AutoclaveValidation

PHARMACEUTICALENGINEERINGJULY/AUGUST2002
Copyright ISPE 2002
Practical Guide to Autoclave
Validation
by Raymond G. Lewis, PE
In addition to
potential
business
liabilities, there
can be significant
costs associated
with an autoclave
validation
process. The
practical
experience that
this article is
based on may
provide
assistance in
ensuring an
effective, efficient
validation
process for
steam
sterilization.
Introduction
T
hi s arti cl e i s based on practi cal experi -
ences gai ned by the author whi l e con-
ducti ng hundreds of val i dati on test runs
on dozens of autocl aves of vari ed manufacture.
I t i s pri mari l y i ntended that personnel who
perform val i dati on testi ng on autocl aves may
benefi t from these experi ences, and that i t wi l l
assi st i n ensuri ng a hi gh l evel of compl i ance i n
the val i dati on process. The arti cl e al so may be of
benefi t i n sel ecti ng an appropri ate val i dati on
strategy and/or cycl e. Personnel unfami l i ar wi th
steam steri l i zati on pri nci pl es or autocl ave val i -
dati on coul d use the materi al as a basi c trai ni ng
tool and i t may be a good refresher for more
experi enced personnel . A l i st of defi ni ti ons and
references are provi ded at the end of the arti cl e.
Sterility Assurance Level
The l evel of mi crobi al i nacti vati on can be de-
scri bed by an exponenti al functi on, Steri l i ty
Assurance Level or SAL. For exampl e, a SAL of
10
-6
means that the probabi l i ty of a si ngl e vi abl e
mi croorgani sm bei ng present on a steri l i zed
i tem/product i s one i n one mi l l i on after the i tem
has undergone a steri l i zati on process. A SAL of
10
-3
means that the probabi l i ty of a si ngl e vi abl e
mi croorgani sm bei ng present after steri l i zati on
i s one i n one thousand.
The SAL requi red i s determi ned by the i n-
tended use of the i tem/product. Steri l i zati on
processes associ ated wi th parenteral s and medi -
cal devi ces that pose a si gni fi cant ri sk i n terms
of the probabi l i ty and severi ty of an i nfecti on
(e.g., i mpl ants, steri l e fl ui d pathways, products
i ntended to come i nto contact wi th compromi sed
ti ssue) general l y have been steri l i zed to an SAL
of 10
-6
. Medi cal devi ce products not i ntended to
come i nto contact wi th breached ski n or compro-
mi sed ti ssue are general l y steri l i zed to a SAL of
10
-3
.
The remai nder of thi s arti cl e i s wri tten as-
sumi ng that a SAL of 10
-6
i s requi red.
Log Reduction
Achi evi ng a 1-l og reducti on means to decrease
the mi crobi al popul ati on by a factor of 10. The
bi oburden i s the number and type of vi abl e
mi croorgani sms contami nati ng an i tem. A ster-
i l i zati on cycl e that provi des a SAL of 10
-6
effec-
ti vel y means that the mi cr oor gani sms that
coul d be present (i .e., bi oburden) are ki l l ed,
and an addi ti onal 6-l og reducti on safety factor
has been provi ded. The fol l owi ng provi des an
exampl e of a cycl e achi evi ng a SAL of 10
-6
.
Bi oburden (worst case) = 134 CFU (col ony
formi ng uni t).
To reduce the mi crobi al popul ati on from 134
to 1 = l og (134) = 2.13 (i .e., a 2.13-l og reduc-
ti on i s requi red to reduce the
popul ati on from 134 to 1).
Appl yi ng an addi ti onal 6-l og
reducti on wi l l theoreti cal l y
reduce the mi crobi al popu-
l ati on from 1 to 0.000001.
Thi s provi des a SAL of 10
-6
or
a one i n one mi l l i on prob-
abi l i ty of a si ngl e survi vi ng
mi croorgani sm.
Total l og reducti on = 2.13 + 6
= 8.13. Therefore to provi de
a SAL of 10
-6
wi th a
bi oburden of 134 CFU re-
qui res a steri l i zati on cycl e
that provi des an 8.13 l og re-
ducti on.
Figure 1. Emptychamber
temperature mapping
(Photograph provided courtesy
of Kuhlman Technologies Inc.)
AutoclaveValidation
JULY/AUGUST2002PHARMACEUTICALENGINEERING
Copyright ISPE 2002
Thermal Resistance Characteristics
The thermal resi stance of speci fi c mi croorgani sms i s charac-
teri zed by D-val ues and Z-val ues. A D-val ue i s the ti me i n
mi nutes, at a speci fi c temperature, to reduce the survi vi ng
mi crobi al popul ati on by 1-l og. A Z-val ue i s the temperature
change requi red to resul t i n a 1-l og reducti on i n D-val ue.
Other ti me measurement vari abl es pertai ni ng to thermal
resi stance are F-val ues and F
o
-val ues. An F-val ue i s the
number of mi nutes to ki l l a speci fi ed number of mi croorgan-
i sms wi th a speci fi ed Z-val ue at a speci fi c temperature. An F
o
-
val ue i s the number of mi nutes to ki l l a speci fi ed number of
mi croorgani sms wi th a Z-val ue of 10C (50F) at a temperature
of 121.1C (250F).
Common Misconception and Equivalent
Sterilization Time
I t i s not uncommon to encounter the concept that 121.1C
(250F) i s the temperature requi red for steam steri l i zati on.
Thi s understandi ng i s not enti rel y correct. Extensi ve empi ri cal
studi es were conducted and one of the cri ti cal vari abl es (tem-
perature) was pre-sel ected. I t i s not surpri si ng that the tem-
perature sel ected was an obvi ous round number i n the tem-
perature range of i nterest (250F). The F
o
-val ue equati on can
be used to determi ne the rel ati ve steri l i zati on ti me at other
temperatures as per the fol l owi ng (wi th Z-val ue = 10C):
F
o
= 10
(T 121.1) /10
where T = temperature ( C) and F
o
= equi val ent steri l i za-
ti on ti me (mi n.)
1
Tabl e A provi des some exampl es and the rel ati onshi p fol l ows
i n graphi cal form i n Fi gure 2.
As i s demonstrated by the data above, steri l i zati on can be
achi eved usi ng any of these temperatures. The l ower the
temperature the l onger the steri l i zati on cycl e requi red. Thi s i s
an i mportant concept to consi der because there are occasi ons
where the temperature needs to be careful l y sel ected. An
exampl e i s a l i qui d that cannot wi thstand hi gh temperatures.
I deal l y, the hi ghest temperature that the l oad can wi thstand
i s sel ected, si nce thi s wi l l provi de the shortest possi bl e cycl e.
Variables Required to Determine an
Ideal Sterilization Cycle
An i deal steri l i zati on cycl e presumes an i deal steri l i zi ng
envi ronment (i .e., saturated steam wi th no ai r). The i deal cycl e
can be determi ned wi th the fol l owi ng three vari abl es: bi oburden,
D-val ue, and requi red SAL. The fol l owi ng provi des some ex-
ampl es:
a) Gi ven: Bi oburden = 75 CFU, D-val ue = 0.5 mi n./l og at
121.1C, Requi red SAL = 10
-6
Then: Log (75) = 1.88
Log Reducti on requi red = 1.88 l og + 6 l og = 7.88 l og
I deal Cycl e at 121.1C (250F) = (7.88 l og)(0.5 mi n./
l og) = 3.94 mi nutes
b) Gi ven: Bi oburden = 1,215 CFU, D-val ue = 1.6 mi n./l og at
121.1C, Requi red SAL = 10
-6
Then: Log (1215) = 3.08
Log Reducti on requi red = 3.08 l og + 6 l og = 9.08 l og
I deal Cycl e at 121.1C (250F) = (9.08 l og)(1.6 mi n./
l og) = 14.53 mi nutes
Overkill Approach
Determi ni ng the bi oburden and D-val ue for al l i tems to be
steri l i zed i n a l oad can be qui te ti me consumi ng and costl y. As
a resul t, for i tems that are not heat sensi ti ve, an overki l l
approach i s general l y empl oyed.
An overki l l approach avoi ds col l ecti ng bi oburden and D-
val ue data by assumi ng worst-case condi ti ons. A bi oburden of
10
6
of a hi ghl y heat resi stant spore formi ng bacteri a (Baci l l us
stearothermophi l us) i s uti l i zed. The D-val ue at 121.1C for
these bacteri a i s general l y sl i ghtl y above 2 mi nutes, and
therefore usi ng 2.5 mi nutes i s a good worst-case val ue.
Wi th a bi oburden of 10
6
, to achi eve a SAL of 10
-6
requi res a
12 (6 + 6) l og reducti on. Under i deal condi ti ons, the l ength of an
overki l l steri l i zati on cycl e at 121.1C i s therefore (12 l og)(2.5
mi n./l og) = 30 mi nutes.
Bioburden and D-Value Approach
For i tems that are heat sensi ti ve and cannot wi thstand an
overki l l approach, i t i s necessary to col l ect bi oburden and
possi bl y D-val ue data. Thi s wi l l dramati cal l y shorten the
steri l i zati on cycl e requi red. For exampl e, i f the bi oburden i s
l ow (e.g., 10 CFU) and even moderatel y resi stant (e.g., D-val ue
= 0.5), an i deal 30-mi nute overki l l cycl e at 121.1C can be
repl aced by an i deal cycl e of 3.5 mi nutes (7 l og x 0.5 mi n./l og).
Al ternati vel y, the steri l i zati on temperature coul d be reduced
Figure 2. Equivalent sterilization time.
Figure 3. Ideal cycle time.
AutoclaveValidation
PHARMACEUTICALENGINEERINGJULY/AUGUST2002
Copyright ISPE 2002
to 112C and yet onl y requi re sl i ghtl y l ess than a 30 mi nute
i deal cycl e. I t may be a si gni fi cant advantage to reduce the
steri l i zati on temperature and/or ti me.
A compromi se approach may someti mes be uti l i zed where
bi oburden data i s col l ected, but D-val ue studi es are not per-
formed. A worst case D-val ue of 2.5 coul d then be empl oyed.
Thi s approach wi l l provi de a somewhat shortened cycl e and
avoi ds the ti me and cost of D-val ue studi es. Fol l owi ng our
exampl e wi th a bi oburden of 10 CFU, the i deal cycl e at 121.1C
can be shortened from a 30-mi nute overki l l cycl e to a 17.5-
mi nute cycl e (7 l og x 2.5 mi n./l og).
Fi gure 3 shows the steri l i zati on ti me requi red at 121.1C for
an i deal cycl e to achi eve a SAL of 10
-6
at varyi ng l evel s of
bi oburden (D-val ue = 2.5 mi n.).
Vacuum and Non-Vacuum Cycles
Previ ousl y, thi s arti cl e has addressed i deal cycl es that pre-
sume an i deal steri l i zi ng envi ronment. I n terms of the l ength
of cycl e requi red, one can onl y approach i deal cycl es for i tems
that are easi l y steri l i zed. Most often, i tems/l oads wi th l ess
than i deal condi ti ons are encountered.
There are three basi c types of cycl es as fol l ows:
a) Hard Goods (Vacuum):
Sui tabl e for i tems easy to steri l i ze si nce ai r removal and
steam penetrati on are hi ghl y effecti ve. Exampl es are many
types of gl assware and l arge di ameter pi pi ng. A typi cal hard
goods cycl e may draw one vacuum pri or to i ntroduci ng
steam, reachi ng the desi red steri l i zati on temperature, and
begi nni ng the steri l i zati on dwel l peri od. A typi cal pressure
vs. ti me graph for a hard goods cycl e i s shown i n Fi gure 4.
b) Wrapped Goods (Vacuum):
Uti l i zed for i tems di ffi cul t to steri l i ze si nce ai r removal and
steam penetrati on are harder to achi eve. Exampl es are
gowns, l ong l engths of tubi ng, and tanks/vessel s/apparatus
wi th smal l i nl et/outl et ports and/or vent fi l ters. A typi cal
wrapped goods cycl e may draw three or more vacuums pri or
to reachi ng the desi red steri l i zati on temperature and begi n-
ni ng the steri l i zati on dwel l peri od. A post steri l i zati on
vacuum al so i s usual l y drawn to evacuate the steam from
the l oad i tems. Often the l ength of ti me to pul l and rel ease
the vacuums exceeds the l ength of the steri l i zati on dwel l . A
typi cal pressure vs. ti me graph for a wrapped goods cycl e i s
shown i n Fi gure 5.
c) Li qui ds/Gravi ty Di spl acement (Non-Vacuum):
I tems that contai n l i qui ds general l y cannot have a deep
vacuum pul l ed or the l i qui d wi l l be drawn out of the i tem.
Li qui d cycl es general l y just heat up and cool down and do
not uti l i ze vacuums. These i tems may requi re a l engthy
cycl e ti me especi al l y where the l i qui d vol ume i s l arge
because the l ength of ti me requi red to heat up and cool down
the l i qui d may be consi derabl e. Another term for a l i qui d
cycl e i s gravi ty di spl acement as the ai r i s di spl aced by
gravi ty (i .e., removi ng ai r by i ntroduci ng steam i nto the top
of a chamber and di spl aci ng the ai r, whi ch i s heavi er than
steam, by removi ng the ai r from the bottom of the chamber).
A typi cal pressure vs. ti me graph for a l i qui ds cycl e i s shown
i n Fi gure 6.
Basic Validation Approach
Installation Qualification (IQ)
The I Q process i s i ntended to demonstrate that the autocl ave
as i nstal l ed meets al l speci fi cati ons, i s i nstal l ed properl y, and
that the supporti ng programs needed for ongoi ng operati on
(e.g., standard operati ng procedures, mai ntenance program,
etc.) are i n pl ace.
An I Q may i ncl ude the fol l owi ng checks:
Mechani cal Equi pment Speci fi cati ons (chamber, val ves,
traps, strai ners, fi l ters, regul ators, vacuum pump, heat
exchanger, condenser, etc.)
Control and I nstrumentati on Speci fi cati ons (programmabl e
l ogi c control l er, operator i nterface, pri nter/recorder, control
val ves, transducers, pressure and temperature transmi t-
ters, resi stance temperature devi ces, swi tches, l evel sen-
sors, i nterl ocks, photocel l s, etc.)
Si te Speci fi cati ons/Uti l i ti es (power, groundi ng, surge pro-
tector, uni nterrupti bl e power suppl y, breakers, water, ai r,
cl ean steam, pl ant steam, drai n, shutoff /i sol ati on val ves,
el ectri cal di sconnect swi tches, etc.)
Drawi ngs Veri fi cati on (P&I D, mechani cal , el ectri cal )
Constructi on Materi al s/Materi al s i n Product Contact
Approval Documentati on (e.g., pressure vessel , el ectri cal ,
etc.)
Change/Spare Parts
Bi l l of Materi al s
Vendor Speci fi cati on Sheets
Purchase Orders
Factory Performance Tests
Commi ssi oni ng Report
Preventi ve Mai ntenance Program
Standard Operati ng Procedures (operati ng, mai ntenance,
cal i brati on)*
Operati ng and Mai ntenance Manual s
Pi pi ng I nstal l ati on Veri fi cati on (sl ope, dead l egs)
Wel d I nspecti on/Surface Roughness Documentati on/Met-
al l urgi cal Documentati on
Control System Documentati on (system confi gurati on/bl ock
di agram, fl ow sheets, di spl ay/report l ayouts, requi red i nter-
l ock consi derati ons, general process l i mi ts, condi ti ons for
operati ng over range, hard copy and el ectroni c appl i cati on
code l i sti ng, ti mi ng di agram, system securi ty, i nput/output
poi nt l i sti ng, data moni tori ng, al arms, software i nventory
Table A. Equivalent sterilization time.
Temperature F
o
Equivalency to 121.1C (250F)
115C (239F) 0.25 min. 1 minute at 115C provides the same lethality as 0.25 minutes at 121.1C
120C (248F) 0.78 min. 1 minute at 120C provides the same lethality as 0.78 minutes at 121.1C
121.1C (250F) 1 min. 1 minute at 121.1C provides the same lethality as 1 minute at 121.1C
122C (251.6F) 1.23 min. 1 minute at 122C provides the same lethality as 1.23 minutes at 121.1C
125C (257F) 2.45 min. 1 minute at 125C provides the same lethality as 2.45 minutes at 121.1C
AutoclaveValidation
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Copyright ISPE 2002
and versi on, software confi gurati ons, parameter l i sti ngs,
software devel opment and testi ng records, change control ,
vendor qual i fi cati on, modul ar software devel opment docu-
ments, detai l ed modul e functi onal speci fi cati ons, etc.)
I nstrumentati on and I nput/Output Dry Loop and Wet Loop
Checks**
PI D Tuni ng**
I nstrument Cal i brati ons**
* Operati ng Procedures can onl y be fi nal i zed after Perfor-
mance Qual i fi cati ons tests are compl eted when val i -
dated l oad confi gurati ons and cycl es are known.
** Note: i n some approaches, these checks are captured as
i ni ti al Operati onal Qual i fi cati on acti vi ti es.
Operational Qualification (OQ)
The OQ process i s i ntended to demonstrate that the compo-
nents of the autocl ave operate properl y and that the autocl ave
i s deemed ready for performance or l oad testi ng.
An OQ may i ncl ude the fol l owi ng checks:
Oper ati onal Tests (oper ator /super vi sor y/mai ntenance
modes, doors, abort and emergency stop, al arms, program-
mabl e parameters, menu navi gati on, securi ty, power-up
and shutdown, operator i nterface di spl ay checks, i nterl ock
overri de control , procedure sel ect/start control , step ad-
vance control , swi tch and i nterl ock tests, etc.)
Power Loss Recovery Test
Source Code Revi ew
Fi l ter Steri l i zati on
Leak/Ai r Removal /Steam Penetrati on/Vacuum Hol d Test*
Jacket Mappi ng
Saturated Steam Check
Empty Chamber Tests
* The Bowi e Di ck test i s desi gned to test ai r removal , the
absence of ai r l eaks and steam penetrati on i nto a porous
l oad. I t uses a test pack of fabri c wi th speci fi c di mensi ons
or there are commerci al , use once packs avai l abl e. I t has
been wi del y empl oyed i n Europe. I n North Ameri ca, a
Vacuum Hol d Test has often been empl oyed. European
Standard EN 554 speci fi es that i f a steri l i zati on process
i ncl udes ai r removal from the product, a steam penetra-
ti on test shal l be carri ed out at the commencement of
each day the autocl ave i s used. Al though a vacuum hol d
test may be l ess sensi ti ve than a Bowi e Di ck test, the
author assumes that a vacuum hol d test can be consi d-
ered as a sati sfactory al ternati ve i f stri ct acceptance
cri teri a are appl i ed. Thi s assumpti on i s based on steam
penetrati on/l ethal i ty i n the worst case l oad i tems bei ng
demonstrated and that the vacuum hol d test therefore
demonstrates absence of l eaks and that the val i dated
condi ti ons that resul ted i n l ethal i ty are bei ng met on an
ongoi ng basi s.
Empty Chamber Distribution Tests (Figure 1)
The basi c objecti ve i s to show the chamber provi des a uni form
steri l i zi ng envi ronment. I n the opi ni on of the author, col d
spots i n autocl aves are rarel y encountered. Someti mes col d
thermocoupl es are mi si nterpreted as col d spots (refer to fol -
l owi ng secti on Ti ps).
Three consecuti ve successful runs are performed for each
cycl e type wi th typi cal acceptance cri teri a as per the fol l owi ng:
Throughout the dwel l ti me, al l temperatures measured i n
Figure 4. Hard goods cycle.
Figure 5. Wrapped goods cycle.
the chamber are wi thi n a 3C band (steri l i zati on temperature
+ 3C).
2
Note: the dwel l set-poi nt -1C/+2C i s often used.
Throughout the dwel l ti me al l temperatures measured i n
the chamber do not fl uctuate by more than 1C.
2
Throughout the dwel l ti me, al l temperatures measured i n
the chamber do not di ffer from each other by more than 2C.
2
The steam i s at a temperature correspondi ng to i ts vapor
pressure.
2
The i nterval of ti me between the attai nment of the steri l i za-
ti on temperature i n the hottest and col dest parts of the
chamber does not exceed 15 seconds for chambers of not
more than 800L and not to exceed 30 seconds for l arger
chambers.
2
Ti med measurements shal l be control l ed to an accuracy of
1%.
2
Requi red pre-certi fi cati on and post-certi fi cati on of the data
l ogger ensures that the temperature measurement system
i s accurate to wi thi n 0.5C.
The vacuum hol d test shoul d achi eve a vacuum l evel of 2.5
psi a (wi th vacuum pump) and mai ntai n the vacuum (wi th-
out further vacuum bei ng i ni ti ated) wi thi n 0.4 psi over a
peri od of fi ve mi nutes.
Performance Qualification (PQ)
Loaded Chamber SteamPenetration Tests
Loaded chamber steam penetrati on runs are then conducted
on every l oad. Note: thi s i s a very ti me consumi ng process,
especi al l y i f you have a si gni fi cant number of i tems to be
steri l i zed. I t i s necessary to determi ne whi ch l oad i tems are the
most di ffi cul t to steri l i ze and whi ch l ocati on(s) wi thi n the
i tems presents the worst-case condi ti ons.
There are two commonl y used methods for determi ni ng the
worst-case i tems/l ocati ons, thermocoupl es, and steam i ntegra-
tors. Steam i ntegrators are commerci al l y avai l abl e stri ps that
provi de a quanti tati ve i ndi cati on of the exposure to steam. The
AutoclaveValidation
PHARMACEUTICALENGINEERINGJULY/AUGUST2002
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amount of steam exposure can be determi ned by measuri ng
the movement of a chemi cal i ndi cator on the i ntegrator
stri p. The author recommends uti l i zi ng steam i ntegrators
si nce they are desi gned to measure steam exposure and
thermocoupl es can resul t i n mi sl eadi ng data (i .e., measur-
i ng temperature wi thout taki ng i nto account whether there
i s any ai r present).
Determi ni ng whi ch l oad i tems are the most di ffi cul t to
steri l i ze and whi ch l ocati on(s) wi thi n the i tems presents the
worst-case condi ti ons can be a daunti ng task. Wi th a l arge l oad
contai ni ng a wi de vari ety of di fferent types of i tems, the
number of possi bl e test l ocati ons seems to approach i nfi ni ty. I t
al so can be di ffi cul t to get the thermocoupl e and/or steam
i ntegrator i nto the i tem wi thout adversel y affecti ng the i tem's
abi l i ty to be steri l i zed and/or rui ni ng the i tem (a concern wi th
expensi ve i tems).
One must eval uate an i tem on a case-by-case basi s and
determi ne how best to chal l enge the i tem. Often the i tem must
be seal ed somehow to return the i tem to a state that represents
equi val ency wi th respect to steam penetrati on. No attempt wi l l
be made to provi de an exhausti ve commentary here, but rather
provi de a few basi c techni ques for answeri ng questi ons that
i nevi tabl y ari se:
What i s the most di ffi cul t poi nt to steri l i ze i n a hose of
uni form di ameter? Common sense can somti mes assi st,
di ctati ng i n thi s i nstance that the most di ffi cul t to steri l i ze
poi nt i s i n the center of the hose.
How do you get a 10-foot l ength of thermocoupl e and/or
steam i ntegrator i nto the mi ddl e of a 50-foot hose? You can
put a sl i ce/cut i nto the mi ddl e of the hose and i nsert the
thermocoupl e/i ntegrator through the sl i ce. Note: the cut
must be seal ed or you wi l l not be chal l engi ng the hose
properl y. You can use si l i con to seal the cut. Al ternati vel y,
i f two 25-foot l engths of the hose are avai l abl e you can joi n
the two l engths wi th a connector and i nsert the thermo-
coupl e i nto the connector. The connector then must be
seal ed. The advantage here i s that you dont rui n the 50-foot
hose. The connector techni que can be used for smal l di am-
eter tubi ng where the hose i s too smal l to i nsert a thermo-
coupl e and/or steam i ntegrator.
What i s the worst-case l ocati on wi thi n a bottl e, fl ask, or
cyl i nder? Thi s has been shown to be i n the center, near, but
not at the bottom.
How can you mi ni mi ze the number of runs requi red to
chal l enge a l oad? Usi ng steam i ntegrators can hel p mi ni -
mi ze the number of runs requi red to chal l enge a l oad. There
are a l i mi ted number of thermocoupl es avai l abl e, but as
many i ntegrators as desi red can be pl aced i n the l oad.
Load Configurations
Another vari abl e of concern i s whether fi xed l oad confi gura-
ti ons or fl exi bl e l oad confi gurati ons are desi red. A fi xed l oad
confi gurati on means that the l oad to be steri l i zed wi l l be
i denti cal for al l future processi ng runs and that the l oad i s
pl aced i n the chamber i n exactl y the same way for al l future
processi ng runs.
I n the opi ni on of the author, the l ocati on of an i tem i n the
chamber does not i nfl uence i ts abi l i ty to be steri l i zed (assum-
i ng that the l ocati on change does not i nvol ve a change i n l oad
densi ty). Thi s observati on i s based on the experi ences of the
author i n conducti ng hundreds of val i dati on test runs on
dozens of autocl aves of vari ed manufacture. However, one
shoul d proceed as i f the l ocati on wi thi n the autocl ave i s a
vari abl e of concern. One can el i mi nate thi s vari abl e by rotati ng
the i tems wi thi n a l oad from run to run and thereby attempt to
demonstrate posi ti onal equi val ency.
For most l oads, agai n i n the opi ni on of the author based on
experi ence, the number of i tems i n the chamber does not
i nfl uence an i tems abi l i ty to be steri l i zed (unl ess the l oad
becomes so dense that steam penetrati on/ci rcul ati on becomes
an i ssue). One shoul d proceed as i f thi s i s a vari abl e of concern.
You can successful l y val i date a l oad whi l e encompassi ng thi s
si tuati on by performi ng mi ni mum and maxi mum l oad studi es.
The fol l owi ng provi des an exampl e of fi xed vs. fl exi bl e l oad
confi gurati ons:
Exampl e l oad:
- three (3) fl asks
- four (4) graduated cyl i nders
- 24 pl asti c bottl es wi th vent fi l ters
Fi xed Load/Fi xed Posi ti on:
I n thi s si tuati on, al l of the l oad i tems are pl aced i n the
autocl ave, each ti me i n the same posi ti on for each i tem, and
a di agram of the l oad confi gurati on i s avai l abl e i n the
procedures so that the operators can reproduce the l oad for
every processi ng run. Thi s si tuati on wi l l requi re the l east
val i dati on runs, but offers no fl exi bi l i ty i n l oad confi gura-
ti on.
Fi xed Load/Vari abl e Posi ti on:
I n thi s si tuati on, al l of the l oad i tems are pl aced i n the
autocl ave, but the l ocati on of the i tem i n the autocl ave can
vary and onl y a l i st of the l oad i tems i s requi red for the
procedures. The val i dati on runs must demonstrate posi -
ti onal equi val ency by rotati ng the i tems from l ocati on to
l ocati on duri ng the test runs. I t may be possi bl e to accom-
pl i sh thi s wi th the same number of val i dati on runs as above
and offers the operators some fl exi bi l i ty i n l oadi ng the
autocl ave. Thi s can be an advantage especi al l y for l arge
l oads contai ni ng numerous i tems.
Vari abl e Load/Vari abl e Posi ti on:
I n thi s si tuati on, any or al l of the l oad i tems (i .e., any
combi nati on of from 0 to 3 fl asks, from 0 to 4 cyl i nders, from
0 to 24 bottl es) can be pl aced i n the autocl ave i n any posi ti on
i n the autocl ave and onl y a maxi mum l oad l i st i s requi red
for the procedures. The val i dati on runs must demonstrate
posi ti onal equi val ency by rotati ng the i tems from l ocati on to
l ocati on duri ng the test runs. The val i dati on runs al so must
demonstrate that the cycl e i s adequate for both a maxi mum
Figure 6. Liquids cycle.
AutoclaveValidation
JULY/AUGUST2002PHARMACEUTICALENGINEERING
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l oad and mi ni mum l oad confi gurati on. The mi ni mum l oad
tests are done wi th onl y one i tem i n the autocl ave, that i tem
bei ng the l oad i tem demonstrated as bei ng the most di ffi cul t
to steri l i ze. Thi s method wi l l requi re the greatest number of
val i dati on runs, but offers the operators a great deal of
fl exi bi l i ty i n l oadi ng the autocl ave. Thi s can be a si gni fi cant
advantage i n many si tuati ons.
Loaded Chamber Biological Challenge Tests
After determi ni ng the worst-case i tems and worst-case l oca-
ti ons wi thi n i tems, these i tems are then chal l enged wi th
bi ol ogi cal i ndi cators (spore stri ps and/or vi al s for pl acement
wi thi n l i qui ds). A thermocoupl e shoul d be pl aced al ong wi th
each i ndi cator, as the temperature data wi l l be requi red to
extrapol ate the cycl e to achi eve the SAL of 10
-6
.
Tests are conducted unti l a cycl e ti me resul ts i n three
consecuti ve runs where the bi ol ogi cal i ndi cators show no growth.
I f i t i s i mportant to achi eve the shortest possi bl e cycl e, thi s
process can consume a great deal of ti me as to determi ne the
success/fai l ure poi nt l i kel y requi res obtai ni ng fai l ed test re-
sul ts al ong wi th successful test resul ts. I n addi ti on, i t takes
ti me to determi ne whether the i ndi cators exhi bi t growth (after
two days of i ncubati on you can be reasonabl y confi dent whether
there i s growth or not i n most cases). I f a few mi nutes of
possi bl y unnecessary ti me added to the cycl e i s not a si gni fi cant
i ssue, i t can be advantageous to attempt to predi ct a cycl e ti me
that you feel wi l l pass. Thi s can save consi derabl e ti me and
val i dati on costs.
Once one has achi eved three consecuti ve runs resul ti ng i n
no growth and therefore demonstrati ng a 6l og reducti on
(assumi ng you were usi ng i ndi cators of 10
6
spores/stri p), the
fol l owi ng equati ons/exampl e show how to extrapol ate the ful l
cycl e requi red to achi eve the SAL of 10
-6
:
La = [12 x (Fo/R)] - Fo
where La = the addi ti onal l ethal i ty (F
o
) requi red
12 = used to extrapol ate a 12-l og reducti on
Fo = the mi ni mum accumul ated F
o
val ue from the bi o-
l ogi cal chal l enge runs at the end of the cycl e
R = the l og reducti on demonstrated (i .e. l og [spore
popul ati on])
Fi = 10
(T-121.1)/10
where Fi = the i nstantaneous F
o
val ue
T = the mi ni mum temperature expected duri ng the
addi ti onal l ethal i ty peri od (Note: thi s tempera-
ture shoul d be taken as the temperature achi eved
at the end of the dwel l peri od at the chal l enge
l ocati on where the mi ni mum accumul ated F
o
val ue
resul ted)
Ta = La/Fi
where Ta = the addi ti onal ti me requi red
C = Ta + D
where C = total dwel l peri od ti me requi red
D = the dwel l peri od ti me whi ch resul ted i n the dem-
onstrated reducti on
Exampl e Cal cul ati on:
The bi ol ogi cal chal l enge runs were performed usi ng spore
stri ps that were enumerated at 1.21 x 10
6
spores/stri p.
Therefore R = l og (1,210,000) = 6.08
The mi ni mum accumul ated F
o
val ue (at the end of the
cycl e) from the bi ol ogi cal chal l enge runs was 30.2 mi nutes.
Therefore Fo = 30.2 mi nutes
La = [12 x (30.2/6.08)] - 30.2 = 29.4 mi nutes
The temperature i n the col dest i tem at the end of the
dwel l peri od was 119.4C
Therefore T = 119.4C
Fi = 10
(119.4-121.1)/10
= 0.676
Ta = La/Fi = 29.4/0.676 = 43.5 mi nutes
The bi ol ogi cal chal l enge runs were conducted wi th a
dwel l peri od of 45 mi nutes. Therefore D = 45 mi nutes
C = 43.5 + 45 = 88.5 mi nutes (note: thi s number shoul d be
rounded up)
Therefore the dwel l peri od must be 89 mi nutes to achi eve
a 12-l og reducti on.
Three consecuti ve successful bi ol ogi cal chal l enge runs
are performed for each l oad wi th typi cal acceptance
cri teri a consi stent wi th the empty chamber di stri buti on
test acceptance cri teri a and al l bi ol ogi cal i ndi cators used
duri ng the test cycl e must show negati ve growth.
Tips
1. I f you are goi ng to draw a vacuum(s), ensure that the l oad
i tems can wi thstand the vacuum(s). You dont want to be the
person who has to report that the new $10,000 tank i s now
as fl at as a pancake.
2. Rotate thermocoupl es from run to run. Thi s avoi ds mi si n-
terpreti ng thermocoupl es that read sl i ghtl y l ower tempera-
tures (i .e., col d thermocoupl es) as col d spots or col d i tems.
3. Label the thermocoupl es by number usi ng a smal l stri p of
autocl ave tape. Thi s wi l l greatl y assi st wi th ensuri ng that
you are properl y recordi ng what thermocoupl e was pl aced i n
each l ocati on and wi l l save val i dati on ti me.
4. I f you are performi ng a l arge number of test runs (e.g., over
the course of several weeks), stri ke a compromi se between
post-cal i brati on veri fi cati on of thermocoupl es after every
run and at the end of the enti re testi ng peri od. I f you wai t
unti l the end of the testi ng peri od, you run the ri sk that al l
of the runs are of no val ue due to not meeti ng the veri fi cati on
acceptance cri teri a. I f you veri fy after every run, you wi l l
add consi derabl y to the l ength of ti me requi red to compl ete
the testi ng. The author has found that performi ng the
veri fi cati on every few runs or every few days i s a reasonabl e
compromi se.
5. Be cauti ous wi th the acceptance cri teri a you empl oy for
post-cal i brati on of thermocoupl es. I f the cri teri on i s too
ti ght (e.g., al l thermocoupl es must meet the acceptance
cri teri a), you may l ose a l ot of runs i f one or two thermo-
coupl es cease functi oni ng or are outsi de of the temperature
tol erance after the run(s).
6. Take great care wi th documenti ng the val i dati on test runs.
The documentati on shoul d i ncl ude: a di agram showi ng the
l ocati on of al l l oad i tems wi thi n the autocl ave chamber, the
i tems contai ni ng thermocoupl es, i ntegrators and bi ol ogi cal
i ndi cators, the preci se l ocati on/number of each thermo
AutoclaveValidation
PHARMACEUTICALENGINEERINGJULY/AUGUST2002
Copyright ISPE 2002
coupl e, i ntegrator and bi ol ogi cal i ndi cator wi thi n each i tem,
the pri ntout from the data recorder, the pri ntout or chart
from the autocl ave, the ti me that the dwel l peri od begi ns
and ends (as per the data recorder ti me), and the resul ts for
each i ntegrator or i ndi cator. Each document shoul d be
cl earl y l abel ed wi th the date, test run number, etc. I f you fai l
to generate good documentati on whi l e conducti ng the runs,
you wi l l not be abl e to recover when anal yzi ng the data/
putti ng together the report, and you wi l l end up wi th
i nadequate or poor qual i ty data to support the val i dati on
process.
7. A thermocoupl e shoul d al ways be pl aced besi de the drai n
temperature sensor (usual l y a drai n temperature sensor i s
used to control the temperature wi thi n the autocl ave).
Cautions
1. I f you are usi ng a non-vacuum cycl e to steri l i ze a non-l i qui d
l oad, you are taki ng a si gni fi cant ri sk. Some regul atory
bodi es wi l l si mpl y not al l ow processi ng of non-l i qui d l oads
wi th non-vacuum cycl es.
2. Some regul atory bodi es are extremel y concerned that al l
poi nts wi thi n the l oad achi eve steri l i zati on temperature
when starti ng the dwel l peri od. Thi s may mean that you are
not drawi ng enough vacuums or that modi fi cati ons to the
i tems bei ng steri l i zed are necessary to al l ow more effi ci ent
steam penetrati on.
3. I f you are not usi ng bi ol ogi cal i ndi cators to val i date your
cycl e, you are taki ng a si gni fi cant ri sk. Usi ng temperature
data al one means that you are assumi ng i deal condi ti ons
where i t i s not justi fi ed.
4. I f you are pl aci ng a smal l quanti ty of water wi thi n l oad
i tems to assi st wi th steri l i zati on, you must have appropri -
ate procedural control s i n pl ace to ensure ongoi ng consi s-
tency wi th the amount of water present duri ng the val i da-
ti on runs and al l subsequent processi ng runs.
Summary
The requi rements to val i date steam steri l i zati on processes
have been documented for many years. For exampl e, perhaps
the most hi stori cal l y si gni fi cant reference gui de, the PDA
Techni cal Monograph No. 1 Val i dati on of Steam Steri l i zati on
Cycl es was publ i shed i n 1978. Nonethel ess, steam steri l i zati on
val i dati on remai ns a si gni fi cant i ssue to regul atory bodi es,
parti cul arl y for processes associ ated wi th hi gh ri sk i n terms of
the probabi l i ty and severi ty of an i nfecti on. Fai l ure to ad-
equatel y address thi s requi rement can pl ace the publ i c at ri sk
and l ead to regul atory ci tati ons/acti on.
I n addi ti on to potenti al busi ness l i abi l i ti es, there may be
si gni fi cant costs associ ated wi th the val i dati on process. Large
numbers of ti me consumi ng and costl y test runs may be
requi red, and i f appropri ate consi derati on i s not gi ven to
empl oyi ng the correct approach, unnecessary ongoi ng opera-
ti onal costs may resul t.
I t i s hoped that the practi cal experi ence that thi s document
i s based on wi l l provi de assi stance i n ensuri ng an effecti ve,
effi ci ent val i dati on process for steam steri l i zati on and that the
end resul t provi des the best possi bl e val i dated cycl e to meet the
needs of the speci fi c appl i cati on.
Definitions
SAL: steri l i ty assurance l evel .
SAL of 10
-6
: the probabi l i ty of a si ngl e vi abl e mi croorgani sm
bei ng present i s one i n one mi l l i on.
Bioburden: the number/type of vi abl e mi croorgani sms con-
tami nati ng an i tem.
Overkill Approach: a steri l i zati on approach based on as-
sumi ng worst-case condi ti ons (a bi oburden of 10
6
of a hi ghl y
heat resi stant bacteri a).
Log Reduction: reduce the survi vi ng mi crobi al popul ati on by
1 l og or decrease the survi vi ng popul ati on by a factor of 10.
12-Log Reduction: the l og reducti on requi red achi evi ng over-
ki l l and a SAL of 10
-6
.
CFU: col ony-formi ng uni t.
D-value: ti me i n mi nutes, at a speci fi c temperature, to reduce
the survi vi ng mi crobi al popul ati on by 90% (one l ogari thmi c
reducti on).
Z-value: temperature change requi red resul ti ng i n a 1-l og
reducti on i n D-val ue.
F-value: the number of mi nutes to ki l l a speci fi ed number of
mi croorgani sms wi th a speci fi ed Z-val ue at a speci fi c tempera-
ture.
F
o
-value: the number of mi nutes to ki l l a speci fi ed number of
mi croorgani sms wi th a Z-val ue of 10C (50F) at a temperature
of 121.1C (250F).
1 F
o
:the equi val ent of 1 mi nute at 121.1C (250F).
Dwell Period: the ti me peri od that begi ns when the autocl ave
temperature has reached the set-poi nt and ends when the
ti mer has expi red.
Worst case items: i tems i n the l oad whi ch are the most
di ffi cul t to steri l i ze (as determi ned by steam penetrati on stud-
i es).
Worst case location: the l ocati on wi thi n an i tem that i s the
most di ffi cul t to steri l i ze (as determi ned by steam penetrati on
studi es).
Gravity Displacement: a method of removi ng ai r by i ntro-
duci ng steam i nto the top of a chamber and di spl aci ng the ai r,
...steam sterilization validation remains a significant issue to regulatory bodies,
particularly for processes associated with high risk in terms of the probability
and severity of an infection.

AutoclaveValidation
JULY/AUGUST2002PHARMACEUTICALENGINEERING
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whi ch i s heavi er than steam, by removi ng the ai r from the
bottom of the chamber.
Vacuum Cycle: a steri l i zati on cycl e that draws one or more
vacuums to remove ai r pri or to starti ng the dwel l peri od.
Pre-vacuum: a vacuum drawn pri or to starti ng the dwel l
peri od to remove ai r.
Post-vacuum: a vacuum drawn after the dwel l peri od has
fi ni shed to remove steam.
Hard Goods Cycle: a steri l i zati on cycl e desi gned for i tems for
whi ch ai r removal i s not di ffi cul t and therefore general l y one
pre-vacuum i s drawn.
Wrapped Goods Cycle: a steri l i zati on cycl e desi gned for
i tems for whi ch ai r removal i s di ffi cul t and therefore general l y
three or more pre-vacuums are drawn.
Liquids Cycle: a cycl e desi gned for l i qui d l oads that general l y
uses gravi ty di spl acement rather than drawi ng a vacuum.
Bowie Dick Test: a test desi gned to veri fy that an autocl aves
vacuum phase i s removi ng a suffi ci ent amount of ai r pri or to
the i ntroducti on of steam i nto the chamber and tests for ai r
l eaks i nto the chamber.
Empty Chamber Tests: tests wi th an empty chamber essen-
ti al l y desi gned to demonstrate that an autocl ave provi des a
uni form steri l i zi ng envi ronment.
Steam Penetration Tests: l oaded chamber tests desi gned to
determi ne the worst-case i tems and worst-case l ocati ons wi thi n
a l oad.
Biological Challenge Tests: l oaded chamber tests desi gned
to chal l enge the worst-case l ocati ons (wi thi n worst case i tems)
wi th bi ol ogi cal i ndi cators to demonstrate the effecti veness of a
steri l i zati on cycl e.
Steam Integrators: commerci al l y avai l abl e i ndi cators that
provi de an i ndi cati on of exposure to steam.
Fixed Load: a l oad confi gurati on where the quanti ty and
l ocati on of i tems wi thi n the chamber are fi xed.
SIP: steam-i n-pl ace or steri l i ze-i n-pl ace (often used i nter-
changeabl y al though the l evel of mi crobi al destructi on achi eved
may di ffer).
References
1. Val i dati on of Steam Steri l i zati on Cycl es: PDA Techni cal
Monograph No. 1 (1978).
2. Steri l i zati on of Medi cal Devi ces Val i dati on and Routi ne
Control of Steri l i zati on by Moi st Heat: European Standard
EN 554 (1994).
About the Author
Raymond G. Lewis, PE i s a Val i dati on Man-
ager for I ndustri al Desi gn and Constructi on
I nc. (I DC) i n Portl and, Oregon. I n thi s posi -
ti on, he i s responsi bl e for i nternati onal cGMP
regul atory i ssues i nvol ved i n the desi gn, con-
structi on, and val i dati on of pharmaceuti cal ,
bi otech, and medi cal devi ce faci l i ti es. He hol ds
a degree i n chemi cal engi neeri ng from the
Uni versi ty of Saskatchewan and a Certi fi cate i n computer
sci ence from the Uni versi ty of Regi na. Lewi s has 16 years of
experi ence i n the pharmaceuti cal /bi otechnol ogy i ndustry i n
val i dati on, engi neeri ng, producti on, faci l i ty servi ces, and com-
puter operati ons.
I DC, 2020 SW 4th Ave., 3rd Fl oor, Portl and, OR, 97201.