sterility Flashcards
sterility
(WHO) absence of viable microorg
* Conditions that guarantee absolute sterility is too harsh for API
Relies on procedural measures for sterility
prevent contamination of biological materials
○1) Clean room technology
2) GOOD MANUFACTURING PRACTICES
§ Environment (door, walls, floor) , equipment, product □ Personnel, practices and training, engineering, weather
§ Avoid microbial contamination holistically: address supportive elements
□ Optimise both individually and collectively to provide greatest confidence in overall process
sterility assurance level (SAL)
PROBABILITY of 1 viable microorg in a certain number of drug products
acceptable safety level: 1 in 10^6 prob non-sterility
sterilie means no __
○ Without Microorganisms
○ Endotoxins inside limit
§ Pyrogens
○ No detectable particles
sterility prep for
Parenteral, EYE, Irrigation
sterilisation process vs process development
NOT REPLACEMENT
○ Bioburden minimised in manufacturing step BEFORE sterilisation
high bioburden critical
○ High risk of contamination with viable microorg
§ Must be removed in initial process (API, excipients)
○ High risk of contamination with pyrogens
pyrogens
§ Substances that produce fever
Eg: endotoxins: lipopolysaccharides produced by gram-ve, e.coli
inactivation factor
degree which viable organisms reduced by the sterilisation treatment applied
overkill for sterilisation
sterilisation process applied to reduce the Biological Indicator (particular microorg) by
factor of 10^12
○ Not always feasible, few API can withstand harsh conditions
○ Intensity and duration of sterilisation treatment too high for some pdts
SAL vs log 10 reduction
SAL: probability of microorg of surviving
Log reduction: efficiency of sterilization method
tests that affect qty of viable microorg
1) sterility test
2) endotoxin test
3) bioburden
4) visible/ non-visible particles
1a) validation of sterility test (suitability test)
1) final pdt + known qty (<100 cfu) of known microorg – anaerobe, aerobe, fungi
- Medium: trypticase soy broth // fluid thioglicollate medium
2) +ve control: only microog
3) -ve control: only final pdt
4) incubate for 3-5days
5) compare lvl of turbidity
- see if pdt has antimicrobial prop (leads to a false neg)
- turbidity of pdt COMPARABLE to +ve control
1b) validation of sterility test (growth promotion test) ensures
- Culture media must meet established performance criteria
*Ensure accuracy of results, for key QC tests, procedures will depend on culture
- Acceptability of each batch of medium
- Medium is “fit for purpose”
- Medium produce CONSISTENT results
steps for growth promotion test
1) Can support growth of <100 viable microorg (SPECIFIC)
a. Indicator microorg to grow vs contaminants etc
b. does it support growth of microog
2) Culture media assessed for sterility a. Based on incubation parameters (time, temp) of the method b. No contaminants
choice of microorg for test
1) environmental contamination
2) those that may have been found in final pdt
2) endotoxins test
eg: injectable
1) Limulus Amoebocyte Lysate Test
a. Reaction between LPS and clottable protein (substance)
i. Amoebocytes (blood cell of horseshoe crab)
b. Endotoxins presence lead to clot into gel like
2) Gel clot method
a. Qualitative method
b. Used for water
c. Sensitivity depends on lysate (but higher) 0.015-0.250 EU/mL endotoxins unit
3) Turbidimetric/ chromogenic method
a. Quantitative method based on ONSET time
b.Dedicate equip
c. sensitivity based on lower limit quantification
endotoxins limit
a) ELC = k (endotoxins kg)/ M (dose kg/hr)
b) MAX endotoxins possible in sample
i) Depends on dose of drug, time, weight, age
<5 EU/kg/h (usually)
Maximum valid dilution
a) MVD = ELC/ method sensitivity
b) Determine how much you can dilute the sample so you still can detect the endotoxins
c) Based on meter SENSITIVITY
safety factor
Safety factor = RANGE OF dilution b. MVD – 1st dilution fitting the spectrum
3) bioburden/ IPC testing
- Conc of microorg in material
(total org /ml or /g)- Microbial control during manufacturing for pdt quality & safety
- Sterile biologic drug pdt
○ Manufactured by sterile filtration
○ Aseptic filtration
○ Processing
Controls microbial load for each step for OVERALL microbial control
steps for bioburden test
1) Bioburden sample conducted before sterile filtration (in-process microbial control)
* Maximum bioburden: in control of sterile filtration step
* Accepted limits for
○ Number of CFU
○ Sample test vol (Not More Than of 10 CFU/100ml)
potential process design and control strategy risk mitigation measures (BIOBURDEN TESTING)
1) Reduce microbial load prior to and on sterile filter
a. Remove bioburden with pre-filters
2) Limit hold times and room temp storage
3) Implement aseptic handling techniques when appropriate
4) Select and validate sterile filter mem with high microbial retention capabilities
a. > 10^6 CFU/ cm3
5) Incr effective filter SA by using larger single filter/ multiple filters in series
6) Limit batch vol to be sterile filtered
7) Test integrity of sterilizing filters PRE-/ POST use
- Test and control bioburden THROUGHOUT manufacturing process
- Raw materials introduction too
Reduce bioburden breakthrough!
- Raw materials introduction too
calculate bioburden
LogN = logN0 - K*t
N (n.o. of cells surviving at time t) = SAL of 10-6
D-value (decimal reduction value), exposure required in sterilisation process to reduce pop by 90%
4) Visible/ non-visible particles
Sub-visible/ visible:
* Particulate contamination of IV solutions
○ Extraneous, mobile
Undissolved particles (x gas bubbles)
sources of visible particles
○ EXTRINSIC: environment contamination
§ Equipment
§ Primary packaging (stainless steel, hair, fibers, glass, rubbers)
○INTRINSIC: formulation (API, excipients, gas bubbles unstable and ppt)
effects of these visible particles
○ mechanical obstruction of lung (from inhalation)
○ Injection site reaction, phlebitis, granuloma
○ Lower therapeutic conc
2 tests for visible particles
Light obstruction particle count test
Microscopic particle count test
Light obstruction particle count test
- Preferred for inj, parenteral infusions of sub-visible particles
1. Make sample go through thin tube
2. Go through with laser
3. Receiver at the other end
4. Tells how much particles of various sizes - Apparatus is calibrated using dispersions of spherical particles of known sizes between 10-25um
- Determine size and number of particles (of that size)
light obstruction particle test formulation considerations
- Large vol: single units tested
- Small vol: <25ml, need combine 10 or more units until total of 25ml
- Powders: dissolved in particle free water (sterilised) / solvent –not contaminated
number of particles per container
○ < 6000 particles per container. Size of >10um
○ <600 particles per container. Size >25um
- free of visible particles
(method 2) Microscopic particle count test
- Suitable binocular microscope
○ Equipped with ocular micrometer calibrated with objective micrometer
○ Mechanical stage can hold, transverse entire filtration area od mem filter
○ Ocular micrometer circular diameter graticule
§ Large circle divided by crosshairs into quadrants
§ Transparent and black reference circles (10um and 25um) in diameter- To detect visible particles, aided illumination or comparable automated methods (>100um)
- Sample in plate, spread thin layer
- Take picture
- Image modification – colour
a. Until able to see the particles
b. Can see shape too - Count the particles + size
- To detect visible particles, aided illumination or comparable automated methods (>100um)
STERILE MANUFACTURING
STERILE PREPARATION
○ Sterile preparation: ensure there’s no microbial contamination on anyone/ anything involved with healthcare practices (surgeries, drug manufacturing)
considerations of sterile prep
§ Safety – adverse toxicological concerns
§ Sterility – free from microbiological contamination
§ Non pyrogenic – endotoxins from contamination
§ Particle free – visible particle contamination
§ Stability – chemical, physical, microbiological
§ Compatibility – formulation, package, other diluents
§ Tonicity – isotonic w/ biological fluids
sterilisation methods
1) dry heat
2) moist heat (autoclave)
3) aseptic filtration
4) gamma irradiation
5) ethylene oxide/ nitrous oxide, hydrogen peroxide fog
6) lyophilisation
aseptic process simulation
Validate aseptic process using microbiological growth medium (instead of product) that closely approximate those used during drug product process
how to closely simulate the same exposure that the product will undergo
exposing the microbiological growth medium to
◊ product contact surfaces of equipment, container systems, critical environments, and process manipulations
◊ imitate as closely as possible the routine aseptic manufacturing process
◊ take into account various interventions known to occur during normal production
And WORSE-CASE SCENARIO
qualify process at beginning
1) assess machine assembly, standard interventions
2) handling, storage of components
3) surrounding environ
4) routine procedures
5) interpretation of results
importance of qualifying process
- point in time test
- need valid all sub-processes
- risk assessment+ worst case scenario for evaluation
1) aseptic filtration process
Numerous fiber/ size
Load specifications
- performed using 0.22 µm filter
- For thermolable objects
- Remove pyrogens
remove microorg from fluid stream w/o adversely affect pdt
1) At minimum concentrations of 10 7 cfu /cm 2
2) Try to stimulate at high bioburden (if successfully drop) = means works for lower bioburden too = “worse case scenario”
aseptic filtration is suitable for/ not suitable for
NOT FOR: Pdts in final container
* But can be filtered through sterile filter of nominal pore size 0.22 micron / less
○ Remove bact, mould
○ Not remove ALL virus, mycoplasmas = additional heat treatment required
aseptic process simulation (liquid)
1) compounding/ sol preparation
* Buffer instead of the final product
* Sterilization of the medium (no false +/-)
* Medium should come in contact with every surface/piece of equipment involved in the process
○ Have sufficient contact time
* Routine test like filter functionality assessment, holding time, sampling
2) Filling * Container and caps, equipment: must be cleaned and sterilized * Medium should come in contact with the whole container+cap assembly (shaking) * Container must be transparent to allow inspection (photosensitive compounds) * Size must be consistent with the product and the process
false + > -ve better
False +ve (affects manufacturer, that their pdt not working well)
>
false -ve (affects pt that this pdt actually does not works )
2) Lyophilization sterilisation process
s — > g
- Lyophilization/ freeze drying
- water is removed from a product after it is frozen and placed under a vacuum,
○ ice to change directly from solid to vapor
(without passing through a lq phase)
- water is removed from a product after it is frozen and placed under a vacuum,
-dissolve drug, excipients
- 0.22 micron filter
- container
1) freeze
2) primary drying, (sublimation)
3) secondary drying (desorption)
- stopper (hydraulic/ screw rod stoppering)
FREEZING = COLD
a. Quick freezing of small vol
b. Crystals (polymorphism) size/ shape affect final pdt
i. Suff cold, no lq inside
Bact dies
1* DRYING (SUBLIMATION) = HEAT
a. Dry under VACUUM
b. Aq vapour removal
c. Endpoint = temp rising (means all water vapourised and pdt is being heated instead, stop heat, prevent degradation)
i. Suff vaccum = sublimation done properly
2* DRYING (DESORPTION) = VACUUM
a. Desorption of water under vacuum
i. For water of crystallizations
ii. Force water molecules to gain energy and leave
End point = water content < 1%
why vacuum used
force water of crystallisation out
a) Under low humidity environ
Maintain stability of pdt (hygroscopicity)
lyophilisation simulation
§ vacuum applied could be comparable or less than the real one for the same time of the real process (WORSE CASE SCENARIO)
○ Simulated lyophilization (lower time and vacuum)
§ Vacuum and time vs boiling out aerobic microorg
§ Anaerobic microorg: air instead of inert gas (higher cost)
(APS) for powders
○ Filling machine can do two consecutive filling (powder/placebo and medium)
○ Medium should be enough to dissolve the powder
○ Possible contamination
placebo and negative control for APS (powders)
○ PLACEBO
○ Mechanical properties similar to the final product
○ Easy to sterilise (validate method)
○ Soluble in the medium (Lactose, Mannitol, PEG, NaCl)
○ No effect on the medium (Growth promotion test)
○ NEGATIVE CONTROL
○ Medium without placebo (contamination)
○ Necessary for the simulation
aseptic processing vs terminal sterility
Terminal sterilisaiton
- Use of lethal treatment on microorg
(heat, radiation, chemical)
Aseptic processing
- Removal or separation of microorg
adv and disadv for terminal sterilisation
terminal
(adv): Relatively easy to reproduce and validate
(disadv): Not for all materials
(120*C not suitable for most drugs)
(usually for packaging and medical devices)
adv and disadv for aseptic processing
(adv) Fewer issues with materials
(disadv)
Higher risk of contamination
More variables in process
- harder to control
- manufacture in sterile conditions
3 eg culture medium for culture testing
§ Fluid thioglycolate medium (FTM)
□ Anaerobic, some aerobic bact
§ Soybean casein digest medium (SCDM)
□ For fungi and aerobic bact
§ Sabouraud dextrose agar (SDA) for yeast
Incubation for sterility test
(days and temp)
§ 14 days
□ For slow growing organisms
At 32.5 (higher temp (above normal conditions) and 22.5 *C (Room temp) respectively
Prior to examination
results of sterility test
§ Any turbidity in culture media indicate growth – must be investigated for cause
□ Contamination
□ Culture media not sterile?
sampling for sterility testing
must be representative of the batch
□ Number of units in batch
□ Vol of lq per container
□ Method of sterilisation
□ Manufacturing requirements of regulatory
- FDA: may need test larger sample size
- Larger sample: more representative of batch
◊ Higher possibility of failure
◊ Better to have false positive than false negative (affect pt safety)
2 types of sterility test
1) mem filtration
2) direct inoculation
Membrane filtration sterility testing
Regulatory method of choice for FILETRABLE pharmaceutical pdts
Membrane filtration sterility testing process
1) Filtration through 0.45um mem filter in filtration canister
1. Not too small as may remove the potential pathogens left inside
2. Remove components (AB/ preservative) that may cause turbidity/ inhibit growth
(affect results
2) Culture medium
3) Incubation
4) CFU counting
Direct inoculation sterility testing key points
- Low sensitivity because of small vol of pdt
- Neutralisation may be necessary if pdt has
- Antimicrobial prop
- Preservatives
- Cloudy sample affect reading of microbial growth
- False +ve
Direct inoculation sterility testing steps
1) Aseptical removal of sampling
1. Takes small sample, this may not be representative of the final batch
2. Eg: saline bag is 500mL, only sample 5mL
a. False -ve
2) Inoculation in culture medium
3) Incubation 14days
Which method (no longer used) was used to estimate the number of viable bacteria in the sample after membrane filtration sterility test?
MPN (most probable number) method to estimate viable n.o. of bact in sample
1. By inoculating broth in 10-fold dilutions
a. Extinction dilution
b. What conc don’t have microbes anymore
How can sterility testing be conducted for medical devices?
Also explain how it should be carried out if medical devices have hollow spaces and pipes.
1) Device to be tested is in direct contact with test media
1. Throughout incubation period
2. Microorg in/ on device to grow and proliferate
2) Rinse with sol for pdt with hollow space, pipers
3) dry heat (common method) specification
180C for 30min
170C for 1hr
160*C for 2hr
Dry heat: Temp > 220*C used for sterilization and depyrogenisation of GLASSWARE
dry heat pros and cons
pros:
Destroys pyrogens for materials that may be damaged by moist heat (GLASSWARE)
Pdt impenetrable to moist heat (powder, petroleum pdt, sharp instruments)
Easier to operate, maintain
Slow rate of heat penetration
cons: heat sensitive pdt
mechanism for dry heat
- Static-air type (oven)
- Forced air type
a. Ventilator, fan to keep air moving, transfer of energy
b. Motor-driven blower
i. Circulates heated air throughout chamber at high velocity
ii. More rapid transfer of energy from air to instruments
4) Moist heat
Autoclave, gravity steriliser specifications
Temp 121*C
Time 15mins
Moist heat pros and cons
Uses biological indicator to verify sufficient heat/ pressure
moist heat destroys microorg by irreversible coagulation and denaturation of enzymes and structural proteins
* Moisture to transmit heat better, conducts
*SAL 10^-6
(cons) Heat/ moisture sensitive products
mechanism for moist heat
1) Direct steam contact at required temp and pressure for specified time
* Parameters:
○ Steam
○ Pressure (to achieve the high temp)
§ Dry saturated steam and entrained water
○ Temp 121, 132*C
○ Time
§ Min exposure period
§ Depend on object and material
2) 3 types of steam sterilisers (autoclave)
* Gravity displacement autoclave
○ Circulation steam from below
* High-speed prevacuum steriliser
○ Vacuum
○ Add steam
§ circulates
* Steam flush pressure pulsing process
○ Go in high P
○Not continuous
application of F and D vlaue to find suitable temp and duration
3) D-value (time to reduce surviving pop by 90%/ log10)
* For direct comparison of heat resistance microorg
- F concept/ F value: measure equiv time that a monitored article is exposed to desired temp
- Temp not same throughout steam
○ Chamber temp > rise> pdt
- Temp not same throughout steam
- 121C is Fo (For steam sterilisation F value specific for Z value of 10C and a T0 value of)
○ Z value: (temp incr needed to reduce the D- value by a factor of 10)
○ T0: reference temp, 121*C usually for steam sterilisation
○ T: product temp
(F0: means if put at 116C for 15mins, same effect as if put pdt at 121C for only 4.7mins)
5) Gamma irradiation pros
Containers and packaging may remain intact (suitable for commonly used materials)
Low temp sterilisation
Disrupt nucleic acids
Suitable penetration and high dose rate
Medical pdts and packaging materials
5) Gamma irradiation cons
Not good for some heat sensitive products
* Aq pdts with proteinaceous component
○ Vaccine, biologics
* Polyethylene (gloves)
Oxidation, delamination, cracking
mechanism of gamma radiation
1) Sterilisation by ionising radiation
1. Cobalt 60
2. Cesium 137
3. Electron accelerators
2) Disrupts nucleic acid
Goes through several layers
eg Ozone (gama radiation)
Consists of O2 with loosely bound 3rd O atom
* Powerful oxidant ○ Destroys microorg * But highly unstable ○ Half life 22 mins at room temp ○ ADVANTAGE: § No need other process to remove residue
6) CHEMICAL sterilisation eg
Ethylene oxide/ nitrous oxide,
Hydrogen peroxide fog
Depend on gas used different saturation and permeation
adv of chemical sterilent
Less invasive
Good for heat sensitive (medical device, surgical supplies)
-which may have compatibility issues
- ethylene oxide, formaldehyde, hydrogen peroxide, peracetic acid at 60*C
Wide range of efficacy: mycobacteria, MRSA, clostridium
Non toxic (by-pdt water vapour, oxygen)
cons of chemical sterilent
Requires tight wrap to avoid leaks
* Contaminates adjacent areas
Low penetration (may need penetration enhancers)
* Affects bact mem/ external layer for H2O2 to enter
* Lauryl carbamate
Flammable and explosive
Eye pain, sore throat, breathing, blurred vision, dizziness, NV, headache, convulsions, blisters, coughing
Carcinogenic (possible)
eg peracetic acid (as a chemical sterilent) activity against
- Active against bact, fungi, yeast (many orgs + fast!)
- Highly BIOCIDAL oxidiser
○ Efficacy in presence of organic soil
○ Endoscopic tubes
- Highly BIOCIDAL oxidiser
steps for chemical sterilent
- Used at 35% in combi + anticorrosive agent (SULFONATE)
- Dilute to 0.2% w/ filtered water (0.2mm)
- Temp 50*C
- Process time of 12mins
○ Need anticorrosive agent
§ May need to put smth on pdt to protect from corrosion
§ **can remove
○ Affect by organic residue
§ Need HIGH CONC to clear
○ Wide range of efficacy (microog cover)
Gas plasmas (chemical sterilent) MOA
1) Gas plasmas generated in enclosed chamber under deep vacuum
1. Using radio freq
2. Microwave energy
2) Excite gas molecules and produce charged particles
3) Form of free radicals
React w/ biological compounds
eg Hydrogen peroxide plasma (as chemical sterilent) efficacy
- Wide range of efficacy: mycobacteria, MRSA, clostridium
- Non-toxic by pdts (H2O, O2)
- BUT low penetration ( Need enhancer)
Hydrogen peroxide plasma steps
- Deep vaccum to pull lq hydrogen peroxide (30-35% conc) from disposable cartridge
- VHP (vaporised hydrogen peroxide) carried into sterilisation chamber by carrier gas
a. Air (negg/ pos pressure) - Operates at 37-44*C
a. Cycle time: 75mins
b. Lower temp, suitable for temp sensitive
- VHP (vaporised hydrogen peroxide) carried into sterilisation chamber by carrier gas
Pyrogens
- Substances that induce fever when inj into mammals
○ Endotoxins – LPS from gam -ve bact
○ Non-endotoxins: other microbial sub
§ Gram +ve, viruses
§ Pyrogens from yeast, fungi
○ Non-microbial pyrogens: rubber, microscopic plastic particles, metal compounds in elastomers
Absence of endotoxins impt
so need use __
impt for sterility for parenteral
Large scale methods of sterilisation (ONLY DRY HEAT) don’t have capability to destroy endotoxins
test for pyrogens by
1) animal (rabbit pyrogen test for broad range)
2) animal (endotoxins = bact endotoxin test)
3) in vitro broad (monocyte activation test)
4) in vitro endotoxin (Recombinant factor C rFC)
why 14 days incubation
Because slow growing organisms present? Need to check if they are present
So medium must not have other organisms inside
missing sterile pdts
Sterile powder
- penicilin G (potassium)
- Ceftazidime
injections
IV bags
Multi-dose vials (MDV)
Patient-controlled analgesia
epidural
irrigations
albumin
Plasma protein fraction Ppf
immunoglobulin
Ophthalmic
filters for sterile filtration
- not suff as sterilisation in final container
- steam sterilisation preferred
- double filter layer/ 2nd filtration
- final sterile filtration as close to filling point
-verify integrity, fibre shedding from filters minimal
- not remove any ingredients from formulation
