Validation of Dry Heat Sterilizer (DHS)

1.0 OBJECTIVE

The objective of this protocol is to establish the documentary evidence to demonstrate that the Dry Heat Sterilizer (Hot air oven) cycle is capable to destroy/inactivate the challenged Endotoxin.

2.0 SCOPE

The protocol is applicable for Dry Heat Sterilizer, which is installed in Microbiology laboratory of Quality control department.

3.0 RESPONSIBILITIES

Sr. No	Activity	Responsibility
1	Protocol Development	QC
2	Execution of this protocol	QC, QA
3	Evaluation of results	QC
4	Final Approval	AGM - QA

4.0 PREREQUISITES

In order to efficiently conduct validation of the DHS Validation by using Endotoxin Indicator, ensure that the following requirements are fulfilled

• Approved Testing Procedure for BET
• Approved SOP for Operating Instruction of Hot Air Oven (Dry Heat Sterilizer)
• Endotoxin Indicator
• Cleaned BET tubes – 12 X 75 mm
• Depyrogenated assay tubes 10 X 75 mm
• Depyrogenated dilution tubes 13 X 100 mm
• Lysate – Sensitivity 0.03EU/ml
• LRW – LAL Reagent Water
• Calibrated Heating Block
• Calibrated Micropipettes
• Endotoxin Free, sterile Micro tips
• Timer
• Test tube rack

5.0 EQUIPMENT / SYSTEM DESCRIPTION

1. Location: Washing Area of Microbiology Section of Quality control department
2. Equipment: The equipment covers the DHS & BET testing facility equipments.
3. System Description: Dry heat sterilizer is used to depyrogenate the tubes and other glasswares which is used for testing Endotoxins and Pyrogens. The dry heat sterilizer (hot air oven) is heated at 250°C for 1 hour to destroy the Endotoxins.

6.0 IDENTIFICATION OF CRITICAL CONTROL / MONITORING PARAMETER:

Before proceeding for validation of Dry Heat Sterilizer by using Endotoxin Indicator, following parameters to be monitored.

• Record the Depyrogenation cycle
• Verify the Lysate and CSE manufacturer's certificate of analysis
• Confirm the labeled Lysate Sensitivity

7.0 VALIDATION TEST

A. Confirmation Of Labeled Lysate Sensitivity

1. Reconstitute CSE in LAL Reagent water (LRW) as per manufacturer's instruction as laid down in the bottle and certificate supplied by manufacturer, and vortex for 5 - 10 minutes
2. Prepare a series of CSE dilution with LRW to yield 1EU/ml and shake the dilution on vortex for 5 minutes.
3. Prepare a 4l by diluting a volume, which is equivalent to 4l concentration from 1 EU/ml to LRW. Shake the dilution on vortex for 5 minutes. (4l to be back calculated from l concentration. i.e. l is the Lysate sensitivity)
4. Prepare CSE dilutions from 4l solutions of at least four concentrations equivalent to 2l, l, l/2 and l/4 by diluting a series of CSE solution with water for BET (LRW).
5. Now reconstitute the Lysate by opening the aluminium seal of Lysate with the help of sterile blade
6. Collect Lysate powder into the bottom of the vial by tapping on a hard surface. And then open the cork slowly.
7. As per manufacturer's instruction, add LRW before use and replace the cork immediately.
8. Take 24 depyrogenated assay tubes and label the tubes by numbering and arrange quadruplicate in stand and the proceed as per following table -

Sample	CSE dilution used	LRW	Lysate in ml	No. of replicates
2 l	100 ml of 2l	-	100 ml	4
l	100 ml of l	-	100 ml	4
l/2	100 ml of l/2	-	100 ml	4
l/4	100 m of l/4	-	100 ml	4
WNC	-	100 ml	100 ml	4

9. Pipette 100 ml diluted CSE i.e. 2l, l, l/2, and l/4 separately into depyrogenated borosilicate test tubes (10 mm X 75 mm) and labeled accordingly. For WNC (Water negative control) use 100 ml LRW separately and perform the test in quadruplicate.
10. Add 100 ml of reconstituted Lysate into each tube and mix gently.
11. Incubate the reaction mixture for a constant period at 37° ± 1° for 60 ± 2 minutes, avoiding vibration.
12. After incubation, take each tube and turn directly from the incubator and invert it through approximately 180°C in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed
13. The test is not valid unless the lowest concentration of the standard solutions shows a negative result in all replicate tests
14. The endpoint is the last positive result in the series of decreasing concentrations of Endotoxin. Calculate the mean value of the logarithms of the end-point concentrations and then the antilogarithm of the mean value using the following expression:

 

Acceptance Criteria

The geometric mean end-point concentration is the measured sensitivity of the Lysate solution (EU/ml or IU/ml). If this is not less than 0.5l and not more than 2l, the labeled sensitivity is confirmed and can be used for further validation.

B. Endotoxin Challenge Test

1. The vial contains 95,000,00 Eu/vial
2. Add 5 ml LRW (95,000,00 Eu/vial ÷ 5 = 19,00,000 Eu/ml) (Stock) and vortex for 15 minutes.
3. Take dilution tubes 15 x 75mm /13 x 100mm label A
4. In tube A Add 0.1 ml (stock) + 1.8 ml LRW = 1:19 (A), 1,00,000 Eu/ml
5. Multiply stock volume as per require ampoules for eg. Add 0.2 ml (stock) + 3.6 ml LRW = 1:19 (A), 1,00,000 Eu/ml (volume will be 3.8 ml).
6. Clean 18 assay tubes (10 X 75mm) to be used for Endotoxin challenge test, as per SOP .
7. To each tube, add 100 ml (0.1ml) Endotoxin Indicator from tube (A).
8. Dry the tubes in a Laminar Air Flow hood overnight.
9. Tear off pieces of aluminium foil enough to be folded double and cover the two assay tubes.
10. Place Endotoxin challenged tubes in double layer on foil. Wrap foil around tubes so that they are completely covered.
11. Carry out the same wrapping procedure for remaining tubes.
12. Keep two tubes as a positive control (do not pass through depyrogenation cycle). Mark as a PPC (unbacked).
13. Rest tubes exposed to depyrogenation cycle at 250°C for 1 hour as per the following locations.

Fig: - Schematic Location for Endotoxin Exposure

14. Load the oven with full load of glasswares, which is used for routine Depyrogenation cycle.
15. Now start the depyrogenation cycle as per SOP for depyrogenation of glassware at 250°C for 1 hour.
16. After Depyrogenation cycle, transfer the tubes to microbiology room and allow to cool at room temperature.
17. Aseptically remove the wrapped aluminium foil and place the tubes in test tube rack according to their exposed locations.
18. Add 1.0 ml LRW to each of backed tubes and vortex for 15 minutes.
19. Take 0.1 ml (100 ml) from each backed tubes add 0.1 ml (100 ml) of lysate and proceed the test.
20 For testing Positive Control
20.1 Add 1.0 ml LRW to the unbacked tubes and vortex for 15 minutes.
20.2 Prepare a dilution of 1 : 10,000 from the unbacked tubes as shown below dilution.

0.1  ml of (10,000) Eu/ml) + 1.9 LRW                         1:20                 (I)         (500Eu/ml)

0.1 ml (I)                           +  1.9 ml LRW                   1:400               (II)        (25Eu/ml)

0.1 ml (II)                          +   2.4 ml LRW                  1:10,000          (III)       (1Eu/ml)

20.3 For positive control, take from tube (III), i.e. 0.1 ml of (III) + 0.1 ml of lysate.
20.4 Take 44 depyrogenated tubes label the tubes by numbering and proceed as per the following table

Tube No	To be labeled as	LRW in ml	1 Eu/ml	Sample in ml	Lysate in ml
1, 2,3, 4	WNC	100	-	-	100
5, 6, 7, 8	WPC	50	50	-	100
9, 10, 11, 12	PPC	-	100	-	100
13, 14, 15, 16	PNC (Location 1)	-	-	100	100
17, 18, 19, 20	PNC (Location 2)	-	-	100	100
21, 22, 23, 24	PNC (Location 3)	-	-	100	100
25, 26, 27, 28	PNC (Location 4)	-	-	100	100
29, 30, 31, 32	PNC (Location 5)	-	-	100	100
33, 34, 35, 36	PNC (Location 6)	-	-	100	100
37, 38, 39, 40	PNC (Location 7)	-	-	100	100
41, 42, 43, 44	PNC (Location 8)	-	-	100	100

21.0 For carrying out standard curve, prepare dilution from tube (III) 1Eu/ml as follows:

0.5 ml (III tube, 1 Eu/ml)          +   0.5 ml LRW                       = 0.5 Eu/ml                 (a)

0.5 ml from (a)                                    +   0.5 ml LRW                       = 0.25 Eu/ml               (b)

0.5 ml from (b)                                    +   0.5 ml LRW                       = 0.125 Eu/ml             (c)

0.5 ml from (c)                                    +   0.5 ml LRW                       = 0.06 Eu/ml               (d)

0.5 ml from (d)                                    +   0.5 ml LRW                       = 0.03 Eu/ml               (e)

0.5 ml from (d)                                    +   0.5 ml LRW                       = 0.015 Eu/ml             (f)

0.5 ml from (d)                                    +   0.5 ml LRW                       = 0.007Eu/ml              (g)

21.1  Take 24 depyrogenated assay tubes and label the tubes by numbering and arrange quadruplicate in stand and the proceed as per following table -

Sample	CSE dilution used	LRW	Lysate in ml	No. of replicates
2 l	100ml of 2l	-	100 ml	4
l	100 ml of l	-	100 ml	4
l/2	100 ml of l/2	-	100 ml	4
l/4	100 m of l/4	-	100 ml	4
WNC	-	100 ml	100 ml	4

21.2 Pipette 100 ml diluted CSE i.e. 2l, l, l/2, and l/4 separately into depyrogenated borosilicate test tubes (10 mm X 75 mm) and labeled accordingly. For WNC (Water negative control) use 100 ml LRW separately and perform the test in quadruplicate
21.3 Add 100 ml of reconstituted Lysate into each tube and mix gently.
21.4 Incubate the reaction mixture for a constant period at 37° ± 1° for 60 ± 2 minutes, avoiding vibration.
21.5 After incubation, take each tube and turn directly from the incubator and invert it through approximately 180° in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed.
21.6 Now calculate the recovered Endotoxin concentration from Positive Endotoxin Indicator control by following expression –
Endotoxin Concentration = Lysate sensitivity x reconstituted volume x dilution
Minimum Log reduction = log Endotoxin concentration of the unbacked control vials – log Endotoxin concentration of the backed vials.

8.0 ACCEPTANCE CRITERIA

1. Test Results are valid if recovery of Endotoxin in non-exposed (PPC) tubes is within two-fold dilution of the labeled claim.
2. The Depyrogenation cycle is valid if the PPC and WPC are positive and WNC and PNC are negative and there is >3 log reduction in Endotoxin in the depyrogenated tubes for three successive cycles.

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