HEATING BLOCK VALIDATION

PROCEDURE
   
1. Take 5 calibrated thermometer and give them No. L1, L2, L3, L4, L5 as mentioned in annexure.

2. Keep five thermometer in test tubes at different location inside well of heating block as per location chart enclosed.

3. Operate Heating block as per S.O.P.

4. Compare temperature of each thermometer with respect to temperature adjusted one on heating block  at every 15 minutes interval upto 1 hours.

5. Record the result as per annexure.

ACCEPTANCE CRITERIA

Temperature on display should be within 37°C + 1°C.

The results obtained at all location of heating block should be within 36 to 38°C.

Change the location by placing thermometer in other than previously selected location to cover maximum well during validation in a calendar year.

FREQUENCY

Every month

HVAC SYSTEM VALIDATION TESTS

1. AIR FLOW PATTERN

• Take the titanium tetra chloride stick.

• Burn the stick.

• Place the burning stick in front of running Air Handling Unit (AHU).

• Observed the flow of air with the help of smock distribution in the room.

• Make chart diagram of flew of air in the room as annexure-I for each room.

2. AIR FLOW VELOCITY AND CHANGE PER HOUR

• Scan the area of  the HEPA Filter, with the  anemometer  probe, 6 inches from the filter face.

• For  ease of experiment, divide the area of the HEPA Filter into 4 equal, hypothetical grids.

• Record the velocity readings taken at the center of the grids, and at the junction of dividing lines (center of HEPA Filter)  in 5.5.

• Calculate the Average Velocity (V in feet per minute ) as :
                                               
          V  = (V1+V2+V3+V4)/4           
                                                                                 Vi  = Velocity observation at each point 

• Measure the dimensions of each air inlet i.e. HEPA Filter, in  feets and record it.

• Calculate the Area (A in square feet) of each Air inlet as product of the  length and the width as :

                  A  =  l x w                     

   where  l    =  length of inlet
               w  =  width of inlet

• Calculate the Total Air Volume ( T in cubic feet per minute ) supplied in each zone, by using the formula :

               T  = A x V                 

 where  A  =  Area of particular Air inlet in          square feet
             V  =  Average air velocity at particular air inlet in feet per minute

• Calculate the total volume of the room by multiplying length of room, breadth of room and height of the room.
                            = L x B  x  H

• Total air change is divided by total volume of the room will give the air change per hour. Fill the record in the form as annexure-II.

3. FILTER LEAK TEST

• Place the velometer at the front of AHU  unit.

• Check the velocity of air to the all corner of the AHU. The air velocity should be within the higher limit of HEPA filter.

• If there is air velocity is more than higher limit change the gas cut to prevent the air leakage.

4. PARTICLES COUNT

• On the air system before one hour of test operation. Take the suitable particle counter and operate it to check the particles in the room at non working operation.

• Collect the information from particle counter and fill them in the format.

• Operate the particle counter when work is on progress in the area. The particles should be count when more than one hour work has been progressed in the area. Record the data in the format.

• Operate the particle counter for all the room maintaining grade A, grade B, grade C & grade D.

5. VIABLE MONITORING

• Expose Plate Count Agar  and  Saboraud  Dextrose  Agar media plates  in  sterile  manufacturing area (Fabrication , Vial Filling ,  Vial  Sealing & Sterile  passage) as  per  location plan given on  the back of paper sheet. Similarly expose the plates in Change Room and Degowning twice in a week and once in a week in Vial Washing , Bung Washing and Component Preparation.

• Monitor the microbial load  on the  surface  of  the  sterile  manufacturing  area  by  swab sampling  and testing. Carry out  swab  sampling  daily in Vial  Filling, Vial Sealing,  Sterile Buffer and Blending III; and twice in a week in Sterile Passage, Change Room and Degowning. Similarly  carry out the swab sampling once  in a week in Change Room, Degowning, Vial Washing ,Bung Washing and component Preparation. Record the results.

• Place the media strips or petridics in the air sampler on operate the air sampler as per standard operating procedure of the equipment.

• Record the data in the format.

• Monitor  the  microbial  load  on  surface  of  hand  gloves  of  the  operators  daily  once  in  each working  shift  at  random  during  activity and  record  the  result. Record the data in the format.

• In  case  of  repeated  failure  during  two  observations the  corrective  action  shall  immediately be  planned  and  implemented.

• Monitor  the  critical  functions  of  HVAC system , Water System.  and  personnel's  behavior  in sterile  and  investigate  cause  of   adverse  results  immediately after  observation.

6. FILTER INTEGRITY TEST (DOP TEST)

• Generate DOP Aerosol using Aerosol generator, by subjecting DOP to 20 psi air pressure. Direct   test   aerosol at  the supply duct in the Air Handling System.                                                                                                                           • Switch  the  photometer   "ON"   and  allow  to  stabilise for  five min.                                                                                • Ensure that 100 % upstream concentration is achieved at all the  terminal HEPA filters.

• Scan the filter matrix and perimeter by passing the receptor probe 1 inch from the filter surface , in overlapping strokes traversing  at  approximately  10 fpm to check for  leaks, if any.
           
• Test all the HEPA filters as per the above steps and record the observations in the format.      

7. PRESSURE DIFFERENCE

• Attached all concerning room (Under Test) to the manometer which are attached the wall of adjacent area.

• On the air system in side tested area and wait to stablise the pressure in the area.

• Observed the pressure difference from all room and from room to room.

• Record the data in the format.
           
8. RECOVERY ( TEMP. & HUMIDITY)

• Off the HVAC system and checked the humidity of the area.

• If humidity of the area within in specification. Increase the humidity by spraying hot water in the are up to 75%.

• Wait to stablise the humidity in the area about 75%.

• Operate the HVAC system and note the time. Wait to stablise the humidity in the area within the specification limit.

• Note and record the time in the format.

• For the recovery test increase the temperature of the area by using hot air  blower in the area and increase the temperature 40 ºC.

• Operate the HVAC system and note the time.  Wait to stablise the temperature  in the area within the specification limit.

• Note and record the time in the format.

9. TEMP. & HUMIDITY UNIFORMITY TEST

• Place the calibrated thermometer on the different location.

• Operate the HVAC system and note the time. Wait to stablise the temperature  in the area within the specification limit.

• Check and record the temperature of the area in format.

• Place the calibrated hygrometer  on the different location.

• Operate the HVAC system and note the time.  Wait to stablise the humidity  in the area within the specification limit.

• Check and record the temperature of the area in format.

10. FRESH AIR DETERMINATION

• On the concerned AHU and wait to stabiles the air pressure in room.

• On the fresh air dumper for fresh air and observed and calculate the intake air by the dumper in the room. Observed and calculate the total air change in the room.

• The intake fresh air is divided by the total air change in the room and multiply by 100 to calculate the % fresh air intake on each cycle by the HVAC system in the tested room.

• Record the data in the performance format record.

AUTOCLAVE VALIDATION IN PHARMACEUTICALS

Validation of an Autoclave for pharmaceutical use will be considered qualified for consistent and reliable performance (validated) on successful completion of the following –

• Bowie – Dick Test for steam penetration (3 trails).

• Empty Chamber Heat distribution studies (3 trails) with temperature mapping probe at different locations of the sterilizer chamber.

• Loaded chamber heat Distribution & penetration studies (3 trails) for each sterilization load of fixed loading pattern:-
1) Sterile area garments (20 number Garments packs, Each pack contains 01 Nos. Boiler suit, 01 Nos. Headgear, 02 Nos booties, 01 pairs gloves)

2) Glassware (S.S Mannifold holder (06 holder) 02 Nos, Sampling unit of Compressed air 02 Nos, 500 ml sampling bottles 10 Nos, 250 ml sampling bottles 25 Nos, 04 Nos S.S Bin.)

3) Media (SCDA Medium – 500 ml 09 Nos. Conical flask, SCDM – 100 ml 20 Nos. tubes, FTM – 100 ml 10 Nos. tubes, MSA – 250 ml 01 Nos Conical flask, CA – 250 ml 01 Nos Conical flask, BGA – 250 ml 01 Nos Conical flask, BSA – 250 ml 01 Nos Conical flask, MCA – 250 ml 01 Nos Conical flask, Peptone Water – 500 ml 06 Nos. Conical flask.)

With temperature mapping probes along with Biological Indicator (Geobacillus stearothermophillus spore vials containing 10^6 or more spores per vials) inside the innermost possible layer of the load subjected for sterilization.

• Estimation of the F0 Value achieved during the sterilization hold period at each temperature mapping probe.
To qualify these tests the equipment should fulfill the acceptance criteria described in the individual test procedures. After completion of the qualification tests all the data generated will be compiled together to evaluate ability of the steam sterilizer to sterilize different components at the set parameters and set loading pattern.

A). BOWIE-DICK TEST FOR STEAM PENETRATION

Objective of this test is to ensure that the vacuum pulses applied the sterilization Hold period are sufficient to remove the entrapped air so as to facilitate rapid and even steam penetration into all parts of the load and maintaining these conditions for the specified temperature holding time (17 minutes at 121 deg.C)
If air is present in the chamber, it will collect within the Bowie – Dick test pack as a bubble. The indicator in the region of the bubble will be of different color as compared to the color on the remaining part of the test paper, because of a lower temperature, lower moisture level or both. In this condition the cycle parameters to be reviewed and the normal sterilization cycles to be modified accordingly.
Bowie – Dick cycle should be normally preceded by a warm – up cycle, as the effectiveness of air removal may depend on all parts of the sterilizer being at working temperature.

PROCEDURE

1. Record the set parameters for the Bowie – Dick test cycle in the annexure.

2. Place one Bowie – Dick test pack near the drain of the sterilization chamber.

3. Select cycle Bowie – Dick on the control panel & operate the steam sterilizer.

4. The print out taken during the Bowie – Dick test cycle & the Bowie – Dick test indicator should be preserved.

5. Compile the observation made during the qualification test for complete evaluation of the system.

ACCEPTANCE CRITERIA

The Bowie – Dick Test indicator should show a uniform color change, non – uniform change and/or air entrapment (bubble) spot on the pattern indicates inadequate air removal from the sterilization chamber.

B). EMPTY CHAMBER HEAT DISTRIBUTION STUDIES

Objective of this test is to ensure that, the sterilizer is capable of attaining a temperature of 121 deg.C during the sterilization hold period with steam pressure of 1.1 to 1.2 kg/cm2.
Temperature spread with in the range of 121 deg.C to 124 deg.C during sterilization cycle will demonstrate the uniform heat distribution within the chamber.
Any location where the temperature indicator is placed, not achieving minimum sterilization temperature of 121deg.C through out the sterilization temperature hold will be considered as cold spot.

PROCEDURE

1. Record the set parameter for the sterilization cycle to be operated during the test for empty chamber heat distribution study, in the Annexure.

2. Pass minimum 16 no. Temoerature mapping probe into chamber through the port of the sterilizer. Seal the port with silicone sealant so that steam leakage does not take place. Suspend the probes in the chamber in different position so that probes do not touch any metallic. Record the position of the probes in a respective schematic form.

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

4. Operate the steam sterilizer and also start the data logger to record actual temperature within the sterilization chamber with respect to time.

5. When the sterilization cycle completes, 1) Collect printout of the sterilizer and preserve as Annexure. 2) Download the data-analysis and printing. Record the temperatures observed at different locations in the Annexure.

6. If the empty chamber heat distribution study is acceptable perform three consecutive runs to demonstrate cycle and sterilizer reproducibility.

7. Compile the data generated during the qualification test for complete evaluation of the system.

ACCEPTANCE CRITERIA

There should be uniform distribution of heat in the sterilizer chamber during the sterilization hold period and the temperature at each temperature mapping probes should be within the range of 121 deg.C to 124 deg.C during the sterilization hold period.

C). LOADED CHAMBER HEAT DISTRIBUTION AND PENETRATION STUDIES

Objective of this test is to ensure that, the steam is sufficiently penetrating into the innermost portions of the load subjected for sterilization to achieve desired temperature of 121 deg.C during the complete sterilization hold period with steam pressure of 1.1 to 1.2 kg.cm2.
If Sterilization temperature (121 deg.C) is not achieved through out the cycle, load configuration or size of the load has to be reviewed and cycle to be repeated.
Temperature spread within the range of 121 deg.C to 124 deg.C during sterilization hold period indicate that, uniform heating process which is achieved in the empty chamber heat distribution study is not affected by load. There could be the possibility of lag period for attaining 121 deg.C during heat penetration runs as the probes are placed deep into the load.
Any location where the temperature indicator is placed, not achieving minimum sterilization temperature of 121deg.C during sterilization temperature hold period will be considered as cold spot.

PROCEDURE

1. Record the set parameter for the sterilization cycle to be operated during the test for loaded chamber heat penetration study in the Annexure.

2. Pass minimum 16 no. Temperature mapping probe into chamber through the port provided. Seal the port with silicone sealant so that steam leakage does not take place. Place the probes inside the load components, which are supported to be most difficult points for steam penetration, also place biological indicator along with temperature mapping probe (12 Nos.). Record the position of the probes and biological indicators in a representative schematic form.

3. Connect the probes to a suitable data logger, which can scan and print the actual temperature with respect to time.

4. Operate the steam Sterilizer and also start the data logger to record the actual temperatures within the sterilization chamber with respect to time.

5. When the sterilization cycle completes, 1) Collect printout of the sterilizer and preserve as Annexure. 2) Download the data-analysis and printing. Record the temperatures observed at different locations in the Annexure. 3) Aseptically collect the exposed biological indicators and send the indicators to microbiology lab for further incubation and observed the results.

6. If the load penetration study is acceptable perform three consecutive runs to demonstrate cycle and sterilizer reproducibility.

7. Compile the data generated during the qualification test for complete evaluation of the system.

ACCEPTENCE CRITERIA

There should be uniform distribution & penetration of heat in the load subjected for sterilization during the sterilization hold period and the temperature at each temperature mapping probe should be within the range of 121 deg.C to 124 deg.C during the complete sterilization hold period.

D) BIO CHALLENGE STUDIES

OBJECTIVE

The steam sterilization process, when challenged with Geobacillus stearothermophillus Biological indicator spore vial, spore population of NLT 10^6 spores/vial, should reduce bacterial load by mean of Sterility Assurance Level (SAL) 10^6
On incubation of the loaded biological indicator, if growth is observed, then the sterilization cycle parameters to be reviewed.

PROCEDURE

1. Determine the initial counts of  biological indicator.

2. Collect the exposed indicator (during the loaded chamber heat distribution & heat penetration studies) by using sterile forceps and scissors in a 100 ml beaker and then send to microbiology lab for incubation (Incubate the vial at 55 to 60 deg.C for 48 hours)

3. Keep one vial as a negative control provided by the Mfg of biological indicator as well as one vial as a positive control (unexposed vial biological indicator).

4. Observe any growth (purple color – sterile, yellow color – Non sterile) in the vial daily. Record the observations on daily basic in the Annexture.

5. Compile the data generated during the qualification test for complete evaluation of the system.

ACCEPTANCE CRITERIA

No bacterial growth should observed during the incubation period of 48 hours at 55 to 60 deg.C.

E) ESTIMATION OF F0 VALUE

OBJECTIVE

The calculated F0 value should not be less than the biological F0 value at all temperature mapping locations during the sterilization hold period.

PROCEDURE

1. Record the temperature at all temperature mapping probes during the sterilization hold period in the Annexure.

2. Calculate the F0 value at each temperature mapping probe by using the equation as below.

3. Record the F0 value (Results) in the Annexure.

4. Compile the data generated during the qualification test for complete evaluation of the system.

CALCULATION

F0 = dt S10(T-121)/z

Where
dt = Time interval between two following temperature measurements (1 minutes).

T = The observed Temperature at that particular time.

Z = The change in the heat resistance of Geobacillus stearothermophillus spores as temperature is changed (10 deg.C).

ACCEPTANCE CRITERIA

The calculated minimum F0 value (by equation) should be more than biological F0 value for the biological indicator vial exposed for the bio-challenge studies.

The biological F0 value for the specific biological indicator spore vial is calculated as per the following equation

F0 = D121 (Log A – Log B)

Where

D121 = D value of the of the biological indicator at 121 deg.C.

A = Biological indicator concentration or spore population.

B = Desired level of non – sterility. (10 deg.c.).

VALIDATION OF SHELF LIFE FOR 70% V/V IPA

VALIDATION TEST

Validation of diluted disinfectant storage conditions are done by following methods

      ·  Surface swab test Method
      ·  Settle Plate Method

1. Prepare 70% v/v solution of Isopropyl Alcohol.

2. Carry out the sterility of diluted IPA solution by Membrane filtration method.

3. Store the disinfectant solution at room temperature, and analyze the sample on the day of preparation, after 24 hrs, 48 hrs, 72 hrs and 96 hours by Surface swab Test, Settle Plate Method.

SURFACE SWAB METHOD

Remove following culture slant from the refrigerator and allow it to attain room temperature:-

      ·  Staphylococcus aureus
      ·  Escherichia coli
      ·  Pseudomonas aeruginosa
      ·  Salmonella, and
      ·  Wild culture

1. Inoculate loop full of the culture from each slant separately into 50 ml of sterile Soyabean casein digest medium and incubate at 32.5 ± 2.5°C for 24 – 48 hrs.

2. Transfer 1.0 ml of the broth culture into 9.0 ml of sterile saline solution (0.9% sodium chloride solution) to obtain a test dilution of 10-1.

3. Transfer 1.0 ml of the 10-1 dilution into 9.0 ml of sterile saline solution to give 10-2 dilution.

4. Similarly serially dilute the culture suspension to obtain dilution of 10-3, 10-4, 10-5  and 10-6.

5. Plate 1.0 ml of the culture suspension from dilution 10-3, 10-4, 10-5 and 10-6 in duplicate into sterile petri dishes.

6. Pour approximately 15-20 ml of sterile Soyabean Casein Digest Agar cooled to about 45°C in each plate. Incubate at 32.5 ± 2.5°C for 24 to 48 hours.

7. Count the number of colonies on each plate and Select the dilution, which gives a count of not less than 10-5.

8. Apply 1.0 ml of each culture suspension containing cell concentration not less than 10-5 cfu/ml, separately on the floor approx 25 cm2 area, and allow to air dry.

9. After drying, take a surface swab as per latest SOP for Swab Testing, and carry out the determination of total aerobic count per cm2 withing 4 hours of sampling.

10. Immediately clean the floor with IPA 70% v/v solution and allow to stand for 30 minutes to facilitate the action of disinfectant solution on the challenge test organisms.

11. After 30 minutes, take a swab and detect the bacterial count as per SOP for swab testing.

12. Repeat the same procedure, step no. 9 to 12 to determine the efficacy of 70% IPA solution after 24 hrs, 48 hrs, 72 hrs and 96 hours at room temperature.

SETTLE PLATE METHOD

1. Expose the pre-incubated sterile Soybean casein digest agar plate for 2 hours.

2. After plate exposure time, immediately clean the floor with IPA 70% v/v solution.

3. After proper cleaning, spray the area with IPA 70 % v/v solution and immediately close the room 30 minutes to facilitate the action of disinfectant solution.

4. After 30 minutes of contact time, immediately expose the plate as per latest SOP for plate exposure and detect the bacterial count per location per 2 hours.

5. Repeat the same procedure, step no. 1 to 4 to determine the efficacy of IPA 70% v/v solution after 24 hrs, 48 hrs, 72 hrs and 96 hours at room temperature

ACCEPTANCE CRITERIA

Diluted disinfectant solution, which is stored at room temperature, is effective when the test result of surface swab and Settle plate shows 90% reduction of the challenged microorganisms.

*  10-3 or 10-1 means 10 to power -1

HOLD TIME STUDY PROTOCOL FOR STERILISED GARMENTS FOR THEIR STERILITY

PROCEDURE

1. Prepare a Dacron bag contains 1 pair of garments used in  sterility testing area, and place 10 cut pieces (6x5 cm) of old dress in-between the dress.

2. Perform the sterilization of the bag as per the SOP for sterilization of dresses.

3. After sterilization place the Dacron bag in Garment cubicle for Hold time study.

4. Collect one cut piece from the dress bag aseptically and direct immerse into the sterilized Soyabean Casein Digest medium and check for the sterility, this sample shall be treated as initial sample. (0 Hour)

5. Similarly collect the dress pieces from the hold bag at regular intervals of 24 hr, 48 hr, 72 hr, 96 hr, and 120 hr and perform the sterility test.

6. Incubate the samples at the specified temperature for 14 days (20-25°C for 7 days and 30-35°C for 7 days).
Note: 0 Hrs starts when the sterilized garments placed in Garment cubicle after sterilization.

ACCEPTANCE CRITERIA

This study is carried out to establish the hold time of sterilized garments after sterilization.
Microbial determination : No growth should be observed in sterility test.

CONCLUSION

After complete evaluation of the hold time study for sterilized garments used for sterility testing a final hold time study summary report shall be prepared which should essentially contain discussion and conclusion which clearly determine the hold time period for sterilized garments.

VALIDATION PROTOCOL FOR HOLD TIME STUDY OF SWAB TEST SAMPLES

PROCEDURE

1.  Prepare 0.9% saline solution and dispense 10 ml quantity in test tube and put one sterile cotton swab in it and sterilize in autoclave at 15 lbs pressure and 121°C for 15 minutes or use pre sterilized swab tubes and fill with 10 ml sterile 0.9% saline solution.

2. Swab 5x5 cm2 area using parallel  overlapping stroke with slow rotation of swab.

3. Repeat the sampling with crossing first stroke at 90 degree angle. After taking the swab put the swab stick back into 0.9% saline solution tube.

4. After taking swab write the location and date of swab with marker on tube.

5. Bring the tubes to quality control laboratory and gently vortex for 1 minute. Perform the bio-burden testing using 1 ml of solution by membrane filtration.

6. Mark and incubate the plates at 22.5 ± 2.5 °C for 3 days followed by 32.5 ± 2.5 °C for 2 days inverted position.

7. Store the tubes containing the remaining solution with swab stick at 2-8 °C.

8. After 24 hours take the swab tubes and gently vortex for 1 minute.

9. Perform the bio-burden testing again using 1 ml solution from the tubes.

10. Mark and incubate the plates at 22.5 ± 2.5 °C for 3 days followed by 32.5 ± 2.5 °C for 2 days inverted position.

11. After incubation count and observe the plates of both studies for number of colonies on colony counter or under light source with the help of marker.

ACCEPTANCE CRITERIA

There shall be no increase in bio-burden on holding at 2-8°C for 24 hrs.

CONCLUSION

After complete evaluation of the hold time study for swab a final hold time study summary report shall be prepared which should essentially contain discussion and conclusion which clearly determine the hold time period for swab test samples.

VALIDATION OF PURE STEAM

Pure steam is used in various operations in pharmaceuticals but its use in sterilization is very common in pharmaceutical sterile manufacturing.
Pure steam system should be qualified. A WHO guide to good manufacturing practice (GMP) requirements clearly says to perform the performance qualification of pure steam. At the time of performance qualification of pure steam generation system, sample shall be taken from each steam user point and analyzed for three consecutive days. Purified water system must be qualified before starting the qualification of pure steam.

SAMPLING OF PURE STEAM

Sampling for Bacterial Endotoxin Test and chemical tests should be done separately. Depyrogenated tubes or bottles should be used for taking sample for bacterial endotoxin test. Allow the steam to drain for minimum one minute. Open the cap of bottle and fill the bottle with steam condensate by holding the bottle in the holder. Gloves should wear into the hands while sampling the pure steam. Tighten the cap of the bottle and mark with the sampling information. It sample is not analyzed within 2 hours of sampling, store the sample at 2-8 °C.

ANALYSIS OF PURE STEAM

Pure steam should be analyzed for following tests:-

1. Non-Condensable gases:- Non condensable gases are air and carbon dioxide those do not condense with the steam. These are generated due to their presence in the purified water that continuously circulates in the water distribution system. Non condensable gases should not be more than 3.5%.

2. Steam Dryness value:- Dry steam has more energy than the wet steam. Wet steam has water with it and does not have heat energy as dry steam. Dryness of steam is determined by the latent heat. Dryness of the pure steam should not be less than 90%. High moisture content can cause the loss in energy of steam and that may cause the longer sterilization time.

3.  pH:- Steam condensate is analyzed for pH value at 25 °C . It should be between 5-7.

4. Conductivity:- Conductivity should be tested with calibrated conductivity meter at 20 °C. Conductivity should not be more than 1.3 µS/cm.

5. Microorgansims:- Steam condensate is tested for microbial contamination using pore plate method. There should not any microbial contamination in steam condensate.

6. Endotoxin Test:- Determine the endotoxin in the pure steam condensate and it should not be more than 0.25 EU/ml as in water for injection.