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Microbiological Analysis of Material & Drug Product

Procedure & Guideline (SOP) for Microbiological Analysis of Raw Material (API & Excipient), In process sample and Finished drug product in pharmaceuticals.

Microbiological Analysis of Drug Product / Material

1.0   Purpose:

    • The purpose of this SOP is to define a procedure for Microbiological analysis of Raw material, in process samples and finished products.

2.0   Scope:

    • This Standard Operating Procedure is applicable at Microbiology Department of pharmaceutical drug manufacturing plant.

3.0   References – Microbiological Analysis:

    • Guideline: Aseptic Technique for Microbiological Testing. (View)
    • SOP-Microbiological limit test strategy (View)

4.0   Responsibility:

    • Officer or Executive of Microbiology Department shall be responsible for preparation of new or revision of existing SOPs.

 

    • Head of the Department / Designee of respective areas & QA shall be responsible for reviewing the SOPs.
    • Unit Head and Head-Quality shall be responsible for approval of SOP.
    • QA shall be responsible for distribution and control of SOPs to various departments.

5.0   Abbreviations & Definitions:

ATCC American type culture collection
CC Change Control (View SOP)
CFU Colony Forming Unit
LAF Laminar Air Flow (View SOP)
NCTC National Collection of Type Cultures
SCDA Soyabean Casein Digest Agar
TAMC Total Aerobic Microbial Count
TYMC Total Yeast Mould Count
µm Micrometer
    • Standard Operating Procedure (SOP): A written authorized procedure, which gives instructions for performing operations.
    • Media Negative control: Negative control performed to ensure the media sterility.
    • Test Negative control: Negative control performed to verify testing conditions and to ensure the chosen diluent sterility.
    • CFU: Visible outcome of growth of micro-organisms arising from a single or multiple cells.
    • Colony: A colony is a pile or mass of a sufficiently large number of cells, growing on or in solid medium that they are visible to the naked eye.

6.0   Procedure – Microbiological Analysis:

  • Recommended Solutions and Culture Media for Microbiological Analysis:

                        [Note: This section is given for information.]

    • Buffered Sodium Chloride–Peptone Solution

Buffered Sodium Chloride–Peptone Solution pH 7.0
Potassium Dihydrogen Phosphate 3.6 g
Disodium Hydrogen Phosphate Dihydrate 7.2 g
Sodium Chloride. 4.3 g
Peptone (meat or casein). 1.0 g
Purified Water. 1000 ml
    • Sterilize in an autoclave using a validated cycle.

    • Casein Soybean Digest Broth:

Casein Soybean Digest Broth
Pancreatic Digest of Casein 17.0 g
Papaic Digest of Soybean 3.0 g
Sodium Chloride 5.0 g
Dibasic Hydrogen Phosphate 2.5 g
Glucose Monohydrate 2.5 g
Purified Water 1000 ml
    • Check the pH if necessary adjust the pH, after sterilization the pH is to be 7.3 ± 0.2 at 25 C.
    • Sterilize in an autoclave using a validated cycle.

    • MacConkey Broth for Microbiological Analysis:

MacConkey Broth
Pancreatic Digest of Gelatin 20.0 g
Lactose monohydrate 10.0 g
Dehydrated Ox Bile 5.0 g
Bromocresol Purple 10 mg
Purified Water 1000 ml
    • Check the pH if necessary adjust the pH, after sterilization the pH is to be 7.3 ± 0.2 at 25C.
    • Sterilize in an autoclave using a validated cycle.

    • MacConkey Agar Solution:

MacConkey Agar
Pancreatic Digest of Gelatin 17.0 g
Peptones (meat and casein) 3.0 g
Lactose monohydrate 10.0 g
Sodium Chloride 5.0 g
Bile Salts 1.5 g
Agar 13.5 g
Neutral Red 30.0 mg
Crystal Violet 1 mg
Purified Water 1000 ml
    • Check the pH If necessary adjust the pH, after sterilization the  pH is to  be 7.1 ± 0.2 at 25C.
    • Boil for 1 minute with constant shaking, then sterilize in an autoclave using a validated cycle.

    • Rappaport Vassiliadis Salmonella Enrichment Broth:

Rappaport Vassiliadis Salmonella Enrichment Broth
Soya Peptone 4.5 g
Magnesium Chloride Hexahydrate 29.0 g
Sodium Chloride 8.0 g
Dipotassium Phosphate 0.4 g
Potassium Dihydrogen Phosphate 0.6 g
Malachite Green 0.036 g
Purified Water 1000 ml
    • Dissolve, warming slightly.
    • Sterilize in an autoclave using a validated cycle, at a temperature not exceeding 115C.
    • The pH is to be 5.2 ± 0.2 at 25C after heating and autoclaving.

    • Xylose Lysine Deoxycholate Agar:

XLDA
Xylose 3.5 g
l-Lysine 5.0 g
Lactose Monohydrate 7.5 g
Sucrose 7.5 g
Sodium Chloride 5.0 g
Yeast Extract 3.0 g
Phenol Red 80 mg
Agar 13.5 g
Sodium Deoxycholate 2.5 g
Sodium Thiosulfate 6.8 g
Ferric Ammonium Citrate 0.8 g
Purified Water 1000 ml
    • Check the pH if necessary adjust the pH after heating it is 7.4 ± 0.2 at 25  C.
    • Heat to boiling, cool to 50C, and pour into Petri dishes.
    • Do not heat in an autoclave.

    • Cetrimide Agar Solution for Microbiological Analysis:

Cetrimide Agar
Pancreatic Digest of Gelatin 20.0 g
Magnesium Chloride 1.4 g
Dipotassium Sulfate 10.0 g
Cetrimide 0.3 g
Agar 13.6 g
Purified Water 1000 ml
Glycerol 10.0 ml
    • Heat to boiling for 1 minute with shaking.
    • Adjust the pH, so that after sterilization it is 7.2 ± 0.2 at 25C.
    • Sterilize in an autoclave using a validated cycle.

    • Mannitol Salt Agar Solution for Microbiological Analysis:

Mannitol Salt Agar
Pancreatic Digest of Casein 5.0 g
Peptic Digest of Animal Tissue 5.0 g
Beef Extract 1.0 g
d-Mannitol 10.0 g
Sodium Chloride 75.0 g
Agar 15.0 g
Phenol Red 0.025 g
Purified Water 1000 ml
    • Heat to boiling for 1 minute with shaking.
    • Check the pH if necessary adjust the pH, after sterilization the pH is to be 7.4 ± 0.2 at 25C.
    • Sterilize in an autoclave using a validated cycle.

    • GN Broth Solution for Microbiological Analysis:

GN Broth
Plypeptone peptone 20.0 g
Glucose 1.0 g
Sodium citrate 2.0 g
Sodium deoxycholate 0.5g
Di-potassium hydrogen phosphate 4.0 g
Mono potassium dihydrogen phosphate 1.5 g
Sodium chloride 5.0 g
Purified Water 1000 ml
    • Adjust the pH so that after heating it is 7.0 ± 0.2.
    • Mix and allow to stand for 15 minutes. With continuous stirring, bring gently to the boil and maintain at boiling point until solution is complete.
    • Cool to 50, mix, pour into petri dishes and cool rapidly.

  • Preparation of the Microbiological Analysis Sample:

    • The Microbiological Analysis procedure for sample preparation depends on the physical characteristics of the product to be tested. Unless otherwise specified, use 10 g or 10 ml of the sample.
    • If none of the Microbiological Analysis procedures described can be demonstrated to be satisfactory, a suitable alternative procedure must be developed.

    • Water- Soluble Products for Microbiological Analysis

    • Take 10 g or 10 ml (1:10 dilution) of the sample to be examined in buffered sodium chloride-peptone solution pH 7.0 or Phosphate buffer solution pH 7.2 or Soyabean-Casein digest broth and mix with the dilute to make 100 ml.
    • If necessary, adjust to a pH of 6 to 8.
    • Further dilutions, where necessary, are prepared with the same diluent.

    • Nonfatty Product Insoluble in water

    • Suspend the product (10 gm or 10 ml) to be examined (usually a 1 in 10 dilution is prepared) in buffered sodium chloride-Peptone solution pH 7.0, Phosphate buffer solution pH 7.2, or Soyabean-Casein Digest broth.
    • A surface-active agent such as 1g per L of polysorbate 80 may be added to assist the suspension of poorly wet able substance.
    • If necessary, adjust to a pH of 6 to 8. Further dilutions, where necessary, are prepared with the same diluent.

    • Fatty Products Microbiological Analysis

    • Dissolve in isopropyl myristate sterilized by filtration, or mix the product to be examined with the minimum necessary quantity of sterile polysorabte 80 or another non-inhibitory sterile surface-active regent heated, if necessary, to not more than 40°C or, in exceptional cases, to not more than 45°C.
    • Mix carefully and if necessary maintain the temperature in a water bath.
    • Add a sufficient quantity of the prewarmed chosen diluent to make a 1 in 10 dilution of the original product.
    • Mix carefully, while maintaining the temperature for the shortest time necessary for the formation of an emulsion.
    • Further serial 10-fold dilutions may be prepared using the chosen diluent containing a suitable concentration of sterile polysorbate 80 or another non inhibitory sterile surface-active reagent.

    • Fluids or solids in Aerosol Form for Microbiological Analysis 

    • Aseptically transfer the product into a membrane filter apparatus or a sterile container for further sampling.
    • Use either the total contents or a defined number of metered doses from each of the containers tested.
    • Amount Used for Test: Unless otherwise directed, use 10 g or 10ml of the product to be examined taken with the proper precautions.

    • Microbial Enumeration Tests: Test for Total Aerobic Microbial Count

    • Pour-Plate Method – Microbiological Analysis
    • For TAMC: Prepared the sample following a method described above.
    • Transfer aseptically 1 mL of the sample to each medium at least two petri plates about 9 cm in diameter, add 15 to 20 ml of agar media, previously maintained at not more than 45°C (If larger Petri dishes are used, the amount of agar medium is increased accordingly) and gently mix the plates for the equally distribution of the sample and allow for solidification.
    • After solidification, incubate the plate of soybean casein digest agar at 30 to 35°C for 3 to 5 days for TAMC and the plate of Sabouraud dextrose agar at 20 to 25°C for 5 to 7 days for TYMC.
    • After incubation period check the plates for the growth and count the number of CFU and take the arithmetic mean of the counts per medium, calculate the total number of CFU/ ml or gm.

    • Media Negative Control

    • Perform the negative control by adding the 15 to 20 ml of Agar media previously maintained at not more than 45°C to sterile Petri dishes and allow for solidification.
    • After solidification incubates plate of soybean casein digest agar at 30 to 35°C for 3 to 5 days for TAMC and the plate of Sabouraud dextrose agar at 20 to 25°C for 5 to 7 days for TYMC in inverted position.
    • After incubation period negative control plates should not show the growth.
    • If growth occurs in the negative control plates test is invalid.
    • Failed negative control needs the investigation.

  • Microbiological Analysis of Products for Specified Pathogens:

    • Sample Enrichment:

    • Diluted sample(1 in 10 dilution) which is used for TAMC and TYMC, Prepare a sample using 10 ml of diluted sample or the quantity corresponding to 1gm or 1ml, to inoculate into 90 ml sterile Soyabean-casein digest broth,
    • Mix well and incubate at 30 to 35°C for 18 to 24 hours for Escherichia coli, Pseudomonas aeruginoga , Staphylococcus aureus, Shigella (if required) and diluted sample(1 in 10 dilution) which is used for TAMC and TYMC, corresponding to 10 gm is incubate at 30 to 35°C for 18 to 24 hours for Salmonella species.

    • Microbiological Analysis for Escherichia coli:

    • After incubation period shake the container, transfer 1 mL of Soybean–Casein Digest Broth to 100 mL of MacConkey Broth.
    • Incubate at 42 to 44°C for 24 to 48 hours.
    • After incubation period Subculture on a plate of MacConkey Agar and incubate at 30 to 35°C for 18 to 72 hours.
    • Interpretation: Growth of pink non-mucoid colonies indicates the possible presence of Escherichia coli. This is confirmed by identification tests.
    • The product complies with the test if no colonies are present or if the   identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification Test: Biochemical test or identification by automated methods can be used for confirmatory identification.
    • Transfer well-isolated suspected colonies from MacConkey agar plate to 5 mL of 5% Peptone water or MacConkey Broth or a suitable medium contained in a test tube.
    • Incubate the tube at 42- 44°C for 24 hours.
    • Add 0.5 mL of Kovac’s reagent to the tube, shake well and allow to stand for one minute.  If a red colour is observed in the reagent layer, indole is present.
    • The preparation being examined passes the test if such colonies are not seen or if the confirmatory biochemical tests are negative.
    • Test Negative control: To verify testing conditions perform a negative  control using the chosen diluent in place of the test preparation. There must be no growth of microorganisms.

  • Microbiological Analysis for Salmonella:

    • After incubation period transfer 0.1 mL of Soybean–Casein Digest Broth  to 10 ml of Rappaport Vassiliadis Salmonella Enrichment Broth.
    • Incubate at 30 to 35°C for 18 to 24 hours. Sub culture on a plate of Xylose Lysine Deoxycholate Agar, and incubate at 30 to 35°C for 18 to 48 hours.

    • Interpretation

    • The possible presence of Salmonella is indicated by the growth of well- developed, red colonies, with or without black centers on Xylose Lysine Deoxycholate Agar: This is confirmed by identification tests.
    • The product complies with the test if colonies of the types described are not  present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control  using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.

    • Identification Test:

    • Biochemical test or identification by automated methods can be used for confirmatory identification.
    • Transfer separately a few of the suspect colonies to Triple sugar iron agar in tubes, using surface and deep inoculation. (This can be done by first inoculating the surface of the slope and then making a stab culture with the same inoculating needle and incubating at 30 – 35 °C temperature for 24 hours).
    • The presence of Salmonella is provisionally confirmed if, in the deep inoculation but not in the surface culture, there is a change of color from red to yellow and usually a formation of gas, with or without production of Hydrogen sulphide in the agar.
    • Precise confirmation may be carried out by appropriate biochemical test such as Urea broth.
    • Transfer separately a few of the suspect colonies to 10 ml of Urea broth contained in test tube and incubate at 30 – 35 °C temperature for 24 hours.
    • Upon incubation absence of red colour in Urea broth tube indicates presence of Salmonellae.
    • The preparation being examined passes the test if, colonies of the type described do not appear or if the confirmatory biochemical tests are negative.

    • Test Negative control:

    • To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. 
    • There must be no growth of microorganisms.
    • Failed negative control needs investigation.

  • Microbiological Analysis for Pseudomonas aeruginosa:

    • After incubation period Subculture on a plate of Cetrimide Agar, and incubate at 30 to 35°C for 18 to 72 hours.
    • Interpretation: Growth of greenish colony indicates the possible presence of P. aeruginosa. This is confirmed by identification tests.
    • The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification Test: Biochemical test or identification by automated methods can be used for confirmatory identification.
    • Oxidase test – Place 2 or 3 drops of a freshly prepared 1 % w/v solution of N, N, N’N’-Tetramethyl-4-phenylene diamine dihydrochloride on filter paper and smear with the suspect colony; if a purple color is produced, the test is positive. Alternatively readymade impregnated discs can also be used.

  • Microbiological Analysis for Staphylococcus aureus:

    • After incubation period Subculture on a plate of Mannitol Salt Agar, and incubate at 30 to 35°C for 18 to 72 hours.
    • Interpretation: The possible presence of aureus is indicated by the growth of yellow or white colonies surrounded by a yellow zone. This is confirmed by identification tests.
    • The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.

    • Identification Test: Coagulase Test:

    • Note: Biochemical test or identification by automated methods can be used for confirmatory identification.
    • With the aid of an inoculation loop, transfer representative suspected colonies from surfaces of Mannitol Salt Agar to individual tubes, each containing 0.5 mL of mammalian, preferably rabbit or horse plasma with or without suitable additives.
    • Incubate in a water bath at 37°C temperature, examining the tubes after 3 hours and subsequently at suitable intervals up to 24 hours.
    • If coagulation in any degree is observed, the test is positive.
    • Carry out a positive control by adding a volume of broth containing 10 to 100 cells of Staphylococcus aureus, prepared from a 24 hours.
    • Culture in soyabean casein digest medium of Staphylococcus aureus (ATCC 6538 / NCTC 10788) to a tube containing 0.5 mL of plasma.
    • Carry out a negative control by incubating an un-inoculated tube containing 0.5 mL of plasma.
    • For the test to be valid the positive control tube should show coagulation within 3 hours of incubation and there should be no coagulation in negative control tube even after 24 hours of incubation.
    • Perform the negative control.

  • Microbiological Analysis for clostridia:

    • Prepare a sample using a 1 in 10 dilution (with a minimum total volume of 20ml) of not less than 2g or 2 ml of the product to be examined.
    • Divide the sample into two portions, of at least 10 ml. Heat one portion at 80 °C for 10 minutes and cool rapidly. Do not heat the other portion.
    • Use 10 ml or the quantity corresponding to 1g or 1 ml of the product to be examined of the portions to inoculate suitable amounts of Reinforced Medium for Clostridia.
    • Incubate under anaerobic condition at 30 to 35°C for 48 hours.
    • After incubation period Subculture from each container on a plate of Columbia agar and incubate under anaerobic conditions at 30 to 35°C for 48 to 72 hours.
    • Interpretation: The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of Clostridia.
    • This is confirmed by identification test. The product complies with the test if colonies of the tests described are not present or if confirmatory identification tests are negative.

  • Microbiological Analysis for Candida albicans:

    • Prepared the product to be examined as described in point no. 7.2 and use 10 ml or the quantity corresponding to not less than 1g or 1ml to inoculate 100ml of Sabouraud Dextrose Broth and mix.
    • Incubate at 30 to 35°C for 3 to 5 days.
    • After incubation period Subculture on a plate of Sabouraud Dextrose Agar and incubate at 30 to 35°C for 24 to 48 hours.
    • Interpretation: Growth of white colonies may indicate the presence of Candida albicans. This is confirmed by identification tests.
    • The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.

  • Microbiological Analysis for Shigella:

    • Use the quantity corresponding to 10g or 10ml of the product to inoculate a suitable amount (determined as under validity of the test method) of Soyabean casein digest broth at 30°C to 35°C for 18 to 24 hours.
    • After incubation period transfer 1.0 ml of Soybean–Casein Digest (Enrichment) Broth to 100 ml of GN Broth.
    • Incubate at 30 to 35 °C for 24 to 48 hours. Sub culture on a plate of XLDA, and incubate at 30 to 35 C for 18 to 48 hours.
    • After incubation period Subculture on a plate of XLDA medium and incubate at 30 to 35 °C for 24 to 48 hours.

    • Interpretation:

    • The possible presence of Shigella is indicated by the growth of red colored translucent colonies, without black Centre on XLDA: This is confirmed by identification tests.
    • The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.
    • Test Negative control: To verify testing conditions perform a negative control using the chosen diluent or media in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification test: Biochemical test or identification by automated methods can be used for confirmatory identification.

  • Microbiological Analysis Sampling and Test Procedure:

    • Sampling for microbiological testing shall be performed prior to collecting samples for other purposes.
    • Sampling personnel shall wear clean, lint free smocks, gowns or uniforms specified for the area where sampling is to occur.
    • Sterile gloves shall be worn and sanitized just before sampling.
    • Non-sterile material shall be sampled under controlled environment, such as Laminar Air Flow hood or room with HEPA filtered air.
    • All microbiological samples shall be collected using clean, sterile, pyrogen-free implements, such as forceps, scoops, pipettes, hoses and sample thieves. Utility system samples shall be collected from existing hoses attached to the ports, if available.
    • Sanitize sampling tables and hoods using a validated sanitizing agent prior to use.
    • Sanitize exterior of sampling implement packages and containers with a validated sanitizing agent prior to use with a clean lint-free wipe/cloth.
    • Open packages and sample containers just before collecting the sample in order to minimize exposure to the environment.

    • Sample container lids shall be held such that the interior surface of the lid faces downward to avoid contamination falling into it.

    • If the sample container lid cannot be held while sampling, it may be placed on a sanitized/sterilized surface with the interior lid surface facing down.
    • Microbiological samples for non-sterile dose facilities shall be collected into sterile sample containers.
    • Gloves shall be changed between samples or sanitized with an approved sanitizer and allowed to dry.
    • Sampling areas shall be cleaned and sanitized after each sample is taken and after completing sample collection.
    • Transport samples in a covered container to the appropriate Microbiology/ Environmental Monitoring Laboratory, as soon as sampling is completed.
    • The testing procedure as per USP/Ph Eur/ IP shall be followed.
    • If the material is official in USP/Ph Eur both, testing shall be done as per USP & Ph Eur both.
    • If results complies as per respective specification then release the batch Product before packing shall be sampled as finished drug product.

  • Frequency:

    • Microbiological analysis shall be performed for batches and /or validation batches (as per respective specification and ATP).
    • After review of the microbiological analysis data of these batches if the data found satisfactory then the microbiological analysis shall be performed for one in ten batches for each finished product.
    • For domestic products the MLT shall not be part of release specification.
    • Any product, if it is exported as per the requirement, the specification will be checked for microbiological tests and analysis will be performed for those batches.
    • Any major changes in the formulation shall be reviewed and if required, microbiological analysis will be done for those batches.
    • For export products MLT shall be part of release specification if required (i.e.as per requirement or registration in respective country).
    • Acceptance criteria (Limit): As per respective specification.

  • Microbiological Analysis Practices in Laminar Flow or Biosafety Cabinet:

    • Approved sanitizing agents shall be used to wipe down the walls and bench top before using laminar flow or biological safety cabinets and allowed to remain for the contact time prescribed for the sanitizing agent.
    • The exterior surfaces of all materials, parts, samples and equipment placed into the hood shall be sanitized with an approved agent prior to beginning tests.
    • Personnel shall wear clean smocks or gowns and sterile gloves when testing within hoods.
    • Gloves shall be changed if hands are removed from the hood/cabinet and contact non-sanitized items outside of the hood. Gloves shall be sanitized again when hands are placed within the hood.
    • Minimize the time samples and media containers are open under the hood/cabinet by opening a container just before collecting a sample or inoculating media and closing the container immediately after use.
    • Assure that open containers are within the clean airflow.
    • Do not over crowd the hood/cabinet work bench while working.
    • Place material near/closer to the horizontal laminar air flow hood filter face or to the rear of vertical biological safety cabinets.

 

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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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