1.0 OBJECTIVE:

The protocol is designed to validate that any Bacteriostasis and Fungistasis activity inherent in the products; to be tested does not adversely affect the reliability of the test and the test procedure to be followed is suitable for testing the products as per the pharmacoepial methods.

2.0 SCOPE:

The execution of protocol shall be done in the microbiology lab of QC department.

6.0 PROCEDURE (METHODOLOGY):

6.1 Overview of the Procedure:

6.1.1 Perform the sterility method suitability test at least three independent replicates of the higher strength batches or whenever there is a significant change in the experimental conditions of the test and whenever there is change in the process of manufacture of the product.

6.1.2 In case of new product perform the sterility test before performing the sterility method suitability test or simultaneously with the test. The suitability of the test method shall be established preferably at the product development stage. If development facility is not available, then test should be stabilized during exhibit batches before manufacturing of commercial batches.

6.1.3 When sterility method suitability test are performed simultaneously, sample shall be release based on the data of first conclusive method suitability trial.

6.1.4 Interim summary report shall be prepared after every trial of product.

6.1.5 Summarize the method suitability data after completion of initial trials and conclusions shall be referred during routine testing and/or regulatory submission. Finding of trials and modifications done during study should be documented with appropriate rationale and conclusion.

6.1.6 Perform the sterility method suitability test of the product as per respective GTP (Current version of the GTP to be used).

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1.0 PURPOSE

This protocol is designed to establish sufficient data, to assure that the Vertical Autoclave is suitable for Sterilization of the defined load patterns when operated in accordance with the established standard operating procedure.

2.0 SCOPE

This Performance Qualification Protocol is applicable for the Vertical Autoclave installed in Microbiology section

5.0 VALIDATION MATRIX

5.1 Frequency of periodic monitoring is 1Year ± 30Days

5.1.1 Following test matrix is prepared for the requalification  of Vertical Autoclave.

Note: Incase of new load introduction one developmental run shall be taken.

Note: - Any Change in Load pattern/process/items three consecutive run are required successful validation.

5.2 Pre-validation test requirements:

5.2.1 Following instruments shall be required for the validation of Vertical autoclave used in the Microbiology Area.

• Calibrated data loggers

• Calibrated Temperature mapping probes.

• Geobacillus stearothermophillus i.e. Biological Indicator Strips/Ampoules

5.2.2 The autoclave shall be qualified after verification of the documented evidence (obtained as per the methods outlined in this protocol) assuring that the equipment is meeting the desired performance attributes consistently.

6.0 Empty Chamber Heat Distribution Studies

6.1 Objective

• The Autoclave is capable of attaining a temperature of 121.0 °C during the sterilization hold period of 15 minutes.

• Analyze the temperature profile and identify the cold spot

6.2 Proedure

• Set the temperature of 122°C for 15 minutes sterilization cycle.

• Prepare at least 08 Nos. calibrated temperature mapping probe with location and channel tag.

• Pass 08 No. temperature mapping probe into chamber through the validation port of the Autoclave.  Seal the port with silicone sealant so that steam leakage does not take place.  Suspend the probes in the chamber in different position as per the drawing no. 1 and ensure that probes do not touch any metallic surface.

• Connect the probes to a suitable data logger, which can scan and print the actual temperature observed at different locations with respect to time.

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1.0 OVERVIEW OF THE STUDY

The 70% IPA Self life Study is based on the ability to reduce the microbial population within 10 days study. Here the known concentration of microorganism is mixed with the disinfectant of respective Concentration. And the recovery of the microorganism is checked at 10 Days.

2.0 OBJECTIVE:

The Objective of this validation protocol is to increase self life of 70% IPA (Isopropyl alcohol) disinfectant solution used for sanitization/cleaning purpose in the company.

3.0 SCOPE:

This protocol is applicable to the 70% IPA solution (disinfectant solution) used for sanitization/cleaning at the site.

7.0 PROCEDURE (METHODOLOGY):

7.1 Preparation of solution

      70 % IPA: Take 70 ml IPA and Add 30 ml Water to Make 100 ml Solution.

7.2 Requirement:

• 70% IPA solution,

• Normal Saline (0.9% NaCl solution),

• SCDA (Soyabean casein digest agar),

• SDA (Sabouraud Dextrose Agar)

• Glassware’s

• Microbial Culture suspension of

• E.Coli ATCC ,

• P.aeruginosa ATCC,

• S.aureus ATCC,

• C.albicans ATCC.

• Incubator 20-25° & 30-35°C

7.3 Preparation

7.3.1 After incubation observe the plate and record the count in the annexure & calculate each Microorganism record the same.

7.3.2 Incubate the SCDA plate at 30-35°C for 5 days and SDA plates at 20-25° for 5 days.

7.3.3 Sample: Take Petri dish and Add 1 ml 70% IPA solution and 1 ml of culture suspension and pour 20 ml SCDA cooled 45°C & SDA cooled 45°C.

7.3.4 Positive control – Take Petri dish and add 1 ml of culture suspension and pour 20 ml SCDA cooled 45°C & SDA cooled 45°C.

7.3.5 Now carry out the analysis as routine practice.

7.3.6 Carry out serial dilution of each microorganism up 10⁵.

7.3.7 Further dilute this solution upto of respective microorganism till solution will give the final concentration of 10⁵ cfu/ml,

7.3.8 Take 1 loopful of Respective microorganism Slant and add 9 ml normal saline to make volume 10 ml. (stock solution)

7.3.9 Prepare SCDA and SDA solution as per their respective preparation method & sterilize it.

7.3.10 Prepare Normal saline solution (0.9% NaCl solution): Take 0.9 g Sodium chloride and make volume up to 100 ml purified water.

7.3.11 Store all this solution to ambient lab temperature

7.3.12 Prepare 3 different 1-liter sample of 70% IPA solution.

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1.0 OBJECTIVE:

The purpose of this Method validation protocol is to established the assurance that the method and material for the Bacterial Endotoxin Test meets the requirements and acceptance criteria.

Successful completion of this method validation will provide the necessary documented evidence to assure that the method and material used in Bacterial Endotoxin Test meets the criteria in accordance Manufacturing site procedures and complies with all cGxP requirements.

2.0 SCOPE:

This method validation protocol shall be applicable for validation of procedure for Bacterial Endotoxin Test of injectable and ophthalmic preparations by Gel Clot Method at Manufacturing site. The procedure for testing Bacterial Endotoxin by gel clot method as described in respective SOP and shall be validated as per the procedure described in this SOP.  This SOP includes the requirements for the test as per BP/EP and USP.

3.0 PURPOSE:

The purpose of this protocol is to describe the test Procedure to be evaluated in order to validate the method of Bacterial Endotoxin Test by gel clot method.

7.0 Test parameters to be evaluated:

7.1 Determination of Non-Interfering Dilution (NID)

7.2 Enhancement / inhibition test

8.0 Method verification approach:

As the method not validated, three run of method validation on non-interfering dilution which is less than Maximum Valid Dilution shall be carried out to verify the method suitability.

9.0 Procedure:

9.1 Preparation Control Standard Endotoxin:

9.1.1 Reconstitute the Lyophilized CSE with required quantity of LRW (as mentioned in the COA) to obtain mentioned concentration in EU/ML.

9.1.2 Prepare a series of two-fold dilutions of the Control Standard Endotoxin in LAL reagent Water to give concentrations of 4l, 2l, l, l/2, and l/4, where, l is the labeled lysate sensitivity.

9.1.3 Store the reconstituted vial of CSE, as per the instruction given by CSE manufacturer.

NOTE: Always ensure for confirmation of labelled lysate sensitivity has been performed for any new lysate or CSE received.

9.2 Calculation of Maximum Valid Dilution (MVD):

9.2.1 Maximum Valid Dilution (MVD) is the maximum acceptable dilution of a product for the BET resulting in detection limits not exceeding the product specific Endotoxin Limit.

9.2.2 Calculate MVD using the following formula,

MVD = (Endotoxin limit × Concentration of sample solution) /(l)

Where, l is the labelled lysate sensitivity.

                   Endotoxin Limit X Concentration of sample

   MVD = ---------------------------------------------------------------

                                               Lysate Sensitivity

                                 0.4EU/µg X 3000µg/mL

For Ex.:  MVD = - ------------------------------------------- = 9600

                           0.125 EU/ml

Prepare sample dilution for MVD/128 and dilute up to MVD/2 as per the dilution pattern given below,

9.3 Dilution Scheme for Sample:

Preparation of sample shall be done as per sample requirement.

For example: If a sample concentration is 3000µg/mL than consider it as solution A.

Perform further dilution at or less than 1:4800 dilution with LRW

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1.0 OBJECTIVE:

The purpose of this Method validation protocol is to established the assurance that the method and material for the sterility testing meets the requirements and acceptance criteria.

Successful completion of this method validation will provide the necessary documented evidence to assure that the method and material used in sterility testing meets the criteria in accordance manufacturing site procedures and complies with all cGxP requirements.

2.0 SCOPE:

This method validation protocol shall be applicable for validation of procedure for sterility testing by Membrane Filtration Method at Manufacturing site. The procedure for testing sterility by membrane filtration method as described in respective SOP and shall be validated as per the procedure described in this SOP.  This SOP includes the requirements for the test (bacteriostasis and fungistasis) as per IP/BP/EP and USP.

3.0 REASON FOR VALIDATION:

To evaluate the correct material and method for the sterility testing Diclofenac Injection 25mg/3ml

7.0 PRINCIPLE:

7.1 Sterile injectable and ophthalmic preparations which can be filtered through a membrane filter are tested for their sterility by Membrane Filtration Method. The preparation under examination is filtered through a membrane filter capable of retaining bacteria, yeast and molds. The membrane is flushed to remove any traces of preparation. One half of the membrane is then incubated with a culture medium primarily supporting growth of anaerobic bacteria and aerobic bacteria and also the other half is incubated with a culture medium primarily supporting growth of fungi and aerobic bacteria for a specified duration and at a specified temperature. No development of any visible evidence of microbial growth in these media until the end of incubation indicates sterility of the preparation under examination.

7.2 Each prepared lot of culture medium which is used for testing sterility of preparations by Membrane Filtration Method is evaluated for its sterility and growth promoting properties before, or in parallel, with the test of sterility on the preparation under examination. However, it is necessary to evaluate the performance of these culture media under the conditions of the test to ensure that is satisfactorily eliminated This is to ensure continuation of Growth Promoting Properties of culture media under the conditions of the test by Membrane Filtration Method and thereby to validate the test procedure.

7.3 The preparation undergoing sterility testing by Membrane Filtration Method as explained in 5.1 is diluted and then filtered through a bacterium retaining membrane filter. The filter is then flushed with a flushing fluid (0.1% w/v solution of bacteriological peptone) to remove any traces of drug preparation from the membrane filter.  The membrane filter is then aseptically cut into two parts and each part of membrane is placed in two different culture media (SCDM/FTGM).

7.4 Stasis test:

The stasis test is intended to demonstrate that the media inoculated with the test preparation will support growth for the full incubation period. It is also demonstrating that growth promoting qualities of media are retained and that preservative inhibitors (if any) remain stable for the full test period.

8.0 FREQUENCY OF VALIDATION OF STERILITY TESTING PROCEDURE:

8.1 The validation of testing procedure for sterility testing by Membrane Filtration Method shall be performed: -

1. When the test for sterility by Membrane Filtration Method is to be performed on a new product.
2. Whenever there is a change in the formulation with respect to variation in any of the component or variation in the process of manufacturing.
3.  Whenever there is a change in the experimental conditions of the test.

8.2 The validation of test procedure for sterility testing by Membrane Filtration Method as described in this Standard Operating Procedure shall be performed three exercises. However, if feasible, the said validation shall be performed on three different lots of drug product under examination.

8.3 Stasis Test: If the media are found to support growth of the test microorganisms, then this test need not be applied to every sample. It should be repeated periodically on the relevant categories of products or when product is reformulated.

9.0 PROCEDURE FOR VALIDATION OF TEST METHOD:

9.1 Development of method for Method suitability test:

9.1.1 Prior to initiate method suitability test, trials to be taken for method development to finalize following parameters:

1. Method of analysis (Open membrane filtration method)
2. Media, dilution fluid
3. Type, make and catalogue number of membrane filter / sterility test canister

Record the observation in Annexure I, titled “

9.2 Method suitability test for sterility test:

9.2.1 Prepare suspensions of the following microbial cultures as per the procedure described in SOP “Procedure for Preparation and Preservation of 10-100 cfu’s /0.1 ml of culture suspension”.

a) Aerobic Organisms:

Staphylococcus aureus ATCC 6538

Bacillus subtilis ATCC 6633

Pseudomonas aeruginosa ATCC 9027

b) Anaerobic Bacterium:

Clostridium sporogenes ATCC 19404 / ATCC 11437

c) Fungi:

Candida albicans ATCC 10231

Aspergillus brasiliensis ATCC 16404

9.2.2 Media going to use in sterility testing should be GPT qualified.

9.2.3 Preparation of Set I (Absence of Test Preparation):

9.2.3 Perform all the steps of the test for sterility by Membrane as per SOP “Procedure for sterility testing of injectable and ophthalmic preparations by Membrane Filtration Method”.

9.2.4 Thus, aseptically filter through the specified membrane filter the specified quantity of diluting / neutralizing fluid, apply the vacuum, flush the membrane with the flushing fluid (0.1% w/v solution of bacteriological peptone) two times each with the specified volume of flushing fluid and a third time with the specified volume of flushing fluid to which has been inoculated Not More Than 100 CFUs of one of the following microbial cultures shown in section 6.2.3.3. and follow the below procedure.

1. Open membrane filtration method:

Aseptically cut the membrane filter into two parts and transfer one half of membrane in specified volume of Fluid Thioglycollate Medium and other half in specified volume of Soyabean Casein Digest Medium.

9.2.5 In a similar manner as above prepare Set I of containers separately containing different microbial cultures as follows:

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1.0 OBJECTIVE:

The purpose of carrying out Performance Qualification of HPHV Steam Sterilizer is to establish documented evidence that the equipment is capable to consistently perform when operated as per Standard Operating procedure in accordance with manufacturer operation manual, thereby establishing its reproducibility. Successful completion of this protocol and approval of the validation report will conclude that the Steam Sterilizer meets all the acceptance criteria and is ready for its intended use.

2.0 SCOPE:

The scope of this validation is limited to the validation of HPHV Steam Sterilizer process for different type of sterilization loads.

5.0 PERFORMANCE QUALIFICATION TEST PLAN

5.1 Performance Qualification Test Matrix

1.0 OBJECTIVE:

The objective of this protocol is to establish the hold time study for machine parts, rubber stoppers and seals load stored under LAF unit in aseptic storage area after sterilization in autoclave.

2.0 SCOPE:

This protocol is applicable for the establish the hold time of the machine parts, rubber stoppers and seals load after completion of autoclave sterilization in Production Area.

5.0 PRE-REQUISITES

• Ensure the training shall be imparted to participants.
• Ensure the machine parts and rubber stoppers are prepared and sanitized as per respective SOP.
• Ensure the machine parts and rubber stoppers are validated.
• Ensure the machine parts and rubber stoppers are prepared and sanitized as per respective SOP.
• Ensure the seals load is validated.
• Readiness of sampling aids..

5.1 Equipment/ material use for the study

• Sterilizer.
• LAF.
• Machine parts accessories.
• Rubber stoppers and seals.
• Swabs.
• Mobile LAF

6.0 Trainings

The validation team member shall be trained on the protocol execution of activity and report compilation.

7.0 PROCEDURE (METHODOLOGY):

7.1 Sterilization and sampling procedure for machine parts and rubber stoppers:

7.1.1 Procedure:

7.1.1.1 Clean and sanitize machine parts as per respective production SOP.

7.1.1.2 Load the machine parts and rubber stoppers load as per validated load pattern in sterilizer trolley.

7.1.1.3 After completion of sterilization cycle, unload the machine parts and rubber stoppers load aseptically from autoclave.

7.1.1.4 Store the sterile item under mobile LAF in sterilize material autoclave unloading area.

7.2 Sterilization and sampling procedure for seals load:

7.2.1 Procedure:

7.2.1.1 Clean and sanitize of seals as per respective production SOP.

7.2.1.2 Load the seals load as per validated load pattern in sterilizer trolley

7.2.1.3 After completion of sterilization cycle, unload the seals from autoclave aseptically.

7.2.1.4 Store the sterile item under mobile LAF in sterilize material holding area.

7.3 Surface Swab Sampling for Bioburden Testing

7.3.1 Procedure:

7.3.1.1 Take the sterile saline test tubes and the pre-sterilized cotton swabs.

7.3.1.2 Transfer the swabs sampling kit to the area to be sampled for swab test as per procedure outlined in respective SOP.

7.3.1.3 Collect the swab samples from the predefined swab location of machine parts by covering 5 × 5 cm² of the part as per the swabbing pattern given in the standard operating procedure and put the swab inside the test tube consider this sample as initial 0 hrs. sample.

7.3.1.4 Gently swab 25 cm² of the surface of articles and for uneven/ crooked surfaces, an approximation shall be done.

7.3.1.5 Swab out 5X5 cm² area in the direction specified in figure below. Use sterile stainless-steel template to measure the area of sampling whenever applicable

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1.0 OBJECTIVE

To validate the calculation sheets, prepared by using Microsoft Excel, used in calculations of analytical results in quality control laboratories.

2.0 SCOPE

The scope of this protocol is to verify the validation of calculation sheets, prepared using Microsoft Excel for calculations of analytical results as per requirement.

10.0 Attchements:

Annexure-I: Excel calculation sheet validation form

Annexure-II: Change Required Form for Excel Sheet Validation

Annexure-III: Calculation Sheet for Excel Sheet Validation

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1.0 OBJECTIVE:

The objective of this protocol is to lay down a procedure for establishing the hold time period for prepared dehydrated media during which the media remain stable under predefined environmental conditions.

2.0 SCOPE:

The validation protocol is applicable to all the microbiological culture media (15 Numbers) used in the microbiology laboratory

5.1 Media Check Parameter:

Parameters to be evaluated in this study are

1. physical checks,
2. pH measurement
3. Growth promotion & inhibitory

5.2 Equipment/Instrument details:

Main Requirements in this study is:

• Microbiological culture media
• Microbial culture suspensions
• pH meter
• Autoclave
• Water bath
• Laminar air flow
• Inoculation loop
• Spreader
• Sterile Petridish and test tubes
• Micropipette and sterile tips.
• BOD Incubators
• Colony counter

5.3 Required Media and Microbial cultures:

The following Media to be studied:

• Soyabean Casein Digest Broth (SCDM)
• Soyabean Casein Digest Agar (SCDA)
• Sabouraud Dextrose Agar (SDA)
• Manitol salt Agar (MSA)
• Cetrimide agar (CA)
• Rappaport Vassiliadis Salmonella Enrichment medium (RVSEM)
• Xylose Lysine Deoxycholate Agar (XLDA)
• Reinforced Clostridial Broth (RIMC)
• Columbia Agar (COA)
• Sabouraud  Dextrose Broth (SDB)
• EE broth Mossel (EEBM)
• Violet Red Bile Glucose Agar (VRBGA)
• Plate Count Agar (PCA)
• MacConkey Broth (MCB)
• MacConkey Agar (MCA)
• R2A Agar      
• Escherichia coli ATCC 8739
• Staphylococcus aureus ATCC 6538
• Pseudomonas aeruginosa ATCC 9027
• Salmonella typhimurium ATCC 14028
• Bacillus subtilis ATCC 6633
• Clostridium sporogenes ATCC 11437
• Micrococcus luteus ATCC 9341
• Candida albicans ATCC 10231
• Aspergillus brasiliencis ATCC 16404

6.0 Procedure:

The study shall be applicable for broth and solid media as follows:

• Media shall be prepared as per the (SOP-QC-9A40-R6) and recorded in Media preparation record.
• Distribute the liquid media in sterile test tubes and pour plates of the selective media prepared.
• All the media test tubes and plates shall be labeled with Name of the medium, Date of preparation, In-house lot number of the media and signature.
• Store the prepared media at 20-25° C throughout the testing period.
• Test the broth media at intervals of 1, 3, 5, 7, 10, 12, 15, 18, & 20 days.
• Test the solid media at intervals of 1, 3, 5, 7, 10, 12, 15, 18 & 20 days.

6.1 pH Measurement:

• Check the physical appearance of broth and solid media and record the observations in Annexure-1.
• Check the pH at pre defined interval up to 20 days of the broth media.
• Check the pH at pre defined interval up to 20 days of the solid media.
• Record the pH data in Annexure 2.
• Note:
• If the day of testing interval is on weekly off/ holiday the testing should be carried on next working day.

  6.2 Growth promotion test and inhibitory test:

• Prepared medium must be inoculated with  microbial inoculums of less than 100 cfu for testing growth promoting properties and not less than 100 cfu for testing inhibitory properties wherever applicable (Refer Annexure- 4).
• Growth promotion testing (GPT) of the media should be done with the microorganisms as listed in Annexure -4. Perform GPT by surface spread technique.
• Maintain negative control of each lot of media prepared and incubate accordingly.
• For enumeration media (i.e. SCDA, SDA, R2A and PCA) perform test by melting the media in water bath on the day of test.
• Record the observations of growth promotion and inhibitory tests in Annexure -3.

6.3 Acceptance Criteria:  

• Media plates should not exhibit dryness or shrinkage.
• pH of the media should be within limits as mentioned in Annexure-2.
• For enumeration media, growth obtained must not differ by a factor greater than 2 from the calculated value obtained for a standardized Inoculum.
• For selective media, characteristic growth of microbial colonies should be observed. as mentioned in Annexure -4.
• No growth should be observed in the media plates and tubes tested for showing inhibitory properties of the media.
• No growth should be observed in the Negative control.

Note:If any test parameter fails to comply with its specification then testing on remaining intervals need not be performed.

7.0 Abbreviations:

• cfu: Colony Forming Unit
• ATTC : American type Culture Collection

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