Endotoxin Testing

Question 1

define pyrogen

Answer 1

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

Question 2

what is an endotoxin

Answer 2

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

Question 3

what is endotoxin testing for

Answer 3

lipopolysaccharide from gram - bacteria

Question 4

where is the LPS found

Answer 4

found in membrane of a gram - bacterium

Question 5

describe the importance of endotoxins

Answer 5

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

Question 6

what is septic shock

Answer 6

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

Question 7

what is the pattern of the sequence of events from endotoxins

Answer 7

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

Question 8

what does injection of fairly small doses of LPS result in

Answer 8

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

Question 9

what is the most likely contaminant to cause sepsis

Answer 9

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

Question 10

explain how endotoxins cause septic shock

Answer 10

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

Question 11

give examples of infectious gram - bacteria that can produce endotoxins

Answer 11

neisseria meningitidis
Escherichia coli
salmonella

Question 12

how can endotoxins still contaminate sterile drugs

Answer 12

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

Question 13

how are endotoxin levels measured

Answer 13

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

Question 14

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

Answer 14

non sterile powders, components and water

Question 15

how much endotoxin is dangerous

Answer 15

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

Question 16

why is it important to understand endotoxin levels

Answer 16

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

Question 17

what is the maximum human dose/endotoxin limit

Answer 17

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

Question 18

what is the established threshold endotoxin limit

Answer 18

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

Question 19

why does endotoxin limit have a time unit

Answer 19

because mechanisms in the liver and blood can neutralise endotoxin

Question 20

what is the endotoxin limit for a product administered intrathecally

Answer 20

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

Question 21

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

Answer 21

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

Question 22

what 3 pieces of information are required to calculate endotoxin limits

Answer 22

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

Question 23

how do we test for endotoxins

Answer 23

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

Question 24

what are the different types of LAL tests

Answer 24

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

Question 25

what is the basic mechanism of the LAL gel clot test

Answer 25

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

Question 26

what is used as LAL reagent

Answer 26

amoebocyte lysate

Question 27

describe how amoebocyte lysate is used as LAL reagent

Answer 27

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

Question 28

describe the process of performing the gel clot LAL assay

Answer 28

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

Question 29

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

Answer 29

- limulus amoebocyte lysate - standardised endotoxin - endotoxin free water - endotoxin free glass or plastic ware - about £500 for basic semi quantitative test

Question 30

how is the LAL assay improved

Answer 30

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

Question 31

why is the LAL chromogenic method used

Answer 31

frequently overcomes interference problems

Question 32

describe the process of the LAL chromogenic method

Answer 32

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)

Question 33

when can the LAL chromogenic/turbidimetric method not be used

Answer 33

cannot use if test product is already coloured or turbid

Question 34

describe the process of the LAL turbidimetric method

Answer 34

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

Question 35

which method for LAL is the most reproducible and accurate

Answer 35

kinetic chromogenic