In-process and finished product QC tests for parenterals:-

Parenterals are defined as sterile dosage forms which are administered to the patient by a route other than oral.In other words,these dosage forms bypass GIT(gastrointestinal tract) and liver and enter directly into systemic circulation. They exhibit maximum bioavailability(100%).

Parenterals are classified into four types:

Based on route of administration Based on the volume Based on the formulation Based on the type of product BASED ON THE ROUTE OF ADMINISTRATION:

Intra venous Intra muscular Intra dermal Intra thecal Intra arterial Intra peritoneal Intra medullary BASED ON THE VOLUME:

Large volume Small volume BASED ON THE FORMULATION

Solutions Suspension Emulsions Dry powders which form solutions Dry powders which form suspensions Concentrated liquids which are diluted before injection. PURPOSE BASED

Injections Infusions Irrigations EVALUATION OF

PARENTRALS:

In process quality control tests

Environmental control Finished product quality control tests

Leakage test (or) Sealing verification Sterility test Pyrogen test Uniformity of weight Uniformity of content Extractable volume Safety test Clarity test/ Particulate matter test. ENVIRONMENTAL CONTROL:

Environmental control is always needed during the preparation of Parenterals so it is necessary to design and implement in-process control and monitoring.

In-process monitoring and control may includes

Environmental Particulate

Microbiological

Filter Integrity Testing

ENVIRONMENTAL PARTICULATE MATTER:

Environmental Particulate monitoring should be carried out using appropriate air Particle Counting

devices to check that the general environmental and work station air remain in conformity with specification.

This method is used to identify large pores, other openings in the membrane filter

MICROBIOLOGICAL MATTER 

As appropriate to the type of manufacturing process, consideration needs to be given to the following Microbiological Monitoring and Control.

Exposure of "Settle Plates" (Petri dishes of nutrient agar) at critical positions within the general Clean Room environment and at the controlled work station(s).

Use of Air Sampling devices to determine the number of viable organisms per cubic metre (or cubic foot) of air in the room, and within the work station(s).

Use of Contact Plates, or Swabs, to check the microbiological quality of surfaces. 

FINISHED PRODUCT QUALITY CONTROL TESTS :

LEAKAGE TEST

As the containers are exposed to various environmental conditions, uneven expansion occurs and as a result the cracks occurs and it leads to the entry of microbes or particulate matter .

This test is done mainly for the ampoules which have been sealed by fusion method to ensure that no leakage is present in them.

There are two methods for the identification of leaks in parentrals they are

1)methylene blue dye test.

2)spark test.

METHYLENE BLUE DYE TEST :

Method:

Leakage test is done in a vacuum chamber.

The ampoules are dipped in 1% solution of methylene blue in vacuum chamber and vacuum is applied.

When this vacuum is released, the coloured solution will enter the ampoules with defective

sealing and the Vaccum is applied for another 15-30min.

Result : The presence of dye in the ampoule, confirms the leakage and hence rejected.

NOTE: This test is not suitable for the vials and bottles because the methylene blue solution may enter through the caps or may diffuse through the rubber closure.

SPARK TEST:

Electrical probe is introduced into the vial or the bottle and when the blue light appears the vial or bottle is said to have a hole.

STERILITY TEST:

The test is designed to identify the presence of viable forms of bacteria, fungi and yeasts in substances, preparations or articles which are required to be sterile. Principle:

when the suitable growth medium is provided and kept at favourable temperature condition, the organism grows and it is indicated by a turbidity in clear medium.

The culture media used are:

Fluid thioglycolate medium Soyabean- casein digest medium Special media for penicillin's and cephalosporin's Any other medium as directed by the manufacture. Fluid thioglycolate medium: It is primarly intended for the culture of anaerobic bacteria.

Special media for penicillin's and cephalosporin's:

The commonly used soya bean-casein digest medium and fluid thioglycollate is taken and the required amount of penicillinase required to inactivate the penicillin is calculated and added to the sample that is being tested.

The inactivation can be checked out by using Staphylococcus aureus as the challenge.

Typical microbial growth is to be observed as a confirmation that the penicillinase concentration is appropriate. 

Steps involved in sterility testing

Selection of the sample size.

Selection of the quantity of product to be used.

Method of testing.

Observation and results.

Selection of the sample size:

Number of items in the batch

Min.No.of items to be tested

NMT 100 containers

10% or 4 containers which ever is greater

NMT 100 but NMT 500 containers

10 containers

NMT 500 containers

2% or 20 containers which ever is less

2. Selection of the quantity of product to be used.

Quantity of each container of injectable preparation 

Minimum quantity to be used for each culture medium 

Less than 1ml

Total contents of a container

1ml/ more but less than 40ml

Half the contents of a container

40ml/ more but less than 100ml

20ml

100ml or more

10% of the contents of a container but not less than 20ml

Antibiotic liquid

1ml

Other preparations soluble in water(or) in isopropyl myristate

The whole contents of each container to provide not less than 200mg.

Insoluble preparations ie creams and ointments to be suspended / emulsified

The whole contents of each container to provide not less than 200mg.

The two methods for the sterility testing are:

Membrane filteration

Direct inoculation

MEMBRANE FILTERATION METHOD:

The method is preferred in the following cases

An oil or oily preparation Liquid product where the volume in a container is 100ml or more. Method:

Filtration of the sample through a membrane filter having the nominal size of 0.45µ and a diameter of 47mm.

After filteration the membrane is removed aseptically from the metallic holder and divided into two halves.

The first half is transferred into 100 ml of culture media meant for fungi and incubated at 20˚ to 25 ˚c for not less than seven days.

The other half is transferred into 100ml of fluid thioglycolate medium and incubated at 30 to 35 ˚c for not less than 7 days.

Observe the growth in the media.

Fluid –A: it has 1g peptic digest dissolved in 1 liter of water

Fluid – B: it has 1ml of polysorbate 80 added to

the fluid A

The various types of filters used are :

Cellulose nitrate filters for aqueous , oily solutions , weakly alcoholic

Cellulose acetate filters are mainly used for strongly alcoholic

DIRECT INNOCULATION METHOD

The medium is prepared and then it is sterilized.

Then the sample to be tested is poured into the medium plates as per the prescribed volumes.

Then it is incubated and the growth of the microbes is examined for not less than 14 days.

The visual testing is done by comparing the test results with the positive and negative controls.

OBSERVATION AND RESULTS:

At the end of the incubation period the following observations are possible:

No evidence of growth; hence the preparation being examined passes the test for sterility. If

there is evidence of growth, retesting is performed using the same number of samples, volumes to be tested and the media as in the original test. If no evidence of microbial growth is then found, the preparation being examined passes the test for sterility. If There is again evidence of the microbial growth then isolate and identify the orgainism. If they are not readily distinguishable from those growing in the containers of the first test then the preparation being examined fails the test for sterility. If they are distinguishable from the organisms of the first test then again do the test using twice the number of samples. The prepration being examined passes the test for sterility in case there is no evidence of microbial growth. In case there is evidence of growth of any micro organisms in second re –test, the preparation being examined fails the tests for sterility. PYROGEN TEST:

Pyrogens are fever producing metabolic products of micro organisms.

The presence of pyrogens in parenteral preperations is evaluated by a qualitative fever response test in rabbits.

Test for pyrogens can be carried out by in-vitro and in-vivo methods.

A) Rabbit test (in-vivo)

B) LAL test (Limulus amoebocyte lysate) (in-vitro)

A)RABBIT TEST:

Principle: 

The test involves measurement of rise in body temperature of the rabbits following the intravenous injection of a sterile solution of the substance to be tested.

The body temperature of the rabbits increases if pyrogens are present in the injected test solution.

SELECTION OF TEST ANIMALS:

Healthy , adult rabbits of either sex, each weighing not less than 1.5kg.

Do not use any rabbit for main test if:

1) It shows a temperature variation greater than 0.20c between two successive readings noted during the determination of initial temperature .

2) And it’s temperature is higher than 39.80c or lower than 380 c.

EQUIPMENTS REQUIRED FOR THE TEST:

All glass ware, syringes, needles and thermometer must be thoroughly washed with water for injection and heated in a hot air oven at 2500c for 30 minutes or at 2000 c for 1 hr.

Retaining boxes for rabbits.

TEST:

The test is carried out on a group of three rabbits.

Procedure:

1.Preparation of the sample:

Dissolve the test substance with a pyrogen-free saline solution. Warm the solution to about 38.50c before the injection.

2)Determination of initial temperature of rabbits:

Insert a clinical thermometer into the rectum of each rabbit and normal readings of body temperature are taken prior to the injection of test solution.

Two such readings are taken at an interval of 30 minutes and the mean is calculated.

This mean reading is taken as the initial temperature of the rabbits.

3)Determination of the response of rabbits:

The test solution is injected into the ear vein of each rabbit.

The volume of injection is not less than 0.5 ml/kg of the body weight.

This volume varies according to the test substance and is prescribed in the individual monograph.

Record the temperature of each rabbit at an interval of 30 minutes for 3 hours after the injection.

This is the maximum temperature recorded for that rabbit.

The difference between maximum temperature and initial temperature is taken as its response.

If this difference is negative, it is taken as a zero response.

Interpretation of results:

If the response of any individual rabbit is less than 0.60c and if the some of the responses of the 3 rabbits is more than 1.40 c, the preparation being examined passes the test.

If the response of any rabbit is 0.60c or more or if the sum of the responses of the 3 rabbits is more than 1.40 c, then the same test is repeated on another 5 rabbits.

If not more than 3 of the 8 rabbits show individual responses of 0.60c or more and if the some of the responses of the 8 rabbits is not more than 3.70c,the preparation being examined passes the test.

ADVANTAGE OF RABBIT TEST:

It is used to identify the presence of a wide range of pyrogens.

DIS ADVANTAGE OF RABBIT TEST:

It is a time - consuming method.

B) LAL (LIMULUS AMOEBOCYTE LYSATE) TEST (IN-VITRO):

This is a sensitive test used to detect endotoxins from gram-negative bacteria .therefore it is also

called as bacterial endotoxin test.

The endotoxins upon reaction with lysate form an insoluble gel clot.

The lysate is obtained by the lysis of amoebocyte (blood cells) of the horseshoe crab,LIMULUS POLYPHEMUS.

The sensitivity of lysate is expressed in terms of endotoxin units(EU).

PRINCIPLE:

The test is based on the formation of a gel in the presence of bacterial endotoxins and the lysate solution .

The lysate consists of a proclotting enzyme and coagulogen which are required for the reaction to occur.

Types: 

1) The gel clot test

2) The turbidimetric test 

3) The kinetic chromogenic test 

1) The Gel clot test:

It is based on the formation of a solid gel clot. 

Procedure:

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 formation of gel clot.

The formation of solid gel confirms the presence of endotoxin.

If the solid gel is not formed,it indicates the absence of endotoxins and the test solution passes the test.

2) The turbidimetric 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 endotoxin concentration.

This test is used for water systems and simple pharmaceutical products.

3)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 quantity of para-nitroanilide produced is directly proportional to the endotoxin concentration.

Advantages of LAL test over rabbit test:

It is easy to perform

It is a rapid method

It is an economical method

It is more sensitive test than rabbit test. Even the minute amounts of endotoxin can be detected by LAL test.

Disadvantage of LAL test:

It is used to identify the presence of endotoxins only.

UNIFORMITY OF CONTENTS:

Unless otherwise stated in the individual monograph, suspensions for injection that are presented in single dose containers and that contain less than 10mg or less than 10 per cent of active ingredient comply with the following test.

For suspensions for injection containing more than one active ingredient carry out the test for each active ingredient that corresponds to above conditions.

The test for uniformity of contents should be carried out only after the content of active ingredient in a pooled sample of the preparation has been shown to be within accepted limits of the stated content.

Determine the content of active ingredient of each of 10 containers taken at random, using the suitable analytical method of equivalent accuracy and precision.

The preparation under examination complies with the test if the individual values thus obtained are

all between 85 and 115 % of the average value.

The preparation under examination fails to comply with the test if more than one individual value is outside the limits 85 to 115% of the average value or if any one individual value is outside the limits 75 to 125% of the average value.

If one individual value is outside the limits 85 to 115% but with in the limits 75 to 125% of average value, repeat the determination using another 20 containers taken at random.

The preparation under examination complies with the test if in the total sample of 30 containers not more than one individual value is outside the limits 85 to 115% and none is out side the limits 75 to 125% of the average value.

UNIFORMITY OF WEIGHT:

For powders for injection that are required to comply with the test for uniformity of content of all active ingredients ,the test for uniformity of weight is not required.

Remove any adherent labels from a container and immediately weigh the container and its contents.

Empty the container as completely as possible by

gentle tapping, rinse if necessary with water and then with Ethanol (95%) and dry at 1000 c to 1050 c for 1hr.

If the nature of the container precludes such treatment, dry at a lower temperature to constant weight.

Allow to cool in a desiccator and weigh.

The difference between the weights represents the weight of the contents.

Repeat the procedure with a further 19 containers and determine the average weight.

Not more than two of the individual weights deviate from the average weight by more than 10% and none deviates by more than 20%.

Pharmaceutical practical guide

4. IPQC TESTS FOR INJECTABLES:a) Drug contents determination.b) Assay of active ingredients.c) pH.d) Pyrogen test.e) Stability test.f) Leakage test.g) Check up of particulate matters.

Documentation

Master formula records

Name of the product________________________________________

Name and Weight of API ____________________________________

Name and Weight of Ingredient _______________________________

Description of equipment ____________________________________

Statement of theoretical yield_________________________________

Process and packaging procedure_____________________________

A description of container____________________________________

closure and packaging material _______________________________

In process control during processing ___________________________

In process control during packaging____________________________

Precaution to be taken______________________________________

Environmental control

Product_______________________________ lot no.__________________________

Room________________________________ date exposed_____________________

Media__________________________

Date Time Incubation temparature Humidity in case of hygroscopic substances’

Plate no duration Location No of colonies

Batch Manufacturing Records

Name of the company:-_______________________________________

Address:-___________________________________________________

Name of the product _________________________________________

Active pharmaceutical ingredient ______________________________

M F R No. __________________________________________________

Batch No._____________________ Batch size ____________________

Mfg. date _____________________Date of expiry_________________

Requisition slip

S no Ingredients

Item code

Standards

ATR no Label Claim Qty required

Qty issued

Preparation of equipment & containers

Description of containers _______________________________________

Q/C report of container ________________________________________

Date ________________________ Equipment used__________________

Cleaning agent used ___________________________________________

Cycle of washing: _____________________________________________

Sign. Of officer_____________________________________________

If sterilized by dry heat or autoclave

Articles Date Time when oven started

Desired temp attained

Temperature Time when oven switched off

Filtration & filling:-

Equipments used for filtration ___________________________________

Date__________________________ Time_________________________

Result of test or analysis of solution_______________________________

Equipment used for filling_______________________________________

Date________________________________________________________

Sign. Of officer____________________________________________

Time Filling started Filling completed

IPQC records

1. Visual inspection:

Description ________________________________________________________

Total no of filled, sealed & sterilized containers rejected __________________

Nature of defects ____________________________________________________

Name of worker who examined

(i). ________________________________________________

(ii). _______________________________________________

(ii). _______________________________________________

Batch Packaging & Labeling Records

Product name_______________________ Batch no _______________________

Strength___________________________ batch size ______________________

Category___________________________ mfg date _______________________

MFG no____________________________ exp date _______________________

Batch Packaging & Labeling Records

Description of packaging______________________________________________

Pre-coding of labels & printed packaging materials,

examined & verified by _______________________________________________

No. of pre-coded ____________________________________________________

(ii). Printed packaging material received __________________________________

Result of bulk finished products ________________________________________

Sign. Of officer _____________________________________________________

Reconciliation of labeling and packaging material

Quantity of material received___________________________________________

Quantity of material destroyed__________________________________________

Quantity of material used _____________________________________________

Quantity of material returned___________________________________________

Date of release____________________ quantity release____________________

Signature of supervisor ______________

Pharmaceutical practical guide

(http://thepharmacistpharma.blogspot.in/2011/03/quality-control-of-sterile-products-5.html)

PYROGEN TESTINGPyrogens produce symptoms of fever, chill, joint pain, malaise, headache and other complaints following IV injection within 30-120 minutes which may subside within 10-12 hours. Pyrogens are the heat stable, filterable and soluble substances of 0.05-1.0 micrometer size and arise from microbial contamination. Chemically these are lipopolysaccharides from the outer cell wall of the bacteria, thus, the term endotoxins is also used interchangeably but not correct entirely. Both G+ and G- bacteria produce pyrogens, however, the pyrogens of G- bacteria are more potent. The pyrogens are heat stable up to some extent, thus, withstand normal sterilization temperatures.

DepyrogenationDepyrogenation is the removal of pyrogen. This is achieved by the following methods.Inactivation - Application of very high dry heat (2500P) for not less than 30 minutes is the desired method for rendering material pyrogen free.Removal of pyrogen by distillationDetection and quantification of Pyrogens1) In-vivo pyrogen (rabbit) testIn-vivo pyrogen test involves the evaluation of the presence of pyrogens in parenteral sample by quantitative fever response produced in rabbits. The principle is based on the fact that the human and rabbits are equally responsive to pyrogen injected intravenously on a dose per weight basis. This test requires the following.Test animals: healthy adult rabbits (of either sex) weighing not less than 1500 gm(1.5kg). The animals have been properly maintained in terms of environment and diet prior to the performance of test. The animalsare screened for their temperature.Their control temperature must not differ more than 1°C from each other. Any individual animal having temperature 39.8°C or less than 38.0°C is excluded from the test.The rabbit-retaining boxes are required to house the rabbits. These boxes "hold" the rabbits so that the temperature can be noted easily during test. The specific directions given in the individual monograph must be followed for the products.The sample to be tested is injected with a slower rate to the animals. The dose of the sample if not specified should be smaller than 10 ml/kg. Special preliminary steps are required and thus, consideration must he given for the products requiring; 1) dilution, 2) pH adjustment, and 3) isotonicity adjustme

PROCEDURE

The control (baseline) temperature of three rabbits is determined. The sample is injected into the ear vein of each of three rabbits which are held in the retaining boxes. A dose of 10ml/kg of body weight is used unless specified in the individual monograph. The temperature of each rabbit is determined at 1, 2, and 3 hours subsequent to the injection of sample. The difference between the initial and final temperatures of each rabbits is noted. Any increase in temperature is taken to be the response of sample injected.

Interpretation of the resultsThe material under examination meets the requirements for apyrogenicity if no rabbit shows an individual rise in temperature of 0.6°C or more above its respective control temperature OR the sum of the temperature rise of 3 rabbits does not exceed 1.4°C. If the results are not within the limits, the test is repeated for additional 5 rabbits and the result is considered for the eight rabbits. After repeating, the material under examination meets the requirements if not more than 3 out of eight rabbits show individual rise in temperature of 0.6 °C OR the sum of rise in the temperature in eight rabbits does not exceed 3.7°C.Sometimes the difference of initial and the final temperature is negative. If the difference is negative, the result of the rabbit test is counted as zero response and the sample is considered apyrogenic.

Advantages of Rabbit TestThe human and rabbits are equally responsive to threshold levels of the pyrogens.2) Limulus Amebocyte Lysate Test

The limulus amebocyte lysate test is also called as in-vitro pyrogen test (USP XXI Specified new test). Officially it is termed as bacterial endotoxin test (BET). The test principle is based on the clotting of lysate of amebocyte (an enzyme obtained from the horse shoe crab) in the presence of pyrogens. The extract from the blood cells of horse shoe crab, Limulus Polyphemus contains an enzyme and protein system called "Limulus- Amebocyte Lysate" (LAL) which reacts with pyrogens so that an assay mixture increases in viscosity and opacity until an opaque gel is formed.Amebocyte + Pyrogen ~ Opaque gelThe reaction accomplishes within 15-60 minutes, depending on concentration of pyrogens after mixing. The concentrated pyrogens make the gel more turbid and thick.

RequirementsLimulus-Ambocyte Lysate is prepared by bleeding healthy mature specimens by heart puncture. The amebocytes are carefully concentrated, washed and lysed by osmotic effects. Prior to perform the LAL test, lysate assay is carried out with purified endotoxins and are accepted if it detects 0.001ug/ml or less concentration of the purified endotoxins.The glassware, such as glass test tubes (10 x 75mm) used in the test must be thoroughly cleaned, dry and heat sterilized. A buffer solution of potassium phosphate 2mEq/ml is used to adjust the pH of test sample at 7. The alcoholic content in sample is to be removed as it causes precipitation of lysate. If the sample contains proteins, it produces gel thus the proteins must be diluted to appropriate concentration before the test.

Similarly other interfering substances present in sample must also be removed before the test.

Procedure

The pH of test sample if specified is adjusted. The test solution and standardized LAL are separately mixed in equal parts (0.05-0.2ml). The mixture is incubated immediately at 36-

38°C for 1 hour in assay tube. The assay tube must be remained undisturbed completely because agitation may irreversibly destroy the gel leading to a false negative result. The test tube is observed after the specified time and is examined for the formation of opaque gel. Formation of gel represents a positive test endpoint reaction. The test is performed using a commercial LAL test kit. This kit contains a lyophilized LAL, and E. coli endotoxin and pure water asstandards and these later two are used to check the sensitivity of the test.

Advantage of LAL test1. It is in-vitro and does not require animal handling, thus is more convenient2. It is 10 times more sensitive than that of the in-vivo rabbit test3. It is economical4. It consume less time, i.e., 1 vs 3 hours required by rabbits test5. It requires less laboratory facilities and minimum equipments6. It requires less test volume7. It is more accurate

QUALITY CONTROL OF STERILE PRODUCTS (3)

Instruments for evaluation of particulate matterSystem Working Principle RemarksVisual Based InspectionAutoskan Light Scattering Non-DestructiveEisai Ampoule Inspection(AIM) SystemLight blockage (Shadow) Non-DestructiveSchering PDS/A-V System Light Scattering Non-DestructiveElectronic Particle CountersCoulter Counter Change in Electric Resistance Destructive; Large errors in measuring flakes and fibers;Not recommended by FDA for parenteralsHIAC (High AccuracyInstruments)Light Blockage Destructive; High Efficiency,Easy calibration;Recommended by USP;ExpensiveMet-One Climet ParticleCounterLight Scattering Destructive; Measures 6 particles sizes at a time;Excellent large particle detectionClimet Instrument Light Obstruction Non-destructive

CLARITY TESTING (DETECTION OF PARTICULATE MATTER)Particulate matter can be detected in parenteral product by two methods, including visual inspection and electronic particulate counting.

A) Visual methods1) Visual inspection by naked eyeIn visual inspection, each injectable is inspected visually against white and black backgrounds. The white background aids in diction of dark colored particles. The light or reflective particles will appear against the black back ground. Some visual-enhancing aids can increase the efficiency. A magnifying lens at 2.5 × magnification set at the eye level facilitates the inspection. Microscopic examination enhances detection of particulate matter in injectables. Visual inspection gives the qualitative estimation of the particulate matter. Acceptance Standards is that each container checked must not contain any visible particulate matter.

II) Automated visual inspectionThe automatic systems are also called as the electron particles counter. The electronic particles counter evaluates the particles in injectables automatically. However, this method requires destruction of the ampoule/container for the particle examination.Electronic particles counting may be based on any one of the following principles: a) change in electrical resistance, b) light blockages principle and c) light scattering. Some of the automated systems for visual particle inspection include Autoskan, Eisai Ampoule inspection machine, Schering PDS/A-V system, etc. Autoskan System.The Autoskan system is based on light scattering principle whereby the particle in the path of a light source causes the scattering of light. The scattered light is measured and provides the corresponding information regarding the presence of particulate in the sample. This is a non-destructive test.

EISAI AMPOULE MACHINE SYSTEMThe Eisai ampoule machine (AIM) system is based on the light blockage principle. The particle size dimensions are determined with the shadow created by the particle under light source. Assessment of the shadow is the indication of the presence of particulate matter. This is also a non-destructive test.Schering PDS/A-V systemThe Schering PDS/A-V System is based on light scattering by particle if present in the sample. This is also a non-destructive test.

B) PARTICLE COUNT METHODSParticle count methods are the USP specified microscopic methods, which require the use of optical microscope and automatic microscope.

I) Optical Microscopic MethodThe optical microscopic method requires magnification of 100 .10x. One eyepiece mustbe equipped with graticule. A graticule have a series of circles of different diameters,usually in a “under root 2 progression”. The graticule is in circular diameter used to sizethe particulate. The micrometer is graduated in 10 micro meter increments.

A circular diameter graticuleIi) Automated Particle CountersThe automated particle counters are based on the light obscuration, light scattering method and the electrical resistance methods. Coulter Counter counts the particles in a sample based on the change in the electrical resistance. Particle size detection limit in this instrument is from 0.1 to 1000 micrometer.The powder sample requires pretreatment such as dispersion in an electrolyte to form a very dilute suspension. The Suspension is usually subjected to ultrasonic agitation to avoid

particle agglomerates. A dispersant may also be added to aid particle deagglomeration. Passage of particle causes the change in electrical resistance in between the electrodes which is proportional to the volume of particle. The change in resistance is converted into voltage pulse which is amplified and processed electronically and split into the particle size distribution into many different size-range.Glass TubeOrifice

Principle of coulter counter

This is a destructive test and large errors in measuring flakes and fibers are expected.This test is not recommended by FDA for parenterals.Illustration for a coulter counterHigh Accuracy (HIAC) InstrumentHigh Accuracy (HIAC) Instrument is based on light blockage principle. The test is highly effective for counting the both solid and liquid suspended particles. The instrument is calibrated easily and the test is recommended by USP. This is destructive test method and is expensive.P Light sourceDigitalValue

Principle of light blockage

Met-One Climet Particle CounterMet-One Climet Particle Counter is based on light scattering principle. The particles are assessed and counted in the sample based on the principle of light scattering. The instrument measures 6 particles sizes at a time and has the excellent ability for the detection of large particle. The test is destructive.

Depending on the instrument used, the sample may be presented as the liquid as suspension or air suspension. The light emitted by a helium-neon laser is incident on the sample particle. Light-particle interaction results in scattering of light. The photo detector converts the signals corresponding area/volume diameter of the particle.Instrument based on light scatter principle

BIOLOGICAL HAZARDS REPORTED OF PARTICULATE MATTER

The particles may localize in lungs, liver, spleen and myocardial tissues and may lead to thrombosisParticles may lead to myocardial infarction due to embolic fibers.Injection of solution contaminated with particulate matter causes granulomas and emboli in lungs Compendial requirementsDue to having a potential for blockage of the capillaries, an injection must be free from the visual evidence of particulate contamination. Thus, according to the Compendial requirements, each final container of the injectable must be inspected individually and the container must show evidence of contamination with visible foreign material

QUALITY CONTROL OF STERILE PRODUCTS (1)

The in-process quality control test includes the leak and clarity testing. The quality control of finished product required the pyrogen and sterility testing.Leakage testLeakage test is employed to test the package integrity. Package integrity reflects its ability to keep the product in and to keep potential contamination out”. It is because leakage occurs when a discontinuity exists in the wall of a package that can allow the passage of gas under pressure or concentration differential existing across the wall. Leakage differs from permeation, which is the flow of matter through the barrier itself.

Followings are the leak test methods.

A) VISUAL INSPECTIONVisual inspection is the easiest leak test method to perform. But this method is least sensitive. The method is used for the evaluation of large volume parenterals. To increase the sensitivity of the method, the visual inspection of the sample container may be coupled with the application of vacuum to make leakage more readily observable. This method is simple and inexpensive. However, the method is insensitive, operator dependent, and qualitative.Sometimes, the method is used in combination with pressure and /or temperature cycling to accelerate leakage to improve sensitivity.

B) BUBBLE TEST

The test package is submerged in liquids. A differential pressure is applied on the container. The container is observed for bubbles. Sometimes, surfactant added liquid is used for immersion of test package. Any leakage is evident after the application of differential pressure as the generation of foaming in immersion liquid. The method is simple and inexpensive. The location of the leaks can be observed in this method. However, it is relatively insensitive and the findings are operator dependent and are qualitative. The optimized conditions can be achieved using a surfactant immersion fluid along with the dark background and High intensity lighting. Generation of a differential positive pressure of 3 psi inside the vial and observation of any leakage using magnifying glass within a maximum test time of 15 minutes.

C) DYE TESTS

The test container is immersed in a dye bath. Vacuum and pressure is applied for some time. The container is removed from the dye bath and washed. The container is then inspected for the presence of dye either visually or by means of UV spectroscopy. The dye used may be of blue, green, yellowish-green color. The dye test can be optimized by use of a surfactant and or a low viscosity fluid in the dye solution to increase the capillary migration through the pores. The dye test is widely accepted in industry and is approved in drug use. The test is inexpensive and is requires no special equipment required for visual dye detection. However, the test is qualitative, destructive and slow. The test is used for ampoules and vials.

D) VACUUM IONIZATION TEST

Vacuum ionization test is useful for testing leakage in the vials or bottled sealed under vacuum. This test is used for online testing of the lyophilized products. High voltage, high frequency field is applied to vials which to cause residual gas, if present to glow.Glow intensity is the function of headspace vacuum level. The blue glow is the indicative of vacuum while the purple glow indicative of no vacuum. The sensitivity of the method is not documented. This test is on-line, rapid and is non destructive test. However, the proteins present in the test sample may be decomposed. This method is used for the lyophilized vials of biopharmaceuticals.

CLARITY TESTING

Clarity testing is carried out to check the particulate matter in the sample.

Particulate matter

Matter of biological or non-biological origin and with observable length, width, and thickness, e.g., bacteria, fungi, dust, dirt, fibers, plastic, rubber, lint etc. It may be any matter, mixed accidentally during manufacturing in the parenteral product which does not belong to the product. Particulate mater may be tiny pieces of lint, glass, dust, rubber, metal fibers, hair, microbes or unidentified and can make the product impure, unclean or unfit for use.

Sources of particulate matter

Particulate contamination particularly of cellulose fibers, dust, cotton fibers, hair, dandruff and loose skin from human origin as well as microbial contamination may arise from the following main sources.1. Material arising from the drug: undissolved substances and trace contaminants etc.2. Material arising from vehicle or added substances: These may include those material not filtered out during a clarification process before to filling the final container.3. Materials present in the final container: Material already present in container and which were not removed by rinsing prior to filling4. Materials falling by chance into the final container during the filling process5. The container or closures which may be deposited in the produce during sterilization, e.g. carbon black, whiting, zinc oxide and clay6. Packaging components: Including glass, plastic, rubber, I/V administration sets, etc.7. Environmental contaminants: Including air, work tops, insects’ parts8. Processing equipments: Including glass, stainless steel, rubber, or filter fiber, etc.9. Personnel: Including skin, hair, and clothing etc.

Particle size

Particles present in injectable are non-reactive, apyrogenic, sterilized. However, by virtue of their size may biologically hazardous. The particulate matter may be capable of blocking the blood vessels with severe results on induction into body with injection.A person with 20/20 vision under inspection conditions is able to detect particles of size range 40 – 50 μm. However, it is universally accepted that the particles size of 50 μm is detected visually by an unaided eye.Particle size greater than 7 μm diameter is considered to be more threatening. Pulmonary capillary are approximately 7 μm in diameter, thus particle of this much size entrapped in vascular bed resulting in multiple pulmonary infarction

To achieve maximum sensitivity in the method, once an appropriate dye has been selected, a laser source is then chosen which is tuned, or may be tuned, to provide a wavelength of a convenient intensity which will induce substantially maximumfluorescence in the dye. A preferred combination is an argon ion laser, such as a Spectra Physics Model 164 argon laser, 514.5 nm, in conjunction with eosin Y dye. A typical power setting is 0.2 watt.

Container samples are immersed in a selected dye solution and may be subjected to a processing cycle which simulates extreme processing conditions. Considerable overstressing conditions may sometimes be employed to provide a safety factor overand above normal production conditions. These stresses may include pressure, vacuum, autoclaving, pasteurization or freezing in a cryogenic dye bath. Sample containers are uniformly rinsed prior to leak evaluation. In the case of plastic containers,the dye may be absorbed onto the outer surface and is not removed by rinsing. However, this has no effect on the laser beam in the detection of leaks. The method has a further advantage in that several aligned containers can be inspectedsimultaneously. In addition, containers holding solutions of macromolecules as for example, Plasma Protein Fraction or albumin, can be successfully examined by the method of this invention without the interference of the Tyndall effect which would beencountered with such solutions if light beams from ordinary (non-coherent) light sources were used. The method is also useful even though the solution in the container or the container wall itself is not perfectly clear or colorless.

The invention may be better understood by the detailed description which follows. 

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The following serves to illustrate the very high degree of sensitivity in the method of the present invention by the use of eosin Y dye in combination with an argon laser.

LIMIT OF DETECTION

A. In Beakers

A one percent solution of eosin Y dye was prepared and diluted with distilled water so as to provide one liter quantities having four different concentrations. The solutions were placed in rigorously cleaned 500 ml. beakers and examinedindividually in a dark room when positioned approximately a foot from the light port of an argon gas laser emitting at a frequency of 514.5 nm and a power of 0.2 watt. The excitation frequency of a 0.001 percent solution of eosin Y had previously beendetermined to be 518 nm.

______________________________________ Dye Concentration Beaker No. μl of 1% dye ______________________________________ 5 0.010 9 0.005 3 0.001 2 0.0005 13 No dye ______________________________________

Seven individuals chosen at random, having no previous experience with the procedure, were asked to examine the beam of yellow light passing through each beaker of solution (while wearing yellow filter goggles) and to list the numbers of thebeaker in the order of decreasing intensity of light. All seven listed the numbers correctly in the sequence of 5, 9, 3, 2 and 13. They all expressed assurance that the intensity of the light beam passing through the solution having a concentration of0.001 μl of 1% dye per liter was quite strong and readily detectable. The beam of light passing through the blank (no dye) appeared as a faint red beam due to the Raman emission of water. Thus the sensitivity level for detection of eosin Y by thismethod is at least a thousand fold greater over the detection of the dye in ordinary light.

B. In Glass Bottles Containing Various Medicaments

The limit of detection of eosin Y by an argon laser was investigated on glass bottles containing a variety of medicaments.

1. 0.9% Saline in a 500 ml. bottle.

2. 5% Dextrose in a 500 ml. bottle.

3. 5% Dextrose-5% Ethanol in a liter bottle.

4. 8% Amino Acid Solution in a 500 ml. bottle.

This solution is a pale, straw-colored solution.

5. Plasmanate.RTM. Plasma Protein Fraction in a 250 ml. bottle. This is a 5% solution of predominately albumin and smaller amounts of α and β -globulins.

The solution is pale yellow.

6. Distilled water in a 500 ml. bottle.

A one percent solution of eosin Y dye was diluted with distilled water so as to give a standard solution containing 0.1 μl. of 1% dye/ml. Using a microsyringe, appropriate microliter quantities were injected through the stoppered bottles soas to give the concentration of 1% dye in the above-indicated volume of product. The bottles were then examined as described in part A.

TABLE I ______________________________________ Concentration Positive Product of 1% dye in μl. Detection ______________________________________ 1 0.01 Yes 0.001 Yes 2 0.01 Yes 0.001 No 3 0.02 Yes 0.01 No 0.001 No 4 0.02 Yes 0.01No 5 0.15 Yes 0.10 No 0.05 No 6 0.01 Yes 0.005 Yes 0.001 Yes 0.0005 No ______________________________________

Even though the sensitivity level falls off when the containers with yellow colored solutions were examined, as a consequence of background fluorescence caused by the product in the solution (Products 4 and 5), the degree of sensitivity is stillsufficiently great to make the method very useful for testing integrity of containers in which such products are stored.

C. In Plastic Containers

Substantially square-shaped plastic containers made of biaxially-oriented polypropylene were used in this study. Since it had previously been observed that the outside walls became stained by eosin Y dye, a number of closed liter sizedcontainers containing distilled water were first placed in a bath of 1 percent eosin Y, care being taken not to expose the closure to the dye. After heating in this bath at autoclave temperatures for a period of time, the containers were opened andthoroughly rinsed inside and out with hot distilled water. The water which was originally in the containers during the dye bath treatment was shown to be free of the dye when examined by the argon laser.

The rinsed containers whose outside walls were stained pink were then filled with known concentrations of eosin Y dye and examined by the argon laser as described in part A above.

TABLE II ______________________________________ Concentration of Positive 1% dye in μl. Detection ______________________________________ 0.0005 No 0.001 No 0.002 No 0.005 Yes 0.01 Yes ______________________________________

LEAK DETECTION PROCEDURE

A dye bath is prepared by dissolving in the proportion 10 g. eosin Y dye for every liter of distilled water. Preferably a surfactant such as 5 grams of sodium lauryl sulfate per liter of dye solution is also included which aids in thepenetration of the dye through any leaks in a container.

Samples of filled and sealed containers are placed upright in a stainless steel tank and covered by about 3 cm. above the tops of the containers with the 1 percent eosin dye solution. The tank is covered with a stainless steel cover andautoclaved at temperatures, pressures and dwell times which simulate commercial production conditions. The tank and contents are cooled to about room temperature and the dye is pumped into a reservoir for possible reuse. The containers are rinsedthoroughly with tap water and dried.

As shown in FIG. 1, each container 10, which may have a suitable closure 12, and containing a medical solution 14, is positioned before a beam 16 being emitted from an argon laser 18 in a dark room. The laser beam emitted has an excitationfrequency of 514.5 nm and the power of the instrument is set at 0.2 watt. When viewed through glasses having the appropriate cutoff filters necessary to remove stray light of the excitation frequency, the beam of light 20 passing through the containerwill appear yellow if dye is present in solution 14, indicating the presence of a leak either in the container or the closure. If beam 20 appears faintly red, no dye is present and the container and/or closure are judged to be free of leaks.

The USP compendial requirements has recommended the following tests for parenteral products 1

 

1. Weight variation or content uniformity2. Particulate matter in injections3. Bacterial endotoxin test4. Pyrogen test5. Sterility test

1. Weight variation or content uniformity test

This test is intended for sterile solids used for parenteral preparation. The weight of 10 individual sterile units is noted and the content is removed from them and empty individual sterile unit is weighed intern. Then net weight is

calculated by subtracting empty sterile unit weight form gross weight. The content of active ingredient in each sterile unit is calculated by performing the assay according to the individual monographs. The content in 10 sterile units is calculated by performing the assay. The dose uniformity is met if the amount of active ingredient is within the range of 85-115.0% of label claim  as determine by the content uniformity method or weight variation method. The dose uniformity is also met if the potency value is 100% in the individual monograph or less of label claim multiplied by average of limits specified for potency in individual monograph divided by 100 provided that the relative standard deviation in both the cases is equal to or less than 6.0%.If one unit is outside the range of 85-115.0%, and none of the sterile unit is outside the range of 75-125.0% and if the relative standard deviation of the resultant is greater than 6.0% then, the fore mentioned test is carried for 20 more sterile units. The sterile units meet the requirements if not more than one unit is out side the range of 85-115%, no unit is outside the range of 75-125.0% and the calculated relative standard deviation is NMT 7.8%.

2. Particulate matter in injections

The preparations intended for parenteral use should be free form particulate matter and should be clear when inspected visually. Two methods are described by USP according to the filled volume of the product to be tested.For large volume parenterals (LVP's), a filtration followed by microscopical examination procedure is used. For small volume parenterals (SVP's)a light obscuration based sensor containing electronic liquid-borne particle counter system is used.The USP standards are met if the LVP's under test contain NMT 50 particles per ml of 10 µm, and NMT 5 particles per ml of 25µm in an effective linear dimensional fashion.The USP standards are met if the SVP's under test contain NMT 10,000 particles per container of 10 µm, and NMT 1000 particles per container of 25µm in an effective spherical diameter.

3. Bacterial Endotoxin test

LAL (Limulus Amebocyte Lysate) test is used to characterize the bacterial endotoxin that may be present. The USP reference standard contains 10,000 USP endotoxins per vial. The LAL reagent is used for gel-clot formation.The test is performed using stated amounts of volumes of products, standard, positive control, negative control of endotoxin. The tubes are incubated at 37±1C FOR 60±2 minutes.When the tubes are inverted at 180C angle, formation of firm gel confirms positive reaction. While formation of a viscous gel that doesn't maintain its integrity or absence of a firm gel confirms negative reaction. The

test is invalid if the standard endotoxin or positive product control doesn't show end point within ±1 two fold dilution from label claim sensitivity of LAL reagent or if the negative control shows gel-clot end point.

4. Pyrogen test

It is performed by using rabbits as test animals.Initially 10 ml/kg body weigh of animal is injected through rat vein at 37±2C within ten minutes from start of administration.The temperatures are recorded at 1, 2 and 3 hours after injection. The requirements of USP are met if the rise in temperature of individual rabbit is NMT 0.6C and the sum of rise in temperature of three rabbits is NMT 1.4C. If any one rabbit shows a rise in temperature of 0.6C and sum of rise in temperature of three rabbits exceeds 1.40C then the test is repeated using 5 rabbits. The requirements are met if 3 out of 8 rabbits shows an individual rise in temperature of NMT 0.6C and sum of maximum rise in temperature of 8 rabbits is NMT 3.7C.

5. Sterility test

Growth promotion medium and incubation conditions are selected based on the test microorganism according to USP and is listed in table 1. The sterility test is done using direct transfer and membrane filtration techniques. Membrane filtration technique is suitable for liquids, soluble powders with bacterio static or fungi static properties, oils, creams and ointments. Sterility test by direct transfer is performed by aseptic transfer of specified volume from test container (table 2) to culture medium and incubated for 14 days and visual observation of medium is done on 3rd, 4th, 5th, 7th, 8th and 14th day. A membrane filter with porosity of 0.45µm with diameter of 47mm with flow rate of 55-75 ml of water per minute at a pressure of 70 cm of mercury should be used.The test meets the requirements when no growth is observed and if growth is observed then the test is repeated in the second stage and generally second stage is repeated with double the number of specimens tested in first stage when the test was found to be conducted under faulty or inadequate aseptic techniques.

USP sterility tests growth promotion microorganisms

Medium Test microorganisms Incubation

Temperature (C) Conditions

Fluid thioglycollate Bacillus subtillis (ATCC No. 6633)

30 to 35 Aerobic

Candida albicans (ATCC No. 8482)

30 to 35

Bacteroides vulgatus (ATCC No. 8482)

30 to 35

Alternative thioglycollate

Bacteroides vulgatus (ATCC No. 8482)

30 to 35 Anaerobic

Soybean-casein digest Bacillus subtillis (ATCC No. 6633)

20 to 25 Aerobic

Candida albicans (ATCC No. 10231

20 to 25

Liquid quantities for USP sterility test

Container content (ml)

Minimum vol taken from each container for each medium

Minimum volume of each medium

Use for direct transfer of volume from each container (ml)

Used for membrane representing total volume from the appropriate number of containers

Number of containers per medium

Less than 10 1ml or entire contents if less

15 100 20 (40 if each does not contain

than 1 ml sufficient vol for both media)

10 to less than 50

5 m l 40 100 20

50 to less than 100

10 ml 80 100 20

50 to less than 100 intended for i.v. administration

Entire contents - 100 10

100 to 500 Entire contents - 100 10

>500 500 ml - 100 10