3. Tests for parenterals
Finished product quality control tests
Mainly seven tests for paenterals
1. Sterility test
2. Pyrogen test
3. Leaker test
4. Particulate test
5. Clarity test
6. Closure integrity test
7. Content uniformity or weight variation test
3
4. Sterility test
Sterility is defined as freedom from the presence of viable
micro-organisms
Sterility test is defined as the microbiological test applied to sterile product to
show that the product manufactured and processed under specification guided by
cGMP
It is destructive test so it is impossible to test every item for sterility
4
5. If bacteria or fungi placed in the medium which provide nutrition &
Water and kept at a favourable temperature, organism will grow & their
Presence can be detected by their turbidity in the clear solution
Steps involved in sterility testing
1. Selection of the sample
2. Selection of the quantity of the product to be used
3. Method of testing
4. Observation & Results
Principle of the test
5
7. Mechanisms of sterilization
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D-Value( Decimal Reduction time) – Time in minutes at any defined temp to
destroy 90% of viable organisms
Z- Value (Thermal destruction time) – Number of degrees of temperature change
to produce tenfold change in D-value
F- value – Lethality value
1. Dry heat sterilization
For thermostable substances ( above 140°c)
Act by oxidizing the proteins
a) Tyndallization
Applicable for drugs unstable at 150°c
Heating 80-100°c for 1 hr in 3 successive days
b) Pasteurization
1. Holder pasteurization- Milk heated at 145°F (62.8°c) held for 3o min & quickly
cooled
8. 8
c) Hot air oven
Used for glasswares, metalware, anhydrous oils
Temperature(°c) Time(min)
170 60
160 120
150 150
140 180
2. Moist heat sterilization
More rapid & efficacious
Applicable for rubbers closures, ampoules, syringes, surgical dressings
Denaturation & coagulation of proteins
Autoclave conditions
9. 9
Temperature(°c) Stream pressure Ib/sq
.inch
Holding
time(min)
115-118 10 30
1121-124 15 15
126-129 20 10
135-138 30 3
3. Radiation sterilization
For thermolabile materials
Act by damaging nucleic acids
a) Ionizing radiation
X-rays & Gamma rays cause mutations & destroy microbes by stopping
reproduction
Creates free H+, OH, OOH radicals
b) Nonionizing radiation
UV –light (253.7nm) cause damage to DNA by dimerization of adjacent thymine
molecules
10. 10
4. Fitration sterilization
For thermolabile materials
a) Depth filters- For sterilization of gases
b) Membrane filters-
Pore size= 0.45 Um( Millipore grade HA)
0.22Um ( Millipore grade GS)
Integrity & performance is evaluated by Bubble point pressure method
Sterilization methods Biological indicators D-value
Dry heat Bacillus atrophaeus, B.subtilis
var niger
NLT-2.5
Moist heat B.Stearothermophillus NLT-1.5
Ethylene oxide B.Atrophaeas NLT-2.5
Ionizing radiation B.Pumilus 0.15-0.20
Filtration Pseudomonas diminuta -
12. Sample must be representative of the whole of the bulk materials & lot of final
containers
Random sampling is taken from the final container
Minimum sample size related to batch size ( BP 2001)
Selection of the sample
12
14. Selection of quantity of the product to be used
Depends mainly on the volume or the weight of the container
Minimum samples to be used in each culture medium in the test for sterility
are: (BP 2001)
14
15. Methods used for sterility testing
Methods used
1. Membrane filtration 2. Direct inoculation
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16. Media used for sterility testing
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1. Fluid thioglycolate medium
For aerobic and anaerobic bacteria
Incubation of the media: 14 days at 30-35°c
2. Soyabean casein digest medium
For culture of fungi
Incubtion media- 14 days at 20-25°c
Nutritive properties of media
1. Aerobic : Staphylococcus aureus, Bacillus subtilis, Pseudomonas
aeruginosa
2. Anaerobes : Clostridium sporogenus
3. Fungi : Candida albicans, Aspergillus niger
18. Direct inoculation method
Suitable for samples with small volumes
Volume of the product is not more than 10% of the
volume of the culture media
Suitable for aqueous solutions, oily liquids.
Creams, ointments
Appropriate quantity of the preparation to be
examined is transferred directly to the culture medium
and incubate for not less than 14 days
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19. Observation and Result
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No evidence of growth- Passed the test for sterility
Evidence of growth- Re-testing is performed foe the same number
of sample, media and volume – if no evidence of growth then passed
then test for sterility
If evidence of growth then isolate and identify the organism
Retesting is performed with twice the no of sample
1. No growth-passed the test
2. If growth occurs-Failed the test for sterility
20. Pyrogen testing
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Introduction
Pyro-(Greek-Fire), gen (beginning)
Fever producing metabolic by-products of microbial growth and death
Bacterial pyrogens called endotoxins gram negative bacteria produce more potent
than gram positive
Characteristics of endotoxins
1.Thermostable
2. Water soluble
3. Unaffected by bactericides
4. Non volatile
21. Pyrogen testing
21
Physiological effects of pyrogen
1. Elevate circulating levels of inflammatory cytokines
2. produces fever, blood coagulation, hypotension. Lymphopenia
3. shock
4. characterized by cardiovascular dysfunction
5. vasodilation
6. vasoconstriction
7. endothelium dysfunction
8. Multiple organ dysfunction or failure
23. Rabbit test
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Why the Rabbit ?
1. Reproducible pyrogenic responce
2. Other species not predictable responce
3. Similar threshold pyrogenic response to the humans
4. Economic
Requirements
Healthy rabbits of weight 1.5 kg
Newziland Albino or Belzium white strain
24. Rabbit test
24
Preliminary test (Sham test)
Performed for conditioning and training the rabbits
IV injection 10mL/Kg body weight of sterile pyrogen free saline solution
Rectal temp is recorded
Any animal showing temp variation of 0.6°c is nat used in main test
Main test
Carry out the test in a group of 3 rabbits
Sample preparation- Dissolve substance to be tested in a pyrogen free saline
solution warm it to 38.5°c before administration
25. Rabbit test
25
Procedure
Inject the solution under examination into the marginal ear veins of each rabbit within
10 min ( Inj volume NLT-0.5mL/Kg & NMT-10mL/Kg body wt)
Record the rectal temp of each animal at ½ hr intervals for 3hrs after injection
The difference between the initial temp & the highest temp recorded for the rabbit is
taken as its responce
27. Bacterial Endotoxin test (LAL test)
Limulus - genus of horseshoe crab
Amoebocyte - blood cells
Lysate - blood extract
Developed by F. Bang and J. Levin in 1960's
The method is based on the F. Bang's studies who observed in 1956 that gram-
negative bacteria, even if killed, cause the blood of the American horseshoe crab
(Limulus polyphemus) to turn into a semi-solid mass
American or japanese horseshor crab (Limulus polyphemus) are used
27
28. Bacterial Endotoxin test (LAL test)
28
Principle of the test
Addition of solution containing endotoxin to the solution of lysate produce
turbidity
Rate of reaction depends on the conc of endotoxins, PH, temp
Reference standard is the freeze dried
The test is based on the primitive blood clotting mechanism of horse shoe
crab
29. Bacterial Endotoxin test (LAL test)
29
The endotoxin limits for pharmaceutical product is calculated by
formula K/M:
Where, where K is the threshold pyrogenic dose of 5 EU/kg, 0.2 EU/kg
for intrathecal injection, and M is the maximum dose in units/kg
The accepted limits of endotoxins in pharmaceutical products
30. Bacterial Endotoxin test (LAL test)
30
Three techniques
There are three basic methods commercially available and currently approved by FDA
for end-product release testing:
1.The gel-clot, based on gel formation:
The formation of a gel-clot indicates the presence of endotoxin in a sample .
The maximum sensitivity is 0.015 EU/mL
2. The turbidimetric test:
Based on the development of turbidity after cleavage of an endogenous substrate.
Tubidimetric systems depend upon an increasing conc of insoluble coagulin released as
the test progresses. The maximum sensitivity is 0.001 EU/mL
3. The chromogenic assay:
Based on the development of color after cleavage of a synthetic chromogen
complex. The maximum sensitivity is 0.005 EU/mL
31. Bacterial Endotoxin test (LAL test)
31
Advantages
1. Invitro test
2. More sensitive
3. Quantitative result
4. Less time consuming
5. Less expensive
Limitations
1. Specific for gram negative pyrogens only
2. Clotting enzymes are heat labile. PH sensitive
Applications
1. Pharmaceuticals - Parenteral dosage forms,LVPs, SVPs
2. Biologicals - Blood products, plasma fractions, vaccines
3. Medical device- Nebulisers
4. Validation of dry heat sterilization
5. Diagnosis of disease caused by gram negative bacteria