Particulates and pyrogens

what are pyrogens?

-              ever-inducing substances, that are difficult to destroy

what are the properties of pyrogens?

-              Non-volatile

-              Thermostable

-              Water soluble

-              High molecular weight (106 Da)

-              Lipopolysaccharides from cell wall of gram-negative bacteria

-              The most potent pyrogens are from gram-negative bacteria

Lipid potency is enhanced by protein/polysaccharides

what are pyrogen sources

-              Solvents

-              Medicament – a substance used for medical treatment

-              Excipients

-              Manufacturing apparatus

what is the hazard of pyrogens?

-              Physiological responses

o   Reddening of injection site

o   Pain in legs and trunk

o   High temperature

o   Multiple organ failure, leading to death

what are pyrogen tests applied to?

-              Applied to all injections over 15ml and powders for reconstitution

o   Especially infusions or large volumes, direct IV injection for seriously ill patients

what is the rabbit test?

o   Inject rabbits with formulations and observe a rise in temperature

o   If at least 3 rabbits have a combined increase in temperature of at least 2.65°C, the product fails

§  The fertile response of rabbits is similar to humans but

·      They are expensive and time consuming

·      Difficult to quantify

·      Some injections cause the same response eg insulin

what is the cumulus amoebocyte lysis test LAL?

o   In-vitro test for bacterial endotoxin based on the primitive clotting mechanism (amoebocytes) of the American horseshoe crab (lumulus polypemus)

o   Advantages – quick, sensitive, quantifiable, inexpensive

o   Disadvantages – pH, cations etc can affect results

o   Enzymes + endotoxins  gel formed 

o   LAL + test solution  coagulation

Sensitivity of 1 picogram/ml

what is particulate matter?

-              mobile undissolved substances unintentionally present in parenteral solutions

what are raw ingredients?

-              – drug, solvent, material not filtered out at the clarification stage of manufacture prior to filling

what are the origins of particulate matter?

raw ingredients

final container

environment

container and closure -

Deposition of closure components during sterilisation eg Zn oxide, clay (coat with lacquer however this can cause flaking)

o   Reaction of formulation with container eg flaking of glass (important to have care with the choice of container)

o   Rubber fragments due to coring by needles

o   Glass fragments on opening of ampoules

what are the hazards of particulate matter?

-              Vascular occlusion – directly by particles over 7.2µm could block arterioles/capillaries indirectly via the formation of emboli

-              Inflammation response

-              Neoplastic response

-              Antigenic response – patient becomes sensitised to material and has an allergic response to material if reintroduced into the body at a later date

how does the size of the particulate affect the injection?

§  Large particles >590µm block the needle

§  Particles >8µm lodge in the lung

§  Particles 3-5µm taken up in the spleen and liver

Particles <3µm may agglomerate

what do hazards of particulates depend on?

size of particles

o   Site of occlusion

o   Shape and surface characteristics of the particle – affects adherence

o   Nature of the particle

Host response

what is the probability of an adverse reaction proprotional to?

-              the total number of particles introduced

what does GMP require for injections?

-              Visual inspection – GMP requires visual inspection of each container by trained personnel

what are the disadvantages of visual inspection?

§  Only larger particles are detected by the human eye

§  Subjective

what are the advantages of visual inspection?

§  Detects gross contamination and incompatibilities in mixtures eg CaCl2 and K2PO4

§  Non-destructive

what is optical microscopy

-              USP method where 25ml of solution is filtered and particles are collected on the filter membrane and counted

o   Particles > 10µm: < 50 particles/ml

Particles >25µm: < 5 particles/ml

what are the disadvantages of optical microscopy?

§  Labour intensive so training is required

§  Special facilities are required

§  Difficulties with oily and viscous solutions

Small sample

what are the advantages of optical microscopy?

§  Identification of particles

how are electrical sensing zone methods?

-              (Coulter counter):

o   BP 1993 method ‘limits test for particulate matter)

o   Particles >2µm: < 1000 particles/ml

o   Particles >5µm: <100 particles/ml

o   Based on Ohm’s law (V=IR)

o   Measures the equivalent spherical diameter (ESD = volume)

Counts and sizes particles

what are the advantages of electrical zone methods?

§  Not dependent on operator technique

§  Readily detects particles of relevant size

Reliable and reproducible

what are the disadvantages of electrical zone methods?

§  Non-conducting solutions require addition of NaCl

§  Bubbles can contribute to false counts

§  Destructive

§  Small samples

§  No indication of the nature of the particle

what is the light blockage method?

o   BP method

o   Particles >2µ: <500 particles/ml

o   Particles >5µm: <80 particles/ml

counts and sizes particles

what are the advantages of light blocking method?

rapid and accurate

what are the disadvantages of the light blocking method?

§  Affected by shape and transparency

§  Variation between commercially available instruments

for the light blocking method, what is a decrease in light intensity proportional to?

the cross sectional area