biowaiver, if the weak acids are highly soluble at pH 6.8: because they will be ionised. 
• Class 3: high solubility: only eligible for a biowaiver, if the drug is very rapidly dissolving i.e. higher solubility, so it should dissolve quite quickly. 
• Class 4: poor solubility and permeability. BRICK DUST
• Biowaiver: when the drug doesnt have to undergo bioavailability studies. 
• BCS solubility: drugs are only considered highly soluble, if the largest dose is soluble in 250 mL of water over a variety of pH ranges i.e. 1-7.5. Compounds with solubilities below 0.1mg/mL face major obstacles, even often compounds with solubilities below 10mg/mL. 
• BCS permeability: drugs are only considered highly permeable, if the drug demonstrates 90% absorption of the administered dose. 
• Usually drugs with a BSC class 1 can be formulated as immediate release or controlled release formulations. But drugs are rarely BSC class 1 because: 
  • up to 40% of NCE discovered are poorly soluble.
  • drugs are being made via combinatorial and high thoroughput screening methods, which usually dont take into account solubility. 
  • Generics: usually the drugs are poorly soluble when off patent which requires high cost methods to fix this. or they often infringe the formulation patents which results in legal issues. 
  • Innovator companies are relying more on formulation patents to extend the product life i.e. poor solubility.

Poorly soluble drugs issues in vivo:

• decreased bioavailability
• Sub-optimal dosage 
• increased chance of food effects
• increasing dose: we might not get responses proportionate to the increase in dose. 
• increased issues during disease states esp. GIT diseases. 
• more frequent incomplete release from the dosage forms. 
• higher inter-patient variability. 
• Patient non-compliance: due to a non-efficient dosage platform.

Poorly soluble drugs issues in formulation:

• Severely limited choice of delivery technologies i.e. no imediate release or controlled release formulations.
• increasingly complex dissolution testing.
• limited or poor correlation with in vivo testing. 
• harsh excipients use 
• use of extreme acidic or basic conditions to enhance solubilisation 

NB there are fewer BCS 1 compounds due to: increase in molecular size and hydrophobicity of drugs, advances in drug discovery technologies. 

Solubility enhancement: 

• Particle size reduction
• soluble salts / salt forms
• solid dispersions: drug is placed in an inert water soluble carrier
• self-emulsifying systems: microemulsions to improve solubility 
• solubilisation by surfactants 
• nanoparticles
• cyclodextrin: ring of sugar molecule with the poorly soluble drug in the cavity. 
• pH of diffusion layer
• pro-drugs
• co-solvents
• lipid-filled capsules
• liposomes: spherical lipid bilayer
• lyophilisation: freeze drying 

Permeation enhancement: 

• Permeation enhancer 
• Absorption enhancing excipients
• efflux inhibitors: lipids and surfactants are inhibitors of P-gp.
• lipid-filled capsules
• GI motility consideration 

• CYCLODEXTRINS: They have a hydrophylic outer surface and a lipophylic core. No covalent bonds are formed or broken during drug-cd complex formation. drug-cd complexes can be formed via supersaturation followed by agitation of the solution, or via adding a mass of drug to the cd and kneading it together to form a paste. the paste is then dried and sieved. Hydrophylic polymers such as hydrxymethylpropyl cellulose can be added to reduce the amount of cd added. Downside to cds: toxicity and stability issues. Advantages: enhance solubility, bioavailability, stability of the drug. As well as can be used to reduce irritation in the GIT (reduce the local [ ] of the free drug below the irritancy level). 
• Amorphous solid dispersions: amorphous states of the drugs are usually more soluble, but less stable. Therefore, they are usually done with polymers via the following methods. spray drying using solvents or supercrtical fluids. OR hot melt extrusions: polymer is softened, API added, mixed together as they flow through the extruder to from strands of polymeric glass embeded with API. Glass strands are then milled into a powder. 
• Polar excipients: Polyethylene glycol (liquid PEG as a co-solvent in liquid based products or dispersion enhancing / wetting agent in solid dosage forms often encorporated by solvent evaporation or freeze drying, PEG will often be used in combination with other surfactants such as sodium lauryl sulphate, stearic acid). Gelatin (naturally derived collagen extract with both positive and negative charges, used as a granulating aid to improve the wettability of products). Sugar glasses ( e.g. inulin which is a naturally occuring fructose polymer, mixed with a drug solution followed by freeze drying to form a sugar glass. this makes the dissolution profile of the lipophilic compound closely related to the profile of inulin. sugar glasses also protect the API from physical and chemical degradation. Lipids (also used as polar excipients but also for self-emulsifying systems). 
• Particle engineering: reduces particle size, increases surface area, reduces diffusion layer thickness and the saturation solubility. By far the biggest effect is the surface area change. via recrystallisation (involves use of solvents which is a complex method), or grinding / milling / spray drying to reduce the size via mechanical stress (this can induce degradation or thermostress is a thermolabile product is used). NB larger drug particles have mimimal absorption in the absorption window, unlike nanoparticles. 

Benefits of nanoparticles:

• greater bioavailability: higher Cmax and AUC.
• less variability with food: smaller difference between fed and fasted state: because we dont have to rely on the effects of food, because the drug has a larger SA. 
• dose proportionality is increased i.e. dose proportional to AUC: this was a problem with poorly soluble drugs: as they lack dose proportionality as well as dose release in the body. 

How are nanoparticles made:

Traditional ways of communition: grinding and milling. Although there can be nanomilling of particles, this will often damage the particle.

Supercritical fluids is the newer approach: reduces particle size by altering pressure and temperature in solvents such as C02. C02 or water can adopt the properties of a gas and liquid at the same time, at a temperature above their thermodynamic critical point: useful aspect. They can diffuse through solids like a gas and dissolve materials like liquids which is useful in recrystalisation at greatly reduced particle sizes. At near-critical temperatures, SCF's are highly compressible allowing moderate changes in pressure and temperature to greatly affect mass, density etc. Manipulating pressure: can make gases highly diffusive, low viscosity, low surface tension to be imparted onto liquid preparations.

Self-emulsifying systems:

• Soft or hard gelatine capsules with liquids: i.e. emulsions containing droplets which can vary in size. Not only do they improve solubilisation, they also improve release and absorption properties due to the already dissolved drug in the formulation and the small droplets which provide a large interfacial surface area.  Non-ionic surfactants can be used which allows high degrees of solubilisation and reduces precipitation of the drug out of the microemulsion in vivo i.e. it is important that the drug remains partitioned into the o / w droplets following dilution with the aqueous medium. Tweens (polysorbates) and labrafil with high hydrophyle and lipophyle balances, are used to ensure immediate o/w droplet formation during manufacture. Co-solvents / surfactants include ethanol, propylene glycol, polyethylene glycol. They are an important tool in the manufacture of lipophilic drugs, as they increase their bioavailability by increasing their permeability and enhance their lymphatic absorption. NB some highly lipophilic drugs administered orally have found to gain access to the systemic circulation, via the lymphatic system, hence avoiding first pass metabolism. 
• Solid products can also be developed: where the suspension is dried to obtain physically stable drug particles in a powder form. 
• SEDDS, SMEDDS, SNEDDS. Self emulsifying drug delivery system. micro-emulsifying and nano-emulsifying drug delivery system. They can be used for BSC classes 2-4. 

• Eating lipids in the diet will simulate secretion of bile acids, which generates mixed and colloidal micelles. Similarly taking self-emulsifying drugs releases bile acids. This is an important step in the absorption of poorly soluble drugs i.e. via the intestinal lymphatic system, and then into the circulatory system, avoiding first pass metabolism. It can also have a solubilisation and absorption effect as triglycerides and surfactants react with the wall of the GI tract. 
• Absorption of lipids: emulsion droplets  in the lumen of the gut  breakdown to form dissolved monoglycerides and fatty acids. They are then taken up into enterocytes (absorption cells in the SI) via diffusion. Production of microproteins with the drug known as chylomicrons (fat droplets containing te drug) are then taken up into the lacteal (lymphatic vessels of the SI). Usually drugs with high Log P of 4-5 or higher. as well as <2grams (-2 tablets). 

Common parenteral indications:

• control onsent of actions, serum peak levels etc
• where adequate concentrations are required e.g. meningitis with aminoglycosides to penetrate the BBB
• biological effect isnt possible orally i.e. poor absorption or degradation e.g. insulin
• unconsious / uncooperative state: nil by mouth state
• compliance: i.e. to improve it e.g. for mental health patients where injections are better
• Rapidity is key: e.g. fluid resucitation
• Fall back route when the desired route is unavailable.
• local effect is needed e.g. dental anaesthetic

Percutaneous / needle type ROA:

• Subcutaneous
• intravenous
• intramuscular
• intra-articular
• intra-dermal
• intra-synovial
• intra-cardiac 
• intra-arterial 

• Intravenous: sterile soutions, emulsions, suspensions, reconstituted sterile solids. usually buffers needed e.g. citrate, phosphate etc. solubility affects dose volume: completely solubilised. Co-solvents may be added to improve solubility or improve stability e.g. glycerin, ethanol, propylene glycol etc. no particles except for some nutritional lipids. Hypertonic solutions possible via slow administration and use of NaCL, KCl, dextrose etc. to adjust tonicity: i.e. making them isotonic. 
• Intramuscular: Prolongued release of oily and low solubility doses. needs to be soluble at site of injection. Partition coeffecient needs to be high. higher blood flow= higher absorption (flow is higher is deltoid arm muscles than lateral thighs due to higher vascuarity and fat content, age and disease also affects flow). can be affected by degradation of the drug at site of injection e.g. proteases. Main precaution: avoid blood vessels, done by pulling back plunger. Thighs generaly tolerate a higher volume and major blood vessels can be avoided: 5ml in gluteal and 2 ml in deltoid regions. 
• Subcutaneous: rapid, predictable, but slower than IM. often used for self medication due to lack of swabbing and infection issues. 0.5-1.5 mL may be administered. poorly absorbed and fragile drugs typically water soluble and non-irritating. 
• Intra-pertioneal: into a cavity or organ. e.g. dialysis. major route portal circulation (liver) i.e. subject to first pass metabolism. larger water soluble drugs are more slowly absorbed than smaller lipid soluble drugs. Major precautions: haemorrhages due to punctures. 
• Intra-spinal: drug goes into cerebrospinal fluid and the brain via the BBB. normally less than 20 mL. 
• Intra-ventricular: into lateral ventricles of the brain e.g. meningitis. 

• These are reusable and needle free injectors. 
• some are spring powered with thousands of uses and others use high pressured gas to pass the drug through the skin.
• usually at a subcutaneous, intra-muscular or intra-dermal levels. 

Microneedle patches:

• as the strateum corneum is the main skin barrier. this uses short needles to deliver the drug through the skin in a minimally invasive manner. for small molecules, proteins and nanoparticles. 
• they increases the skin permeability by forming micron-scale pathways  in the skin
• they actively drive the drug into the skin during microneedle insertion
• they target the strateum corneum but they tend to also pierce the epidermis and superficial dermis too. 
• examples include drug coated needles (drug is coated around metal spheres which are pierced into the skin), or the use of dissolving microneedles (where the drug is enclosed in polymers which will dissolve and leave much smaller wounds in the skin). 

• strateum corneum is the barrier to drug delivery via the skin route.
• daily dose of drug which can be administered transdermally is 5-25 mg. this ROA is limited to potent drugs.
• Formulation enhancers can be used to either or both modify the drug / delivery vehicle or the strateum corneum. 
• Besides formulation enhancers there are powered enhancements methods: iontophoresis, phonphoresis and electroporation patches. 
• Transdermal penetration routes are either through the strateum corneum, or through the shunt routes known as sweat ducts, hair follciles or sebaceous glands. Usually is through the strateum corneum, except for iontophoresis drug delivery which is via the shunt routes where there is minimal electrochemical resistance. 
• Stateum corneum structure: brick and mortar structure. has a rich layer of terminally differentiated keratinocytes known as corneocytes. has a intercellular lipids matrix which contains the main lipid classes ceramides, cholesterol and free fatty acids. Skin also contains esters and triglycerides. The lipid structure of the skin is different to other biological membranes it has an extruded lipid phase behaviour.  
• Extruded lipid phase behaviour: hydrocarbon chains are arranged into a crystalline lamella gel and liquid crystal phase domains within the lipid bilayer. the first few layers rearrange into broad intercellular lamella. 
• Water is also involved which is an essential plasticiser. and natural moisturising factor to maintain suppleness. 
• Drugs usually get through the skin inter-cellularly more than transcellularly: so its the lipids between the cells which provide the barrier. the lipids are quite rigid due to cholesterol, ceramides etc. Drug also has to pass through a hydrophylic region and vice verca. 
• So the drug needs adequate solubility within the lipid domains of the strateum corneum, as well as sufficient hydrophylic nature to allow partitioning into the viable tissues of the epidermis. therefore the Log P needs to be between 1-3. 

• Molecular weight should be less than 1000 daltons, preferrable 500 daltons. 
• Melting point should be less than 200 degrees.
• Log P should be between 1-3.
• No or few polar centres like carboxyllate or zwitterionic structures: you want to avoid ionised groups. 
• We want it to mimic injected drugs so must have a half life of less than 6-8 hours. Mimics IV drips, so maintains drugs with a short half life. 
• 50 cm 2 maximum patch size: this limits the drug diffusion. 
• 5-20 mg a day is usually the maximum feasible dosage. 

Modifying the drug vehicle:

• Drug selection: selecting right drug with the right properties 
• Pro-drug: such as esterification to remove ionised groups. Esters are good! we dont want to hydrolyse esters!
• Ion pairs, complexes: to make the drug uncharged. No charges! 
• Chemical potential: altering thermodynamics of the systemi.e. making a saturated or supersaturated drug [ ]
• Eutectic systems used in the skin: to lower the melting point 
• Liposomes, vesicles used to modify strateum corneum and carry the drugs through: these include transferosomes. 

Modifying the starteum corneum:

• Hydration increases water amount in the skin: causes swelling of the strateum corneum
• Lipid fluidisation
• Bypass / removal of lipid layers
• iontophoresis 
• phonphoresis
• electroporation

Chemical potential: 

NB thermodynamic activity is a measure of the effective [ ] of the drug in solution.

• This involves the formation of saturated and supersaturated solutions.
• Saturated and supersaturated solutions would increase the thermodynamic activity of the drug.
• This is done either by solvent evaporation from the warm skin surface or by the use of co-solvents such as water: this penetrates into the skin and enhances permeation. It also acts as an anti-solvent for the drug which increases drug flux by 5-10%.
• These systems and inherently unstable and may crystallise, therefore anti-nucleating agents are added to improve drug stability.
• The starteum corneum already has anti-nucleating agents such as ceramides, cholesterol, triglycerides which will stabilise the super-saturated solution.  

Eutectic systems:

• Depress the melting point of the drug to lower than or around the skin temperature. A eutectic mixture involves two or more components which together have a lower melting point, than when used individually. 
• Various eutectic systems also contain a strateum corneum penetration enhancer as the second component e.g. terpenes, menthol or fatty acids. Examples of permeation enhancers: 2-propanol (solvent), poloxamer (surfactant polymer), sodium methyl hydroxybenzoate (preservative), stearates and propylene glycol (lipids). 

• Penetration enhancement can be expressed by the enhancement ratio. Which looks at enhancement after : compared to enhancement before. 
• It involves disruption of the intercellular lipid lamellar structure: which is an extruded lipid behaviour, interaction with intercellular proteins of the starteum corneum and improving the partitioning of the drug with a co-enhancer or co-solvent penetrating the SC.
• HYDRATION: most important method which happens in most products, safest and best ways. It alters the drug solubility and partitioning. Increases skin hydration, swelling, and opening of the SC structure, leading to increased penetration. Can be occlusive or oil in water emulsions which donate water particles to the SC. 
• LIPOSOMES AND LIPID PARTICLES: this involves lyposomes hydrating or altering lipid layers where lipids similar to the SC are more likely to rapidly enter and fuse with the SC:
  • Transferosomes: these are deformable liposomes which contain surfactants e.g. sodium cholate, which fuse into the strateum corneum, squeezing their way between the lipids to disrupt the lamellar structure.
  • Ethosomes: these are liposomes with a high alcohol content which fluidise the SC.
  • Niosomes: these are vesicles composed of non-ionic surfactants. 
  • Solid lipid nanoparticles: these are carriers for enhanced skin delivery of sunscreen, vitamin A and E etc 
• KERATIN AND LIPID DISRUPTION: these increase the permeability by disordering the SC:
  • disrupt keratin e.g. urea and surfactants.
  • fluidise lipids e.g. azone, alcohols, terpenes and fatty acids
  • These are more aggressive ways.
  • they either mix with the lipids changing the solubility or
  •  they extract the lipids forming channels within them e.g. terpenes or
  • at high [ ] they pool within the lipid domains to create permeable pores that provide less resistance for polar molecules
  • SAR's have been developed: using unsaturated fatty acids you would need longer hydrocarbons compared to using saturated fatty acids.
  • Penetration enhancers = skin irritation

• Increased partitioning and solubility of the drug:  shifting the solubility parameter of the skin closer to that of the drug increases solubility and hence drug flux.
• Iontophoresis:patch with a battery attached: performing electrophoresis to drive the drug through the skin. i.e. uses low voltage current to increase permeation. If both charged drugs  using 2 electrodes and weakly charged/uncharged drugs using an electro-osmotic flow of water. Rate of delivery of the drug can be controlled using the microprocessor and patient: maximum current used is limited by skin irritation. Higher the voltage, reaches nerve endings: irritation. 
• Electroporation: Allows the temporary disruption of the lipid layers using short high voltage pulses, which can then eventually reseal: temporary opening. Quite closely spaced micro-electrodes are used which can only reach the strateum corneum: avoids muscle pain, reduces skin irritation.
• Phonophoresis: ultrasound used to help penetration of drugs and other compounds into the skin. ultrasound is an oscillating pressure wave at a sound to high to be heard. used for small lipophylic drugs. The ultrasound forms bubbles in the skin which the drug passes through. Besides ultrasound, pulse laser can also be used. 

Benefits of inhalation medication:

• Rapid onset of action
• Avoids first pass metabolism
• Avoids GIT degradation
• lower dosage avoids adverse drug reactions
• Accurate dose adjustement and titration to needs of patient is ideal for PRN medication
• small volume ranging from 25 to 100 mL
• tamperproof container 
• protection from air or moisture instabilities 
• Systemic benefits include: when chemical / physical interaction with other medication is occuring, when dose exhibits eratic pharmacokinetics, breakdown in the GIT, for acute and breakthrough pain. 

Respiratory tract anatomy: nose breathes in moist and warm air which filters out large particles i.e. >15 um, cillia traffics to the mouth to swallow to GIT or cough / sneeze out large particles.

Upper respiratory tract: nose, throat (pharynx) and larynx. Lower respiratory: trachea, bronchi, bronchioles and alveoli.

Epigglotis protects the airways when swallowing. Blow reflex reaction isolating the nasal pathway. Nose spray where you blow out via your mouth, tube in the nose which releases the drug. Prevents you swallowing the drug or inhaling it. 

Factors affecting particle deposition: Dry powder (diameter, density, shape, charge and chemical characteristics). Liquid aerosols (velocity, propellant, particle size, size distribution). Deposition occurs by impaction, sedimentation and brownian diffusion. Increasing density decreases minimum size for deposition.  Particles below 0.1 microns are more likely to be deposited by diffusion. Increasing size raises the possibility of deposition by impaction or sedimentation. Larger sizes are more likely to be deposited in the upper respiratory pathways. 

• particle size and velocity affect deposition. 
• Inertial impaction: when particle inertia / momentum renders particle unable to change direction with flow.
• Gravitational sedimentation: this occurs when air velocity is low. air velocity is low when you hold your breath. greater when long residence time and high settling velocity. 
• Brownian diffusion is significant when it comes to deposition of small particles i.e. <0.1 um which are less affected by inertia and gravity. 
• Electrostatic interaction: charge on particle induces the opposite charge on the lung wall, and results in an acceleration of the particle in its proximity. 
• Interception: when size of airway approaches size of particle. 

NB for effective deposition by sedimentation / impaction in traditional inhalers: less than 10 um, usually 2-8 um. 

• sprays: useful for upper respiratory tract. usually using metered dose pumps, deposited in the nose or in the back of the throat. OR using bidirectional flow: isolates the nasal circuit from the lungs-enabling particle size, flow rate and direction to be optimised. While exhaling into the device, the nasal palate of the patient closes off, which secludes the nasal cavity completely reducing unwanted loss of drug. OR controlled aerosol: uses controlled particle dispersion using electronic atomisers for particle size control.
• pressurised metred dose inhalers: now contain hydrofluroalkanes, instead of CFC's as a liquid propellant. fast moving microfine solution or suspension, which the patient inhales slowly and deeply into the lungs. can be breath actuated or battery powered or manual valve operation. 
• super fine particle inhalers: for small airway disease using HFA's too.
• Nebulisers: drug is found in a polar solvent i.e. usually water. large / less convenient. smaller aerosolizations developments.
• Dry powder inhalers: growing to replace some pMDI's. No propellants needed. dry powder fluidises when the patient inhales. Drug my be carried out on microparticles, which then shears off to  penetrate  more deeply in the lungs. Quick and powerful, deep as possible inhalation needed to mobilise the powder. It can be a powder reservoir, drug in a blister disk or *****,  capsule to meter the dose. Aerodynamic behaviour is key to dispersion of agglomerates and disposition of drug. It could be just drug alone, or drug with lactose crystals. Active DPI's are being developed with pneumatic, impact force and vibratory dispersion to standardise sheer and turbulence for controlled relese.

NB traditional air jet nebuliser: compressed air or oxygen exits a narrow orifice at high velocity. When air exits, there is negative pressure. This negative pressure draws liquid out from a tube. The liquid which is drawn up fragments into aerosols with large droplets.  i.e. >40 um. The larger droplets are removed via impaction. The smaller particles continue on to be inhaled into the lungs. 

Ultrasonic nebulisers: smaller devices. aerosols are created on a piezoelectric crystals, which vary based on voltage input. as the pieozelectric crystals agitate, the liquid agitates forming aerosols. Large aerosol particles are removed by impaction. ultrasound is a form of kinetic energy. 

Vibrating mesh ultrasonic nebulisers: As the piezo contracts and expands driven by the voltage, the mesh is thrust into the liquid and moved forward. This alternating momentum, causes the release of finer droplets. Very efficient: all the liquid is converted into an aerosol. Can be converted into smart device using electronic software to personalise the medication. 

Particle size and aerodynamic diameter is important for DPI: Particles in the 1-5 micron range have an efficient alveolar absorption. Larger particles deposit in the oral / pharynx region. Smaller particles don't deposit well and are innefficient, until they reach the 0.1 um range. Aerodynamic diameter describes the dynamic behaviour relating gravitational settling and inertial impaction. This is reduced by: decreasing size, decreasing density and increasing it's shape factor: reduce aerodynamic diameter. Size has the highest effect on aerodynamic diameter. Aerodynamic size related factors: Crystalinity (polymorphs with different stability, solubility and bioavailability AND different crystal habits i.e. crystals growing habits). Hygroscopy (the particles are typically hygroscopic, and the lungs have very high relative humiity: particle size will grow, density will decrease overall as the crystal is more dense than water, altered adhesive and cohesive properties, irreversible aggregation i.e. recrystallisation in the lungs).  

How are particles made for DPI's: Traditionally jet, pin and ball milling. When the traditional methods fail to produce the desired size: spray-drying (more spherical-but amorphous particles) or super-critical fluids. Excipients are not always used for DPI's, when they are used they serve as carriers to improve dispensing, metering and to reduce cohesion by occupying high energy sites of micronized particles. Excipients are limited by low buffering capacity of the lungs and irritation: usually lactose as it is highly crystalline and has a smooth surface (low energy which avoids agglomeration). OR lipids.  Excipients aren't used to improve solubility, they are used to improve dispensing i.e. example with insulin, improves its breakdown to monomers. 

• Enhance solubility: co-solvents largely ethanol, enhances the solubility of surfactant propellants. Inverse micelles or liposomes are also used.
• Wettability: increase the wettability: since metred dose inhalers are the drug dispersed in a solution / suspension. surfactants finely divide actives / excipients
• Stabilise suspension: surface active agents (lecithin, oleic acid, sorbitan trioleate) adsorb to particles as steric barrier to agglomeration. Need to shake inhaler before use to ensure mixed.
• Lubricate valve: surfactants or simple lubricating oil e.g. food grade silicone is used to minimise friction which may impaire valve function. 
• Flavouring: menthol often used as a taste masker.
• Antioxidants: ascorbic acid
• Absorb moisture: MDI's arent sealed systems and they are prone to excess mositure seaping through. 
• Preservatives: benzalkonium chloride- can cause allergies. Phenyethyl alcohol can be used as a preservative and odour combat. 

• Small airway diseases i.e. less than 2mm in diameter such as chronic asthma and COPD are inadequately treated using traditional inhalers.
• Majority of particles from traditional inhalers deposit in the upper respiratory tract by impaction and sedimentation processes. 
• Smaller particles are less likely to deposit in the upper respiratory tract, and thus will pass into the distal / peripheral airways. These however were believed to be deposited poorly and end up being exhaled. True for particles >0.5-1 micrometres. Which was the limit of traditional inhalers.
• There is clinical evidence that superfine particles will improve diseases. 
• Superfine particles is a mixture of ultra fine <100 nm (0.1um) and extra fine <1 um particles developed with HFA's pMDI's. Why does this occur? look at below

Effect of particle size on lung diposition:

• Deposition by impaction and sedimentation are both directly proportional to the particle size and are most effective for large particles i.e. >1 um. 
• Inertial Impaction: particle with sufficient momentum or inertia doesn't change direction with the airflow in a curved airway, and thus impacts. 
• Gravitational Sedimentation: by gravity is related to the resistance time in the airway and its terminal settling velocity, increased by holding breath. 
• This results in most of the dose not being deposited i.e. large losses to the GIT, and upper respiratory tract. 
• This is improved for particles < 0.1um. 
• Diffusional deposition is inversely related to the particle size and is more effective for small sub-micron particles. Little deposition with particles >1um. deposition increases, as particle size decreases below 0.1 um.  

Clinical evidence of superfine particle size:

• Comparable effects, or some studies show superior effects.
• There is a reduction in the daily dose of corticosteroids needed, as more is absorbed. 
• Greater asthma control and quality of life, with lower variability between patients.
• Some studies also show an improved therapeutic ratio with smaller particles. 

Protein biologics vs small molecule drugs:

• Versatility: they replace diseased tissue, as well as modify.
• Unspecific binding: to molecular structures other than the specific target can cause toxicity including tumorogenecity: not applicable to therapeutic proteins i.e. they are highly specific.
• Blood levels of drug and duration of action: in man not appropriate, often a problem in small molecule drugs: not applicable to mAB as they tend to have longer circulation times. 
• Less frequent dosing: long circultaion times compared to small molecule drugs i.e. weeks vs hours.
• risk of DDI's: lower or not applicable.
• Different structures for each indication: not applicable to the similarly structured mAb: same molecule for lots of different indications.
• Inappropriate molecular target: if this occurs i.e. choosing a dirty target can lead to toxicity. applies to both.
• Immunogenic effects: higher risk for therapeutic proteins, therefore use humanized proteins. 

• The biologics are now slowly coming off patent i.e. their patents are expiring.
• The innovator manufacturers who owned the patents: have all the manufacturing data required to make the biologics i.e. the process is the product, unlike other generic drugs that come onto market- its not that simple.
• Generic companies: only have the final product and probably would have different processes: they need to work out how to get the final biologics.
• This is why its called BIOSIMILARS and not biosame. 

NB bioequivalence in a protein, is different compared to when the meaning is applied to small molecules. In small molecules: 2 drug molecules become bioavailable at the same rate and at the same extent after administering the same dose. In biologics, as no drug or biologic is 100% safe, management of risks is important in demonstrating bioequivalence. This includes appropriate use of animal models to demostrate efficacy and safety. But there is a risk: proteins in animals, are different to proteins in humans. 

• Ribosome synthesised into  polypeptide chains which are unfolded intially.
• These polypeptides can be folded into intermediates and then native state structures. Native structures can oligomerise, form fibres or crystals. All along this pathway: proteins can dissagregate, form disordered fragments, form amyloid fibrils. 
• Bottom line protein has lots of different conformational states and multimeric states we need to be aware of. 
• Structural role of amino acids in proteins: 3 types of non-covalent bonds:
  • Hydrophobic bonds: hydrophobic side chains can be aliphatic or aromatic. mainly found in the core of proteins, but when the protein distabilises they form bonds together forming aggreggates, and colloidal instability.
  • Charged side chains: salt bridges between oppositelly charged side chains. 
  • Polarity: hydrogen bonding between charged and neutral side chains i.e. OH and C=O. 
  • NB many poteins are stable at their isoelectric point i.e. net charge of 0: but this may encourage aggregation. Because at the isoelectronic point, thats the pH at which the net charge of the protein is 0, the negative and positive charges balance out, reducing repulsive electrostatic forces, attraction forces dominate causing aggregation and precipitation.  
• Protein aggegation process: protein unfolds to form some sort of intermediate, which may go back to the native state, or continue unfolding to form a very unstable, highly energised primary structure. To reduce its energy, it binds to something so: aggregates. In native proteins: we also have to think about surfaces involved in protein manufacturing: which can encourage aggregation. E.g. a hydophobic surface: internal hydrophobic side chains become exposed, bind to hydrophobic surface, form an aggregate. 
• Limit to particulate matter in injections: matter can be detected using microscopes or light obscuration. 

• Chemical degradation of exposed susceptible side chains: oxidation, deamidation, hydrolysis etc.
• This results in many different conformers of protein structures i.e. chemical degradation of proteins, leads to conformational changes.
• Many lead to instability resulting in aggregation: exposure of hydrophobic regions, exposure of cysteine residues forming disulphide bridges etc
• Factors inducing conformational changes / instability:
  • extremes of pH: acidic pH causes denaturing of protein structure which includes insulin 
  • shear forces: shaking a vial, instead of swirling
  • air / water interfaces formed by foaming, shaking, agitation etc 
  • adsorption to solid surfaces e.g. hydrophobic surfaces 
  • freezing, drying and re-hydration: during manufacturing and reconstitution
  • elevated temperatures and pressure

How to stabilise proteins:

• Amino acids: preferrential hydration and exclusion. decrease protein-protein interactions. increase solubility, reduce viscosity and turbidity by reducing the aggregation type effects.
• Polymers: Competitive adsorption. steric exclusion around the protein. prefferential exclusion and hydration.
• Polyols: preferential exclusion. Accumuation in hydrophobic regions. Polyols have mutiple hydroxyl groups.
• Salts: preferential binding. Interaction with protein bound water. Binding of the salt instead of the protein. 
• Surfactants: Competitive adsorption at surfaces. reduce denaturation at air / water interfaces. similarly interfere with ice / water interface upon freezing. 
• Anti-oxidants: sacrificial to protect the protein:, discharge leak testing of finished vials.
• Anti-microbial preservatives: required if multi-use product. 
• Acylation of the protein: acylation with fatty acid to increase binding affinity to serum albumin resulting in longer acting insulin, glucagon and interferon.
• PeGylation: to reduce plasma clearance rate and reduce frequent administration. However, some binding proteins may be less active when pegylated. This is the attachment of polyethylene glycol. 
• Surface engineering: removal of sites on proteins likely to cause aggregation. e.g. mutate the surface, which alters the aa sequence.  

Preferential interaction:

• Common thermodynamic mechanism: which helps us understand if an excipient will denature the protein or protect it.
• DENATURANTS: interaction with polypeptide backbone of protein: e.g. urea which has most aa with hydrogen bonding. There is higher interaction when the protein is unfolded: causes the protein to unfold. +ve [ ] between local and bulk domain. 
• PROTECTANT: e.g. sucrose which is an osmolyte and isnt found in the local domain of the protein: if the protein unfolds, it forces back into the folding position. -ve [ ] between local and bulk domain. 
• NB: difference in preferential interaction can be related to free energy change. 

Preferential exclusion:

• Thermodynamic process which explains stabilisation by excipients. 
• Degree of preferential exclusion and and increase in chemical potential are directly proportional to the protein molecule surface area exposed to the solvent. 
• System minimises the effect of minimum preferential exclusion by favouring the state with the lowest surface area i.e. lowest chemical energy. 
• Sucrose or polyols are preferrentially excluded from the protein, they are found in the bulk domain instead of the local domain of the protein: this draws water out of the protein via osmosis. Allows the protein to bind more tightly, reducing its chemical potential, and saving energy. 

• Low temperature: extends protein shelf life, cold denaturation / unfolding can be  reversible as the temperature drops- but  as temperature drops, solvent properties change: dielectric constant, acid / base ionisation, diffusion rates, solubility of hydrophobic residues, and hydrogen bonding energies: this affects the protein structure and how the energy minimises.
• Feezing: provides a lower temperature but repeated freezing and thawing can cause aggregation by pH and [ ] changes, and by provision of nucleating points on ice water interfaces for aggregation. During the freezing process it can cause unfolding: but when frozen it is stable.
• Cryoprotection: (protection from freezing process) by sugars, polihydric alcohols, oligosaccharides, aa largely work by preferential exclusion.  They do this by lowering cold denaturation temperatures (before the proteins start to unfold) and stabilising osmotic stresses. Whereas surfactants interfere with ice water interface. 
• NB ice crystal formation and freeze [ ]: as the temperature is lowered, you get a super cooling effect, ice crystals forms and ice nuclei form: solutes in the fluids concentrate, as the water leaves into ice crystals, and this results into freezing point depression, where the temperature is lowered to get freezing, eventually freezes. Slowly cooling: bigger ice crystals. Rapidly cooling: smaller ice crystals. Eventually you end up with a eutectic solid or maximally cryoconcentrated matrix of ice crystals and excipients, with the proteins trapped between the ice crystals. 
• Cautionary note: concentration gradients can form during freezing, and tend to remain if thawing occurs, and then no mixing occurs. Vial shape, size and materials affect freezing. Important to gently swirl the vials after thawing to ensure the gradient freezing effects have been mixed out. 

• aka freeze drying.
• lyophilised protein formulations tend to have greater long term stability than protein solution formulations. 
• However, proteins undergo reversible confromational changes when being lyophilised. This exposes otherwise buried regions and renders them prone to aggregation. This can also occur similarly during reconstitution, because the solutes are at a high [ ]: resulting in great pH changes, ionisation changes etc. Therefore refrigerate lyophilised proteins to reduce denaturation rates and as they are hygroscopic i.e. sealed to avoid water vapour absorption. 
• Lyophilisation background: Lyophilisers or freeze dryers freeze the product and reduce the water and pressure. eutectic mixtures form and they freeze at lower temperatures than water or form sugar glasses on freezing (if contains sugar). You get the eutectic temperature when everything is frozen.  When it is frozen, water seperated into ice crystals, increases solute [ ] and lowers the freezing temperature.
• Water in the ice crystals is removed during sublimation of water vapour from ice directly: without any melting.
• NB: proteins have a shelf life: manufacture needs to predict the shelf life of the protein: one way of doing this is accelerated stability testing: accelerate instability of the proteins, and test different formulations media on their stability effects. Complete denaturation at extreme temperatures and pressures: this wouldnt tell us much: only tells us it unfolds and aggregates. We want to know how well it remains stable at normal temperatures and increased pressure: 20, 30, 40 degrees at high pressure. Refolding window: is where proteins can be unfolded or refolded without completely denaturing: this window can be used during stability testing. 

• Recombinant human insulin enginnering: insulin is a monomer (51 aa) little protein structure. It exits naturally in a hexameric structure of 6 insulin monomers, through oligomerisation. The hexamer adopts the structure of a globular protein. Two axial Zn ions are coordinated by the 6 histidine side chains, to the polypeptides. Engineering of structure and formulation has been used to achieve long and rapid acting insulins. 
• Fast acting prandial insulin engineering: Hexameric insulin has much more difficulty going through barriers therefore: mutations of one or more aa in the protein sequence results in protein disruption: conversion to dimeric and monomeric. At this size scale it diffuses faster and improves transport. Resulting in faster acting at subcut administration, faster absorption at mucosal barriers and rapid response from infusion pumps. 
• E.g: intermediate acting insulin: NPH insulin; neutral pH insulin precipitated by protamine. protamine is a highly cationic peptide which leads to protein aggregation. It aggregates insulin, for slower release. protamine = cationic peptide = aggregates insulin. 
• E.g: long acting insulin: formed by isoelectric precipitation: it is isoelectric at pH 5-6 for the normal wild insulin. Insulin chains are then extended i.e. insulin beta chains. this shifts the isoelectronic point to pH 7 ish. When its unbuffered in a pH 4 formulation: it doesnt aggregate, but at pH 7 in the body it does. In the body: the extensions are removed by exopeptidases.  Once its in the body it aggregates and forms precipitates, and then it breakdowns. Remember: at the ip the net charge of the protein is 0, because the negative charges and the positive charges balance out. This reduces the repulsion and increases attraction between proteins which increases aggregation.
• Another way of doing long acting insulin: exploit albumin binding. the fatty acid side chain is added to the insulin via acylation, increasing its stablity and  circulating time in the body, as it binds to albumin. Albumin is recirculated in the blood by recycling receptors, resulting in longer circulation times. Modifying the fatty acid side chain, reduces the potency of insulin but can use higher units in practice. 
• Eating: results in peaks and troughs of glucose [ ] in the blood. fast acting: works around meal times. intermediate acting and lond acting: covers the whole day. 
• Insulin therapeutic variability: short and rapid acting insulin is less variable because of prandial administration, shorter duration of action with reduced risk of hypos. Early NPH insulin formulated with protamine creates suspensions with crystals, therefore speed of dissociation and absorption varies in the same patient day to day. Subsequent advances in insulin engineering were made to reduce variability. 

• Long acting insulins are completely soluble and reduces variability but: glargine requires dissolution of isoelectric precipitates formed after injection and breaking down of the extended beta chains  by exopeptidases resulting in variability, thus requiring greater care and consistence of administration. Detemir is less variable resulting from fatty acid modification: it reversibly stabilises hexamers which avoids precipitation and it binds to albumin, so insulin absorption rates are minimally affected by blood flow which increases circulating levels. So acylation is better than isoelectric precipitation; less variable because doesnt have to rely on exopeptidases. 
• NB: Regimens of insulin include the basal bolus: short acting insulins given with means and long acting insulins given OD / BD. This is for patients with variable lifestyles and who can cope with administering the injections, and blood glucose monitoring. Biphasic twice daily insulin: these are a combination of short and intermediate acting insulin. These are usually for patients with predictable lifestyles, but stereotypical meal intakes. Long acting OD insulin: for patients unable to cope with demands of multiple daily injections and blood glucose [ ] monitoring, where the purpose is not tight glycaemic control. 
• Sick day rules for insulin: always take your insulin, dont take short acting insulin if vomitting. eat and drink well. monitor your cBG and ketone levels every 3-4 hours. 
• Next generation insulins: novel insulin structures are still being explored. E.g: fast acting prandial made to be more rapid i.e. afrezza (inhaled insulin). long acting basal formulations are more prolongued i.e. degludec 42 hours. greater thermal stability for hot climates. 

• production of mAb: select B cell / lymphocytes  clone- they are the cells which produce mAB: the problem with them is that they don't grow very well . Fuse with myeloma cell to form a hybridoma.  Optimise hybridoma growth and mAb production using robotics. 
• production of mAb next step: human antibody development: human hybridomas are difficult, therefore mouse hybridomas are used- immunogenic anti-mouse reactions and rapid clearance occur however, and they also lack the human fc receptor function, thus there are no long circulation times and connection to the immune system.
• Therefore, recombinant mAb enginnering has been used over the past 2 decades- chimeric mAb is used and the constant regions are replaced with the human genes, leaving the mouse variable region in place (antigen binding region), next step was the replacing of the mouse antigen binding region with a  humanised form(mouse antigen binding loops), finally a fully human antibodies are now being used which are produced by mice. The antibodies are then combined with myeloma cells to form hybridomas, which are cultured to form mAb.
• Fully human recombinant antibodies: the 4 mouse genes have been replaced by human transgenes, in a transgenic mouse: mouse which has had its genome altered through genetic engineering techniques. The mouse is then immunised to raise immune response. B cells are then chosen, hybridoma production by being fused with myeloma cells and they are developed in bioreactor cell culture to produce human antibodes. The antibody Fc receptor function can also be engineered. 
• Some background: antibodies are proteins used by the immune system to identify foreign objects for the immune system. Each antibody recognises a specific antigen via antigen binding. Antibodies have the variable and constant domains. Monoclonal antibodies are identical antibodies as they were produced by one type of immune cells e.g. b cell all clones of a single parent cell. They all have identical antigen binding sites. Step 1: immunise the mouse. Step 2: screen the mouse for antibody production. fuse myeloma cells with the immune cells to form hybridomas. Step 3: production of mAb. Only b cells that have been fused with the myeloma cells will survive in the culture as b cells from the spleen have a limited survival time. 
• Types of Mab: Murine: these are derived straight from mice, when given to humans they develop anti-mouse antibody response. Chimeric: they combine the antigen binding parts of the mouse (variable region) with the effector parts (constant region) of a human i.e. they substitute the fc region of the mouse with that of a human. Humanised: these are mAb with complimentary determining regions (i.e. variable regions) from a mouse so that the antibody can bind to the antigen, crafted onto  a human constant region containing the fc part.
• Variable region recognises the antigen. Fc region allows the antibody to switch on the immune system against the antigen.  Constant region: determines the mechanism to destroy the antigen.

• Administration and distribution: After administartion the mAb distribute mainly within the central compartment (extracellular fluid). This is because penetration inside cells is limited by their high molecular weight and hydrophylicity. NB except where transported via receptor-mediated endocytosis / transcytosis. 
• mAb seem to have a volume of distribution of the order of 0.1L / kg: approximately the same as the extracellular fuid volume. E.g: volume of distribution of infliximab at a steady range is 4.5 to 6 L. 
• Degradation and elimination: although the exact mechanism is not fully understood, primary routes are by renal clearance and by proteolytic catabolism by endo-exopeptidases after receptor-mediated endocytosis in the cells of the reticulo-endothelial systems aka mononuclear phagocyte system of the immune system.  
• Pharmacokinetics: Where relatively large loading doses via iv roa given, time profiles result with very high peak [ ]'s. Trough drug level is monitored, rather than the peak level before the next dose is given (measure the remaining amount in their circulation). Their half life's are approximatelly 10-15 days, but some can have a half life of 70 days. Difference between peak and trough levels.
• Antibody recycling: mAb recirculation by brambell receptor (they are capable of transporting Ab e.g. Ig), binding to the fc tail, is essential for maintaing Ig and albumin homeostasis. In adults the FcRn is primary expressed in vascular endothelial cells to keep the proteins in the blood circulation or RES, with lower levels on monocyte cell surfaces, tissue macrophages and dendritic cells. Fc receptor plays a critical role in protecting circulation of mAb however, saturates at high mAb [ ]'s, resulting in an inverse relationship between dose and half life: lower half life for high [ ] / dose antibody OR where high levels of mAb antibodies are seen such as in chronic inflammatory diseases. Recycling results in a half life of approximatelly 14-21 days: pH dependent interaction with the FcRn receptor, preventing renal or RES clearance, allowing infrequent dosing. So it binds in the endosomes at a pH of 6, binding weakens at neutral pH. 

• Loss of response caused by anti-drug antibodies: this can lead to interuption of therapy or replacement with alternative mAb. ADA's: Antibodies produced by the patients attacking and eliminating therapeutic antibodies. 
• Pharmacokinetic binding ADA's: aka BAB's these bind anywhere on the antibody surface except for the active site: they form immune complexes with mAb, increasing clearance rates and dosage requirements, with indirect pharmacodynamic effect: reduced effect and patient response AND Bab's against Fc region of the drug antibody may reduce recycling, as it stops the antibody binding to the FcRn receptor.
• Pharmacodynamic neutralising ADA's: aka NAB's of higher affinity directly interfere with the activity of the drug: by binding to epitopes within or proximal to the active site, blocking the antibody from binding to the target i.e. activation of the immune system. The effect here is more to do with the PK: although it is blocking the PD activity, it results in increases clearance. 
• ADA risk factors affecting efficacy: 
  • mAb related: homology to endogenous proteins (similar to body proteins: which may have antibodies against them), non-glycosylated more immunogenic, sequence peptide affinity for HLA / MHC antigen presenting proteins: human to mouse protein ratio i.e. more mouse ration to human.
  • Genetics: HLA are highly polymorphic and this combines with mAb aa sequence differences. more susceptible genetics.
  • underlying disease: chronic inflammation, too much IGg and fcrn saturated. 
  • other medications: immunomodulators.
  • dosage: may affect peripheral tolerance: too much antibodies and not enough FCRN: loss of peripheral tolerancecan lead to ADA's. 

• As antibody structures are rather large: antibodies can be fragmented into smaller structures using proteolytic enzymes and reducing agents. Smaller antibody fragments are also engineered directly from recombinant human monoclonal antibodies and fused with other effector / active proteins: so just use the binding subunit and combine with another protein body.
• Variable region antibody fragments are used mainly for imaging and diagnostic type applications as they lack Fc interaction with immune system and for long circulation times.  
• Fc fragment engineering: fc engineering maintains the interaction with the immune system and binding site for antibody recycling to enable long circulatory times, but may not be as long in practice yet because of the changes in structure that go on e.g. steric hindrance issues. So the Fc region is taken and antigen binding regions, FcRn binding regions taken up from other proteins are stuck to the Fc region to make hybrid molecules. 

• Routes of entry: via raw materials (water, ingredients, packing), via manufacturing environment (air, water, surfaces, personel), via use and storage (container design failure, consumer handling). 
• Animal derived products: these have microbial risks- viruses, prions>toxins>bacteria.  
  • Excipients e.g. gelatin, collagen.
  • Biologics e.g. vaccines and therapeutic proteins. these are complex culture media containing serum, peptones, cells, tissues etc. cell culture e.g. monoclonal antibodies, stem cells. microbial culture like E.coli e.g. insulin, human growth hormone.
  • Biologics continued: animal products used to grow organisms for vaccines are both anti-bacterial and anti-viral. 
  • Diseased free sources e.g. organs, cell, tissues etc. 
• During storage and use: wide necked containers are more prone than narrow ones. During storage and transit there are flunctuating temperatures and humidity. Containers are designed to minimise entry of microogranisms with the use of secondary packaging. They also tend to minimise contamination when opened by avoiding negative pressure, and minimising use / manipulation.  

• Death: this can also be dynamic i.e. when organisms are dead, breakdown continues. Hazard risk from microbial parts such as toxins e.g.pyrogens: which induce a non-specific rise in body temperature via the hypothalamus. 
• Survival: static i.e. they dont grow and proliferate. risk of infection varies with microorganism and patient. minimum infectious dose differs e.g. cryptosporidial oocysts in water are highly infectious. E.coli is faces is more infective than oral salmonella. prions in parenteral have low infectability but are highly resistant. 
• Growth: dynamic i.e. risks pathogens growing to an infectious dose. results in batch failure through bacterial, micro-fungi / yeast spoilage.
  • Dynamic contamination consequences: microbial numbers increase and may exceed infectious dose. They produce secondary metabolites: 
    • pH changes to acidic or alkaline
    • Texture changes e.g. cracking of emulsions
    • colour change: pigments
    • bad taste, smell, gas production: so hissing sound when lid pops. 
    • toxins e.g. thermolabile proteins, heat stable pyrogens.
  • Eat active: when the active  is consumed, the product is safe but functionless. When active is modified, different pharmacological activities can occur.
  • Eat excipient: consumption or modification of excipients resulting in texture changes,  altered dissolution / release profiles i.e. pk. 

• Pyrogens interact at the hypothalamus in the brain to induce non-specific rise in body temperature. pyrogens can be:
  • endogeneous: i.e. human origin, coming from within. released from damaged cells in the body such as heat shock proteins or cell damage is caused by diseases.
  • exogeneous: outsider source. from a microbial origin during infection, contamination risk from microbial products, hazard risk from microbially spoiled products. The pyrogens are usually heat stable. E.g: Gram negative bacteria LPS shedds releasing: lipid A components are pyrogenic and O antigens / core polysachharides are immunogenic.  
• Pharmaceutical production of pyrogen free products: highly stringent production procedures are required because: 
  • Even in distilled water there is a high rate of gram negative LPS shedding. 
  • Even if such bacteria are subsequently killed, some LPS will remain and cause an inflammatory response upon injection, treatment etc.
  • Even after washing glassware sufficient LPS will remain: therefore water for injection  used must be pyrogen free and containers, glasswares and closures too! Plastics tend to be used these days as they can be extruded at high heat levels, unlike glassware which needs to be autoclaved first. 
  • Examples of acceptable pyrogens limit are based on dose of product and endotoxins limit. 

• These are substances added to products with the intention of increasing the use and shelf-life of the product. Including antioxidants.
• Antimicrobial preservatives: a chemical agent which is capable of reducing the number of viable microorganisms within an object which is safe for its designated use and which will maintain the number of viable microorganisms at or below that level for the shelf-life of the product. E.g. ethylene oxide, formaldehyde, chlorhexidine, glutaraldehyde, Bronopol etc. 
• Preservatives regulation: USP- antibacterial in bacteriostatic and fungistatic [ ] must be added. EP- requires bacteriocidal and fungicidal. Must be present in adequate [ ] at the time of use to prevent the multiplication of microorganisms. Inadvertently introduced into the preparation in use. Antimicrobial preservative effectiveness end point test to determine the point which inhibits microbial growth in product. Because of inherent toxicity to the patient: there are maximum volume and  [ ] which can be used in products. 
• Preservatives biologics: Protein pharmaceutical is often single dose, but because of their cost they are preferred to be in multidose form. However, several proteins are reactive with preservative agents and therefore are only available as single dose units without preservative agents.
• NB: cidal: kills static: prevents growth. 
• Single dose without preservative: Single dose containers and pharmacy bulk packs that do not contain anti-microbial agents are expected to be used promptly after opening or discarded within 3 hours for immediate use products. 
• Large volume single dose containers: these may not contain a preservative, special care must be taken in storing such products after the containers have been opened to prepare a mixture, particularly those that support the growth of microorganisms such as TPN. 
• Although refrigeration slows the growth of most microorganisms, it doesnt prevent it: think of food going bad in the FRIDGE!

• Single-dose containers: sterile medication without preservatives and not required to meet the antimicrobial effectiveness testing requirements. Designed for use with a single patient: cant use with 2 patients! E.g: vials, ampules and pre-filled syringes. 
• Multi-dose containers: sterile medication with preservatives that has met antimicrobial effectiveness testing requirements, or is excluded from such testing requirements. Beyond-use date for an open or entered e.g. needle punctured container is 28 days, unless otherwise specified by manufacturer. E.g: vials.
• Single-patient-use-container: sterile medication is designed to be used multiple times for a single patient. may contain preservatives e.g. insulin pens, unless too toxic for the roa esp. CNS. This means the product is more at risk of microbial growth. e.g. inthrathecal analgesic cartridge.
• NB endpoint testing: point which inhibits microbial growth. There are seperate categories of end point testing for each product type e.g. parenteral and opthalmic, optic and nasal, topically applied, liquid oral dose forms. Different pathogenic organisms to be absent for each route of administration.

• Bacteristat: a chemical agent which can prevent the growth of microorganisms within an otherwise nutritious environment. 
• Bactericide: a chemical agent which will reduce the viability of a population of microorganisms exposed to it. 
• NB terms are meaningless unless specifying the [ ] range over which the effect is manifested.  active [ ] ranges vary for different [ ] groups. same compound may be bacteriostatic at lower  [ ] and bacteriocidal at higher. 
• Sterilant: a chemical agent which is capable of producing sterility within an object or field. 
  • Term can be dangerously misleading. It must be accompanied by a protocol which includes time, temp. and [ ]. It must also be capable of killing all organism forms, including spores i.e. there is no such thing as "quite" sterile. Only the aldehydes and ethylene oxide and sometimes hypochlorite really perform this function. 
• Antiseptic: A chemical agent which can be used as an antimicrobial agent on body surfaces i.e. skin and mucous membranes. 
• Disinfectant: A chemical agent that is too irritant to be used on body surfaces but which can be applied to inert surfaces in order to reduce the level and type of microorganisms to a degree that the object is safe to use. 
• NB: preservatives in intimate contact also need to be of low toxicity and may be used at lower [ ]'s to minimise toxicity. Lower toxicity antimicrobials tend to be used as antiseptics, as higher [ ]'s may be needed to be bactericidal and activity should not be lost on use of the product by dilution and binding. Activity of more toxic microbials used as preservatives needs to be lost on dilution and binding. More than 1 antimicrobial compound may be used to achieve broad spectrum activity against bacteria, fungi and some viruses. 
• NB: The ideal compound should have a broad spectrum of activity against ALL microorganisms, which only applies to toxic aldehydes and ethylene oxide as they are sporocidal. Only a limited number of antimicrobials are licensed for use in medicines. The greater the degree of intimacy between the product and the patient, the fewer antimicrobials due to toxicity issues. 

• critical factors associated with lethal action of chemical anti-microbial agents:
  • Exposure: so not just [ ], but time of contact as well. 
  • Physicochemical conditions: pH, temperature, excipients e.g. polymers.
  • Particular to chemical and organisms: nature of the target organisms and type of preservative.
• Effect of pH upon activity: if the preservation ionises in solution and activity is greater in either the ionised or unionised species, then activity will depend upon the degree of ionisation. Ionisation is affected by pH and pka.
• D value: time for 90% reduction in viability. 
• Q: temperature coefficient: shows the effect of temperature on the antimicrobial activity: the lower the Q the more potent the antimicrobial. 
• Implications of a high Q: large loss of activity upon cooling, large gain in activity upon heating (heating with bactericide, temperature of disinfection, temperature of storage).
• Concentration exponents: this is also known as the dilution coefficient (n). Describes dependence of activity upon [ ] or dilution. gives no indication of activity per se. Using D and n however together can predict activity at any given [ ]. The indication of the loss in biocidal activity and toxicity upon dilution, is important in product design and use. n needs to be considered alongside capacity (binding). NB lower n means it is less affected on dilution e.g. potent antimicrobials: but this can be bad, if the antimicrobial is too toxic for use. Capacity: is the loss of the antimicrobial via adsorption onto surfaces: more potent antimicrobials, with lower [ ] have a poor capacity. 

• Capacity: this is activity losses through binding. In complex materials, total [ ] may not represent activity. Free [ ] better represents activity because:
  • adsorption onto organic debris will reduce effective [ ] and its biological activity and toxicity. 
  • action against target cells will reduce effective [ ].
  • losses proportional to amount of organic mass i.e. low in parenteral administration, medium in antispesis treatment, high in disinfection of drains. 
• Capacity is the ability of antimicrobial to minimise losses of activity through adsorption. this is more apparent with antimicrobials which are more potent i.e. compounds effective at lower [ ] and so easilly lost by adsorption. Losses in activity through adsorption i.e. poor capacity will become more aparent for agents with highest [ ] exponent. Capacity to content with high biomass is inversely related both to activity and concentration [ ].   
• Preservative interactions: adsorptive interactions with formulations will reduce antimicrobial effectiveness. Interaction with excipients (colloids, suspending agents, containers). Partitioning within multiface systems will reduce bioavailability (oil/water emulsions, creams, multiphase polymer formulations). 
• Bioavailability: bacteria grows in aqueous phase, oil/water interfaces. Adsorbed preservative is lost unless affinity to bacteria is greater. Partitioning of preservative into oil phase reduces active [ ]. But this is reversible. If residual [ ] is bacteriocidal then capacity of system is enhanced. 
• Three phase systems: oil-water-surfactant. Bacteria associated with aqueous phase and w/o interface.

Summary of activity loss through adsorption, binding and partition:

• Microorganisms live in the water phase:
  • preservatives bioavailable to kill the microorganisms is not the total [ ]. but it is the free [ ] available in the water phase. 
  • preservative is less available in the water phase when: 
    • [ ] reduced by reversible partitioning into the oil phase.
    • [ ] reduced by sometimes irreversible binding to solid particles, container etc i.e. adsorption, poor capacity. more common in more potent antimicrobials, with a higher n.
    • [ ] reduced in the aqueous phase by reversible binding or interactions with other molecules such as surfactants. 

There are two general approaches to make a sterile product: 

• Produce under "clean" i.e. reduced microbial growth conditions then terminally sterilise in the final container. 
• Produce and assemble under conditions "free" of microorganisms and other particulates. So like adding 2 sterile products together, to make the final product. i.e. asceptic processing.

Microbial contaminants within the working environment:

• raw materials: synthethic / semi: they are mostly carriers, unlike natural products which are full of intrinsic materials.
• water: primary requirement for bacterial growth. water and air are vectors. 
• Manufacturing environment: air, personnel, equipment and facilities. 

Sources of microorganisms: 

Microorganisms are ubiquitous. 

• Resident organisms: soil (gram +ve, endospore forming, fungi), water (gram -ve, yeasts, mould), animals and humans, plants (yeast and mould). 
• Transient organisms: carried by water and air. 

• sterile: free of viable MO. sterile is an absolute term. there is nothing like "quite" sterile. 
• sterilisation: killing or removal of all viable MO. It can leave the dead MO behind but no viable ones. 
• Sterilisation processing traditional methods:
  • Killing: heat sterilisation, radiation, ethylene oxide.
  • Removal: Filtration, centrifugation. 
• Sterilisation standards: these are used to control the number of MO in the manufacturing environment. Validate sterilisation agent and process. Monitor sterilisation process. Regulated by EN and FDA. Must apply to the standards of the market which you are applying the product to. 

• Kill curve: exposure of a population of MO to a sterilant, for increasing periods of time. Then count the number of viable cells present. S0 is before exposure to sterilant i.e. at time 0. There is a decrease in the number of viable cells as a function to the exposure of the sterilant.
• Kill curve is an asymptote curve because it never reaches the x-axis: this is because you get a fixed proportion of the number of cells killed. A log 1 reduction each time = time for 90% reduction in MO. 
  
• Kill curve looks at the log of survivors as a function of time: in most situations unless youve cross contaminated you get a straight line relationship. The slope of the line is a measure of the thermal death rate. 
• Effect of temperature: the lower the temperature, the less the kill rate: the shallower the curve. A steeper curve= high death rate. 
• Effect of different organisms: temperature would affect different MO differently. you can tell by looking at a graph which MO is the most resistant. Q: temperature coefficient: how temperature affects the efficiency of an antimicrobial i.e. is it temperature dependent, would it affect the Log D value. 

Summary of inactivation kinetics:

• demonstrates first order kinetics
• infinite probability of survival: asymptote curve
• Affected by temperature, EO, radiation etc
• Organism specific. 

D value:

• This is the time taken at a fixed temperature to reduce the population by 90%. 
• This can also apply to radiation, EO etc.
• D value units are minutes.
• The lower the value, the more sensitive the MO is to temperature, EO, radiation etc.
• Decimal reduction times: it doesnt matter what points on the graph you choose, as long as you take 2 points which are 1 log cycles apart. 
  
• The d-value of a MO can be influenced by the environment: a subtle change in temperature can make the MO more resistant. 
• The D-value can be influenced by:
  • bacterial species
  • vegetative vs spore form
  • production methods i.e. nutrient environment ( suspension media, culture medium, carrier materials) and treatment dose.

Thermal resistance curve: 

• This is the temperature change required to produce a 90% reduction in D-value: Z-value.
• Z-value is explicit to temperature sterilisation only.
• This is a measure of thermal resistance. It is an indicator of efficiency if we've got something to compare it to: like a standard value. i.e. a range of reference or indicator organisms. 
• E.g: reference organisms: Bacillus stearothermophilus value is 10: moist heat. Bacillus subtilus value is 20: for dry heat.

• When is a product deemed sterile?
• The sterility assurance level indicates sterility.
• S.A.L= 10"-6. 
• Manufacturers usually target a value of 10"-8, just incase something would go wrong.
• A slight difference in resistance between MO has a major impact on processing and manufacturing time. Or even starting off at different number of cells can affect it. This is why it is important to have good manufacturing process, and this is controlled by standards of the manufacturing process.  

Bioburden estimation:

• A population of viable MO on or in a product / primary package.
• This is important to know because the initial population numbers are required to specify sterilisation parameters and inactivation kinetics i.e the kill curve. 
• It is a multistep process to assess how many viable MO are associated with a product:
  • Sample selection
  • Collection of material to test
  • Transport of product to test laboratory and treatment
  • Transfer to culture medium
  • Incubation

Bioburden estimation techniques: 

• Direct method: this is the contact with the culture medium: but limited so we usually use the indirect method.
• Indirect method involves the following:
  • Contact with elluent: this is something which doesnt shock the MO osmotically. e.g detergent: doesnt kill MO, only removes them from surface. although, it has a mild antibacterial activity so NB!
  • Physical treatment: ultrasound or shake bacterial cells with glass beads.
  • Transfer to culture medium
• Selection of a removal technique considerations: ability to remove MO, effect of removal methods on microbial viability, types and locations of MO, nature of product; this influences the natural microbial flora, culture conditions. 
• Selection of culture medium conditions: the type of MO likely to be encountered is dependent upon: nature of the product, methods of manufacture, potential sources of microbial contamination. There is no such thing as a universal growth medium. Each media would have numbers of Culture Forming units, number of colony types etc

• Process validation: the establishment of documentary evidence that provides a high degree of assurance that a specific process will consistently produce a product meeting it's pre-determined specifications. 
• This is divided into installation qualification (tools used in sterilisation) and performance validation (how well the steriliser performs). 
• Performance validation is divided into physical qualification (monitor and measure physical parameters such as temperature, pressure etc to see if a process is working- preferred method) and microbiological qualification (kill curves of MO: usually isnt preferred but with EO, you dont have a choice). 

• An inocculated carrier contained within its primary pack ready for use and providing a defined resistance to the specified sterilisation process. They are like a manufactured pack of endospores. 
• It provides the means of assesing directly the microbial lethality of a sterilisation process. 
• Standardised containers containing selected MO having known stable resistance to sterilisation agents. 
• Used for validation (all forms of sterilisation) and monitoring (EO) of sterilisation processes. 
• In use, the proportion of test organisms surviving the process are measured and related to the expected lethality of the process. 
• BI's are characterised by: factors governing the choice of BI: stability, resistance (high in comparison to product bioburden), non-pathogenic, recoverrability allowing them to grow properly. 
  • strain of test MO
  • reference to culture medium i.e. a reference number of where you purchased it
  • manufacturers name  
  • number of CFU's per test piece
  • D-value
  • Z-value
  • recommended storage conditions: so their resistance doesn't alter 
  • expiry date
  • disposal instructions 
• Recomended test BI:
  • Filtration: brevundimonas diminuta
  • moist heat: bacillus stearothermophillus
  • dry heat: bacillus subtillus
  • irradiation: bacillus pumilus 
  • EO: bacillus subtilus
  • NB if other strains are used they must demonstrate equivalent peformance. 

GENERAL GUIDANCE:

• Balancing the advantages of available methods against the disadvantages.
• No requirements to specify which methods should be used: up to the manufacturer to justify their logisitics i.e. validation after working out their choice.
• choice of method to be used is made at the design / development stage.
• The EMEA decision tree is very reliable and used by most operators to decide on which method to go for. 

SPECIFIC GUIDANCE: 

• terminal sterilisation of product in the final container is preferred to asceptic processing: i.e. manufacturing the product under "clean" conditions and then sterilising the final container.
• Agent / sterilant needs to be in contact with the whole product.
• Process variables are controlled and monitored
• process does not present a hazard to the operators or the environment
• process does not leave toxic residues within the product 

TRADITIONAL METHOD: 

• Removal: FILTRATION and centrifugation 
• Destruction: EO, RADIATION, HEAT (moist or dry)

• This is the passage of a fluid (liquid or gas) across a filter, removing any contaminating solutes. 
• Particles < pore diameter size
• Irregular shape i.e. orientation of the particle
• simultaneous arrival: all particles rushing together may block the pore
• Blocked pore: by a larger particle
• Surface interactions: most filters have a charge, bacteria have a charge i.e. invariably negatively charged 
• Filter voidage: filters are not monolayered. Particles that get through the filter do not have a direct route through the filter. They are convoluted routes so at some point the particle will collide with the filter matrix. The open areas within the filters are known as the voidage, where particles accumulate, so has potential for particles to get stuck here. 
  

Filter types:

• Depth filters: non-fixed pore size (variety of porousities). Inertial compaction (particles collide with the matrix), high retentative capacity, robust, cheap, no sterility i.e. they can not guarantee that you will have a sterile product. 
• Screen filters: aka absolute filters. They have a uniform pore size e.g. 0.8 um or 0.45 um. Direct interception: where the particle size is the size of the filter, easilly blocked, fragile when dry, more robust when wet, expensive (5 times the price of a depth filter), sterility of 0.22 um. 

Filter validity:

• Bubble point pressure test: physical test. place the screen filter in a holder, add water, increase air pressure to get a stream of bubbles. 
• Challenge the filter with brevundimonas diminuta 0.4 um : smallest MO known. 
• Minimum requirement of a 0.22 um sterility filter is that 10"-7 / cm2 cells will be removed. NB how its not 10"-6. 
• Working capacity of the filter: 10"-9-10"-10 cells will be removed. 

• Moist heat: death occurs by a combination of protein coagullation and hydrolysis. very efficient and rapid process. used for aqueous products, devices and dressings. 
• Dry heat: death by oxidative processes. In the absence of moisture, hydrolysis doesn't occur: takes longer than moist heat sterilisation. Used for dry powders, oil preparations, empty glassware and instruments.

DRY HEAT:

• Technology: 2 types: dry heat ovens with a batch process OR sterilising tunnels with a continuous process. 
• Mechanism of heat transfer: 
  • conduction: heat along oven walls
  • radiation: directly from the heating element 
  • convection: most important form: air heats up, heat transfers from the air into the product. used mostly. 
• Critical aspects: product size (the smaller, the large the SA, the quicker). Loading pattern (important not to stack up the products). Air circulation. 
• Cycle: drying (bringing it up to a high enough temperature to remove moisture). heating (to the appropriate sterilising temperature). exposure (for a set amount of time to a particular temp.) cooling down process (can often take a while). 
• E.g of cycle times: at 120-480 minutes.160-120 min. 170-60 min. 180-30 min.

• Technology: autoclave. The autoclave can be self-boiler (producing steam under pressure) OR main stream (similar idea but at a larger scale). Because we heat under pressure we can get to temperatures beyond 100. 
• Mechanism of heat transfer: Its a continuous process-super heated  steam transfers onto product. This creates a small vacuum around the product which pulls in more heat- continues until the product is at the same temperature as the surrounding steam.
• Critical aspects: Air removal (if air is present you don't get the vapour saturation).  Saturated steam and steam under pressure. The steam quality is important: dry saturated is prefered. Temperature must be maintained within + / - 5 kelvin. Time of contact needs to be sufficient enough to given a SAL >10"6. Bioburden level. 
• Autoclave operation: air removal from both the chamber and the product- this occurs via downward displacement where the steam pushes the cold air out via evacuation. Heating to the sterilisation temperature and holding time. cooling-sterile air is pumped in to cool off the product. Drying. 
• Autoclave cycles: most common temperature; 121-124: takes 15 minutes at a pressure of 15. 
  • Fluid cycle
  • porous load cycle: used for fabrics and dressings: recognises the fact that there is trapped air in dressings. 
  • air ballasted cycle: for special products, which are sealed in plastic containers.
• Cycle validation and monitoring: Master temp. recorded (involves test loads (not given to patients) and thermocouples). Tem. record chart (drain probe temperature as it will always be the coolest past due to downward air displacement). 
• Determining the master temperature: drain is the coolest area. a minimum of 12 thermocouples are used, and the process is continued until the thermocouple reads the right temperature. MTR is specific  for 1 type of load: new load= new validation 

• Compendial cycles: the reality! we know we want a SAL of 10"6, a typical cycle is 15minutes long at a temperature of 120 ish. If you run all those conditions you get measured SAL (sterillity assurance levels ) of 10"15, 10"20 etc this is GROSS OVERKILL.
• gross overkill: over processing the product, costly issues, product degradation, economically wasteful and expensive. 
• Therefore we need to meausure the total lethality: F0 is the cell death. it allows lethalities to be compared: by comparing different temperature and time combinations. 
• F0: lethality expressed in terms of the equivalent time in minutes at a standard temperature of 121 delivered to a product in its final container, with reference to MO with a z value of 10. it is a cumulative value i.e. F1+F2+F3 etc as it measures the total lethality. i.e. compares the lethality you have to that of a 121 degrees cycle. 
• Should assume minimum SAL of 10"6
• Minimum F0 for moist heat sterilistaion at 121 is 8 minutes. 

Biological data: 

• F0= D (logN0-logN)
  • D is the d value at the given temperature
  • N0 is the initial number of MO present i.e. bioburden
  • N0 is the number of MO surviving the process- actual or estimated. 

Thermal data: moist heat

• F0= inverse of log (T-121/Z) * dt
  • 121 is the reference temperature for moist heat. for dry its 170. 
  • T is the temperature of heating
  • Z is 10
  • dt is the time of heating 

In summary: it is an alternative to compendial cycles. allows lethalities to be compared. can be used for heat labile products. offers greater flexibility for moist heat sterilisation. The higher the f0 value the more MO are killed i.e. the more effective the sterilisation cycle. 

• Used for disposable single use items and approximatelly 50% of all medical devices including indwelling devices. 
• Chemical sterilisation: alkylation of sulphydryl, amino, hydroxyl and carboxyl groups on Protein and nucleic acid. 
  
• Lethality is affected by [ ], temperature and relative humidity. i.e. in order to be effective it needs a certain moisture content to be present.
• Not same degree of sterility assurance as other methods as a lot of factors affect its sterility. 
• Requires Std Prd load containing suitable biological indicators.
• Toxic residues: because its a chemical.
• Operator safety: highly explosive, so very strict guidelines are in place. 
• Comes as a liquid but tends to be used in its gaseous form for sterilisation as it penetrates products more easilly. 
• Extremely explosive in air: therefore exclude from air by adding N2, C02. 
• Doesnt have a specific target for cells: its like a general blasting of the cells. 
• Critical lethal parametres: 
  • Exposure time: varies significantly depending on product
  • Temperature: 25-65- vaporises at a quite low temperature
  • Humidity: 40-85 RH. bacteria cells are extremely resistant to this chemical in the dry state therefore humidity is required.
  • [ ]: 250-1200 mg / L: depends on product size 
  • B subtilus ( same MO for dry heat). used for both validation and monitoring 

• Process characterisation: Pre-conditioning area (air is removed from the product, and steam enters to increase the RH). steriliser (explosive proof). aeration room (products are held in quaranteen to make sure any residual EO is removed via a vacuum. Can take up to a week: varies). 
• Sterilisation cycle in more detail:
  • Evacuation: ensures complete removal of any remaining air from the chamber and product.
  • Vacuum hold: makes sure chamber is sealed correctly, no air back into the chamber. physical means can be used to monitor the presence of air.
  • Conditioning: temperature, moisture levels raised again.
  • sterilant injection: vapourised EO enters the chamber.
  • Exposure: try and keep a constant level of EO present in the chamber.
  • Sterilant removal: active mechanism, catalytic converter is used to destroy the EO by converting it to water and c02. almost 90% is converted.
  • flushing: sterile air enters part of the chamber and a vacuum removes any remaining air. saw tooth part of the curve.  

• X-ray irradiation: ionising radiation, expensive, low penetrative power, tends to be used for surface items e.g. thin paper. 
• Pulsed light: broad spectrum white light, short pulses, has a UV ouput. used for in-line sterilisation and intravascular product e.g. saline. 
• Microwaves:  intense heating for short cycles. used for solutions in vials, contact lens solutions etc.
• Gas plasma: mixture of ions, free radicals, electrons, neutrons. 50 minutes at 60 degrees. Medical devices alternative to EtO.

The current problems of alternative technologies: 

• unknown lethal effects: so what was the mechanism of death
• kill kinetics is different to traditional processes
• validation compliance
• monitoring can be an issue
• no established regulatory requirements for years: last one was more than 10 years ago.