“MICROBIAL TESTING OF PHARMACEUTICAL WATER &

FINISHED PRODUCTS”

Presented By :

“MICROBIOLOGICAL ANALYSIS OF PHARMACEUTICAL WATER &

FINISHED PRODUCTS”

Presented By : DEEPIKA SHARMA

Presently operating the following major therapeutic

segments:Analgesics Anti Inflammatory

Anti-Bacterials Anti-Ulcerants

Dermatologicals Herbal Drugs Nutrition

Neurotropics Placental ExtractsSkeletal Relaxants

Anti-Arthiritic / Chondroprotective Agents

PHARMACEUTICAL WATERThe quality attributes of water for a particular application are dictated by the requirement of its usage. Sequential processing steps that are used water for different pharmaceuticals are shown in the figure below

Preparation of Media Use purified water for preparation of media.Weigh accurately quantity as mentioned in table, after weighing the required amount take it slowly from the pan without spilling the dehydrated media and transfer it into a clean container wash weighing vessel if required.Add a small volume of purified water and swirl to dissolve completely.Make up the required volume by adding purified water from the side walls of the container.Boil the media if required to dissolve completely using, a hot plate/water bath.

Allow the media to cool to about 250C and check the pH with the help of a pH meter, the pH meter should lie within the range specified on individual container on

dehydrated media adjust if required with 0.1 N HC1/0.1N NaOH.Dispense the media as per the requirement sterilize by loading the media bottles/ flasks in the autoclave as per the validated load cycle diagram by autoclaving at 1210C at 15 lbs pressure for 15 mins.Label the media container/flask/bottle/test tube as follows :

Media Name :Batch No. :

Sterilized Load No. :Use Before Date :

AIMS AND OBJECTIVES

A. Pathogen Testing:To find out the extent of growth of micro organism in:1. Raw (Powdered forms)2. Finished product (Oral doses, k e.g. tablets, capsules & syrups)

By counting number of C.F.U (Colony Forming Unit) using:1. Pour Plate Method2. Heterotrophic PIate Count (Membrane Filtration)

B. LAL (Limulus Amoebocytes Lysate) Testing:To find out extent of endotoxin level in the water sample (W.F.I=Water For injection) by forming gel in depyrogenated tubes using:

1. Gel Clot Method2. End Point Method

Water sample considered:1.1 W.F.I (Water for injection)2.1 CSSP (Clean Steam Purified Water)C. Sterility Testing:

The sterility test is used to detect the presence of viable forms of bacteria, fungi and yeast in or on pharmaceutical preparation.

1. Membrane Filtration Technique 2. Direct Inoculation Technique

Microbial Limit Test For

Finished ProductsThis test provides tests for the estimation of the number of viable aerobic microorganisms present and for freedom from designated microbial species on pharmaceutical articles of all kinds, from raw materials to the finished form.

To find out the extent of growth of micro organism two methods are used :

Pour Plate MethodHeterotrophic PIate Count (Membrane Filtration)

1. Plate Method

1 ml of the final dilution onto each of two sterile Petri dishes. Promptly add to each dish 15 to 20 ml of

Soyabean Casein Digest Agar medium that previously has been melted and cooled to approximately 45°C, &solidify at room temp invert the Petri dishes and incubate for 48hr to 72 hours. Following incubation, examine the plates for growth, count the no. of colonies.

2. Membrane FiltrationUse membrane filters 50mm in diameter and having a normal pore size of not greater than 0.45 um the

effectiveness of which in retaining bacteria .Sterilize and assemble the filtration apparatus.Transfer 10m1 to membrane filter and filter immediately.

Test For Pathogens

ProcedurePre Enrichment:

Transfer 1.0 ml\ 1g of sample in 10 ml nutrient broth tubes & incubate it at 32 ± 2.5°C for

24hrs. After incubation examine tube for growth & if present mix. By gentle shaking .

Test for E.coli :

Primary TestPrepared Mac Conkey broth & distribute it in 100 ml test tube. Transfer 1 ml of the pre enriched medium into the sterile Mac Conkey broth and incubate it at 42 ± 2°C for 48 hours.If any growth occurs, carry out for further identification of E.coli.Streak the growth over Mac Conkey agar, EMB & incubate it for 24hrs at 36 ± 2°C.

E.coli on MacConkey Agar

Gram Stain Characteristic Colonial Morphology

Gram-negative (Cocco bacilli) Brick red may have surrounding zone of ppt bile

Confirmative Test Indole Test

Reagent : Kovac’s reagent. Method : To 5 ml of 24 hr old peptone culture, add 0.5 ml of Kovac’s reagent shake gently and incubated at 43.5 deg .And stand for 1min if red colour ring is observed then indole is +nt, which confirm E.coli presence.

Test for SalmonellaPrimary TestPrepared Tetrathionate Bile Brilliant Green Broth & distribute it in IOml test tube having a cap opening and sterilize it.Transfer I ml of pre enriched sample medium to 10 ml of Tetrathionate Bile Brilliant Green Broth and incubate it for 48 hours at 44.5 ± 2°C.

If growth is observed in the inoculated medium, so streak out the growth on BSA XLDA and BGA and incubate it for 24hrs at 36 ± 2°C.If any characteristics colony is shown on any of the above media, it requires the need for confirmative test.

Selective Medium Characteristic Colonial Morphology

Brilliant Green Agar Small, transparent, colorless or pink to white opaque

XLDA Bismuth Sulphite Agar

Red with or without black centers, black or green.

Environmental Monitoring

MICROBIOLOGICAL ENVIRONMENTAL MONITORING:

Environmental monitoring carried out in non-sterile drug product and drug substance manufacturing areas

Environmental monitoring carried out in :

Capsule mfg. area.Ware houseMicro laboratorySolution preparation roomSVP (small volume parental) illing AreaLVP Bottle Pack AreaGlass Filling Area

Procedure1) Media of EM : Preincubated plates of Soyabean Casein Digest agar medium can be used.

2)Frequency for EM : Environment monitoring shall be performed IN CLASS 100 DAILY IN 10000 TWICE A WEEK IN

100000 OCNE IN A WEEK.3)Sampling area, sapling locations and EM schedule:

•Adjacent to doors•At low level return air grilles•Between HEPA's in clean rooms•an corners of rooms.

Incubation of plates : •Environmental monitoring operator shall ensure that all plates are incubated with in 4 hours from the time plate exposure completed.•Incubate the plates in an inverted position at 20-350C for 72 hours.•Further incubate the same plates in an inverted position at 30-350C for another 48 hours.

Observation:Observe the plates for bacterial and fungal count and note down the observations in environmental monitoring record.The total no. of colony forming unit (CFU) shall be calculated as a sum of bacterial (CFU) and fungal (CFU).

Surface Sampling by SWAB

To determine and identify molds actually growing on the surface sampled, as opposed to the mere presence of mold spores.

BACTERIAL ENDOTOXINS TEST{LIMULUS AMEBOCYTES LYSATE TEST}

TOXIN

Toxin is a substrate, such as a metabolic product of the organism that alters the normal metabolism of host cells with deleterious effects on the host. Toxins produced by bacteria can be divided into two main categories :

Exotoxin Endotoxin

1.Exotoxin

Exotoxins are water-soluble, heat-labile and high molecular weight proteins. Exotoxin may travel from the site of infection to other body tissues or target cells in which they exert their effects. Exotoxins usually are :

Synthesized by specific bacteria (mainly Gram –ve) Heat labile proteins inactivated at 60-800CAssociated with specific diseases and have specific mechanism of action. Highly immunogenic and stimulate the production of neutralizing antibodies called antitoxins. Unable to produce a fever in the host directly.

2.EndotoxinEndotoxin is the structural element of bacteria, called Lipopolysaccharide (LPS). Lipopolysaccharide is contained in the outer membrane of most bacterial cell walls, but the greatest amount found in Gram-ve bacteria.

Under certain conditions, this Lipopolysaccharide is toxic to specific hosts. This LPS is called an endotoxin because it is bound to the bacterium and released when the microorganism lyses.

The toxic component of LPS is the lipid protein, called Lipid A The lipid component exhibits all the properties associated with endotoxicity.Bacterial endotoxins are : Heat stable.Toxic only a high doses (milligram per kilogram amount).Weakly immunogenic.Usually capable of producing general systemic effects: fever (are pyrogenic), shock, blood coagulation,

weakness, diarrhea, inflammation, intestinal hemorrhage and fibrinolysis Bacterial endotoxin has a number of physiological effects following intravenous injections including the following; activation of cytokine system; endithelial cell damage; permeability of vascular leading to a drop in blood pressure; and intravascular coagulation. Incases where the body is subjected to massive doses, such as in the case of gram negative bacterial infection; endotoxin release may cause ultimately death from septic shock.

LAL TESTINGThe limulus Amoebocyte Lysate (LAL) test is used for detection and quantification of gram-negative endotoxin using limulus amoebocyte lysate – A Multi enzyme Complex, is derived from cytoplasm of circulating Amoebocyte cells (WBCs) of Horse Shoe Crab (Limulus polyphemus).

Limulus -- Large North American Horse Shoe Crab. Amoebocyte -- WBC in the blood of crab. Lysate -- A lysed preparation of cells.

PRINCIPLE OF LAL TESTEndotoxin activates a pro-enzyme of LAL having a mol. Wt. of 1,50,000. Activation also depends on the presence of metal ion such as calcium, manganese or magnesium.The activated pro-enzyme (coagulase) reacts with a lower molecular M. protein factor also contained in LAL substance.The lower molecular weight fraction called coagulogen is cleaved by the pro-enzyme into a soluble clot, a precipitate,

Therefore, the coagulation reaction requires three factors in addition to endotoxin. These are:Clotting enzyme.Clotting protein (coagulogen)Certain divalent cations found in LAL reagent.

Endotoxin

Ca++

PROENZYME COAGULASE

COAGULOGEN COAGULIN CONTROL STANDARD ENDOTOXIN (CSE)CSE is commercially marketed; freeze dried, purified (free from protein).

Lysate reagent is standardized against the CSE, so the sensitivity is expressed in Endotoxin Units per milliliter (EU/ml)

STORAGE: CSE powder is to be stored at 2-25 °C. Store dehydrated endotoxin at 2-8 °C up to 4 weeks.

Maximum Valid Dilutions And Minimum Valid Concentration•The maximum valid dilution (MVD) depends on: •the potency (concentration) of the sample. •The endotoxin limits for the precut. •The lysate sensitivity.

Maximum valid dilution is calculated from the following expression:MVD = Endotoxin limits (EU/ml or EU/unit) x potency

Sensitivity of Lysate (EU/ml)

MATERIAL USED:REAGENT REQUIRED FORLAL TESTLimulus Amoebocyte Lysate (sensitivity 0.125 EU/ ml). Endotoxin standard (LPS from E. coil 0113 of 100 EU). LAL reagent Water (LRW).Sample of product to be tested.

PREPARATION OF LALReconstitute the lyophilized lysate by adding 1.9 ml LAL reagent water. Swirl gently but thoroughly for at least 30 sec. Do not shake as the content give rise to foam formation. PREPARATION OF CSE Reconstitute endotoxin by adding 5 ml. LRW. Vortex the vial of endotoxin for at least 15 seconds. Dilute the endotoxin with LRW to a concentration of 1 EU/ml. Using the 1 EU/ml. endotoxin solution prepare a serial to fold dilution series that brackets the end point of lysate.

Procedure Take 100 l of each dilution as prepared above in 10 x 75 mm sterilized test tubes & in each test tube add 100 l of Lysate. Stir each test tube on vortex mixture for a second. Then incubate all the test tubes in a dry heat incubator block at 37+100C for 60+2

OBSERVATION After incubation and end point determination (manually), the sensitivity of the LAL reagent will be confirmed if the test results are positive to with in one two fold dilution of the stated labeled potency. If a gel clot is formed in one of the 0.06 dilution tube, it means that given lysate is over sensitive than the labeled potency. Now, the actual lysate sensitivity is determined as follows :

TABLE : Assay Results – Gel Clot MethodEndotoxin Dilution (EI/ml)

Replicate 0.50 0.25 0.125 0.06 0.03 Endotoxin

1 + + + - - 0.125

2 + + + - - 0.125

3 + + + + - 0.06

4 + + + - - 0.125

GEL CLOT METHODSample + LAL Reagent -------------------------------- > Observe presence or absence of gel (100 micro Iitre)(100 micro litre) 60(+2) min.+Gel: Gel that holds its integrity when tube is inverted at 180 °C -Gel: Clear or viscous liquid or soft gel which flows when tube is inverted.

RESULTS

Each tube in gel clot method is interpreted as either positive or negative.A positive test is defined as the formation of a film gel .

A negative test is characterized by the absence of gel .

LIMITATION OF LAL TESTLAL is dependent only for detection of pyrogen origination from Gram-negative bacteria .

Sensitivity of LAL varies with endotoxin from various microbial sources.

LAL cannot discriminate between living and dead bacteria.

PRECAUTIONS

All the glassware should be thoroughly cleaned and depyrogenated. Reaction temperature should not exceed 36-38°C.pH of the reaction mixture must be in the range of pH 5-7. cis Reaction time should be no longer than one hour.Each test must be accompanied by positive and negative controls Strict aseptic condition must be used.

Test For Sterility

INTRODUCTIONThe sterility test is used to detect the presence of viable forms of bacteria, fungi and yeast in or on pharmaceutical preparation. The working conditions in which the tests are performed should be free from any microbial form & should be monitored regularly by sampling the air & surfaces The tests must be carried out under conditions designed to avoid accidental contamination of

the product during the test. A batch is defined, as a collection of sealed containers that are same in the nature i.e. the risk of the contamination is same for each of the unit in it.

PRINCIPLEThe test is based upon the principle that if bacteria & fungi are placed in the medium, which provides nutritive material & water the organisms will grow and their presence can be indicated by turbidity in the originally clear medium.

CULTURE MEDIAThe following culture media are found to be suitable for the test of sterility:Fluid Thioglycollate Medium (FTGM)After dissolving media in distilled water, distribute it into suitable vessels. Sterilize by autoclaving for 18-20 minutes at 1210C. Cool promptly to 250C C & store at 20-30 0C. Soyabean Casein Digest Medium

After dissolving all ingredients in distilled water, distribute it into suitable vessels. Sterilize by autoclaving for 18-20 minutes at 121 °C.

ProcedureMETHODSMembrane Filtration Technique Direct Inoculation TechniqueMembrane Filtration Technique:A membrane generally suitable for sterility testing has nominal porosity of 0.5, a diameter of approx. 47 mm and a flow rate of 55 to 75 ml of water per minute at pressure of 70 cm of mercury. It is made up of nitrocellulose or nylon.This method is to be performed where the substance being examined is: • Oil,

(b) Ointment that can be put into the solution. FiltrationFilterate aseptically then remove the membrane from the holder, cut the membrane in half with the help of tong & scissors, immerse one half of the membrane in 100ml of soybean casein digest medium (SCDM) & incubate at 20-25°Cfor not less than 14 days. Similarly other half of the membrane in 100ml of Fluid Thioglycollate medium (FTGM) & incubate at 30-35 C for not less than 14 days.

Direct Inoculation Method Aseptically transfer the specified volume of the material from each container to a vessel of culture medium

Mix the liquid with the medium & Incubate in the specified media for not less than 14 days. And check the turbidity for the presence & absence of microbial growth .

Application For bacterial detection Fluid Thioglycollate Medium

For Fungal Detection Soyabean Casein Digest Medium

Incubation Temp. Incubation Period Positive Control

30 – 350C14 days 1. Staphylococcus aureus2. Pseudomonas – aeruginosa

20 – 250C14 Days 1. Bacillus subtitles 2. C.albicans

Observation , Results &

ConclusionBacterial Endotoxin Test:‑Observation After incubation and end point determination (manually), the sensitivity of the LAL reagent will be confirmed if the test results are positive to with in one two fold dilution of the stated labeled potency. If a gel clot is formed in one of the 0.06 dilution tube, it means that given lysate is over sensitive than the labeled potency. Now, the actual lysate sensitivity is determined as follows:

Result: The positive LALgel clot is characterized by the formation of a solid gel , which remains intact in the bottom of the tube upon incursion. From this study it was concluded that:CFU and Endotoxin level of raw, finished product and water sample respectively tested from different poilll nr sources.

No microorganism recovered from any of the water Sample and Products. No microorganism recovered from the raw, finished Product..

Sterility Test : Result: The above sample complies the sterility test.

Environmental monitoring :-Result : for class 1, 00, 000 should not exceed more than 10cfu \plate.The alert limit should not

exceed more than 50 cfu/plate.. action limit of plate exposure for class 10,000 should not exceed more than 50 cfu per plate & the alert limit for the same class should not exceed more than 25 cfu per plate. The action limit for the class 100 should not be more than 01 cfu per plate.