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PY365 Quality Of Sterile Medicines > Endotoxin Testing > Flashcards

Endotoxin Testing Flashcards

1
Q

define pyrogen

A

a substance that produces a rise in body temperature when introduced into the body

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2
Q

what is an endotoxin

A

a pyrogen of bacterial origin
- all endotoxins are pyrogens, but not all pyrogens are endotoxins

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3
Q

what is endotoxin testing for

A

lipopolysaccharide from gram - bacteria

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4
Q

where is the LPS found

A

found in membrane of a gram - bacterium

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5
Q

describe the importance of endotoxins

A
  1. LPS from gram - bacteria causes a wide spectrum of non specific pathophysiological reactions
    - such as fever, changes in WBC counts, hypotension, septic shock
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6
Q

what is septic shock

A

systemic, immunological response to infection which may, in extreme cases, be fatal

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7
Q

what is the pattern of the sequence of events from endotoxins

A

follows a regular pattern- latent period, physiological distress
- tachypnoea (abnormally rapid breathing)
- tachycardia
- altered body temperature
- hypotension
- prostration
- in severe cases, death

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8
Q

what does injection of fairly small doses of LPS result in

A

results in death in most mammals
- how soon death occurs varies on dose of endotoxin and route of administration

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9
Q

what is the most likely contaminant to cause sepsis

A

gram - sources of endotoxins, as they can be found in water

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10
Q

explain how endotoxins cause septic shock

A
  1. LPS activates almost every immune mechanism, as well as the clotting pathway
  2. as a result, LPS is one of the most powerful immune stimuli known
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11
Q

give examples of infectious gram - bacteria that can produce endotoxins

A

neisseria meningitidis
Escherichia coli
salmonella

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12
Q

how can endotoxins still contaminate sterile drugs

A
  1. endotoxins are secreted in tiny quantities by living gram - bacteria
  2. but endotoxin molecules can be released after death/lysis of the gram - bacteria
  3. these dead bacteria/lysates contain endotoxins that remain biologically active
  4. endotoxins are heat stable and pass through sterilising filters
    - so we need to test specifically for endotoxins
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13
Q

how are endotoxin levels measured

A

measured in endotoxin units (EU)- a unit of biological activity of the USP or BP reference endotoxin standard

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14
Q

what are the most likely sources of endotoxin contaminants in pharmaceutical products

A

non sterile powders, components and water

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15
Q

how much endotoxin is dangerous

A

humans can develop symptoms when exposed to as little as 5 EU/kg body weight

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16
Q

why is it important to understand endotoxin levels

A
  1. analysts in pharmaceutical development may eb presented with new compounds or products and asked to calculate an endotoxin limit
  2. must understand the concept of endotoxin limit for a drug given iV, IM or intrathecally in clinical settings
17
Q

what is the maximum human dose/endotoxin limit

A

the maximum amount of endotoxin that must not be reached or exceeded when a pharmaceutical product is given to a patient

18
Q

what is the established threshold endotoxin limit

A

5EU/kg/hr or 350EU per 70kg adult
- to avoid fever/hypotension from IV or IM injection

19
Q

why does endotoxin limit have a time unit

A

because mechanisms in the liver and blood can neutralise endotoxin

20
Q

what is the endotoxin limit for a product administered intrathecally

A

0.2 EU/kg/hr or 14 EU per 70kg adult
- much lower as there are no clearance mechanisms in intraspinal spaces

21
Q

what is the equation that can be used to calculate endotoxin limits

A

endotoxin limit = K/M
- K= threshold pyrogenic dose (5 EU/kg/hr if IV or IM)
- M= dose of the drug in units/kg/hr

22
Q

what 3 pieces of information are required to calculate endotoxin limits

A
  1. route of administration- for IM and IV K= 5EU.kg
    - for IT, K= 0.2 EU/kg
  2. dose per kg of patient- if the dose is given as a whole person dose, it must be adjusted by dividing the dose by the weight of the target population to get dose/kg
  3. dose per hour- if the total dose is administered by an infusion lasting n hours, it must be adjusted by dividing the total dose by the length of administration to get dose/hr
23
Q

how do we test for endotoxins

A

using an in vitro quantitative test
- the limulus amoebocyte lysate (LAL) test

24
Q

what are the different types of LAL tests

A
  1. gel clot
  2. endpoint turbidimetric
  3. kinetic turbidimetric
  4. endpoint chromogenic
  5. kinetic chromogenic
25
Q

what is the basic mechanism of the LAL gel clot test

A
  • endotoxin is added to pro-clotting enzyme to form active clotting enzyme
  • this combines with clotting protein to produce a gel clot
26
Q

what is used as LAL reagent

A

amoebocyte lysate

27
Q

describe how amoebocyte lysate is used as LAL reagent

A
  1. crabs are caught, bled and returned to sea
  2. the pooled blood is centrifuged
  3. the pelleted amoebocytes lysed in water and the released coagulogen diluted to a standardised concentration
28
Q

describe the process of performing the gel clot LAL assay

A
  1. the test sample is diluted to varying degrees, alongside control standard endotoxin
  2. standardised Limulus lysate is added to the tubes of test and control standard endotoxin which are incubated for 60 mins
  3. the tubes are inverted and examined to see if the mixture has clotted
  4. gel clot results are scored as positive or negative
  5. can be qualitative or semi quantitative
  6. for the test to be positive, the gel must be solid during a momentary inversion of the reaction tube
29
Q

what is necessary to have when performing a LAL semi quantitative gel clot assay

A
  • limulus amoebocyte lysate
  • standardised endotoxin
  • endotoxin free water
  • endotoxin free glass or plastic ware
  • about £500 for basic semi quantitative test
30
Q

how is the LAL assay improved

A
  1. many compounds tested interfere with the gel clot assay
  2. development of alternative methods to the gel clot test
    - LAL chromogenic method
    - LAL turbidimetric method
31
Q

why is the LAL chromogenic method used

A

frequently overcomes interference problems

32
Q

describe the process of the LAL chromogenic method

A
  1. LAL mixed with PNA- a synthetic peptide coupled to the colourant, p-alanine
  2. LAL/PNA then added to the test substance
  3. if endotoxins are present in the sample, the subsequent enzymatic reactions of the LAL reagent break the peptide bonds connected to the p-nitroaniline molecules, releasing yellow colour into solution
  4. the more endotoxin present, the more yellow the solution becomes
  5. measure spectrophotometrically at 405nm
  6. can be endpoint (less accurate but quick and cheap) or kinetic (high volume, very reproducible and accurate)
33
Q

when can the LAL chromogenic/turbidimetric method not be used

A

cannot use if test product is already coloured or turbid

34
Q

describe the process of the LAL turbidimetric method

A
  1. measures endotoxin activation of LAL spectrophotometrically
  2. mix sample with LAL
    - time to reach a predetermined absorbance level is inversely proportional to amount of endotoxin in sample
  3. can measure endotoxin levels from a standard curve
  4. can be endpoint or kinetic but still some problems with interference
35
Q

which method for LAL is the most reproducible and accurate

A

kinetic chromogenic

PY365 Quality Of Sterile Medicines (19 decks)

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