Particulates and Pyrogens

what are pyrogens?

fever inducing substances

• not microbial fever → not caused by living organisms

why are pyrogens difficult to destroy?

• non volatile :. stable

• thermostable (up to ~250ºC :. cannot destroy them via heat)

• water soluble :. cannot filter them

• high MW (10⁶ Da)

• lipopolysaccharides (from cell wall of gram negative bacteria)

where do the most potent pyrogens come from?

gram negative bacteria

• potency is enhanced by protein/polysaccharide

what are the sources of pyrogens?

• solvents

• medicament (any medicinal substance)

• excipients

• manufacturing apparatus

what formulation type will be impacted most by the presence of pyrogens?

large volume parenterals (i.e. > 15 mL)

• :. infusions, IV preparations

what are the physiological responses produced if you have been exposed to pyrogens?

• reddening of injection site

• severe pain in legs and trunk

• high temperature

• multiple organ failure :. potential death

which preparations are pyrogen tests applied to?

• large volume parenterals > 15 ml

• powders for reconstitution

what are the different pyrogen tests available?

• rabbit test

• lumulus amoebocyte lysis test (LAL test)

how does the rabbit test for pyrogens work?

• inject rabbits with formulations and observe for a rise in temperature

• if 3 rabbits have a combined increase of > 2.65ºC then the product fails

why are rabbits used in the rabbit test?

their febrile (fever producing) response is similar to humans

what is the disadvantages of the rabbit test?

• expensive and time consuming

• difficult to quantify

• some injections cause an increase in body temperature e.g. insulin

• ethical concerns

how does the LAL test work?

• in vitro test for bacterial endotoxin

• based on clotting protective mechanism of the American Horseshoe Crab

• if the crabs are exposed to the endotoxin then this is detected in their blood and it clots :. gel formed = positive response

• enzymes + endotoxins → gel formed

• LAL + test solution → coagulation

what are the advantages of using the LAL test?

• quick

• sensitive

• quantifiable

• inexpensive

what are the disadvantages of using the LAL test?

pH and cations can affect the results

what is the sensitivity of a LAL test?

1 picogram/mL → equivalent to 1 × 10^-12 gram/mL

what is the difference between microbial, pyrogen and particle contamination?

• microbial contamination: harmful living microorganisms which causes infections or product spoilage

• pyrogen contamination: substances that can trigger fever when introduced into the body, typically endotoxins from bacterial cells

• particle contamination: unwanted solid particles (e.g. dust, fibers) in a product

define particulate contamination

presence of visible and/or sub visible extraneous matter

• sub visible is harder to remove because you cannot see it

what formulation type will be impacted most by the presence of particulates?

large volume parenterals > 15 mL

what are the sources of particulate matter in parenteral solutions?

• raw ingredients: drug, solvent, material not filtered out at clarifications stage of manufacture prior to filling

• final container: material not removed during rinsing prior to filling

• environmental: material falling into the final container during filling e.g. hair

• container and closure (during storage and use): deposition of closure components during sterilisation, reaction of formulation with container, rubber fragments due to coring by needles, glass fragments on opening of ampules

how can needles introduces particulate matter?

• needle = thin, hollow metal tube

• when drawing up the drug from a multi use vial, the needle can carve out a thin slice of the rubber bung

• this thin slice of the rubber bung can be injected into the bag for large volume parenterals

why are all large volume parenterals delivered systemically?

because of their large volume

what are the hazards associated with particulate matter?

• vascular occlusion

• inflammatory response → particle can be pro-inflammatory

• neoplastic response e.g. asbestos can produce a cancerous response

• antigenic response

how can vascular occlusion result from particulate matter?

• direct: particles greater than 7.2 micrometers could block arterioles and capillaries

• indirect: formation of emboli

how can an antigenic response be produced from particulate contamination?

patient becomes sensitised to material :. has an allergic response to material if introduced to the body at a later date

what does the nature of particulate hazard depend on?

• size of particles

• site of occlusion (influences downstream effects)

• shape, surface characteristics of the particle → affects adherence

• nature of the particle

• host response

what size particles may block a needle?

large particles > 590 micrometers

what size particles may lodge in the lungs?

> 8 micrometers

what size particles may take up the spleen and liver?

3-5 micrometers

what size particles may agglomerate?

< 3 micrometers

describe the relationship between the probability of an adverse reaction and the total number of particles introduced

proportional

why is it recommended to discard the first couple mLs from an infusion bag?

removes particles lining the tubes

what are the different methods of detection of particles?

• visual inspection

• optical microscopy

• electric sensing zone method (coulter counter)

• light blockage method

describe the visual inspection detection method for particulates

GMP requires each container of a batch to be examined by trained personnel

what are the advantages of the visual inspection detection method for particulates?

• detects gross contamination

• detects incompatibilities

• non destructive

what are the disadvantages of the visual inspection detection method for particulates?

• only larger particles are detected by the human eye (not sub visible particles)

• subjective

describe the optical microscopy detection method for particulates

• US Pharmacopoeia method

• 25 mL of solution is filtered → particles collected on the filter membrane are counted using a graticule in the microscope

• for particles > 10 micrometers, there should be less than 50 particles/mL

• for particles > 25 micrometers, there should be less than than 5 particles/mL

what are the advantages of the optical microscopy detection method for particulates?

• identification of particles → can see what kinds of particles are in the product :. can improve manufacturing processes based on where particles is believed to have come from

what are the disadvantages of the optical microscopy detection method for particulates?

• labour intensive → training required

• special facilities required to prevent introduction of particles into the product that wasn’t initially there

• difficulties with oil and viscous solutions

• small sample

describe the electric sensing zone detection method for particulates

• old BP method → replaced by light blockage method

• electric current is applied → solution is drawn through an orifice → as particle goes through, the orifice is blocked for a period of time :. can measure change in resistance using electrodes

• based on ohms law (voltage = current x resistance → V = IR)

• measures equivalent spherical diameter → converts change in resistance to equivalent spherical diameter

• counts and sizes particles

• for particles greater than 2 micrometers, there should be less than 1000 particles/mL

• for particles greater than 5 micrometers, there should be less than 100 particles/mL

what are the advantages of the electric sensing zone detection method for particulates?

• not dependent on operator technique

• readily detects particles of relevant size → can detect very small particles

• reliable and reproducible

• very sensitive

• counts and sizes particles simultaneously :. quick and convenient compared to microscopy

what are the disadvantages of the electric sensing zone detection method for particulates?

• non-conduction solutions require addition of NaCl

• bubbles can contribute false counts :. have to be careful with stirring rate

• destroys sample

• small samples

• no indication of the nature of the particle → just size

• if 2 particles are pulled through the orifice at the same time, they are counted as 1

describe the light blockage detection method for particulates

• current BP method

• instead of electric current, light intensity is used

• draw solution through a tube → as particles go between the light source and detector, there’s a decrease in light intensity

• decrease in light intensity is proportional to cross-sectional area of the particle

• for particles greater than 2 micrometers, there should be less than 500 particles/mL

• for particles greater than 5 micrometers, there should be less than 80 particles/mL

what are the advantages of the light blockage detection method for particulates?

• rapid

• accurate

• counts and sizes particles simultaneously

what are the disadvantages of the light blockage detection method for particulates?

• affected by the shape and transparency

• variation between commercially available instruments

if our practice for the removal of pyrogens and particulate matter was not good enough, which patient group would be most affected?

neonates in intensive care

• they have the smallest vasculature

• they receive their nutrition and medication intravenously