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
What are the advantages and disadvantages of APS?
Advantages:
- Define routine procedures, assess manufacturing steps
- Can use non-ideal conditions, create safety margin when system works with non-ideal conditions
Disadvantages:
- ‘point-in-time’ evaluation, need to check which step went wrong
- not QA
- need risk assessment and worst-case scenario
Sterility Method 1: Aseptic Filtration
How is aseptic filtration done?
- What size is the filter, what can it remove, what can’t it remove
Use 0.22um filter
=> ‘bacteria retentive filter’ can remove bacteria and molds, but not all viruses or mycoplasmas (require additional heat treatment)
=> unable to filter ions as well
=> can remove pyrogens
Sterility Method 1: Aseptic Filtration
Aseptic filtration is done with minimum conc. of ____ for which standard organism?
Why is this so?
Minimum concentration of 10^7 CFU/cm2
Use high conc. of standard test organism (Brevundimonas diminuta), so that if system works with high bioburden, it will work when actual bioburden is lower
Sterility Method 1: Aseptic Filtration
What are some considerations for the sterilized filter used?
Integrity of sterile filter
- should be verified for volume and filtrate load
- fibre-shedding from filter should be minimal
- filter should not rupture (filter integrity test)
*Asbestos-containing filters should not be used
Filter should not remove any ingredients from the formulation or release any substances into the formulation
*Final sterile filtration is advised to be done immediately before filling, preferably using double-filter layer or second filtration
How is APS done for liquids (e.g., liquids that undergo aseptic filtration)?
During compounding/solution preparation step:
- Sterile medium should come into contact with every surface/piece of equipment involved in the process (*medium should come into contact longer than final product)
- Include routine tests such as filter functionality assessment, holding time, sampling
During filling step:
- Medium should come into contact with the whole container + cap (shake)
- Container should be transparent to inspect, size must be consistent with the product and process
Sterility Method 2: Lyophilization
What is lyophilization and how is it done?
Lyophilization is the process in which water is removed from the product after it is frozen and placed under a vacuum, allowing ice to change directly from solid to vapor without passing liquid phase.
It is carried out in aseptic conditions
- Dissolve drug and excipient in suitable solvent/diluent, generally WFI
- Sterilize bulk solution by passing it through 0.22um filter
- Fill into sterile containers, partially stopper
- do not close completely as subsequent evaporation
- do not open due to contamination risk - Transport partially stoppered container to lyophilizer
- Freeze the solution in freeze-drying chamber
- Apply vacuum to chamber and heat the shelves in order to evaporate the water from the frozen state
- Complete stoppering of the vials by hydraulic or screw rod stoppering
- *pdt is hygroscopic, stopper in low humidity environment
*Transfer of partially closed containers should be undertaken in Grade A environment
Sterility Method 2: Lyophilization
What are the three separate and interdependent processes involved?
- Freezing
*Take note of possible polymorphism due to crystallization => may affect solubility (physicochemical property) and reconstitution of final pdt - Primary drying (sublimation)
- Done under vacuum with heat
- Temp should be stable during sublimation
- Temp rise indicates end point (all water removed, pdt now being heated) - Secondary drying (desorption)
- Desorption refers to water being adsorbed onto the crystal/particle
- The presence of water can result in stability issues
- Hence desorption of water under vacuum can detach water from the crystal
- End point = water content <1%
What are some advantages and disadvantages of lyophilization?
Advantages:
- Ease of processing a liquid
- Stability in lyophilized form
Disadvantages:
- Expensive
- Requires sterile diluent (for reconstitution)
How is APS done for lyophilization?
Simulated lyophilization
- Container filled with growth medium loaded onto lyostat, vacuum applied (can be comparable or less than the real one for the same time)
=> condition worse than one we expect (e.g., lower time and vacuum) can give safety margin
Issues (affect growth of microorganisms):
- vacuum and time: boiling out of aerobic microorganisms?
- growth of anaerobic organism need to use inert gas instead of O2 (cost?)
What are some considerations when performing APS for lyophilization?
- CIP/SIP (clean in place, sterilization in place) consolidated practice
- since cleaning of system is involved - Set up can be performed before sterilization
- set up procedure require several steps, also set up procedure for cleaning of system - Long holding times can produce product sticking
- Product formulation is usually prepared just before filling
- Batch dimension can be limited by storage tank size (size depend on equipment)
What are two alternatives to lyophilization to obtain sterile powders?
- Sterile recrystallization
- drug is dissolved in a solvent and the obtained solution sterilized through 0.22um membrane filter
- sterile antisolvent added to crystallize the drug particles, which are then filtered and dried aseptically
disadvantage: may have variations between batches - Spray drying
- solution of drug sprayed into dry chamber where it comes into contact with hot steam of a sterile gas (80-100dc)
How is APS done for powder?
Filling step:
- filling machine can do two consecutive filling, medium and placebo powder dissolved in it
*placebo powder:
- should have similar mechanical properties to the pdt
- easy to sterilise
- soluble in the medium (e.g., lactose, mannitol, PEG, NaCl)
- no effect on growth medium (test using growth promotion test)
Negative control - medium without placebo
What are some considerations when performing APS for powders?
- CIP/SIP not applicable since no cleaning of systems involved
- dose/QC parts are aseptically assembled
- set up is longer than for liquid
- during holding times, product can remain in machine without sticking
- blending is usually prepared off-line
- batch dimension is not limitation
- presence of powder requires interventions for cleaning
- machine tuning may require human interventions - operator source of contamination
What medium can be used for APS (powder)?
Soybean casein digest medium or Trypticase Soy Broth (TSB) for aerobic microorganism, fungi, yeast
Fluid thioglicollate medium (FTM) for anaerobic microorganisms
Sabouraud Dextrose Agar (SDA) for yeast
What outcomes are assessed for ASP (powder)?
Visual inspection to spot contamination/turbidity
- Performed after incubation at 20-35dc for 14 days (or at least 7 days with two sep temp)
- Perform at half-time and end of incubation period
Counting and identification
- Check morphology
Supervision via QA
Discuss the limitations of water for injection as a sterile diluent
While WFI minimizes the possibility of toxicity and chemical reactions,
- its use is limited if drug has limited solubility (large volume WFI required)
- instability of aqueous solution
Apart from WFI, what are some alternatives for sterile diluents?
- Fixed oils (corn, cottonseed oil)
- Ethyl oleate, isopropyl myristate
- Ethanol (cannot use at high extent, cause pain, hemolytic at high conc.)
- Dimethyl sulfoxide (work well with low solubility drug, but hemolytic and toxic at low conc.)
- PEG
What are the requirements for sterile injection?
- Sterile and bacteria endotoxin below allowable limits
- Standards for particulate matter
- Restricted use of additives (additives must be compatible)
- Additives include: vehicle, preservatives, tonicity adjusting agents, buffers, antioxidants, dispersing agents and surfactants
Epidurals (intrathecal) must be _____ free as it can cause paralysis
preservative-free
List some sterile products
Sterile powder
Injections
IV bags
Multi-dose vials
Patient-controlled analgesia
Epidural (intrathecal)
Irrigation
Albumin
Plasma protein fraction Ppf
Immunoglobulin (lyophilized)
Ophthalmic
Disinfectants used in which grade areas should be sterile before use?
Grade A and B
*Grade A: high-risk operations e.g., filling and making aseptic connections, should have unidirectional airflow
