Standard Operating Procedure : VALIDATION OF STEAM STERILIZATION

Standard Operating Procedure : VALIDATION OF STEAM STERILIZATION

Purpose:
• It is established for the proper validation of the sterilization process.
2. Scope:
• It is applicable to microbiological Lab.
3. Responsibilities:
• GM Production
• Quality Control Manager
• Microbiologist.
4. Procedure:
• Biological indicators to be used:
 Strain of bacillus stearothermophillus ATCC 7953Ø
• Take 3-4 ampoules of above mentioned biological indicator in container in which the other filled ampoules are placed which are to be sterilized in autoclave.these ampoules of biological indicators are placed at different places of autoclave so that to check the proper sterilization process.Close the access door and continue to operate the apparatus. 
• After completion of the sterilization procedure, and at a noted time with in 2 hours, aseptically add each contents of ampoule in 30 ml of Soybean Casein Digest Medium.Incubate each media at a temperature of 55-60oC. Observe each inoculated medium at 24 hours and 48 hours,and every 1 or 2 days thereafter for a total of 7 days. If after 7 days of incubation there is no growth in the medium the test for sterilization is met.
Quality Record(s)/Form(s)

Analytical Method Validation

.0     Purpose     :   To lay down a procedure for Analytical Method Validation.

2.0     Objective   :  To provide documented procedure for Analytical Method Validation.

3.0     Scope        :   To define role/responsibility of various persons responsible for Analytical Method Validation.

4.0     Responsibility    :

·        Primary        :         Officer QA/ QC

·        Secondary    :         Manager QA/ QC

5.0      Procedure   :

·        General Concepts

Ø      Validation is the act of demonstrating and documenting a procedure that operates effectively.

Ø      The discussion of the validation of analytical procedures is directed to the four most common types of analytical procedure:

R           Identification tests

R           Quantitative tests for impurities content

R           Limit tests for the control of impurities

R           Quantitative tests of the active moiety in samples of drug substance or drug product or other selected components in the drug product.

Ø      Typical validation characteristics which should be considered are:

R           Accuracy

R           Precision

R           Specificity

R           Quantitation Limit

R           Linearity and Range

R           Robustness

·        Method Validation Parameter for the assay of ---:

Ø      Linearity: ----- to be analyzed as per proposed method. The results obtain is used to statistically evaluate for coefficient of determination (r²), standard error of estimate and y intercept.

Ø      Precision: Precision of the chemical method is ascertained by carrying out the analysis as per the procedure and as per normal weight taken for analysis. Repeat the analysis five times. Calculate the % assay, mean assay, % Deviation and % relative standard deviation and %RSD.

Ø      Accuracy: Accuracy of the method is ascertained by standard addition method at 3 levels. Standard quantity equivalent to 80%, 100% and 120% is to be added in sample.

·        Method Validation Parameter for residual solvent by GC for ---:

Ø      Specificity: Resolution of the analyte peak from the nearest peak: Solution of each of the analyte was injected separately and their retention time is noted. The standard working solution containing a mixture of the component being analyze is also injected and each of analyte peaks is check for its resolution from the nearest.

Ø      Precision:

R     Repeatability: Six replicate injections of standard solution for system precision should analyze as per the proposed method and from the chromatograms obtained the percentage % RSD is calculated.

R     Intermediate precision: The purpose of this test is to demonstrate the intermediate precision of the method when method is executed by a different analyst and on different day. Results obtained will be compared.

Ø      Linearity and Range: Solution of analyte solvent, having different concentration should make separate from L.O.Q. concentration, which is 50% to 150%. The result obtained is statistically evaluated for coefficient of determination (r²), standard error of estimate and y intercept.

Ø      LOD & LOQ:

R     The limit of Detection (L.O.D.) was calculated as per below equation:

                    

                      LOD          =              3.3     X       SD

                                                                  Slope 

R     The limit of Quantification (L.O.Q.) was calculated as per below equation:

                                   

                                                LOQ         =              10      X     SD

                                                                                         Slope

Ø      Accuracy / % Recovery (By Standard Addition Method): Accuracy of the method was ascertained by standard addition method at 3 levels.

R     Standard solution quantity equivalent to 50%, 100% and 150% are added in sample.

R     The solutions amount is analyzed by the proposed method and chromatogram obtained.

R     The amount recover by the method is compared to the amount added. Percent deviation is calculated at each levels and a grand average across all the levels are also calculated.

Methanol standard concentration ––  3000 ppm

Acetic acid standard concentration –– 5000 ppm

DMF standard concentration ––          880  ppm

Ø      Robustness:

R     The evaluation of robustness should be considered during the development phase and depends on the type of procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters.

R     If measurements are susceptible to variation in analytical conditions, the analytical condition should be suitably controlled or a precautionary statement should be included in the procedure.

R     One consequence of the robustness should be that a series of system suitability parameters (e.g. resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used.

Process Validation

1.0     Purpose     :   To lay down a procedure for process validation.

2.0     Objective   :  To provide documented procedure for process validation.

3.0    Scope       : To define role/responsibility of various functions responsible for process validation.

4.0     Responsibility    :

·        Primary        :         Officer QA/ QC/ Production

·        Secondary    :         Manager QA/ QC/ Production

5.0      Procedure   :

Validation is the act of demonstrating and documenting a procedure which operates effectively. Process validation is the means of ensuring and providing documentary evidence that processes (within their specified design parameters) are capable of consistently producing a finished product of the required quality.

·        Types of Process Validation:

Ø      Prospective Validation: Validation carried out during the development stage on the basis of a risk analysis of the production process, which is broken down into individual steps; these are then evaluated on the basis of past experience to determine whether they may lead to critical situation.

Ø      Concurrent Validation: Validation carried out during routine production of products intended for sale.

Ø      Retrospective Validation: Involves the evaluation of past experience of production on the condition that composition, procedures and equipment remain unchanged. The source of data for this validation may include batch document, process control chart, maintenance logbook, records of personnel change process capability studies, FP data and stability results.

·        The proposed validation is designed as per the concurrent validation GMP guidelines because of the following reasons:

Ø      All the stages are already commercialized and a large number of batches of each stage are carried out on a large scale.

Ø      All critical quality attributes are established.

Ø      In process checks at each stage for acceptance are established as ‘In-process checks specification’.

Ø      Standard output in terms of quality and quantity is defined and established.

Ø      No failure of process or product at each stage is observed for a large number of batches.

Ø      The process now will be repeated by the standard SOPs for manufacturing, testing, and cleaning at each stage and monitored thoroughly to get output of consistent quality and quantity to create a validation report to comply with concurrent validation provisions.

·        General Concept:

Ø      Three consecutive validations will be performed to prove that the method is validated.

Ø      Whenever a new product is introduced, equipment usage and nature of potential contaminant will be studied to assess whether it poses a challenging study for process validation

·        Description of Process:

Ø      The  manufacturing process comprises of 5 chemical steps, packing and report writing on each step being common for all stages, starting from --. All the stages are taken under purview of the protocol.

R           Stage I:

R           Stage II:

R           Stage III:

R           Stage IV:

R           Stage V l

Ø      Before starting the process equipment should be cleaned and ECR report should be there and all raw materials should be analyzed and attached AR No. report should be attached with process validation report.

Ø      The unit operations with critical process parameter and operating range with in process check is well established as this product is prepared on actual commercial scale. All the three batches that to be taken for process validation is provided in the tabular form.

Ø      Process is well optimized with respect to molar proportion of ingredients, time, temperatures etc.; no parameter needs to be verified in the existing commercial process. Reaction time is the only parameter, which is variable and hence critical. Therefore only time will be monitored as per the frequency given above to establish the consistency of quantity and quality for all V Stages.

Ø      Check that all the stages follow acceptance criteria according to protocol

VALIDATION OF INCUBATORS

 1.0      OBJECTIVE

           To lay down a procedure for validation of incubator in microbiology laboratory.

2.0        RESPONSIBILTY

            Microbiologist / Executive.

3.0        ACCOUNTABILITY

            Quality  Assurance Control

4.0               PROCEDURE

4.1       DETAILS OF the Standard thermometer used for validation of  Lab thermometer.

Ø      Type    : Liquid - Glass lab Thermometer.

Ø      Place the standard thermometer dipped in glycerin in incubator at various locations as mentioned in annexure-II.

Ø      The incubator is set for desired temperature with the knob on control panel of incubator e.g 32.5 ⁰C,  35 ⁰C & 22.5 ⁰C.

Ø      Wait till the temperature reaches at set point & note down the temp. displayed on the small screen of incubator and compare this temp. with standard thermometer kept near the RTD probe.Likewise check the temperature after every 15 minutes up to one hour and note down the readings on the format.

Ø      Record any difference between the displayed temp. & temp. showed by standard thermometer and set the incubator accordingly

Ø      Frequency of validation :  once in a year.

Ø      The observations are noted in the format of annexure-I

             

4.2       PROCEDURE FOR VALIDATION OF D.H.S (HOT AIR OVEN):

Ø      Keep all the apparatus wrapped thrice with aluminum foil inside DHS as locations shown in the diagram.                

Ø      Keep the Spore loaded strips (having spore of B subilis) & Endotoxin indicator indicators having 10,000EU/vial in DHS(hot air oven) kept in 30ml vial wrapped thrice with aluminum foil at locations shown in the diagram keep one vial unbaked as PPC.   

Ø      Set the hot air oven at 250°C ,wait till the temperature reaches upto the set temperature.

Ø      After reaching the set temperature, note the time & temperature, hold for one hour

Ø      After completion of depyrogenation cycle ,switch off the power supply and take out Indicators for testing.

4.3       Procedure for testing reduction of endotoxin as under.

Ø      Take out the baked endotoxin loaded vial from the DHS.

Ø      Reconstitute 1ml of LRW in the baked vial and transfer 100ml of sample to the depyrogenated reaction tube kept in heating block at 37⁰ C and add 100ml  of LAL in the same tube in duplicate.

Ø      Prepare the dilutions for PPC for confirming 10,000EU/vial .

Ø      Note the gel clot after incubation of one hour

4.4.       Procedure for testing of reduction of prepared spore loaded strip(B subtilis):

Transfer (spore loaded) strip from backed vials and are inoculated in100ml sterile SCD media and incubate at 30 – 35⁰C for 7 days to observe for any turbidity, if any, report to Manager QC

5. 0          REFERENCES:         

USP 25 page no.-1890 & 2251

 Annexure 1. Formats for Instrument validation

DILUTION FOR PPC (Use depyrogenated glass-wares for testing as per SOP no.:K/QC/052)

 CSE contains 1,00000 EU Reconstituted  with  1000  ml of LRW distributed 100ml each in 10ml vials.

Keep one vial as positive.

Dilution for PPC
10,000 EU / vial--> 0.1 ml of CSE + 4.9 ml of LRW---> 0.1 ml of above + 3.9 ml of LRW---> 
0.1 ml of above + 4.9 ml of LRW (0.5 EU / ml) --->  500ml of above  +  500ml of LRW (25 EU / ml)-->
500ml of above  +  500ml of LRW (0.125 EU / ml) --> 500ml of above  +  500ml of LRW (0.06 EU / ml)
--> 500ml of above  +  500ml of LRW    (0.03 EU / ml)

FOR SAMPLE : Reconstitute 1 ml in each backed vial vortex for at least ten minutes and take 100ml of sample  + 100ml of Limulus amoebocyte lysate in the reaction tube and incubate for one hour at 37 °C  ± 1°C and record the results.

HEATING BLOCK TEMPERATURE: 37°C ± 1°C    TIME FROM:           TO:           HRS

S. no.	DETAILS	GEL	S. NO.	DETAILS	GEL
1.	Blank	-ve     -ve	10.	Sample (NPC) in duplicate	-ve     -ve
2.	2 l dilution in duplicate (PPC)	+ve    +ve	11.	Sample (NPC) in duplicate	-ve     -ve
3.	2 l dilution in duplicate (PPC)	+ve     +ve	12.	Sample (NPC) in duplicate	-ve      -ve
4.	l dilution in duplicate (PPC)	+ve    +ve	13.	Sample (NPC) in duplicate	-ve      -ve
5.	l dilution in duplicate (PPC)	+ve    +ve	14.	Sample (NPC) in duplicate	-ve       -ve
6.	l/2  dilution in duplicate (PPC)	-ve     -ve	15.	Sample (NPC) in duplicate	-ve       -ve
7.	l/2  dilution in duplicate (PPC)	-ve     -ve
8.	l/4  dilution in duplicate (PPC)	-ve     -ve
9.	l/4  dilution in duplicate (PPC)	-ve     -ve

.

OBSERVATION OF SPORE LOADED STRIP OF B.subtilis

NAME OF MEDIA	SCD media              INCUBATION TEMP. 30-35°C
NO. OF DAYS  ®	1	2	3	4	5	6	7
GROWTH OBSERVED Location:- B.subtilis