The document outlines quality control tests for sterile products, emphasizing the importance of sterility in pharmaceutical dosage forms like parenteral preparations and ophthalmic solutions. It details various classifications, processing methods, and quality control tests including leaker tests, clarity tests, pyrogen tests, and sterility tests to ensure the safety and efficacy of sterile products. Additionally, it describes the materials, equipment, and procedures necessary to maintain aseptic conditions during the production of these products.

1. QUALITY CONTROL TESTS
FOR STERILE PRODUCTS

2. STERILE PRODUCTS
• Pharmaceutical dosage forms with the common characteristic
of sterility i.e. free from contaminating microorganisms.
• Sterile products include the following:
– Parenteral preparations
– Ophthalmic preparations
– Irrigation fluids
– Dialysis solutions
• Sterility in these preparations is essential because they are
placed in direct contact with the internal body fluids or
tissues.

3. OPHTHALMIC PREPARATION
• These are sterile liquids, semi-solids and solid preparations
intended for administration upon the eye ball and/or upon
conjunctiva in conjunctival sac.
• Eye drops
• Eye ointments
• Ophthalmic inserts
• These are sterile liquids to wash open wounds and body cavities.
For single use only.
• 0.9% w/v NaCl solution
• Sterile water for irrigation
IRRIGATION FLUIDS

4. PARENTERAL PREPARATIONS
• Sterile, pyrogen limited i.e. bacterial endotoxin units limit,
preparations intended for administration by injection, infusion, or
implantation into the human body or animal body.
• Greek words:
• ‘para’ means outside
• ‘enteron’ means intestine
• Any drug/fluid whose delivery does not utilize the alimentary canal
for entering into the body tissues.
• Supplied in glass ampoules, bottles or vials, plastic bottles or bags
and prefilled syringes with closures that are made up of plastic or
elastomers.

5. PARENTERAL PREPARATIONS
• Containers should be made from material that is sufficiently
transparent to permit the visual inspection of the contents.
• The type of material for each parenteral preparation is usually
stated in the individual monograph.
• Closures should be equipped with a firm seal to prevent entry
of microorganisms and other contaminants and sufficiently
elastic to allow the passage of needle and reseal to prevent air
borne contamination.
• Components of closure should be chemically compatible with
the packed fluid.

6. PARENTERAL ROUTES OF ADMINISTRATION
• Intra venous
• Intra muscular
• Sub cutaneous
• Intra dermal
• Intra thecal
• Intra peritoneal
• Intra cardiac
• Intra articular
• Intra synovial
• Intra arterial

7. UNIQUE CHARACTERISTICS OF PARENTERALS
• Sterility
• Stability
• Pyrogen free
• Particulate free
• Isotonicity
• CLARITY

8. OFFICIAL TYPES OF INJECTABLES
• According to the USP, injectable are classified into five general types:
– Injection: Liquid preparations that are drug substances or solutions (e.g.,
Insulin injection, USP)
– For injection: Dry solids that, upon addition of suitable vehicles, yield
injectable solutions (e.g., Cefuroxime for injection, USP)
– Injectable emulsion: Liquid preparation of drug substance dispersed in a
suitable emulsion medium (e.g., Propofol, USP)
– Injectable suspension: Liquid preparation of drug substance suspended in a
suitable liquid medium (e.g., Methyl prednisolone acetate suspension, USP)
– For Injectable suspension: Dry solids that, upon addition of suitable vehicles,
yield injectable suspensions (e.g., Imipenem and Cilastatin for injectable
suspension, USP)

9. CLASSIFICATION ON BASIS OF VOLUME
› Small volume parenteral (SVP):
– An injectable that is packaged in containers labelled as containing 100 ml
or less.
– e.g., Verapamil HCl, Furosemide, Propanolol HCl, Diclofenac sodium.
– ampoules, vials, prefilled syringes.
› Large volume parenteral (LVP):
– Injectable solutions usually supplied in volumes of more than 100 ml with
sizes of 250 ml, 500 ml, 1000 ml usually.
– e.g., Calcium solutions, TPN, NaCl solution, Ringer lactate solution,
Dextrose solution, Amino acid, peptide and other protein fraction
solutions.
– glass or plastic bottles, collapsible plastic bags.

10. FORMULATION OF PARENTERALS
› API
› Vehicle
– Aqueous vehicle
› Water for injection
› Sterile water for injection
› Bacteriostatic water for injection
› Sodium chloride injection
› Bacteriostatic sodium chloride injection
› Ringer’s injection
› Lactated Ringer’s injection
– Non aqueous vehicle
› Corn oil
› Peanut oil
› Cottonseed oil
› Sesame oil
› Additives: Anti oxidants, Chelating agents, Preservatives, Buffering agents

11. PROCESSING OF PARENTERAL PREPARATIONS
• Aseptic environment
– Containers are washed with distilled water or WFI and sterilized, rubber closures are
washed with 0.5% sodium pyrophosphate in water.
– Manufacturing area is maintained bacteria-free by use of UV lights,
– High efficiency particulate air (HEPA) filters,
– sterile manufacturing equipment and sterilized work clothing.
– Controlled conditions of temperature and humidity.
• Collection of raw materials
– All the raw material is collected from the ware house after accurate weighing.
• Preparation of Parenteral product
– The ingredients are dissolved according to GMP in WFI,
– another solvent or
– combination of solvents.

13. • Filtration
– The solutions are filtered through a membrane filter until sparkling clear.
• Filling and sealing
– The solution is transferred rapidly into final containers i.e. single-dose or multiple-
dose containers with the least possible exposure.
– Sealing of containers is done immediately after filling.
• Sterilization
– Thermostable product is sterilized preferably by autoclaving.
› 10 lb. pressure (115.5°C) for 30 mins.
› 15 lb. pressure (121.5°C) for 20 mins.
› 20 lb. pressure (126.5°C) for 15 mins.
– Thermolabile substances product is sterilized by exposure to ethylene oxide or
propylene oxide at increased humidity (60%) and temperature (50°-60°C) for 4-16
hours.
– In case of parenteral suspensions and emulsions, the individual components are
sterilized separately because sterilization of suspension or emulsion may alter its
pharmaceutical or therapeutic characteristics.

14. • Evaluation of Parenteral product
– Leaker test
– Clarity test
– Pyrogen test
– Sterility test
– Uniformity of content and weight
• Labeling and Packaging

15. QUALITY CONTROL TESTS FOR EVALUATION OF
FINISHED STERILE PRODUCTS
Leaker test (Dye Bath test)
– Used to detect incompletely sealed ampoules.
– Ampoules are immersed in a dye bath (0.5-1% methylene blue).
– Vacuum and pressure is applied to create a negative pressure inside
ampoules, using vacuum chamber.
– Dye penetrate an opening into the ampoule through capillary action.
– The ampoule is washed from dye then visually inspected for presence of dye.
– Only a tiny droplet can penetrate through a very small opening at ≥685.8
mmHg (27 inches of Hg) of vacuum within 15-30 minutes.
– It is a slow, qualitative and destructive test.
– Vials and bottles are not subjected to this test. They are tested for sealing
efficacy.

16. Leaker test for vials and bottles
– Air is injected through a syringe into a few vials or bottles which are
then submerged in water.
– Incompletely sealed vials and bottles will eject air into the water in
the form of bubbles.
– Clarity test
– Used to detect any particulate matter in the sample.
– Particulate matter in USP is defined as any unwanted, insoluble,
mobile matter other than gas or air bubbles. Such contaminants
include dust, cloth fibers, glass fragments, materials leached from
glass or plastic container or seal.
– 2 methods
› Subjective method or visual inspection
› Limit test for particulate matter

17. Subjective method (Visual inspection)
– In this test transparent or white particles are observed against black
background and black or dark particles are observed against white
background.
– A white and black matt panel is held vertically with an adjustable
lamp holder with white light source and a light diffuser.
– Intensity of illumination is maintained at 2000 lux – 3750 lux for clear
containers and more for colored containers.
– Particles 50 microns or greater can be detected with visual inspection.

18. Limit test for particulate matter
– Used to detect and count particles ≥2 microns and particles ≥5
microns.
– For large volume parenterals, single unit is used.
– For small volume parenterals less than 25 ml in volume, the contents
of 10 or more units is combined in a clean container to obtain a
volume of not less than 25 ml.
– HIAC counter
› A high intensity light source (laser, halogen lamp) is used to illuminate the
particle as it passes through the detection chamber. Light scattering or
obscuration by the particles is detected to count the particles.
– Coulter counter
› Consists of two chambers of electrolyte solution separated by one or more
microchannel with an electrode in either chambers. Sample is added to the
electrolyte solution and drawn through the microchannel. As the particle pass
through the orifice it is detected by increase in electrical resistance.

19. – Microscopic particle counter
› The microscope is equipped with an ocular micrometer which is calibrated with
a stage micrometer, a mechanical stage capable of holding the sample, two
suitable illuminators to provide illumination, and is adjusted to 100 ± 10 X
magnification.
› Used when sample cannot be analyzed by light obscuration because they are
hazy, have high viscosity or contain colloids and surfactants.
• BP Limits

20. › USP Limits

21. • Pyrogen test
– Used to check for the requirement of absence of pyrogens in a sterile
product.
– Pyrogens are fever-inducing substances that can be harmful or even
fatal if administered to humans above certain concentrations.
– Endotoxins are type of pyrogens which are heat stable
lipopolysaccharides (LPS) present in bacterial cell walls, released only
when bacteria die.
– TWO methods
– Rabbit Test
– LAL Test

22. › USP Limits

23. Rabbit Test (In Vivo Test)
– 3 healthy adult rabbits are used to perform this test whose temperatures do not
differ by more than 1% from each other and do not exceed 39.8°C.
– Normal or control temperatures are taken for individual rabbit not more than 40
minutes before the test.
– Syringes, needles and glassware are heated at 250°C for not less than 30 minutes to
make them pyrogen free.
– Warm the sample to be tested to 37°C ± 2°C.
– Volume of injection should not be less than 0.5 ml/kg and not more than 10 ml/kg of
bodyweight.
– Inject into ear veins and record the temperature at 30 minutes intervals 1 to 3 hours
subsequent to the injection.
– Calculate mean change in temperature. If no rabbit shows an individual rise in
temperature of 0.5°C, then the sample pass the requirement for absence of
pyrogens.
– If any rabbit shows a temperature rise of 0.5°C or more, continue the test using 5
more rabbits. Not more than 3 out of 8 rabbits should show an individual
temperature rise of 0.5°C or more or the sum of 8 individual temperatures must not
exceed 3.3°C.

24. LAL (Limulus Amebocyte Lysate) Test (In Vitro Test)
– It is a more sensitive test to bacterial endotoxins.
– Limulus amebocyte lysate is an enzyme found in the extract of blood
cells from horse-shoe crab (Limulus polyphemus).
– This enzyme coagulates in presence of low levels of endotoxins.
– Some products cannot be tested with LAL because API interfere with
the outcome. E.g. vancomycin HCl, meperidine HCl, promethazine
HCl.
– A strategy to overcome this is by diluting the product by more than
two folds.
– Equal volume of LAL reagent and sample (0.1 ml of each) is mixed in a
pyrogen free test tube
– The test tube is then incubated at 37°C for 1 hour.
– After incubation the test tube is inverted at 180 degrees for result.
– If clot is formed the sample fails the test otherwise it pass.

25. Sterility Test
– It is a procedure carried out to detect and confirm the absence of any
viable form of microbes in pharmacopeial preparation or product.
– Sampling
› The sample must be representative of the whole of the bulk material and a lot
of final containers.

26. Quantity of product to be used
› Quantity of the product to be used for sterility testing depends mainly on the
volume or weight in the container.

27. • Two methods
– Membrane filtration method
– Direct inoculation method
• Membrane filtration method
Appropriate for :
› Filterable aqueous preparations
› Alcoholic preparations
› Oily preparations
› Preparations miscible with or soluble in aqueous or oily solvents (solvents with
no antimicrobial effect)
– All steps of this procedure are performed aseptically in a Laminar
Flow Hood.

29. • Direct inoculation method
– In this method suitable quantity of the preparation to be examined is
transferred directly into an appropriate culture medium so that the
volume of the product is not more than 10% of the volume of the
medium.
– Incubate for not less than 14 days.
– It is a suitable method for samples with small volumes.
– It is suitable for oily liquids , ointments and creams.

30. • Results for Sterility Tests
– If there is no evidence of growth , pass the test for sterility.
– If there is evidence of growth, test is re-performed using the same
number or volume of sample and medium as in the original test.
– Now if there is no evidence of growth , pass the sterility test for the
sample. But if again there is evidence of microbial growth , retesting is
done with twice amount of the sample and medium. If there is no
evidence of growth , pass the sterility test and if there is evidence of
microbial growth , the batch is then rejected.

31. Fluid Thioglycollate Medium
› L-Cysteine 0.5g
› Agar granulated moisture ≤ 15% 0.75g
› NaCl 2.5g
› Dextrose 5.5g
› Yeas Extract (water slouble) 5.0g
› Pancreatic Digest of Casein 15.0g
› Sodium Thioglycollate 0.5g
› Thioglycollic Acid 0.3 ml
› Resazurin Sodium Solution (1:1000) 1.0ml
› Water q.s 1000.0 ml
› pH 7.1 ± 0.2
› Temperature Fluid thioglycollate medium is to be incubated at 30-35 °C.
› Storage Unsealed medium 10 days
› Storage sealed medium 1 year
› (provided that medium is capable of cultivation of relevant microbe)
›

32. Soybean Caseine Digest Medium
› Pancreatic Digest Caseine 17.0g
› Papaic Digest Soybean Meal 3.0g
› Sodium Chloride 5.0g
› Diabasic Potassium Phosphate 2.5g
› Dextrose 2.5g
› Water q.s 1000 ml
› pH 7.1 ± 0.2
› Temperature Soya-bean casein digest medium is to be incubated at 20-25 °C.
› Storage Unsealed medium 10 days
› Storage sealed medium 1 year
› (provided that medium is capable of cultivation of relevant microbe)

33. Diluting Fluid A
› Peptic Digest of Animal tissue 1g
› Water q.s 1000
› pH 7.1 ± 0.2
› Sterilization 121°C /20 minute
› Oily

34. Diluting Fluid A
› Sample is oily or lecithin
› polysorbate 80 1ml/1000 ml Fluid A
› pH 7.1 ± 0.2