Validation of sterile product

Guided by-Dr Tejal MehtaHOD of PharmaceuticsInstitute Of PharmacyNirma University Submitted by-

Akansha Sharma14mph101Department of pharmaceutics

Introduction

• Sterile products are the unique dosage forms that are

free from microorganisms, freedom from pyrogens,

freedom from particulates.

• They have high standard of purity and quality.

• The ultimate goal is absence of microbial

contamination.

• Because of these features there are differences in the

design and specifications of premises, equipment, and

processes of sterile products.

Principle involve in validation of sterile product

• To build sterility into a product.

• To demonstrate to a certain maximum level of

probability that the processing

and sterilization methods have established sterility to

all units of a product batch.

• To provide greater assurance and support to all the

results of the end products sterility test

Process Of Microbial Destruction

• Regardless of type of lethality induced by sterilization process whether it be heat, chemical ,radiation micro-organism will die according to a logarithmic relationship between the concentration or population of living cells and the time exposure or radiation dose to the treatment.

D –value principle and logic

• The D value is a single quantitative expression of the rate of killing of micro-organism.

• D value = Exposition time required (in minutes) to

kill 1 log (90%) of the micro organisms.

• A D value of 6 means that it takes 6 minutes to

reduce par a factor of 10 (90%) or 1 log the number

of micro organisms.

Z -value

• Z value = the number of degrees of temperature

required to obtain a variation of 1 log of the D value.

• Z= T2 -T1

• log D1 - log D2

• If the temperature is lowered, bugs are killed moreslowly and the D value increases (because it takesmore time to kill) .

• Conversely if the temperature is higher, faster thebugs die, and lower is the D value

Continue..

• The Z value is the necessary component of the calculation

which allows the comparison of the spore lethality at

different temperatures.

• Generally accepted Z values assumptions are:

• Steam sterilization Z=10 C

• Dry heat sterilization Z=20 C

• The Z value can be determined by one of the two methods

• -By carrying out experiments at different temperatures to

determine the thermal death times.

• -By carrying out experiments to determine D values at

different temperatures

F value

• If the D value is measured at different temperature and

pressure it can be seen that a D value varies with the

pressure. More the D value decreases with a specific

increase of pressure, more powerful is considered the

process.

• F value = a measure of the capacity to inactivate

bacteria in function of the temperature.

• Mathematically the F value is expressed by the rate of

mortality per minute in function of temperature for a

given pressure.

Fo Principle• The Fo value is mentioned both in the USP and in

current good manufacturing practice for large volume parentral.

Concept of Fo

▫ Lethality factor equivalent to time at 121.1°C

1 minute at 121.1°C is equivalent to Fo of 1.

Lethality can accumulate during heat up and cool

down phases.

Typical temperature profile of a heat sterilization

process

• Fo is calculated using the following equation:

• Fo = Δt * 10(T-121.1/Z)

• where:

• “Δt” is the time interval between measurements of temperature (T)

• “T” is the temperature of sterilized product at time (t)

• “Z” is a temperature coefficient which measures the number of degrees required to change the D-value of an organism by 1 log

• When heat labile products will not withstand excessive heat treatment, "D121" value studies of product isolates are necessary to determine the minimum Lethality Factor (F0) that will provide an acceptable assurance of sterilization.

• The minimum "F0" value required by a process can berelated to the "D" value of the bioburden by the followingequation:

F0 = D121 (log A - log B)where:

• "D121" is equal to the time required at 121oC to reduce thepopulation of the most heat resistant organism in the unitby 90%;

Continue..• "A" is the microbial count per container; and

• "B" is the maximum acceptable probability of survival ( 1 x 10-6 for pharmaceutical dosage forms).

• F0 values may be solved using the biological approach.

• F0 = D121(log A − log B)

• For example, if D121 = 1.0 min, A = 106, and B = 10−6, F0is calculated to be

• F0 = 1(log 106 − log 10−6) = 12 min

• Thus, the cycle must be adjusted so that the F0 valuecalculated by physical methods (time and temperaturedata) will be at least 12 min.

Basic Step in validation of sterile product

• Five basic steps are necessary to validate any process

1. Written documentation

2. Manufacturing parameters

3. Testing parameters

4. In-process controls

5. Final product testing

Continue..

• Select or define the desired attributes of the product . example: The product will be sterile.

• Determine the specifications for desired attributes.

• Select the appropriate process and equipments

• Develop and conduct tests that evaluate and monitor the processes.

• Develop and conduct tests that evaluate and monitor the equipment and personnel.

• Examine the test procedures themselves to ensure their accuracy and reliability.

METHODS OF STERILIZATION OF STERILE PRODUCTS• Heat sterilization

▫ Moist heat (steam )

▫ Dry heat

• Gaseous sterilization

▫ Ethylene oxide

• Membrane filter sterilization

• Radiation sterilization

▫ Gamma

▫ Beta

▫ Ultraviolet

▫ microwave

QUALITY CONTROL TESTS FOR STERILE PRODUCTS

• The following tests are performed on the finished sterile product to maintain quality control :

▫ Leakage test

▫ Clarity test

▫ Pyrogen test

▫ Sterility test

Leakage test

• This test is performed by immersing the ampoules in a vacuum chamber consisting of a dye like 1 % methylene blue solution.

• A vacuum of about 27 inches Hg or more is created for about 15 to 30 minutes.

• This negative pressure causes the solution to enter in the ampoule with defective sealing.

• The vacuum is released and ampoules are washed externally.

• The coloured solution in the ampoule confirms the leakage and are discarded.

• High frequency spark test detects pin holes in the ampoules is another means of detecting leakage.

Clarity test

• The parenterals must be free from microorganisms or any particulate matter like glass,fibre,floaters.

• Particulate matter- defined as presence of undesirable,extragenous,mobile and undissolvedsubstances within the sterile products.

• Methods to detect particulate matter:▫ Visual method▫ Microscopic count or membrane filtration method▫ Light obstruction method▫ Coulter counter method

Visual method

• The filled containers are examined by holding the neck of the containers against illuminated screen.

• Black surface is used for light coloured particle and white is used for dark coloured particles.

• If any particle is visible, then that container is discarded.

Microscopic count method

• A measured sample solution is filtered through a membrane filter. The collected particles on the surface of the filter are then counted with the help of a microscope at 100X magnification.

• The whole method is carried out under aseptic conditions

Light obstruction method

• The method uses an electronic instrument that produces a light beam of high intensity.

• The solution is allowed to pass under this bright light.

• If the particle present in the solution passes through, a shadow cast is produced due to the obstruction of light by the particles.

• The particles are measured and counted automatically by the device.

Coulter counter method

• The electronic method is used to detect the particles and also to determine the particle size.

• The sample solution is added to an electrolyte solution.

• This solution is drawn through a small orifice of the device.

• Positive and negative electrodes are present one on either side of the device.

Continue..

• As the particles passes through the orifice, it displaces its

own volume of electrolyte and at same moment an

increase in electrical resistance is observed between two

electrodes.

• The resulting voltage pulses which are proportional to the

particle size are amplified and are measured and counted.

• Particles below 0.2 um can also be detected by this

method.

Pyrogen test

• Test involve measurement of rise in body temperature of rabbits due to IV injection of sterile solution of substance under examination Designed for products that can be tolerated by test rabbits

• Test animal:• Healthy, either sex, same variety, weight NLT 1.5 kg • Diet- complete and balanced diet • Condition- no loss of body weight during week preceding

the test • Temperature- uniform • Do not use the animal for pyrogen test repeatedly once

every 48 hr • Temperature rise by 0.6 degree

Test:

• Use group of rabbits.

• Preparation of sample- Dissolve the substance in sterile pyrogen free saline solution. Warm the liquid under examination to approx. 38.5 degree before injection

• Procedure-

• a) Record temp of all animal at interval NMT 30min, beginning at least 90 min before injection of solution under examination and continue for 3hr after injection.

• b) Record the initial temperature of each rabbit (NMT 40min immediately preceding the injection). It should be variats 0.2degree and not more than that, between two successive animal.

• c) Do not use the animal having temperature more than 39.8 and less than 38 degree.

• d)Inject the solution under examination slowly into MARGINAL VEIN OF EAR of each rabbit over a period not more than 4min. Amount of sample to be use is given in monograph.

• e)volume of injection- NLT 0.5ml/kg and NMT 10ml/kg.

• f)Record the temperature of each animal at half hourly interval for 3hrs after injection.

• g) The difference between initial temp and maximum temp is taken to be response.

• h)when the difference is negative, the result is counted as a zero response.

• Interpretation of result:

• Sum of response of group of 3 rabbits does not

exceed 1.4 degree and if the response of any

individual rabbit is less than 0.6 degree. The

preparation under examination passes the test.

• If the response of any rabbit is 0.6 or more or the

sum of response of three rabbits exceed 1.4, continue

the test using five other rabbits.

• If not, more than three of eight rabbit show individual

response of 0.6 or more and if sum of response of

group of eight rabbit does not exceed 3.7, the

preparation under test passes the test.

LAL (limulus amoebocyte lysate test)

• Test is used to detect endotoxins from gram negative bacteria.• Also called as bacterial endotoxin test.• principle:• The test is based on the formation of a gel in the presence of

bacterial endotoxins and the lysate solution.• The lysate consist of a proclotting enzyme and coagulogen

which are required for the reaction to occur.

• There are 3 main types:• The gel clot test• The tubidimetric test• The kinetic chromogenic test

The gel clot test

• The lysate solution is mixed with an equal volume of the test solution in a pyrogen free test tube.

• The test tube is allowed to stand for about 60 minutes. now the tube is inverted and observed for the clot formation.

• The presence of solid gel confirms the presence of endotoxins.

• It requires positive (known concentration of endotoxin) and negative (water) controls.

The tubidimetric test

• This test is based on the measurement of opacity change in the LAL test due to the formation of gel clot.

• Opacity is directly proportional to the endotoxinconcentration.

The kinetic chromogenic test

• The test is based on the measurement of colour change which is caused by the release of chromogenic chemical, para nitroanilide.

• This p-nitroanilide is a by-product of the clotting reaction during the LAL test.

• The quality of Para – nitroanilide produced is directly proportional to the endotoxin concentration.

• ADVANTAGES OF LAL OVER RABBIT:

• Easy to perform

• Rapid

• Economical

• More sensitive

• Preparation of culture media:

• The culture media used for sterility test must be capable of promoting the growth of a wide range of micro-organisms such aerobic bacteria, anaerobic bacteria, and fungi.

• Two types of media are used:

• Fluid thioglycolate medium and

• Soyabean casein digest medium

• Sampling:

• The selection of samples and the number of samples to be taken from any given batch of sterile product.

• The material must be thoroughly mixed, if the sample is to be taken from the bulk.

• The sample is taken at random from the batch of final container.

• Determination of sample qty to be used in each culture medium:

• IP has recommended the minimum qty of samples to be used in each culture medium.

• Test methods:

Membrane filtration method

Direct inoculation method

• Membrane filtration method:

• Membrane filter of pore size not greater than 0.45 um and diameter of about 47 mm that are effective in retaining micro-organisms are employed for the test.

• The filtrations system and membrane are sterilized.

• The test substance are filtered through the membrane under aseptic conditions.

• If the test substance has antimicrobial properties, the

membrane is washed with 3 successive 100ml

quantity of sterile diluent.

• The membrane is then aseptically cut into two equal

halves.

• One half of membrane is immersed in 100ml of soya

bean – casein digest medium and incubated at 20-25

C.

• The other half of the membrane is immersed in 100

ml of fluid thioglycollate medium and incubated at

30-35 C.

• Both the media are incubated for not less than 7 days.

• Direct inoculation method:

• The quantity of the test substance to be used in each

culture medium is directly inoculated into culture

media under aseptic conditions.

• Mix the inoculated liquid with the medium.

• If the test substance contains antimicrobial properties

then it is neutralized by adding suitable inactivating

substance to the medium.

• Incubate the inoculated media at 20-25 C in case of

soya bean-casein digest medium and at 30-35 C in

case of fluid thioglycolate medium for not less than

14 days.

• Interpretation of results:

• Examine the culture media during and after the incubation period to detect the possible microbial growth.

• The sample passes the test for sterility if microbial growth is not found.

• If there is an evidence of microbial growth, the test containers are kept aside. then repeat the test. if growth is not found then the samples passes the test for sterility.

• If microbial growth is found in the retest, isolate and identify the organisms. If same organisms are found as in the first test, the sample fails the test for sterility.

REFERENCES

• Pharmaceutical process validation,3rd edition, edited

by Robert A Nash, Alfred H Wachter, Marcel Dekker.