Microbial Analysis of Pharmaceutical Microbial Analysis of Pharmaceutical ProductProduct

(Mikapil Injection)(Mikapil Injection)

Guided By Submitted ByGuided By Submitted By

Sr.Executive Mitali jainSr.Executive Mitali jain

Devender Joshi M.sc. IV semDevender Joshi M.sc. IV sem

Pil Psychotrpic India Ltd Company Pil Psychotrpic India Ltd Company

Haridwar (U.K.) Haridwar (U.K.)

CONTENTSCONTENTS

• INTRODUCTIONINTRODUCTION• AIM AND OBJECTIVES AIM AND OBJECTIVES • REVIEW OF LITERATURE REVIEW OF LITERATURE • MATERIALS AND METHODSMATERIALS AND METHODS• Microbial analysis of water Microbial analysis of water • Environmental monitoringEnvironmental monitoring• Sterility TestSterility Test• SUMMARY AND CONCLUSION SUMMARY AND CONCLUSION

INTRODUCTIONINTRODUCTION

Microbial analysis of waterMicrobial analysis of water• Microbiology plays an important & key role in pharmaceutical industry Microbiology plays an important & key role in pharmaceutical industry

particularly injections and tablets. At all the stages during the particularly injections and tablets. At all the stages during the manufacturing of an tablets & injections, almost precaution are to be manufacturing of an tablets & injections, almost precaution are to be taken to prevent external contamination.taken to prevent external contamination.

• Water is the basic & primary need of all vital life processes. With Water is the basic & primary need of all vital life processes. With increasing industrialization & population growth, water sources, increasing industrialization & population growth, water sources, available for various purposes, such as drinking, recreation, available for various purposes, such as drinking, recreation, aquaculture, agriculture, have been adulterated with industrial as aquaculture, agriculture, have been adulterated with industrial as well as animal & human wastes. As a result, it has a formidable factor well as animal & human wastes. As a result, it has a formidable factor in transmission of several diseases. Polluted (sewage) water contains in transmission of several diseases. Polluted (sewage) water contains solids & dissolved Water is the basic & primary need Microbiology solids & dissolved Water is the basic & primary need Microbiology organic compound that impart an offensive odour & serve as an organic compound that impart an offensive odour & serve as an excellent medium for the growth & multiplication of microorganism. excellent medium for the growth & multiplication of microorganism.

• Typical organism found in different types of water belong to fungi, Typical organism found in different types of water belong to fungi, protozoa, algae, bacteria for ex: the causative agent of dysentery, protozoa, algae, bacteria for ex: the causative agent of dysentery, typhoid fever (typhoid fever (Salmonella typhiSalmonella typhi), cholera (), cholera (Vibrio choleraeVibrio cholerae) etc. ) etc. According to 1980 study by the world Health Organization (WHO), at According to 1980 study by the world Health Organization (WHO), at least 30,000 people die everyday in developing countries of the world least 30,000 people die everyday in developing countries of the world because of unsanitary water supplies.because of unsanitary water supplies.

Water in Pharmaceutical industry is of Water in Pharmaceutical industry is of following typesfollowing types

• Raw WaterRaw Water

• Purified WaterPurified Water

• Water For InjectionWater For Injection

Specification of water for injectionSpecification of water for injection

ConductivityConductivity: Not more than 1.3 µs/cm: Not more than 1.3 µs/cm

Total bacterial count: Total bacterial count: Not more than 10 cfu/100mlNot more than 10 cfu/100ml

Total fungal count: Total fungal count: Nil cfu/100mlNil cfu/100ml

Endotoxin limit: 1Endotoxin limit: 1

Raw water/drinking waterRaw water/drinking water↓↓

Softening and dechlorinationSoftening and dechlorination↓↓

Reverse OsmosisReverse Osmosis↓↓

Ultrafilteration (0.5µ, 0.2µ) Ultrafilteration (0.5µ, 0.2µ) ↓↓

U.V. Light TreatmentU.V. Light Treatment↓↓

DistillationDistillation↓↓

Steam collectSteam collect↓↓

Water for Injection (WFI)Water for Injection (WFI)

ObjectivesObjectives

OBJECTIVES -OBJECTIVES -1)1) Environmental monitoringEnvironmental monitoring - To estimate the number of viable aerobic - To estimate the number of viable aerobic

microorganism present in environment .microorganism present in environment .

2)2) Microbial limit testMicrobial limit test - The Microbial Limit Test are to be performed the - The Microbial Limit Test are to be performed the quantitative and qualitative estimations of specific viable microorganism quantitative and qualitative estimations of specific viable microorganism present in sample.present in sample.

3)3) Bacterial endotoxin testBacterial endotoxin test - To lay down the procedure to detect or quantify - To lay down the procedure to detect or quantify the bacterial that may be present in the sample of articles to which the detect the bacterial that may be present in the sample of articles to which the detect presence of viable form of microorganism in the pharmaceutical product test is presence of viable form of microorganism in the pharmaceutical product test is applied by Gel Clot Technique.applied by Gel Clot Technique.

4)4) Sterility testingSterility testing – To detect presence of viable form of microorganism in the – To detect presence of viable form of microorganism in the pharmaceutical product (mikapil injection)pharmaceutical product (mikapil injection)

5)5) Antibiotic assayAntibiotic assay - To determine the concentration and quantity of an - To determine the concentration and quantity of an antibiotic by its effect in inhibiting the growth of susceptible microorganism.antibiotic by its effect in inhibiting the growth of susceptible microorganism.

Bacterial Endotoxin TestingBacterial Endotoxin Testing

• ObjectiveObjective: : To lay down the procedure to detect or quantify To lay down the procedure to detect or quantify the bacterial endotoxins that may be present in or on the the bacterial endotoxins that may be present in or on the samplessamples to which the test is applied by Gel Clot Technique.to which the test is applied by Gel Clot Technique.

• PrinciplePrinciple: : BacterialBacterial Endotoxins Test is the most sensitive Endotoxins Test is the most sensitive and specific test available to detect & measure the presence and specific test available to detect & measure the presence of endotoxin, a fever producing byproduct of gram –ve of endotoxin, a fever producing byproduct of gram –ve bacteria, commonly known as pyrogen. assay of very high bacteria, commonly known as pyrogen. assay of very high sensitivity has been developed using lysates of amebocytes sensitivity has been developed using lysates of amebocytes from the horseshoe crab (from the horseshoe crab (Limulus polyphemusLimulus polyphemus). Endotoxin ). Endotoxin specifically causes lysis of amebocytes. In the standard specifically causes lysis of amebocytes. In the standard Limulus amebocyte extracts are mixed with the solution to be Limulus amebocyte extracts are mixed with the solution to be tested. If endotoxin is present, the amebocyte extract gels are tested. If endotoxin is present, the amebocyte extract gels are precipitates, causing a change in turbidity.precipitates, causing a change in turbidity.

Procedure:Procedure:

50µl of sample is added to the tubes using a calibrated 50µl of sample is added to the tubes using a calibrated micropipette with disposable pyrogen free tip.micropipette with disposable pyrogen free tip.

↓↓100µl of reconstituent LAL reagent is added each tube100µl of reconstituent LAL reagent is added each tube

↓↓Tubes is shaken properly to assure mixing of sample and lysateTubes is shaken properly to assure mixing of sample and lysate

↓ ↓incubation period of 60± minincubation period of 60± min at37 ±1at37 ±1

After incubation tubes are removed from the heating block one After incubation tubes are removed from the heating block one at a time and smoothly rotated 180at a time and smoothly rotated 180

↓↓A positive test define as a firm clot and negative test show no A positive test define as a firm clot and negative test show no

gel formationgel formation

Calculation:Calculation:

MVD : MVD : Maximum Valid DilutionMaximum Valid Dilution

For determining the maximum valid dilution:For determining the maximum valid dilution:MVD = MVD = (Endotoxin Limit) X (Potency of the product)(Endotoxin Limit) X (Potency of the product)

λ (Labelled sensitivity of lysate inEu/ml )λ (Labelled sensitivity of lysate inEu/ml ) Where,Where,

Potency of product = concentration of the product in Potency of product = concentration of the product in unit/mlunit/ml

MVD = MVD = 0.33 Eu/ml X 50 mg/ml0.33 Eu/ml X 50 mg/ml

1.25Eu/ml = 132 1.25Eu/ml = 132

MVD/4 = 33 (30)MVD/4 = 33 (30)

0.1ml of sample + 0.9 ml of LRW- (1) 0.1ml of sample + 0.9 ml of LRW- (1) 0.1 ml of - (1) + 0.2 ml of LRW- (2)0.1 ml of - (1) + 0.2 ml of LRW- (2)

MATERIALS AND METHODSMATERIALS AND METHODS

MICROBIOLOGICAL ENVIRONMENTAL MONITORINGMICROBIOLOGICAL ENVIRONMENTAL MONITORING

ObjectiveObjective: : To estimate theTo estimate the viable aerobic microorganism present viable aerobic microorganism present in environment.in environment.

PrinciplePrinciple : : Microbial environment monitoring is done to check the Microbial environment monitoring is done to check the bioburden of aseptic area of controlled environment. The bioburden of aseptic area of controlled environment. The atmosphere contains all the major group of microbes ranging atmosphere contains all the major group of microbes ranging from algae to the viruses. Fungus spores are important coponent from algae to the viruses. Fungus spores are important coponent of air spores. The microbial flora of air is transient and variable. of air spores. The microbial flora of air is transient and variable. Air is not a medium in which microorganism can grow but is a Air is not a medium in which microorganism can grow but is a carrier of particulate matter , dust and droplets , which may be carrier of particulate matter , dust and droplets , which may be laden with microbes . The no. of kinds of microorganism are laden with microbes . The no. of kinds of microorganism are containing the air are determined by the sources of The containing the air are determined by the sources of The atmosphere of each unit .atmosphere of each unit .

METHODS OF ENVIRONMENT MONITORING

• Active air sampling by air samplerActive air sampling by air sampler

• Passive air sampling by Settle plate methodPassive air sampling by Settle plate method

FigFig:- :- Microbial air samplerMicrobial air sampler

Sterility TestingSterility Testing

Membrane filtration methodMembrane filtration method:-:-

The sterile bulk material in 200ml sterile 0.1% peptone waterThe sterile bulk material in 200ml sterile 0.1% peptone water↓ ↓

Shake to dissolve completelyShake to dissolve completely↓↓

After the content filtered , rinse the membrane with 300 ml portion of 0.1% peptoneAfter the content filtered , rinse the membrane with 300 ml portion of 0.1% peptone↓↓

Cut the membrane into two halves using sterile scissors or forcepsCut the membrane into two halves using sterile scissors or forceps↓↓

Transfer 1 half into FTM and other into SCDMTransfer 1 half into FTM and other into SCDM↓↓

Incubate both tubes of SCDM at 20°c for 14 days & FTM at 30°c -35 °c for same periodIncubate both tubes of SCDM at 20°c for 14 days & FTM at 30°c -35 °c for same period↓↓

During incubation if turbidity, precipitate or other evidence of microbial developsDuring incubation if turbidity, precipitate or other evidence of microbial develops

Microbial Limit Test of WaterMicrobial Limit Test of Water

This Testing is done by two method:-This Testing is done by two method:-a)a) Total Aerobic viable plate count or Total Aerobic viable plate count or

Standard plate count:-Standard plate count:- It is useful in determining the efficiency of It is useful in determining the efficiency of

operation for removing or destroying organism as during operation for removing or destroying organism as during sedimentation filtration chlorination ,the bacterial limit for sedimentation filtration chlorination ,the bacterial limit for raw water is 100 cfu/ml and fungal limit for raw water 10 raw water is 100 cfu/ml and fungal limit for raw water 10 cfu/ml .cfu/ml .

RequirementRequirement:- (1) Incubator:- (1) Incubator

• For bacterial growth that is set at 37cFor bacterial growth that is set at 37c• For fungal For fungal growth that is set at 20c growth that is set at 20c

((2)Glassware2)Glassware

• PetridishesPetridishes

• Test tubesTest tubes

• Conical flaskConical flask

All the glassware sterilized at 121c for 15 All the glassware sterilized at 121c for 15 lbs pressure.lbs pressure.

1)1) Colony counterColony counter

2)2) WeightWeight

3)3) Growth mediumGrowth medium

• Soyabean casein digest agar medium.Soyabean casein digest agar medium.

• Sabourd Dextrose agar medium.Sabourd Dextrose agar medium.

b)b) Pour plate methodPour plate method

Take 10 ml sample dissolve in 100ml SCDA it is called solution-ATake 10 ml sample dissolve in 100ml SCDA it is called solution-A↓↓

Take 1.0ml of solution-A pipette out in Sterile petri platesTake 1.0ml of solution-A pipette out in Sterile petri plates↓↓

Then poured the medium into the plateThen poured the medium into the plate↓↓

SCDA was used for bacterial growth & SDA was used For fungus growthSCDA was used for bacterial growth & SDA was used For fungus growth ↓ ↓

Incubated the plate at 30-35°c for 24 to 48 hour for bacterial growthIncubated the plate at 30-35°c for 24 to 48 hour for bacterial growthIncubated the plate at 20-25°c for 24 to 72 hour for fungal growthIncubated the plate at 20-25°c for 24 to 72 hour for fungal growth

↓↓After incubation colonies were countedAfter incubation colonies were counted

↓↓

Summary and ConclusionSummary and Conclusion

• During Four month training in Psychotropics India Ltd. I went During Four month training in Psychotropics India Ltd. I went analyzing various types of water and studies their uses in analyzing various types of water and studies their uses in pharmaceutical industry.pharmaceutical industry.

• Then the bacterial endotoxin test was carried out in which Then the bacterial endotoxin test was carried out in which the presence or absence of the pyrogen was detected in the presence or absence of the pyrogen was detected in various pharmaceutical product .various pharmaceutical product .

• The sterility test is performed to determine the given The sterility test is performed to determine the given product is totally free from microorganism.product is totally free from microorganism.

• The environmental monitoring was carried out which is The environmental monitoring was carried out which is necessary to check the bioburden of an aseptic area so that necessary to check the bioburden of an aseptic area so that contamination of products being manufactured must be contamination of products being manufactured must be prevented. prevented.