SOPs

Colony counter; Purpose

To establish the procedure for ideal operation and cleaning of Colony Counter, Model: SC6, Stuart UK, in microbiology test.

Colony counter; Scope

This SOP applies for operation and cleaning of Colony Counter, Model: SC6, Stuart, UK in Microbiology Section of General Building at XX Pharmaceuticals Limited.

Definitions/Abbreviations

N/A

Responsibilities

The roles and responsibility is as follows

Executive/ Senior Executive, Microbiology

Perform operation & cleaning.

Manager, Microbiology

Confirm that operation is carried out properly.

Keep the SOP up to date.

Review that SOP is technically informative & application of sound knowledge.

Head of Quality Assurance

Take initiative to Approval of this SOP

Procedure:

Instructions

• Do not press hardly on counting platform.
• Do not use any concentrate cleaning or disinfection solution to outer surface of machine.
• Do not open petridish during colony counting because all micro-organisms are unscrupulous pathogen.
• Cover with a pack of clear discs to protect receiver plate from dust and scratches.

Operation Procedure

• Turn on unit pressing by <ON/OFF> switch located at back of the unit.
• Select plate receiver background as per requirement either dark or white pressing by side panel.
• Select appropriate adaptor for use of less than 90 mm petridish & place it on the receiver plate.
• Adjust sensitivity turning the control knob at clockwise for increasing and at anticlockwise for decreasing.
• Place covered Petridish on receiver plate, using centering adapter if required.
• Press & hold the <correct/reset> key to ensure display is set to zero before counting.
• Mark on each colony with a felt tip pen. Every time a colony is marked, apparatus will register count with a bleep sound and counter advance.
• Press <correct/reset> key once for removal of each count If unwanted counts are made.
• Place first petridish on receiver plate to use averaging facility.
• At end of the count, press the <save> key to store the count in the memory. This will indicated by three dashes on the display. Replace petridish with next and press <save>key to resume the count. Repeat until all dishes have been counted.
• At the end of the run press the <average> key to display the average count. When average facility is active a red LED spot at the top left hand corner of the display will be visible.
• Press & hold the <correct/reset> key until the display returns to zero when count is completed. This will clear the memory of saved counts.
• Switch unit OFF at mains after all the counting is completed.
• Maintain Colony Counter Log Book, Annexure-I for each operation.

Cleaning & Disinfection Procedure     

• Remove plate from instrument for cleaning the receiver plate,
• Make sure that instrument is switch off position.
• Disconnect power plug.
• Remove petridish from platform.
• Clean outside of the equipment initially with dry cloth.
• Disinfect the outer surfaces with 5% Dettol/Savlon solution.
• Finally clean with Purified water.
• Clean after each work.

Maintenance

Inform to supplier or Engineering Department for corrective action, If Colony counter shows any mechanical, electrical or any others problem.

Download Annexure Here

Annexure I: Colony Counter Logbook

Biosafety Cabinet; Purpose

To establish the ideal operation of Bio-safety Cabinet to confirm that the air are free from any microorganisms & any particle in order to favor microbiological test.

Biosafety Cabinet; Scope

This SOP applies for operation of Bio-safety Cabinet, Model: AC2-4E1 in Microbiology Section at XX Pharmaceuticals Limited.

Definitions

• BSC    : Bio-safety Cabinet
• DOP   : Dioctyl Phthalate
• HEPA : High Efficiency Particulate Air.
• IPA     : Iso Propyl Alcohol.
• PAO   : Poly alpha olefin.

Responsibilities

The roles and responsibility is as follows

Executive, Microbiology

Operation, cleaning & disinfection and performance checking of Bio-safety Cabinet

Manager, Microbiology

• Ensure Operation, cleaning & disinfection, performance checking and application of sound technical information.
• Review that SOP is technically informative.

Head of Quality Assurance

Take initiative to Approval of SOP

Procedure:

Instructions

• Do not wipe HEPA filter side of BSC.
• Do not turn on UV light without full cover of slash.
• Move always gently during operation of BSC to minimize air turbulence.
• Care should be taken that water do not enter into HEPA filter through working station of down flow.

Operating Procedure

• Press UV light button to turn on the UV light.
• Wait for 30 minutes.
• Press light button to turn on.
• Pull up front slash at 50% open position. Open position (%) will be indicated on the display.
• If show alarm, adjust position of slash.
• Press FAN button to start up and wait for 3 minutes.
• After 3 minutes, fan will be automatically started and
• Use 70% ethanol/IPA to wipe all the surfaces of workstation, except in front of HEPA filter.
• Use 70% ethanol to wipe all materials (Glassware’s, containers and other articles) before bringing them inside the Bio-safety Cabinet.
• Start the work.
• Use 70% ethanol to wipe again entire platform after finishing work.
• Press light button to turn off.
• Pull down to cover front slash at 0% open position. Open position (%) will be indicated on the display.
• Press UV light button to turn on the UV light
• Wait for 30 minutes to disinfect the whole cabinet.
• After 30 minutes, press UV light button to turn off.
• Disconnect the power plug of the cabinet.

Performance Check

• Check the performance of HEPA filter by monitoring the Particle count and Microbial count once in a year and prepare the report in Annexure-I & Annexure-II
• Check efficiency of HEPA filter by DOP/PAO test once in six months and prepare the report in Annexure-I & Annexure-II

Maintenance

• Inform to the supplier or Engineering Department for maintenance, if Bio-safety Cabinet shows any mechanical, electrical or any others fault.
• After maintenance, check HEPA filter efficiency & monitor Particle Count and microbial count.

Microscope; Purpose

To establish the procedure for ideal operation & cleaning of Microscope for use in microbiological test.

Microscope; Scope

This SOP applies for operation, cleaning and of Microscope, Model: CX21-LED SET1, Olympus, Japan in Microbiology Section at XX Pharmaceuticals Limited.

Definitions/Abbreviations

N/A

Responsibilities

The roles and responsibility is as follows

Executive/ Sr. Executive, Microbiology

Operation, cleaning and performance checking of Microscope

Manager, Microbiology

Ensure Operation, cleaning & disinfection and performance checking.

Head of Quality Assurance

Take initiative to Approval of SOP

Procedure:

Instructions

• Use 2.5% Dettol/Savlon solution for cleaning of objectives lens, eyepieces and condenser.
• Do not use any concentrate alcohol or any others solution.
• Use only immersion oil on slide.
• Wipe properly objective lens to remove immersion oil at the end of use.
• Do not disassemble the microscope without approval of Head of Microbiology Section.

Operation Procedure

• Wipe entire platform with clean cloth.
• Prepare specimen (stain, if necessary) on a glass slide & place it on stage properly under objective lens with aid of adjustment knob.
• Put on switch for light source.
• Rotating light intensity adjustment knob for increase or decrease brightness clockwise or anticlockwise.
• Adjust objective lens (10x, 40x, 100x) according to requirement.
• Use both coarse focus and then Fine focus for better visualization.
• Focus an area on the specimen, which is clearly defined or not over crowded.
• Do not move specimen holder directly by hand this will damage rotator mechanisms of the knob.
• First focus specimen with 10x then with 45x, and then with 100x objective lens (if necessary).
• After work is done wipe again lens & platform with another clean cloth, separately.
• Cover the microscope at end of the work.

Cleaning & Disinfection Procedure        

• Switch off Microscope & disconnect power plug.
• Remove slide from stage.
• Clean outside of chamber with dry cloth.
• Use 2.5% Savlon/Dettol solution to Disinfect outer surfaces of Microscope
• Clean Objective lens smoothly & Ocular lens with clean, soft, fiber free cloth before and after use.
• Clean objective lens with cider wood oil if found any stain on lens.

Maintenance

If Microscope shows any mechanical, electrical or any others problem, inform to supplier or Engineering Department for corrective action.

Incubator; Purpose

To operate, clean & calibrate incubator in order to support the bacterial growth in microbiological test.

Incubator; Scope

This SOP applies for operation, cleaning and calibration of Incubator, Model : INE500 & INE600 in Microbiology Section At  XX Pharmaceuticals Ltd.

Definitions/Abbreviation

[] CAL.1: Calibration Temperature 1

[] CAL.2: Calibration Temperature 2

[] CAL.3: Calibration Temperature 3

Responsibilities

The roles and responsibility is as follows

Executive/ Senior Executive, Microbiology

• Follow the instructions of this procedure correctly.

Assistant Manager, Microbiology

• Confirm that this procedure is kept up to date.
• Confirm appropriate personnel from the section are trained on this procedure.
• Confirm that SOP is technically sound and reflects the required working practices.

Head of Quality Assurance

Take initiative to Approval of this SOP

Procedure

Instructions

• Do not wipe with damped cloth at on position.
• Use only 70% IPA or Ethanol to wipe chamber with any disinfectants
• Never overload the chamber with tested items.
• Do not keep the items those may produce inflammation with air.
• Keep all tested items to avoid the touch inner surface of the chamber.
• Do not move the incubator at on position. Severe vibrations may cause serious damage of the temperature probes.

Operation Procedure

• To the front of the instrument, Press push/turn control key to put on the main power switch. The incubator will start in normal mode with display of the timer, chamber temperature, alarm temperature (red color indication).
• Turn the push/turn control key by holding down SET key, at the clockwise or anti clockwise for setting date, local time, operating temperature, and alarm temperature. After setting, SET key will be released the display briefly flashes the set point.
• The display then changes to the actual current temperature & starts to the setting temperature. The temperature will be automatically augmented at setting temperature and display the setting temperature digitally.
• Observe display temperature until its stable position.
• Hold down the SET key (Approximately 3 Seconds) to select the operation mode, if require. The current operating mode will be flashed on the display. There are three operating mode in the incubator.
  1. Normal Operation
  2. Weekly Programmer
  3. Ramp time Programme Operation­­­

• Select required programme & set as per operation manual.
• Select fan speed to set air changes.
• Turn the push/turn control at clockwise until the fan symbol flashing to move air slider opens & closes the air valve to control supply and discharge of air.
• Check chamber temperature using by a calibrated digital thermometer, when setting Temperature reaches.
• Keep required materials inside incubator.
• Incubator will automatically control Temperature.
• Instrument will automatically adjust the temperature. When the temperature exceed the setting temperature, “off” light will illuminate and if the temperature decrease the “on” light will illuminate.

Cleaning & Disinfection Procedure

• Switch off incubator & disconnect power plug.
• Remove all test flasks & others items from chamber of incubator.
• Clean inside of chamber initially with dry cloth & wipe finally with 70% IPA wetted cloth.
• Clean outside of chamber with dry cloth.
• Disinfect all surfaces with 5% Savlon solution & dry outer surface of the incubator.
• Reload all items into chamber when reach to dry chamber surface.
• At the end of cleaning, connect the power plug and switch on the incubator.
• Clean chamber once in a month.
• Clean outer surface of the incubator every day.

Performance Check

• Check chamber temperature daily once.
• Record chamber temperature in Daily temperature Record, Annexure-I.
• Temperature range ± 2.5° C from set temperature.

Calibration Procedure

• Press and hold down SET key until normal mode light blinking.
• Turn push/turn control key clock wise to reach SETUP light.
• Select Calibration to rotate push/turn control key.

Use three calibration temperatures as below

• 1 : Temperature calibration at low temperature
• 2 : Temperature calibration at medium temperature
• 3 : Temperature calibration at high temperature
• Select required calibration temperature in SETUP key & set corresponding calibration correction to 0.0⁰
• Measure the deviation from selected calibration temperature under the steady conditions, using a reference instrument.
• Set calibration correction in SETUP key. If measured reference temperature is too low, calibration correction setting is negative sign.
• Adjust temperature if require from adjusting value of calibration temperature (CAL). Correction value should not more than ±10C.
• Carry out check measurement using reference thermometer.
• Carry out others two calibration temperatures in the same manner.
• Calibrate incubator once in a year ± 15 days.
• Record calibration in Calibration Record, as per Annexure-V of Engineering SOP

Maintenance

• If Incubator shows any mechanical, electrical or any others problem, inform to supplier or Engineering Department for corrective action.
• After corrective action, recalibrate incubator.

Download All Annexure Here:

Annexure I Daily Temperature Record of Instrument

Annexure II Incubator Operation Log Book

Laminar Air Flow Operating Procedure; Purpose

To confirm that the air are free from any microorganisms and any particle in order to favor of microbiological test.

Laminar Air Flow Operating Procedure; Scope

This SOP applies for operation and cleaning of Laminar Air Flow, Model: AHC-4D1 in Microbiology Section at XX Pharmaceuticals Ltd.

Definitions/Abbreviations

[] DOP: Dioctyl Phthalate

[] HEPA: High Efficiency Particulate Air

[] LAFWS: Laminar Air Flow Work Station

[] LAF: Laminar Air Flow

[] PAO: Poly-alpha-olefin

Responsibilities:

The roles and responsibility is as follows

Executive/ Senior Executive, Microbiology

Operation, cleaning and disinfection and daily performance check.

Manager, Microbiology

• Confirm Operation, cleaning and disinfection and performance checking.
• Confirm that SOP is technically informative & application of sound knowledge.
• Confirm that Laminar Air Flow is suitable for working.

Head of Quality Assurance

Take initiative to Approval of SOP

Procedure:

Instructions

• Do not wipe HEPA filter side of LAFWS.
• Do not move rapidly in a sweeping motion during operation of LAF to minimize air turbulence.
• Do not use any disinfectant containing chlorine-based substances as this may be corrosive of the stainless steel.
• Do not use Bunsen burner & aerosol generating instruments whenever possible as they interfere with airflow.
• Do not turn on UV lamp without the front cover setting.

Operating Procedure:

• Switch on the main power.
• Set the front cover & make sure that the clean bench is fully closed & the interlock is working effectively.
• Press UV/Exit button to turn on the UV lamp. UV can only be turned on when fan and light both are off.
• Wait for around 60 minutes.
• After 60 minutes, remove front cover from LAFWS.
• Turn on Fan/Up button to start fan.
• Press to turn on Light/Down to illuminate light.
• To turn electrical socket of inner cabin, Press Socket/Set button
• Use 70% Ethanol/IPA to wipe the work surface.
• Before continuing the work, Leave LAF for 10 minutes
• After 15 minutes, wipe all materials with 70% ethanol before bringing them inside the LAF Hood.
• Start the work.
• Work in cleaned bench in a slow & controlled manner.
• Move hands in & out of the work zone opening slowly.
• Use 70% ethanol to wipe again the entire platform after end of the work,
• Press Fan/Up button to turn off the fan.
• Press Light/Down Turns off the light.
• Set front cover and press UV/Exit button to turn on the UV lamp to decontaminate the work bench.
• Switch off the UV/Exit to turn off the UV lamp after 30 minutes.

Cleaning Procedure

Work surface and wall

Clean work surface & walls with appropriate 5% Dettol/ Savlon solution.

Exterior surface

• Use a damp cloth to clean the exterior surface, predominantly on the front & top in order to remove dust that accumulated there.
• Use fresh with Sterile Purified Water (which is sterilized at 121⁰C for 15 minutes or filtered by 0.2 µm) to eliminate any remaining of cleaning agent.
• Use MEK (Methyl-Ethyl-Ketone) solution to eliminate stubborn stains or spots on the stainless steel surface. In such cases, wash the stainless steel immediately afterwards with clean water and liquid detergent. Then use polyurethane cloth or sponge for washing.

Microbiological Performance Check

• Check performance of HEPA filter by monitoring Particle count & Microbial count before conducting sterility test.
• For non-sterile preparation, check performance of HEPA filter by monitoring the particle count & microbial count in every six months.

HEPA Filter Integrity Test

• Check efficiency of HEPA filter by DOP (Dioctyl Pthallate)/PAO(Poly Alpha Olefin) test once in  a year.

Maintenance

• If LAF exhibits any mechanical, electrical or any others difficulty, notify to the supplier or Engineering Department for repairs.

Working Guideline in Microbiology Laboratory; Purpose

To confirm that microbiological best laboratory practice and special precautions are maintained in Microbiology laboratory in order to prevent laboratory & personal contamination.

Working Guideline in Microbiology Laboratory; Scope

This SOP applies for maintaining microbiological best laboratory practices and special precautions in Microbiology Laboratory at XX Pharmaceuticals Limited.

Definitions/ Abbreviation

None

Responsibilities:

The roles and responsibility is as follows

 Executive/ Senior Executive, Microbiology

Maintain microbiological best laboratory practices & special precautions.

Manager, Microbiology

Ensure microbiological best laboratory practices, special precautions and application of sound technical information.

Head of Quality Assurance

Take initiative to Approval of SOP

Procedure:

Media Preparation and Quality Control

Working Guideline in Microbiology Laboratory; Media Preparation

• Choose correct media or components in making media based on the use of accepted sources or references for formula.
• Check Certificate of Analysis describing expiry date, storage conditions etc.
• Read instructions on the label carefully before media selection and preparation.
• Follow if any special instruction such as heating, additives & pH adjustment etc.
• Use always purified water for media preparation.
• Record accurate weight of media & make up volume with purified water.
• Dissolve media thoroughly into water prior to dispensing & sterilization.
• Avoid overheating or less heating media.
• Sterilize the media as per label of the container.

Media Storage

• Label media properly with batch or lot numbers, preparation & expiry dates.
• Store media according to manufacturer’s instructions.
• Store prepared media under validated conditions.
• Do not store agar at or below 0⁰ Because freezing could damage the gel structure.
• Protect stored media from exposure to light & excessive temperature.
• Before prolonged storage, place into a sealed package or container to retard moisture loss.
• Re-melt agar media in a hot water bath or by using free flowing steam only once.

Quality Control Testing of Media:

• Check pH & growth promotion to confirm media efficacy.
• Perform limited growth promotion test for each lot, if media is sterilized using a validated method.
• Do not use media if any parameters do not comply.
• Pre-incubate & 100% inspection prior to use the media.
• Maintain double-wrapped condition for those media used in critical environmental monitoring.

Maintenance of Microbiological Cultures:

• Handle with care microbiological cultures due to their pathogenicity and toxicity.
• Use as standard culture from international recognized organization such as American Type Culture Collection (ATCC), National Collection of Type Culture (NCTC) or any other manufacturer.
• Select Standard culture as freeze dried lyophilized condition in vial or ampoules form or ready to use condition with Certificate of Analysis.
• Ensure identification of culture prior to its use.
• Use only permitted method for culture maintenance.
• Maintain storage and sub-culture as per standard operating procedure for standard culture maintenance.
• Be care to prevent extreme sub-culturing working control cultures that increase the risk of contamination.
• Preserve all typical culture at 2 to 8⁰ 
• Inoculate frozen stock monthly or weekly.
• Discard any unused portion to minimize risk of loss of viability & contamination of the stock.

Maintenance of Laboratory Equipment

• Perform standard validation practices such as Installation Qualification, Operational Qualification and Performance Qualification for equipment.
• Calibrate equipment periodically.
• Check performance of all equipment’s on a routine basis.
• Maintain always approved protocol for IQ, OQ & PQ of equipment’s.
• Clean & disinfect all equipment’s as per standard operating procedure for that equipment.

Laboratory Operation

• Isolate items between sterility testing room & microbial limit test and bio-assay room.
• Do not interchange item between those area without sterilization.
• Sanitize carefully both hands with appropriate sanitizer.
• Before entrance into the test room, wear garments, mask and gloves as appropriate.
• Always use sterile garments, gloves and mask into sterility testing area.
• Aseptically handle the items into microbial limit test room.
• Disinfect all items with 70% IPA or any others suitable sanitizer, before transfer the items into testing room. Open growth plate only into bio-assay room or microbial testing room.
• Aseptically collect the sample.
• Identify contaminants at least genus level.
• Use specific sample area to preserve all samples.
• Isolate all contaminated samples to reduce the false-positive results.
• Follow SOP for disposal of used media to discard all contaminants.
• Clean & disinfect all area according to SOP of cleaning and sanitizing Quality Assurance Department.
• Lessen the movement into microbial testing room and bio-assay room.
• After completion of sterility, exit from sterility testing room.
• Wear another set of sterile dress, gloves, mask If require to re-enter into sterility test room.
• Wash both hands with liquid soap or any skin cleanser solution, after completion of test and before exit from Laboratory, Finally disinfect both hands.

Documentation

• Prepare documents in approved format.
• Preserve documents into specific file up to defined period.
• Review & update all standard operating procedure, specification & analytical Method within defined period.
• Verify the data & calculation.

Maintenance of Laboratory Records

• After completion of each work, prepare record in approved format
• Follow approved SOP to reflect how the test is actually performed.
• Keep calibration record after Perform calibration of any equipment’s
• Preserve all records after approval.
• Do not discard any approved record without permission and proper justification.
• Forward test result of raw materials, in-process & finished products to the respected department.
• Achieve and protect all laboratory record against catastrophic loss.

Spillage Management

Spills:

• Report immediately to the reporting authority after spillages of culture.
• Do not touch any spilled cultures & surrounding debris( e. g. glass , cotton or wool plugs) with exposed hands.
• Wear disposable gloves & disinfect area by covering spill with several layers of paper towel/ cloth soaked in a suitable disinfectant.
• Leave for it for 15-30 minutes.
• Using paper towels, sweep spill debris into dustpan.
• Transfer all disposable materials to a suitable container e.g. an roasting bag/autoclave for autoclaving and disposal.
• Decontaminate dustpan either by autoclaving or by soaking (at least 24 hours) in hypochlorite solution.

Broken glass

• Sweep carefully into a suitable container.
• Dispose in a puncture proof container.

Splashes on clothing and skin

• Soak in disinfectant contaminated cloth.
• Treat to splash on skin as soon as possible.
• Wash thoroughly with soap & finally with hot water.
• Disinfect the skin if necessary.

Validation of Moist Heat Sterilizer; Purpose

To validate Moist Heat Sterilizer by Biological Indicator.

Validation of Moist Heat Sterilizer; Scope

This SOP applies for validation of Moist Heat Sterilizer in Sterile Processing area and Microbiology Laboratory at XX Pharmaceuticals Limited.

Definitions/Abbreviation

Validation

Validation is the process to establish documented evidence, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications & quality attributes.

Biological Indicator

Biological Indicator is a defined preparation of viable spores made from Bacillus stearothermophillus & Bacillus subtilis & has a particular spore count per indicator of not less than 10⁴ and not more than 10⁹ spores which is used to monitor the efficacy of sterilization process.

• BI: Biological Indicator
• ATCC: American Type Culture Collection
• NCTC: National Type Culture Collection

Responsibilities

The roles and responsibility is as follows

Executive/ Senior Executive, Microbiology

Preservation of Biological Indicator, perform Moist Heat Sterilizer validation and report preparation.

Manager, Microbiology

Ensure Moist Heat Sterilizer Validation, documentation and application of sound technical information.

Head of Quality Assurance

Take initiative to Approval of this SOP

Procedure

Instructions

• Biological Indicator is usually non-pathogenic but all microorganisms are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
• Discard all items after autoclave.
• At the end of work, leave all used items at designated containers safely.
• Make sure that all personal ornaments, cell phone are left to prevent unauthorized contamination, before entrance into the test room.

Handling of Biological Indicator

• Check expiry date of Biological Indicator (Bacillus stearothermophilus, ATCC-7953).
• Discard it after autoclaving if expiry date is exceeded
• Observe Certificate of Analysis of Biological Indicator.
• Ensure that the paper strip is fully intact.
• Preserve always BI at 25⁰C or as per manufacturer instructions.
• Do not preserve it at freezer.

Validation Frequency

Perform the validation once in six months interval.

Exposure of Indicator:

• Carry out validation as per test schedule.
• Map the chamber with most critical area for exposure of Biological Indicator.
• Define location & label on each Biological Indicator.
• Place spore strip of Biological Indicator of Bacillus stearothermophilus (ATCC 7953) at 8 points as per chamber mapping, table-1.
• Complete sterilization cycle along with product to be sterilized or empty sterilizer.

Analysis of Indicator

After sterilization, Cut cover paper aseptically & transfer paper strip into separate test tube containing 9 ml of sterile Tryptone Soya Broth including a positive control & negative control.

 

 

Incubation of Indicator:

Incubate all strips including positive & negative control at [50 to 55]⁰C for 7 days.

Interpretation of Result

Observe indicator strip after 7 days for growth. Turbidity of culture media indicates positive growth. Sterilizer is valid if the following criteria are met:

• No growth found in exposed all indicator strip
• No growth found in negative control indicator strip.
• Growth found in positive control indicator strip.

Repeat validation if following result is found

• Growth found in one or more than one exposed indicator strip
• Growth found in negative control indicator strip.
• No growth found in positive control strip.

The sterilizer is invalid if the following result are found

• Growth found in one or more than one exposed indicator strip
• No growth found in negative control indicator strip.
• Growth found in positive control indicator strip.

Correction Action:

• If growth found in anyone indicator strip in repeat test, inform test result to concerned department and Engineering Department for corrective action.
• After corrective action, Engineering Department shall inform to Microbiology Lab. for the repeat test.
• Microbiology Section shall perform the complete test.

Report Preparation

Report the validation in Moist Heat Sterilizer Validation Report, Annexure-I.

Download Annexure Here:

Annexure I Moist Heat Sterilizer Validation Report

Validation of Dry Heat Sterilizer; Purpose

To validate Dry Heat Sterilizer by Biological Indicator & endotoxin Indicator.

Validation of Dry Heat Sterilizer; Scope

This SOP applies for validation of Dry Heat Sterilizer in Sterile Processing area and Microbiology Laboratory at XX Pharmaceuticals Limited.

Definitions/Abbreviation

Validation

Validation is the process to establish documented evidence, which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications & quality attributes.

Biological Indicator

Biological Indicator is a defined preparation of viable spores made from Bacillus stearothermophillus & Bacillus subtilis & has a particular spore count per indicator of not less than 10⁴ and not more than 10⁹ spores which is used to monitor the efficacy of sterilization process.

Endotoxin Indicator

Endotoxin Indicator are designed for monitoring depyrogenation process or validation or which may be measured by comparing the levels of endotoxin before and after a depyrogenation cycle using LAL reagent . USP[United State Pharmacopoeia]  suggests that a depyrogenation cycle should be reduce the endotoxin  by at least 1000 fold ( 3- log reduction ) in endotoxic activity as measured by LAL method.

Endotoxin

It is the component of cell wall of certain bacteria which type of bacteria is known as gram negative bacteria. Endotoxin induce strong immune response and enhance release of cytokine.

[] ATCC: American Type Culture Collection

[] BI: Biological Indicator

[] CSE: Control Endotoxin Standard

[] EI: Endotoxin Indicator

[] λ: Lambda(Lysate Sensitivity)

[] LAL: Limulus Amebocyte Lysate

Responsibilities

The roles and responsibility is as follows

Executive/ Sr. Executive, Microbiology

Preservation of all Indicator, perform Dry Heat Sterilizer validation & report preparation.

Manager, Microbiology

Ensure Sterilizer Validation, documentation and application of sound technical information.

Head of Quality Assurance

Take initiative to approval of this SOP

Procedure:

Instructions

• Biological Indicator is usually non-pathogenic but all microorganisms are unscrupulous pathogen. So before handling it, wear protective items such as sterile wear sterile latex free gloves, laboratory coat & eye protection (if required).
• CSE[Control Standard Endotoxin] is pyrogenic in humans. Care should be exercised when handling to avoid ingesting it.
• Use caution if handling hot vials. Wear gloves or wait until vials are cool.
• Before transferring BI[Biological Indicator] from Microbiology Laboratory, disinfect whole surface of all apparatus with the help of 70% IPA or any others disinfectants.
• Leave it at designated containers safely.
• Ensure that waste container is tightly capped until autoclaving.

Handling of Biological Indicator & Endotoxin Indicator :

• Use BI[Biological Indicator] for sterilization cycle validation & EI[Endotoxin Indicator] for Depyrogenation cycle validation.
• Read insert package carefully for instruction of use.
• Check expiry date of Biological Indicator (Bacillus subtilis, ATCC-9372) and Endotoxin Indicator.
• Discard it after autoclaving if expiry date is exceeded.
• Observe Certificate of Analysis of all indicators.
• Ensure BI[Biological Indicator] paper strip & EI [Endotoxin Indicator] vial is intact.
• Preserve always BI at 25⁰C and at [2 to 8]⁰C for EI.
• Do not preserve it at the freezer.

Culture Media Sterilization :

• Prepare required amount of CSDM [Casein Soyabean Digest Medium] for the test & distribute [15 to 20] ml of medium in different test tubes.
• Sterilize media at 121⁰C for 15 minutes.
• Preserve those at [2 to 8]⁰C for use.

Validation Frequency

• Perform the validation once in six months.

Exposure Condition:

• Carry out validation as per test schedule.
• Map chamber with most critical area for exposure of BI[Biological indicator] or EI[Endotoxin Indicator].
• Define location & label each BI or EI.
• Place EI at 4 points & BI at 12 points as per chamber mapping, mentioned on Table-1.
• Complete sterilization cycle along with product or empty sterilizer.
• After sterilization, transfer all indicators to Microbiology Laboratory.

BI[Biological Indicator] Assay

• Perform whole analysis under Laminar Air Flow workstation.
• Aseptically open envelopes of BI[Biological Indicator] test strips.
• Place each test strips including negative control and positive control strips in individual tubes containing [15 to 20]ml of TSB.
• Identify all tubes.

Incubation of Biological Indicator

• Incubate the BI strips at [30⁰ to 35]⁰C for 7 days.

Interpretation of Result

Observe BI[Biological Indicator] tube daily for growth. Growth should occur in positive control tube within 48 hours. The turbidity indicates positive growth of tubes. Sterilizer is valid if following criteria are met:

• No growth found in exposed all tubes.
• No growth found in negative control tube.
• Growth found in positive control tube.

Repeat validation if following result is found :

• Growth found in one or more than one exposed tube.
• Growth found in negative control tube.

Sterilizer is invalid if following result are found

• Growth found in one or more than one exposed tube.
• No growth found in negative control tube.
• Growth found in positive control tube.

EI Assay Procedure of LAL Test(Gel Clot Method) :

• Use the LAL reagent sensitivity 0.125 EU/ml for this assay.
• Reconstitute each EI vial with 1.0 ml LAL water including Positive control & vortex at least five minutes and then dilute 1:8 by using LAL water at least duplicate.
• Make a control series (2 λ, λ, λ/2, λ/4) if a new lot of Endotoxin indicator or LAL reagent is used. Otherwise test at λ (Lysate sensitivity) only.
• Carry out LAL test for exposure vials, positive control and negative control as per Standard Operating Procedure for Bacterial Endotoxin Test.

Incubation of Indicator

• Incubate all LAL test tubes including positive & negative control at [37±1]⁰C for [60±1] minutes.

 

Interpretation of Result

• A more exact calculation of endotoxin reduction is made by finding the end-point of the positive control, by ten-fold & then two fold dilution, & subtracting the logarithms of the exposed vial from the logarithms of the positive control.

Use the logarithm of lamda for the endotoxin concentration in exposed vials when there are negative LAL test results for the undiluted solutions.

The sterilizer is valid if the following criteria are met

• No clot formed in all exposed tube
• No clot formed in negative control tube
• Clot formed in positive control tube

Repeat validation if the following result is found

• Clot formed in one or more than one exposed tube
• Clot formed in negative control tube
• Not clot formed in positive control tube

The sterilizer is invalid if the following result is found

• Clot formed in one or more than exposed tube
• No clot formed in negative control tube
• Clot formed in positive control tube

Corrective Action:

• If endotoxin found in anyone exposed vial in the repeat test, inform the test result to concerned department & Engineering Department for corrective action.
• After corrective action, Engineering Department or concerned department shall inform to Microbiology Lab. to perform the validation again.
• Microbiology Section shall perform the complete test.

Report Preparation

Report the validation in Dry Heat Sterilizer Validation Report by Biological Indicator, Annexure-I and Dry Heat Sterilizer Validation Report by Endotoxin Indicator, Annexure-I

Download All Annexure Here:

Annexure I:  Dry Heat Sterilizer Validation Report by Endotoxin Indicator

Annexure II: Dry  Heat Sterilizer Validation Report by Biological Indicator

Micropipette; Purpose

To initiate that the procedure is suitable for ideal operation, cleaning and calibration of Micropipette for use in microbiology test.

Scope

This SOP applies for Operation, Cleaning & Calibration of Micropipette, Model: 8-105-00-9 & 8-106-00-9 in Microbiology Section at XX Pharmaceuticals Limited.

Definitions/Abbreviations

N/A

Responsibilities

The roles and responsibility is as follows

Executive/ Senior Executive, Microbiology

Operation, Cleaning and Calibration of Micropipette.

Manager, Microbiology

• Ensure Operation, Cleaning, Calibration and application of sound technical information.
• Review that SOP is technically informative.

Head of Quality Assurance

Take initiative to Approval of SOP

Procedure:

Instructions

• Keep Micropipette always at vertical position on the respective stand.
• Never sink Micropipette into water.
• Never reset or adjust the volume.
• Care should be taken during pipetting that solutions do not enter into Micropipette.
• Do not try to volume highly viscous solution by this device.
• Do not use hot solution in this device.

Operation Procedure

• Check is calibration status of Micropipette. If Micropipette is not calibrated, calibrate it before use.
• Set desired volume by turning red colored setting screw button at clock wise for increasing and anticlockwise for decreasing the volume.
• Set micropipette tips on bottom side in tight condition.
• After setting desired volume, observe display digit.
• Draw desired volume of solution by pressing red colored setting screw in single punch.
• Rinse tips least three times with solution at first time.
• Deliver total volume by pressing same button at double punch.
• Draw total volume of the solution.
• Press setting screw for delivery the solution.
• After use, always keep it on the stand at vertical position.

Cleaning Procedure

• Clean outer surface with dry cloth.
• Use 70% IPA solution to disinfect outer surfaces except display of Micropipette.
• Clean Micropipette before & after use.

Calibration Procedure

• Set Micropipette reading as 100 µL for Model: 8-105-00-9 & 500 µL for Model: 8-106-00-9.
• Set tips with Micropipette properly.
• Take distilled water of temperature at 250C±10C into a beaker.
• Weigh & tare another 100 ml beaker.
• Draw 100 µL or 500 µL of distilled water by Micropipette & deliver it into tarred beaker.
• Record weight of distilled water.
• Repeat same procedure up to nine times.
• Calculate the accuracy(% of error) & Precision(% CV) as below :
• Accuracy (% error) = Mean Value – Reference value/Reference value x 100
• Precision (% CV) = Standard deviation/mean x 100.
• Record the result in Micropipette Calibration Record, Annexure-I.
• Calibration Frequency: Perform the calibration once in a year.

Maintenance

If Micropipette shows any error or any mechanical fault, inform to the supplier or Engineering Department for maintenance, after repairing or maintenance, recalibrate it before use.

Download Annexure Here:

Annexure I Micropipette Calibration Record

Antimicrobial preservative effectiveness test; Purpose

Antimicrobial preservative effectiveness test; To confirm that the concentration of Antimicrobial Preservatives used in different drug preparations are able to kill or destroy or inhibit or prevent or terminate the growth of microorganisms and thus make the product stable throughout its declared shelf life.

Antimicrobial preservative effectiveness test; Scope

This SOP is applicable for Oral liquids tests at Microbiology Laboratory in XX Pharmaceuticals Ltd.

Definition/Abbreviation:  

Preservative:

Antimicrobial preservatives are the substances added to different dosage forms or products to protect or defend them from microbial growth or from different microorganisms that are introduced unintentionally during or succeeding in the manufacturing process.

[] ATCC: American Type Culture Collection

[] SDA: Sabouraud Dextrose Agar

[] TSA: Tryptone Soya Agar

Responsibilities:

The roles and responsibilities are as follows:

Lab Attendant

Sample collection

Executive/ Sr. Executive, Microbiology

Sample collection, analysis of preservative test & respective test report preparation.

Manager, QC/Microbiology

• Ensure sampling, analysis, documentation & application of sound technical information.
• Review of this SOP and confirm that the whole procedure is technically sound.

Head of Quality Assurance

• Take initiative to Approval of this SOP.
• To ensure the overall implementation of this SOP

Procedure:

Instructions

• Use Bio-safety Cabinet to Perform tests of Antimicrobial preservative effectiveness of products.
• Before preparation of media and conduct antimicrobial preservative effectiveness test, Wear a mask, Hand gloves and headgear Bring the media to the boil to dissolve completely before autoclave.
• Mix well before pouring the substances.
• Cool the broth media at room temperature (25⁰C) and the agar media at 50⁰C & for use as per the requirement of the test.

Media & Biochemical Preparation

As per Media preparation for Media, preparation SOP prepare the same.

Inoculum Preparation:

• Remove the vial of pellets from refrigerated storage & allow equilibrating to the room condition.
• Warm hydrating & diluting fluids to [34 to 38]⁰C, before use.
• To achieve a concentration of about 10⁸ cfu per ml, transfer pellets to hydrating fluid.
• Immediately place the microbial suspension into a [34 to 38]⁰C incubator for 30 minutes
• To assure complete hydration, immediately following incubation, vortex the hydrated material to achieve a homogenous suspension.

Inoculation of Microbial Suspension to Product

• To give inoculums of 10⁵ to 10⁶ microorganisms per ml of product and mix well, inoculate the product to be examined, each with a suspension of the test organisms.
• The volume of the suspension of inoculums does not exceed 1 percent of the volume of the product.
• Maintain the inoculated product at the temperature range from [20 to 25]⁰C and protected it from light.
• According to the type of the product, Remove 1 ml sample from each container at suitable intervals & determine the number of viable microorganisms using the Pour plate method.

Suitability of counting method for non-sterile products

As per suitability of counting method for suitability of Microbial count, method SOP perform the same.

Test Sample Preparation

• Aseptically accurately measured 1 ml sample to be transferred from each inoculated product container to a market sterile capped dilution tube containing 9 ml of sample diluent & mix well. The ratio of this dilution is 1:10.
• Aseptically accurately measured 1 ml sample to be transferred from 1:10 dilution to a second dilution tube containing 9 ml of sterile diluent and mix well. This is the ratio of this dilution is 1:100.
• Continue this dilution up to 10-5 or as essential levels.

Plating and incubation:

• Take two sterile petri plates then Take 1 ml quantity sample from each dilution.
• Maintain the temperature not more than 45⁰C, then For Bacterial count pour 15 to 20 ml of sterile TSA medium into each plate being at and mix well.
• Maintain the temperature not more than 45⁰C, For Fungal count, pour 15 to 20 ml of sterile SDA medium into each plate being and mix well.
• Pour two plates with TSA & SDA medium each with 1 ml diluents as the negative control.
• After solidification incubate them at [20 to 25]⁰C for 5 to 7 days for the fungal count and [30 to 35]⁰C for 3 to 5 days for the bacterial count.

Acceptance Criteria

• For Oral Liquid & Other than Antacids
• For Bacteria (S. aureus, E.coli & P. aeruginosa)
• Not less than 1.0 log reduction from the initial count at 14 days.
• No increase from the 14 days counts at 28 days.

For Fungi (C. albicans, A. brasiliensis)

• No escalation from the initial calculated count at 14 and 28 days.

For Oral Liquid Antacids Preparation

• For Bacteria (E.coli, P. aeruginosa, S. aureus)
• No upsurge from the initial calculated count at 14 and 28 days.

For Fungi (C. albicans, A. brasiliensis)

• No increase from the initial calculated count at 14 and 28 days.

[Reference of acceptance criteria: USP 41]

Download All Annexure Here:

Annexure I: Preservative Efficacy Test Report Oral Liquid Preparation
Annexure II: Preservative Efficacy Test Report Oral Liquid Antacids Preparation