EVALUATION OF PARENTERAL PRODUCTS

Meera Sharma13HPH026

Evaluation Tests

A) Sterility testB) Clarity testC) Leakage testD) Pyrogen testE) Assay

Sterility Test

It is done by detecting the presence of viable forms of bacteria, fungi and yeast in parenteral products.

The test for Sterility must be carried out under strict aseptic conditions in order to avoid accidental contamination of the product during test.

All glassware required for the test must be sterile. The test for Sterility may be carried out either by:

a) Membrane filtration method

b) Direct inoculation method

PRINCIPLE:If bacteria or fungi are placed in a medium which provide nutrition & water, & kept at a favourable temperature, organism will grow & their presence can be indicated by their turbidity in the clear solution. STEPS INVOLVED IN STERILITY TESTING :1. Selection of the sample2. Selection of the quantity of product to be used3. Method of testing4. Observation & results

Selection of the sample

Sample must be representative of the whole of bulk material & lot of final containers.

Random sampling is taken from the final containers. As per IP 2007 the guideline for minimum number of items recommended to

be tested are:

No. of items in the batch Minimum no. of items recommended to be tested

1. Injectable preparations

Not more than 100 containers 10% or 4 containers whichever is greater

More than 100 but not more than 500 containers

10 containers

More than 500 containers 2% or 20 containers whichever is less

2. Ophthalmic or other non-injectable preparations

Not more than 200 containers 5% or 2 containers whichever is greater

More than 200 containers 10 containers

3. Surgical dressings

Not more than 100 packages 10% or 4 packages whichever is greater

More than 100 but not more than 500 packages

10 packages

More than 500 packages 2% or 20 packages whichever is less

4. Bulk solids

Less than 4 containers Each container

More than 4 containers but not more than 50 containers

20% or 4 containers whichever is greater

More than 50 containers 2% or 10 containers whichever is greater

Selection of the quantity of products to be used Depends mainly on the volume or weight of the container. Minimum samples to be used in each culture medium in the test for

sterility are:

Quantity of each container Minimum quantity to be used in each culture medium

For liquidLess than 1ml Whole contents of the container1ml or more but less than 40ml Half the contents of a container100ml or more 10% of the contents of a container but less

than 20mlFor solidsLess than 50mg The whole contents of a container50mg or more but less than 300mg Half the contents of a container300mg or more 100mg

Methods of testing

Membrane filtration method Direct inoculation method

Membrane filtration method (METHOD 1):

Membrane filtration Appropriate for

Filterable aqueous preparations

Alcoholic preparations

Oily preparations

Preparations miscible with or soluble in aqueous or oily (solvents with no

antimicrobial effect)

Media used for membrane filtration method: FLUID THIOGLYCOLLATE MEDIUM : Specific role of some ingredients primarily intended for the culture of aerobic &

anaerobic bacteria. Incubation of the media: 14 days at 30 -35°C

SOYA-BEAN CASEIN DIGEST MEDIUM Primarily intended for the culture of fungi. Incubation of the media: 14 days at 20 -25°C  

Membrane filter 0.45μ porosity

Filter the test solution

After filtration remove the filter

Cut the filter in to two halves

First halves (For Bacteria) Second halves (For Fungi)

Transfer in 100 ml culture media(Fluid Thioglycollate medium)

Incubate at 30-350 C for not less then 7 days

Transfer in 100 ml culture media(Soyabeans-Casein Digest medium)

Incubate at 20-250 C for not less then 7 days

Observe the growth in the media Observe the growth in the media

Steps:

Suitable for samples with small volumes

Volume of the product is not more than 10% of the volume of the medium

Suitable method for aqueous solutions, oily liquids, ointments an creams

Direct inoculation of the culture medium suitable quantity of the preparation to be examined is transferred directly into the appropriate culture medium & incubate for not less than 14 days.

H.P.I

Direct inoculation method (METHOD 2):

Observation And Results

Culture media is examined during and after at the end of incubation. The following observations are

possible:

• No evidence of growth → passed the test for sterility

• There is evidence of growth → Re-testing is performed for same no. of sample, volume & media

as in original test → No evidence of growth → Passed the test for sterility.

• There is evidence of growth isolate → & identify the organism.

• Re-testing is performed with twice no. of sample

No evidence of growth → Pass the test for sterility.

There is evidence of growth → Failed the test for sterility

Clarity Test It is performed to ensure that the parenterals are free from visible foreign particles. Each parenteral preparation in

its final container is subjected individually to a visual inspection to exclude the possibility of foreign particles. The unlabelled containers are held by the neck against strongly illuminated black ( for dark particles)& white

screen(for light colour particles). The contents of the container are slowly inverted & rotated ,then examined. . It may be dangerous when the

particle size is larger than R.B.C. & may block the blood vessel. This type of products are immediately rejected from the batch.

The limit test for particulate matter is prescribed in I.P. 1996 (A- 125) Applicable for: 100 ml or more volume containers of single dose IV given by IV infusion.

Not applicable for: Multi-dose injections Single dose SVP Injectable solutions constituted from sterile solids .

Sources of particulate matter: Intrinsic contamination: Originally present in products e.g. Barium ions may react or leach with Sulphur ion which are already

present in formulation may produce barium sulphate crystals.

Extrinsic contamination Material comes from outside or environment e.g. coming off the material from body & cloths of

person Entry of particle from ceiling , walls & furniture May be in the form of cotton, glass rubber, plastics, tissues, insect fragments, bacterial contamination, dust, papers etc…  

Methods of monitoring particulate matter contamination :

Methods of monitoring particulate matter contamination Visual method, Coulter counter method, Filtration method, Light blockage method

 

1. Visual method: Simple method Filled container are examined against strong illuminated screen by holding neck & rotating it slowly or inverted it to keep out the foreign matter.

2. Coulter counter method: It is used for detection of particles less than 0.1 micrometer in diameter. Based on electrode resistance. Sample is evaluated between two electrode & if particle found the resistance of electrode is increased.

3. Filtration method: It is used for counting the particles in hydraulic fluids. Sample is passed through a filter & the material collected on the surface of the filter is evaluated under microscope.

Disadvantage: Skilled & trained person is required

4. Light blockage method: Used for hydraulic oils Allows stream of fluid under test to pass between a bright white light source & photoiodide sensor. 

Seal packaging / Leaking test The sealed ampoules are subjected to small cracks which occur due to rapid temperature changes or due to

mechanical shocks.

Vials & bottles are not suitable for this test because the sealing material used is not rigid.

Filled & sealed ampoules

Dipped in 1% Methylene blue solutionUnder negative pressure in vacuum chamber

Vacuum released colored solution enter into the ampoule

Defective sealing

Pyrogen Testing Pyrogen = “Pyro” (Greek = Fire) + “gen” (Greek = beginning). Fever producing, metabolic by-products of microbial growth and death. Bacterial pyrogens are called “Endotoxins”. Gram negative bacteria produce more potent

endotoxins than gram + bacteria and fungi. Endotoxins are heat stable lipopolysaccharides (LPS) present in bacterial cell walls, not present in

cell-free bacterial filtrates Stable to at least 175oC; steam sterilization ineffective Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm) so endotoxins can easily pass

through 0.22μm filters Sources: Water (main), raw materials, equipment, process environment, people, and protein

expression systems if using gram negative bacteria.

Principle: Rabbits are used to perform this test because their body temp increases when pyrogen are introduced into their

bodies by parenteral route 3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are selected Do not use any rabbit

having a temp higher than 39.8 o C Showing temp variation >0.2 o C between two successive reading in the determination of initial temp

Same test is performed within 7 days of actual test Animal showing temp increase over 0.6 o C should be removed from pyrogen testing

Method : Dissolve the substance being examined in, or dilute it with a pyrogen free saline solution Warm the liquid being examined to approx. 38.5o C temp before injection The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body weight Withhold water during test Clinical thermometer is inserted into the rectum of rabbit to record body temp 2 normal reading of rectal temp are should be taken prior to the test injection at an interval of half an hr

& its mean is calculated- initial temp The solution under test is injected through an ear vein Record the temp of each rabbit in an interval of 30 min for 3 hrs The difference between initial temp & maximum temp is recorded- taken as response

No. Of rabbits Individual temperature rise(℃)

Temperature rise in group(℃)

Test

3 rabbits 0.6 1.4 passedIf above test not passed3+5 rabbits taken

0.6 3.7 passed

If above test do not pass then test is performed againIf the above test do not pass then the sample is said to be pyrogenic

Interpretation of result

Bacterial endotoxin (LAL) test ) To detect or quantify endotoxins of gram-ve bacterial origin Reagent: Amoebocyte lysate from horseshoe crab (Limulus polyphemus or Tachypleus

tridentatus). The name of the test is also Limulus amebocyte lysate (LAL) test • Mechanism of LAL Test:

The test is based on the primitive blood-clotting mechanism of the horseshoe crab enzymes located with the crab's amebocyte blood cells endotoxin Initiation of an enzymatic coagulation cascade

Proteneous gel

Test: Equal Volume of LAL reagent and test solution (usually 0.1 ml of each) are mixed in a depyrogenated test-

tube

Incubation at 37°C, 1 hour

Remove the tube - invert at (180°) observe the result

Pass-fail test

LAL test Three different techniques:

1. The gel-clot technique - gel formation

2. The turbidometric technique - the development of Turbidity after cleavage of an endogenous substrate

3. The chromogenic technique - the development of color after cleavage of a synthetic peptide- chromogen complex

Advantages of LAL test Fast - 60 minutes vs. 180 minutes Greater Sensitivity ,Less VariabilityMuch Less False, Positives ,Much Less Expensive Alternative to Animal Model, cheaper, Particularly useful for:

Radiopharmaceuticals and cytotoxic agents Blood productsWater for injection

Commercially derived LAL reagents : Bleeding adult crabs into an anticlotting solution washing and centrifuging to collect the amoebocyte lysate in 3% NaCl lysate is washed and lyophilized for storage activity varies on a seasonal basis and standardization is necessary.  

Assay Assay is performed according to method given In the monograph of that parental preparation

in the pharmacopoeia Assay is done to check the quantity of medicament present in the parenteral preparation

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