a practical approach to steam autoclave cycle development

Journal of Validation Technology124

A Practical Approach to SteamAutoclave Cycle Development

ABSTRACT

The purpose of this article is to define one of the designapproaches that can be used to develop an autoclave cycle.The design approach discussed in this article is based on anoverkill design approach from Parenteral Drug Association(PDA) Technical Monograph Number One and Six-SigmaStatistical Process Control. Many validation engineers inthe Biotech Industry will use a trial and error approach. Weestimate the temperature set point and dwell time. Then, acycle development run or performance qualification (PQ) isperformed to see whether the cycle will work. If the cycledoes not work, we will either increase the temperature setpoint or increase the dwell time. We have seen a cycle tem-perature set point as high as the autoclave’s manufacturerrecommended maximum operating temperature of 130˚C.

Utilizing this cycle development approach in Food andDrug Administration (FDA) or European Medicinal Evalua-tion Agency (EMEA) regulated industry will minimize thecost of maintenance and operation, and reduce the time todevelop the cycle and validate the system. Performing cycledevelopment before process qualification and validation ishighly recommended by both the International Society ofPharmaceutical Engineering (ISPE) and PDA guidelines.

This article is intended to suggest one of the practicaloverkill approaches of developing wrapped goods (not heatlabile load items) cycle for steam autoclave. The goal of thiscycle development is to provide optimum critical parame-ters that ensure the materials or products will be sterilized.Moreover, it develops a master load cycle that can supportusing one cycle for any wrapped goods. Upon successfulperformance qualification (PQ) of one master load cycle;items can be loaded randomly or without a particular loadpattern as long as the items are loaded properly. Master load

cycle strategy – in comparison to traditional individual cy-cles for various loads of the same types – is recommendedby PDA Technical Monograph No. 1.

INTRODUCTION

The purpose of the cycle development is to develop amaster cycle with optimum temperature set point and dwelltime to provide a high sterility assurance level (SAL) ofwrapped goods (dry goods or porous goods). Cycle develop-ment for wrapped goods is more essential than liquid or heatlabile load items. Liquid and heat labile load items havemore defined parameters that require less experimentation todevelop the autoclave cycle than wrapped goods. This cycledevelopment evaluates cycle and load preparation, pre-con-ditioning to improve air removal, heat penetration, and post-conditioning to provide usable items which maintain sterilebarrier integrity. The cycle development determines the otherparameters such as jacket temperature set point, alarm lim-its, and calibration tolerance. It also develops a universalmaster load cycle for qualification. The master load cyclecan support using one cycle for any wrapped goods. Uponsuccessful performance qualification, master load cycle willsupport a mix and match randomized load pattern. Items canbe loaded randomly or without a particular load pattern aslong as the items are loaded properly. Therefore, it will re-duce the operation and maintenance costs since there is onlyone cycle to be validated or used.

APPROACH

1. Perform vacuum leak test and Bowie-Dick test2. Perform empty chamber temperature mapping at

121.5˚C

!

B Y V I C T O R T S U I , P . E . A N D W A R D W I E D E R H O L D , B . S . M . E .

Victor Tsui, P.E. and Ward Wiederhold, B.S.M.E.

February 2007 • Volume 13, Number 2 125

Figure 1Cycle Development Approach

Load 1Perform

TemperatureMapping for HP

and HD

Load 2Perform


and HD

Load 3Perform


and HD

Load 4Perform


and HD

Load 5Perform


and HD

Thermo DataAnalysis

DetermineMaster

Load Items

DetermineMaster

Load CycleOther

Parameters

Complete CycleDevelopment

Perform EmptyChamber

TemperatureMapping at

121.5˚C

Perform ChamberIntegrity Testing

Perform MasterLoad CycleValidation

Confirmation

DetermineMaster

Load CycleTemp.

DetermineMaster

Load CycleDwell Time

CycleDevelopment



3. Develop a list of items that need to be autoclaved4. Perform a series of temperature mapping at

121.5˚Ca. Determine the master load itemsb. Determine the temperature set pointc. Determine dwell timed. Determine other parameters

5. Perform master load cycle validation confirmation

Biological Indicator (BI)

Commercially supplied Biological Indicator (BI)According to USP 29 for moist heat BI:Population (N) of 5x105 to 5x106 spores per carrier D121 of 1.5 to 3.0 minutes

In this case study we used D121 of 1.9 minutes, whichdoes not bracket the entire range. It targets the low endD121 of 1.5 to 3.0 minutes, but allows some ranges for BIvariability and availability. It is greater than 90 %“overkill.”

F0 Calculation

F value is a measurement of sterilization effectiveness.F(Tref,z) is defined as the calculated equivalent time (lethal-ity) at temperature Tref delivered for sterilization. The z valueis the number of degrees of temperature change necessary tochange the D121 value by a factor of 10. D121 value is thetime in minutes required for a one-log reduction of the pop-ulation of a BI under specified lethal conditions at Tref of121.1˚C (denoted 121 for brevity).

F0 or F(T=121˚C, z=10˚C) is normally used to describe the de-livered lethality for steam sterilization.

F0 =⌡10(T-121)/10dt where T is the temperature and t istime. Most data loggers will calculate the accumulated F0value.

F0 = D121 x (log N - log 10-6) where 10-6 equates to theSterility Assurance Level (SAL), desired endpoint

Overkill is denoted as D121 is 1 minute and N is 1 x 106

F0 = 1 x (log 106 - log 10-6) = 12 minutesFor this case study BI used: D121 = 1.9 minutes and N = 1.7x 106

Therefore, F0 = 1.9 x (log 1.7 x 106 - log 10-6) = 22 minutes

Procedure

• Perform Vacuum Leak Test

Vacuum leak test is an essential tool to evaluate systemperformance in an autoclave with vacuum pre-condition-ing. System leaks may contribute to poor heat penetrationwhen inadequate air removal is achieved. Typical specifi-cation is less than 0.5 psi for 15 minutes according toHTM2010 specification. Vacuum leak test should be per-formed routinely; normally at least twice per year orbased on the system’s historical performance. The testshould be performed also after any major maintenance orpart replacement that may impact the chamber integrity(such as drain valve, door gasket, and vent valve). It is es-sential to establish a baseline (good condition) before thecycle development process.

• Perform Bowie-Dick Test

In conjunction with a vacuum leak test or alternately, aBowie-Dick Test or variant air removal test should also bechecked regularly. Bowie-Dick test is a commerciallyavailable test pack (single use) to evaluate air removal andsteam penetration in an empty sterilizer that uses a vac-uum system that is used for porous load sterilization. Themanufacturer of the Bowie-Dick test card will suggest thetemperature range and time. A Bowie-Dick test cyclemust be programmed accordingly.

• Perform Empty Chamber Temperature Mapping

Empty chamber temperature mapping can identify any“cold spot.” At the end of the cycle, observe the amount ofcondensate and adjust the exhaust, vacuum drying, andcooling time longer to reduce the condensate. Also checkthe pure steam supply and ensure the steam trap is in theoptimum position. According to British Standard (BS) EN285, steam quality tests should be performed to measurethe supply steam quality. These tests include dryness, su-perheat, and non-condensable gases. High condensation,particularly pooling, may have a dramatic impact on heatpenetration and possibly sterile barrier integrity. Exces-sive condensate will soak the autoclave bag resulting in anincreased chance of autoclave bag tears.

Perform empty chamber temperature mapping by usingone of the standard (factory pre-programmed) programs,but modify the temperature set point to 121.5˚C anddwell time to 30 minutes. If the jacket temperature isprogrammable, set point should be 1˚C plus calibration



tolerance of temperature sensor (usually ResistanceTemperature Device RTD) less than sterilization setpoint. Example, if the calibration tolerance is 0.5˚C, thejacket temperature set point should be 120˚C. Autoclavechamber wall temperature (jacket temperature) shouldbe set slightly lower or equal to the chamber temperaturein order to prevent superheated steam. The RTD calibra-tion tolerance is added to provide a safety margin tocompensate for instrument error.

As shown in Figure 2 and Figure 3, place temperature element (Thermocouple TC) of the temperature logger(validator) on four corners and the middle of each shelffor heat distribution (HD) data. Observe the amount ofresidual condensate at the end of the cycle and ensurethat the autoclave bag is not soaked with condensate. Ad-just the exhaust time, vacuum drying time, and coolingtime, and repeat the empty chamber temperature map-ping as needed until an acceptable cycle is completed.

Figures 2 and 3Empty Chamber Test TC Placement Diagram and Placement Summary

2

Drain

Load Door

Unload Door

Chamber

71

3

4

5

6

8

9

10

11

12

1 Top of chamber, front left corner 7 Bottom of chamber, back left corner2 Top of chamber, back left corner 8 Bottom of chamber, center3 Top of chamber, center 9 Bottom of chamber, front right corner 4 Top of chamber, front right corner 10 Bottom of chamber, back right corner5 Top of chamber, back right corner 11 Next to drain sensor 6 Bottom of chamber, front left corner 12 Next to Chamber sensor

TC Location TC LocationNo. No.



• Perform Temperature Mapping with All Items

Perform temperature mapping of the loaded chamber.Collect a list of all the items that need to be autoclaved.If there are a lot of items that cannot be included in a sin-gle load, divide them into two or more loads. Use thesame cycle as the above empty chamber temperaturemapping program. Temperature set point of 121.5˚C anddwell time to 30 to 45 minutes (long enough to ensureall items can reach F0 of 22 minutes).

Load the chamber with the items identified earlier. Placethermocouple (TC) inside each item to collect heat pen-etration (HP) data and place a TC on the four cornersand middle of each shelf for heat distribution (HD) data.Again observe the condensate at the end of the cycle andensure the autoclave bag is not soaked with condensate.Repeat the same load one to two times, but randomizethe items into different locations. Load patterns are ran-domized from run to run to test the location-to-locationor item-to-item variability.

Perform temperature mapping for other load items, ifany. In this case study, there were six loads that ran twotimes each.

• Determination of Master Load Items

Analyze the thermal data from heat penetration thermo-couples (HPTC) of all runs to evaluate and determine aworse-case wrapped hard goods cycle.

Identify and select all the worse-case items from differ-ent loads by ranking the time to reach F0 of 12 minutesfrom HP data. Normally, a worse-case item has a highermass or a restricted opening for steam penetration. Forthis particular case study, one of the worse-case items isa 20L-bioreactor (higher mass). This chamber can onlyfit one; otherwise, the maximum number of items shouldbe used for master load items. These items are the worst-case items as listed in Figure 4.

Determination of Temperature Set Point

Sterilization temperature is determined by calculatingthree-sigma from heat distribution (HD) data. All tempera-ture readings from the different loads during the dwell periodare used to calculate the standard deviation. The temperatureset point should be 121.5˚C plus three-sigma. Three timesthe greatest process variance is added to the standard (basereference value for temperature sterilization used in the de-velopment study) 121.5˚C, to ensure the process temperaturewill always remain above the standard. From the case study,the greatest process variance of measured temperature fromtwelve (12) test runs performed at a set point of 121.5˚C wasdemonstrated to be 0.53˚C (see Figure 5: TC04), below thestandard deviation is 0.53˚C. Therefore, the sterilization setpoint is 123˚C (121.5˚C + 1.5˚C).

Figure 4Worst-case Items Ranking

Bioreactor (20L) 35

Manifold tubing (10 mm ID x 4') 31

STM Filter (1/4" 1/4" 1/4" tee) 30

Sampling Assembly (1/4" x 1/4" x 1/4" tee) 30

Harvest/Transfer (diaphragm valve) 30

Dead End Assembly (tubing) 30

2L Flask (inside) 30

Reference tubing (inside) 30

SMA tubing (inside) 28

Items Time to reach 12 F0 (min)



Determination of Dwell Time

Similarly, dwell time is determined by calculating three-sigma. Typical commercial available BI has higher D valuethan 1 minute. For this case study, F0 is required about 22minutes to achieve SAL of 10-6 for BI challenge. With a tar-get F0 of 22 minutes, the worst-case data (QC load run #3,#4) indicates that a dwell time of 26.1 minutes is required toachieve the target (see Figure 6). The standard deviation isabout 1.4 minutes. In order to provide assurance the targetis met with a safety factor, dwell time is 30 minutes (26 + 4minutes).

Determination of Other Parameters

Similarly, process alarm set points can be set based onthe data collected from the cycle development runs. Addingthree times the greatest process (time) variance to the aver-age data point from the cycle development runs can deter-mine the alarm set points (see Figure 7).

From this case study, the set point is 123˚C and the min-imum requirement is 121˚C. The process tolerance is 123˚C- 121˚C = 2˚C. The rule of thumb of 4 : 1 ratio, the calibra-tion tolerance of the controlling temperature sensor (nor-mally drain probe for wrapped goods) should be +/-0.5˚C,which is also PDA recommended. Tolerance of better than

TC 01 TC 02 TC 03 TC 04 TC 05 TC 06 Avg 122.1 122.0 122.2 122.4 122.4 122.2Max 123.3 123.3 123.2 126.8 123.9 123.5Min 119.0 119.4 119.0 119.0 119.6 119.1

Range 4.3 3.9 4.2 7.8 4.3 4.4SD 0.1778 0.3041 0.1332 0.5284 0.1595 0.0578

3-Sigma 0.5333 0.9124 0.3995 1.5853 0.4786 0.1735

SP 122.0 122.4 121.9 123.1 122.0 121.7

Figure 5Temperature Set Point Determination

Avg: Average (˚C)SD: Standard Deviation (˚C)

Max: Maximum temperature (˚C)Min: Minimum temperature (˚C)

SP: Set point (˚C)

Total of 12 runs Heat Distribution TC Data

load topleft

unloadtop left

middlecenter

load topright

unloadtop right

load bottomleft

TC 07 TC 08 TC 09 TC 10 TC 11 TC 12Avg 121.9 122.4 122.2 122.3 122.2 122.2Max 123 124.1 123.3 123.4 123.6 123.6Min 118.9 119.1 119.0 119.2 119.4 119.4

Range 4.1 5.0 4.3 4.2 4.2 4.2SD 0.062 0.4423 0.1403 0.0576 0.0731 0.0415

3-Sigma 0.1861 1.3268 0.4208 0.1727 0.2194 0.1244

SP 121.7 122.8 121.9 121.7 121.7 121.6

Total of 12 runs Heat Distribution TC Data

unload bottom left

bottomcenter

load bottomright

unload bottomright

drainline

adjacent to chamber RTD



Figure 6Wrapped Goods Dwell Time

Filters QC Load 20L BioreactorRun 1 2 3 4 5 6

Min F 16:51:35 11:41:45 17:40:00 10:13:00 18:51:55 10:13:00Dwell start 16:40:38 11:30:56 17:27:14 10:02:18 18:40:34 10:00:28

Time to target 0:10:57 0:10:49 0:12:46 0:10:42 0:11:21 0:12:32

Dwell Time(s) 657.0 649.0 766.0 642.0 681.0 752.0Avg(s) 653.0 704.0 716.5SD(s) 5.7 87.7 50.2

Dwell to 12 (min) 11.2 16.1 14.5

BI F 17:03:00 11:52:00 17:50:00 10:23:00 19:01:00 10:23:00Dwell start 16:40:38 11:30:56 17:27:14 10:02:18 18:40:34 10:00:28

Time to KILL 0:22:22 0:21:04 0:22:46 0:20:42 0:20:26 0:22:32


Dwell to 22 (min) 24.5 26.1 25.9

2L Flasks Tubing QC BasketsRun 7 8 9 10 11 12

Min F 17:09:55 19:30:30 18:56:45 21:55:40 14:05:00 16:41:00Dwell start 17:00:04 19:19:58 18:45:54 21:45:42 13:55:28 16:32:02

Time to target 0:09:51 0:10:32 0:10:51 0:09:58 0:09:32 0:08:58


Dwell to 12 (min) 11.6 12.3 10.5

BI F 17:20:00 19:41:00 19:07:00 22:06:00 14:15:00 16:51:00Dwell start 17:00:04 19:19:58 18:45:54 21:45:42 13:55:28 16:32:02

Time to KILL 0:19:56 0:21:02 0:21:06 0:20:18 0:19:32 0:18:58


Dwell to 22 (min) 22.8 22.4 20.5

Min F: time to achieve 12 F0 (HH:MM:SS)BI F: time to achieve 22 F0 (HH:MM:SS)SD: Standard Deviation(s)Avg: Average(s)



Figure 7Process Alarm Set Point Determination

AIR REMOVAL 0:00:41 123 0:11:20 0:13:23 14

HEAT UP 0:00:07 21 0:09:24 0:09:45 11

STERILIZATION 0:00:01 3 0:29:59 0:30:03 31

EXHAUST 0:00:20 60 0:00:51 0:01:51 2

EQUALIZATION 0:00:11 33 0:00:56 0:01:29 2

TIME LIMITALARM SET

POINT

STANDARD DEVIATION(H:MM:SS)

THREESIGMA

(SECOND)

AVERAGE(H:MM:SS)

SET POINT(H:MM:SS)

SET POINT (MIN)

Figure 8Master Load Cycle Validation Confirmation

TIME (HH:MM:SS)

F0 (Minutes)



0.5˚C (such as 0.1˚C) is very difficult to calibrate in prac-tice. On the other hand, the temperature set point should notbe lower than 122˚C unless the product or material to be au-toclaved is temperature sensitive.

If the jacket temperature is programmable, set pointshould be 1˚C plus calibration tolerance of temperature sen-sor (usually Resistance Temperature Device RTD) less thansterilization set point. For this case study, the jacket temper-ature should be 121.5˚C (123 - 1 - 0.5˚C).

Master Load Cycle Validation Confirmation

Program the master load cycle with the above alarm setpoints and parameters.

Perform a master load cycle run with temperature map-ping and items BI to confirm sterility and process effective-ness. For this case study and a worse-case challenge, thetemperature set point was lowered to 121.5˚C. If not all theBIs pass; perform another master load cycle with BI at themaster cycle temperature set point (123˚C for this casestudy). Calculate to ensure all heat penetration is above theminimum required F0 (22 minutes for this case study). Forthis case study, the temperature set point of 121.5˚C had suf-ficient F0 to kill all BIs. The minimum F0 achieved was 32.2minutes, which provides a maximum BI challenge SLR of 17(See Figure 2). These data indicate that the new master loadcycle will reliably and predictably achieve sterility with anoverkill cycle delivery of a spore log reduction (SLR) wellabove 12.

CONCLUSION

The autoclave is now ready for PQ. From this point, thePQ is much more straightforward. There is only one masterload cycle to be validated. The protocol is similar to the lastmaster load cycle validation confirmation run. Typically, PQrequires performing the same run three times. Randomizethe load pattern for each run. The master load cycle shouldhave high enough safety margins to demonstrate a high de-gree of confidence for sterility in excess of a SAL of 10-6.Moreover, it develops a master load cycle that can supportone cycle for any qualified wrapped goods items to beloaded randomly or without a particular load pattern as longas the items are loaded properly. !

REFERENCES• PDA Technical Monograph No. 1, Validation of Moist Heat

Sterilization Process-Cycle Design, Development, Qualifica-tion and Ongoing Control.

• USP 29<1035> Biological Indicators for Sterilization• Health Technical Memorandum (HTM) 2010 Sterilization

Part 3 Validation and Verification • British Standard EN 285 - Sterilization - Steam sterilizers -

Large sterilizers

ABOUT THE AUTHOR

Victor Tsui, P.E. is Associate Director of Plant Engi-neering at VaxGen, Inc. Mr. Tsui is a professionalchemical engineer. He has a Masters degree in Engineering and over 17 years engineering experi-ence. Victor can be reached by telephone at (650) 255-7148 or by email at [email protected].

Ward Wiederhold is a Validation Consultant for BI/OEquipment and Services, LLC based in the San Fran-cisco Bay area. He has over 15 years experience inthe Pharmaceutical and Biotech Industry. He holds aB.S.M.E. degree from the University of Missouri.

Article Acronym Listing

BI Biological IndicatorBS British StandardD Dwell (time)EMEA European Medicinal Evaluation AgencyFDA Food and Drug AdministrationHD Heat DistributionHP Heat PenetrationHTM Health Technical MemorandumISPE International Society for

Pharmaceutical EngineeringPDA Parenteral Drug AssociationPQ Performance QualificationQC Quality ControlRTD Resistance Temperature DeviceSAL Sterility Assurance LevelSD Standard DeviationSLR Spore Log ReductionSP Set PointTC ThermocoupleUSP United States Pharmacopoeia

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