IC8 Concepts for Sterility Flashcards
Sterility is defined by WHO as the absence o viable microorganism. Why absolute sterility not achievable?
Conditions that guarantee absolute sterility are too harsh for active ingredients
What is sterility assurance level (SAL)?
1 in 10^6 probability of viable microorganism in the drug product
What are the 3 criteria for sterility?
- No microorganism
- Endotoxins within limit
- No detectable particles
Sterility is a requirement for ____, _____, and _____
Parenteral preparations, eye preparations and preparations for irrigation (to clean wound)
Why must bioburden be minimized in manufacturing steps before sterilization?
High bioburden is critical because:
- Higher risk of contamination with viable microorganisms
- Higher risk for contamination with pyrogens
What is inactivation factor?
What is overkill approach?
Degree to which viable organisms is reduced by sterilization treatment applied
Overkill approach - sterilization process to reduce microorganism by 10^12
*Most materials unable to withstand harsh sterilization process to achieve 12 log reduction, intensity and duration of sterilization treatment may be too high to tolerate
Explain the validation of sterility test (suitability test) *it is a final sterility test
**Done after neutralization of the sterile pdt to validate the sterility test
Test sample is spiked with known quantities (<100 CFU) of known microorganisms
Positive control without sample => contaminated with just microorganisms
Negative control => clean
Incubate for 3-5 days (slow growing organisms to be incubated longer)
If level of turbidity of test sample is comparable to positive control = sample is NOT sterile (POSITIVE)
Explain the criteria for culture media used, in validation for sterility test (growth promotion test)
Culture media criteria:
- Acceptability of each batch of medium
- Medium is ‘fit for purpose’
- Medium can produce consistent results
Explain the validation of sterility test (growth promotion test) *it is a final sterility test
Media must support growth:
Used to confirm that each lot of growth media used can support the growth of less than 100 viable microorganisms
Media must be sterile:
The media must be incubated and assessed for sterility according to the incubation parameters (time, temp) established by the method
What might cause false negative and false positive for the validation of sterility test (growth promotion test)?
False negative:
- growth media unable to support growth of the indicator organism
- this affects patients* more dangerous
False positive:
- growth media was non-sterile and contained other microorganisms
- this affects facility/manufacturer
Describe endotoxin test? (LAL test)
Limulus Amoebocyte Lysate Test
- Blood extracted from horseshoe crab contains a clottable protein (derived from lysis of the blood)
- Blood coagulates in presence of bacterial endotoxins
- end point: gel, cloudy
Why is endotoxin undesirable in product?
Endotoxin is a lipopolysaccharide (LPS) found in the outer membrane of gram negative bacteria, it is a pyrogen.
It can lead to release of interleukins and cytokines => pro-inflammatory
How is endotoxin limit concentration (ELC) calculated?
*ELC gives the max amount of endotoxins that can be present in a product
ELC = K/M,
where K = max endotoxins dose, usually 5 EU/kg
M = maximum recommended dose (max dose/kg/h)
How is ELC used to determine the safety factor for dilution of the product?
First, maximum valid dilution (MVD) is determined via:
MVD = ELC / method sensitivity
whereby, method sensitivity has a distinct value for different methods, it measures the ability to establish that such a difference is significant
Safety factor = range of working dilution b/w MVD and first dilution fitting the specs
What does maximum valid dilution (MVD) of 60 mean?
1 part in 60 parts total dilution
If we dilute anymore than this, we cannot determine the endotoxin limit
What is bioburden/IPC (in process control) testing? *test conducted to ensure microbial control during manufacturing, thus ensuring pdt quality and safety
*It establishes the accepted limits for number of CFU and sample test volume
State what is the acceptable microorganism count based on EMA
Test microbial load at sterile filtration step, before aseptic filling and processing
IPC bioburden sample is drawn immediately prior to sterile filtration step, must be within accepted limits for number of CFU and sample test volume
*EMA: not more than 10CFU/100mL
What are some methods to reduce bioburden?
- reduce microbial load prior to and on sterile filter
- limit hold times and room temp storage
- implement aseptic handling techniques
- select and validate sterile filter membrane (high microbial retention capabilities >=10^6 CFU/cm^2)
- increase effective filter surface area - use larger filter/use multiple filters in series
- limit batch volume to be sterile filtered (but small yield of pdt)
- test integrity of sterilizing filters pre and post use
How to calculate bioburden?
LogN = LogNo - k*t
k is the death rate constant (gradient of downslope), No is the initial number of cells, N is the number of cells at time t
Rearrange:
Log (N/No) = -kt
To achieve 1 log reduction,
Log (1/10) = -kD
D = 1/k
To achieve SAL (6 log reduction),
t = D * (LogNo - LogSAL)
Therefore we can calculate time taken to achieve 6 log reduction in microbial load
=> If t is too long, means need to find ways to increase k in order to shorten the time
=> incr k is achieved by reducing bioburden
What are the two particle count tests to evaluate sub-visible/visible particles in product?
- Light obscuration particle count test (*preferred method for injections)
- Microscopic particle count test
Describe the light obscuration particle count test
Suitable apparatus based on principle of light blockage which allows automatic determination of the SIZE and NUMBER of particles
Apparatus is calibrated using dispersions of spherical particles of known sizes between 10um and 25um
How are parenteral samples drawn and used for the light obscuration particle count test?
Large volume parenterals: test single units
Small volume parenterals <25ml: combine contents of 10 or more units and obtain volume of at least 25ml
Powders for parenteral: reconstituted with particle-free water or appropriate solvent
Describe the microscopic particle count test
Microscope equipped with ocular micrometer which consist of a large circle divided by crosshairs into quadrants, transparent and black reference circles 10um and 25um in diameter
When using light obscuration and microscopic particle count test, preparation complies with the test if:
Average no. of sub visible particles present in the units tested
does not exceed 6000 >= 10um per container
AND
does not exceed 600 >= 25um per container
Should be free of visible particles
What is Aseptic Process Simulation (APS)?
Substitute product with microbiological growth medium in order to validate the aseptic process
Involves using microbiological growth promoting media in place of the product to undergo aseptic process, media is then incubated and inspected for microbial growth
Selection of nutrient medium should be made based on dosage form of the pdt, and selectivity, clarity, concentration, and suitability for sterilization of the nutrient medium
