Sterilization - Validation, Qualification Requirements: Mrs Robyn Isaacson

Manufacture of sterile medicines Advanced workshop for SFDA GMP
inspectors - Nanjin! Novem"er #$$%

1
Sterilization - validation,
qualification requirements
Mrs Robyn Isaacson
2
Sterilization - Overview
Objectives
Discuss definition of Sterile
!riefly describe sterilization met"ods
Describe a##roac"es to be used for t"e

validation of a sterilization #rocess usin$
Moist %eat as an e&am#le
Describe requirements for routine monitorin$

and control of sterilization
Review issues t"at are s#ecific to ot"er

sterilization #rocesses
3
Sterile 'roducts - Overview
(ertain #"armaceutical #roducts must be sterile

injections, o#"t"almic #re#arations, irri$ations solutions,
"aemodialysis solutions
)wo cate$ories of sterile #roducts
t"ose t"at can be sterilised in final container *terminally

sterilised+
t"ose t"at cannot be terminally sterilised and must be

ase#tically #re#ared
4
,"at is t"e definition of sterile-
.ree from microor$anisms

In #ractice no suc" absolute statement re$ardin$ absence
of microor$anisms can be #roven
Defined as t"e #robability of / in a million of a

container bein$ contaminated */0
-1
+
)"is referred to as t"e Sterility 2ssurance 3evel

*S23+
Or$anisms are 4illed in an e&#onential fas"ion

5
Definition of Sterile
MI(RO!I23 I52()I62)IO5
5
t
7 5
o
e
-4t
0
200
400
600
800
1000
1200
)ime
5
o

o
f

s
u
r
v
i
v
i
n
$

o
r
$
a
n
i
s
m
s
-2.00
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
No remaining
Log No remaining
No remaining 1000 368 135 50 18 7 2 1 0 0 0
Log No remaining 3.00 2.57 2.13 1.70 1.26 0.83 0.39 -0.04 -0.47 -0.91 -1.34
0 1 2 3 4 5 6 7 8 9 10
6
Microbial Inactivation
-3
-2
-1
0
1
2
3
4
1 2 3 4 5 6
)ime
3
o
$

5
o

o
f

S
u
r
v
i
v
i
n
$

O
r
$
a
n
i
s
m
s
D-value - )ime *or dose+ required to reduce t"e
#o#ulation of or$anisms by / lo$ *or 809+
D-Value
Resistance of an or$anism is referred as its D-value
7
2 sterilization #rocess must deliver a

Sterility 2ssurance 3evel *S23+ of / in a
million */0
-1
+
It is not #ossible to measure /0

-1
)"e required S23 can be ac"ieved by

a##lyin$ a #rocess t"at will reduce t"e
number of or$anisms to zero and t"en
a##ly a safety factor t"at will deliver an
e&tra 1 lo$ reduction
8
:&am#le
.or an initial bioburden of /0
;
t"e
sterilization #rocess will need to
ac"ieve an < lo$ reduction in
viable or$anisms
)"is will require < times t"e D-
value *e=$= if t"e or$anism "as a
D value of ; minutes t"en < & ; 7
/1 minutes will be required to
ac"ieve an < lo$ reduction and
an S23 of /0
-1
+ *'oint >+
Microbial Deat" (urve
-7
-6
-5
-4
-3
-2
-1
0
1
2
0 1 2 3 4 5 6 7 8
)ime
3
o
$

5
o

o
f

S
u
r
v
i
v
o
r
s
?
>
9
(ommonly used met"ods of sterilization
Moist %eat
Dry %eat
@as *:t"ylene o&ide+
Radiation *@amma or :lectron+
.iltration
Ot"ers - A6, Steam and formalde"yde,

"ydro$en #ero&ide
10
Moist %eat
Saturated steam
(ommon cyclesB
/;/C( for /D minutes
/EFC( for E minutes
Ot"er cycles of lower tem#erature and lon$er

time may be used *e=$= //DC( for E0 minutes+
Ased for sterilization ofB
terminal sterilization of aqueous injections,

o#"t"almic #re#arations, irri$ation G
"aemodialysis solutions, equi#ment used in
ase#tic #rocessin$
11
Moist %eat
not suitable for non-aqueousHdry

#re#arations
#referred met"od of sterilization

12
Dry %eat
3et"ality due to o&idative #rocesses
%i$"er tem#eratures and lon$er e&#osure

times required
)y#ical cyclesB
/10C( for /;0 minutes
/I0C( for 10 minutes
/<0C( for E0 minutes
tunnels used for t"e sterilisation of $lass vials

may use muc" "i$"er tem#eratures *E00C+ for a
muc" s"orter #eriod
13
Dry %eat
Ased forB
$lassware and #roduct containers used in

ase#tic manufacture, non aqueous
t"ermostable #owders and liquids *oils+
also used for de#yro$enation of $lassware

*;D0C(+
*'yro$ens - substances found in cell wall of

some bacteria w"ic" can cause fever w"en
introduced into t"e body+
14
:t"ylene O&ide @as
:it"er #ure or in mi&tures wit" ot"er inert

$ases
Requires #resence of moisture
(om#le& #rocess
)y#ical cyclesB
/-;F"ours
;D-/;00 m$H3 $as
;D-1DC(
E0-<D9 relative "umidity

15
:t"ylene O&ide
Ased forB
"eat labile #roduct containers
surface sterilization of #owders
2dequate aeration to reduce to&ic

residues
16
Radiation
@amma rays $enerated by (obalt 10 or (aesium

/EI radionuclidesJ or
2ccelerated electrons from an electron $enerator
;D 4ilo$rays *4@y+ usual dose

dose de#endent on bioburden *resistance of or$anisms
not #redictable+
#rocess must be #ro#erly validated
used forB
dry #"armaceutical #roducts
"eat labile #roduct containers
can cause unacce#table c"an$es

17
.iltration
Removes or$anisms from liquids and

$asses
0=; - 0=;; micron for sterilization
com#osed of cellulose esters or ot"er

#olymeric materials
filter material must be com#atible wit"

liquid bein$ filtered
used for bul4 liquids, $asses and vent

filters
18
6alidation - Overview
Selection of sterilzation #rocess must be

a##ro#riate for #roduct
terminal sterilization is t"e met"od of c"oice
moist "eat *autoclavin$+ is t"e most common

#rocess used for terminal sterilization
#roduct must not be affected by "eat
containerHclosure inte$rity must be establis"ed
items bein$ sterilised must contain water *if

sealed+ or material must allow for removal of
air and #enetration of steam for steam *moist
"eat+ sterilization
19
6alidation - 'rotocol
Requirements for Moist %eat Sterilization
Ot"er #rocesses follow similar requirements
6alidation #rotocol s"ould include t"e

followin$ details for eac" sterilization
#rocess
#rocess objectives in terms of #roduct ty#e,

containerHclosure system, S23 required
s#ecifications for time, tem#erature, #ressure

and loadin$ #attern
descri#tion of all equi#ment and su##ort

systems in terms of ty#e, model, ca#acity and
o#eratin$ ran$e
20
6alidation - 'rotocol
Moist %eat continuedB
#erformance c"aracteristics of all equi#ment

e=$= #ressure $au$es, valves, alarm systems,
timers, steam flow ratesH#ressures, coolin$
water flow rates, cycle controller functions,
door closure $as4etin$ and air brea4 systems
and filters
met"odolo$y for monitorin$ #erformance of

equi#ment and t"e #rocess and labatory
testin$ met"odolo$y
#ersonnel res#onsible for all sta$es and final

evaluation *s"ould "ave e&#erience and
necessary trainin$ and be aut"orized+
21
6alidation - (alibration
3aboratory testin$ s"ould be #erformed

by a com#etent laboratory, met"odolo$y
s"ould be documented
2ll instruments must be calibrated e=$=

tem#erature recorders and sensors
t"ermocou#les
#ressure sensors for jac4et and c"amber
timers
conductivity monitors for coolin$ water
flow metres for waterHsteam
water level indicators w"en coolin$ water is used
t"ermometers includin$ t"ose for t"ermocou#le
reference, c"amber monitorin$ and laboratory testin$
22
6alidation - (alibration
Indicators s"ould be calibrated

#"ysical and c"emcial indicators s"ould be tested
to demonstrate acce#table res#onse to time and
tem#erature
biolo$ical indicators s"ould be tested for count

and timeHtem#erature e&#osure res#onse
for commercial indicators - test certificate wit" count
and D-value and e&#osure res#onse s"ould be
available= Results acce#table if verified in "ouse
#eriodically=
In "ouse indicators must be fully c"aracterized *D-
value, identification+ and a##ro#riate for sterilization
#rocess
2ll indicators s"ould be a##ro#riately stored and
wit"in e&#iry
23
6alidation - (ycle Develo#ment
(once#t of .
o
3et"ality factor equivalent to time at /;/C(
/ minute at /;/C( is equivalent to .

o
of /=
3et"ality can accumulate durin$ "eat u# and cool
down #"ases
Typical temperatre pro!ile o! a "eat #terili$ation proce##
%"at &ol' (e t"e )o o! a cycle at 121*+ !or 15 minte#,
24
.
o
is calculated usin$ t"e followin$
equationB
.
o
7 KtL/0
*)-/;/H>+
w"ereB
Kt is t"e time interval between measurements of

tem#erature *)+
) is t"e tem#erature of sterilised #roduct at time *t+
> is a tem#erature coefficient w"ic" measures t"e

number of de$rees required to c"an$e t"e D-value of
an or$anism by / lo$
25
)"e minimum .
o
required by a sterilzation
#rocess is related to t"e resistance of t"e
bioburden *D-value+
.
o
7 D
/;/
*3o$2 - 3o$ !+
w"ereB
D
/;/
is equal to t"e time at /;/C( to reduce t"e
#o#ulation of t"e most resistant or$anism in eac"
#roduct container by 809 *or / lo$+
2 is t"e number of microo$anisms #er container
! is t"e ma&imum acce#table #robability of
survival *Sterility 2ssurance 3evel , /0
-1
+
26
)wo a##roac"es to sterilization
Over4ill
'robability of survival
Over4ill a##roac" used w"en t"e #roduct

can wit"stand e&cessive "eat treatment
wit"out adverse effects
(ycle s"ould deliver an .
o
of at least /;
)"is will ac"ieve a /; lo$ reduction of
microor$anisms wit" a D-value of / minute
*2ssumin$ eac" #roduct unit contains /0
1
or$anisms a /; lo$
reduction will result in /0
-1
or$anisms #er unit or
#robability of survival *S23+ of / in a million+
*5ormal bioburden is usually muc" lower and t"e or$anisms
normally muc" less resistant t"an t"is+
27
!iolo$ical Indicators
device consistin$ of a 4nown number of

microor$anisms, of a 4nown resistance to a
#articular sterilization #rocess in or on a carrier
and enclosed in a #rotective #ac4a$e=
Or$anisms are in t"e form of endos#ores *not

ve$etative state+ as t"ese are most resistant to
sterilization
Or$anism D-6alue
Geobacillus stearothermophilus
*most common+
/ - ;=<
Bacillus coagulans 0=E
Clostridium sporogenes 0=< M /=F
Bacillus atropheus 0=D

28
S#ore Stri#s - a narrow stri# of
fibrous #a#er im#re$nated wit" a
bacterial s#ore sus#ension
contained in a $lassine envelo#e
S#ore Dots - (ircular #ieces of
fibrous #a#er im#re$nated wit"
t"e s#ore sus#ension
S#ore Sus#ensions - #ure s#ore
sus#ension of t"e desired
c"allen$e or$anism w"ic" can be
inocluated onto t"e surface of a
material
Self contained units containin$ s#ore
stri#s or sus#ensions and t"e
media in w"ic" t"ey are to be
incubated *sim#leHconvenient to
use+
29
'robability of Survival a##roac" used used

for "eat labile #roducts
)"e #rocess is validated to
ac"ieve a destruction of t"e
#resterilization bioburden to
a level of /0
0
*'oint ?+, wit" a
minimum safety factor of an
additional si&-lo$ reduction
*'oint >+
Determination of t"e
minimum .
o
required is based
on t"e bioburden and its "eat
resistance
,"at .
o
would be required for a bioburden of /0
;
*/00+ if D-value was /-
30
6alidation of Sterilization
!asic 'rinci#les
Installation Nualification *IN+
:nsurin$ equi#ment is installed as #er

manufacturerOs s#ecification
O#eration Nualification *ON+
:nsurin$ equi#ment, critical control equi#ment

and instrumentation are ca#able of o#eratin$
wit"in required #arameters
'erformance Nualifcation *'N+
Demonstratin$ t"at sterilizin$ conditions are

ac"ieved in all #arts of sterilization load
'"ysical and microbiolo$ical

31
6alidation - :qui#ment
Installation Nualification
:nsurin$ equi#ment is installed as #er

manufacturerOs s#ecification
considerations for new and e&istin$ equi#ment
s#ecifications for t"e ty#e of autoclave,

construction materials, #ower su##lies and
su##ort services, alarm and monitorin$
systems wit" tolerances and accuracy
requirements
for e&istin$ equi#ment documented evidence

t"at t"e equi#ment can meet #rocess
s#ecifications
32
6alidation - :qui#ment
O#erational Nualification
:nsurin$ equi#ment, critical control

equi#ment and instrumentation are ca#able
of o#eratin$ wit"in required #arameters
)"ree or more test runs w"ic" demonstrate

controls, alarms, monitorin$ devices and o#eration
indicators function
c"amber #ressure inte$rity is maintained
c"amber vacuum is maintained *if a##licable+
written #rocedures accurately reflect equi#ment o#eration
#re-set o#eration #arameters are attained for eac" run
33
6alidation - 'erformance
'erformance Nualification
Demonstratin$ t"at sterilizin$ conditions are

ac"ieved in all #arts of sterilization load
'"ysical and microbiolo$ical

'"ysical
%eat distribution studies on em#ty c"amber

ma&imum and minimum cycle times and tem#eratures
to identify "eat distribution #atterns includin$ slowest
"eatin$ #oints
mulit#le tem#erature sensin$ devices s"ould be used
*t"ermocou#les+
location of devices s"ould be documented and ensure
t"at "eat distribution is uniform
34
'erformance Nualification - '"ysical *;+
%eat distribution of ma&imum and minimum

c"amber load confi$urations
multi#le t"ermocou#les t"rou$"out c"amber *not inside

#roduct containers+ to determine effect of load
confi$uration on tem#erature distribution
tem#erature distribution for all loads usin$ all container
sizes used in #roduction s"ould be tested
#osition of t"ermocou#les s"ould be documented
Slowest to "eatHcold s#ots in eac" run s"ould be
documented, inlcudin$ t"e drain
re#eat runs s"ould be #erformed to c"ec4 variability
tem#erature distribution #rofile for eac" c"amber load
confi$uration s"ould be documented
35
'erformance Nualification - '"ysical *E+
%eat #enetration studies to detect t"e ma&imum

and minimum tem#erature wit"in all loads
all #arts of eac" load must be on contact wit" steam
need to determine lowest and "i$"est tem#erature
locations and slowest and fastest to "eat locations
*measured inside #roduct containers+
need to consider all variables suc" as container size,

desi$n, material, viscosity of solution and fill volume=
(ontainer wit" ma&imum fill volume and slowest to "eat
solution s"ould be used
ma&imum and minimum load confi$urations for eac"

sterilization cycle usin$ routine cycle #arameters
36
'erformance Nualification - '"ysical *F+
%eat #enetration *;+
May be necessary for container ma##in$ for lar$er

volumes - cold s#ot t"en used for #enetration studies
5eed to consider effects of #ac4a$in$ e=$=

overwra##in$
)"ree runs #erformed once cold s#ots "ave been

identified to demonstrate re#roducibility
37
'erformance Nualification - Microbiolo$ical
!iolo$ical c"allen$e studies

used w"en 'robability of Survival a##roac" is used
may not be necessary w"en cycle is P /;/C( for /D minutes
*e&ce#t AS and 2ustralia+
biolo$ical indicators *!I+ containin$ s#ores of Geobacillus
stearothermophilus are most commonly used *considered
worst case+= !Is containin$ ot"er or$anisms may be used
#erformance studies based on #roduct bioburden require a
considerable amount of wor4
indicators s"ould be #laced t"rou$"out t"e load, adjacent to
t"ermocou#les, at cold s#ots and slowest to "eat locations
*identified durin$ "eat #enetration studies+
any $rowt" is unacce#table unless #rocessin$ errors
demonsrated
38
6alidation re#ort must demonstrate requirements

in 6alidation #rotocol "ave been met, any
deviations must be justified
Requalification must be re#eated on an annual

basis *usually one run is acce#table+
2ny c"an$es or modifications must be evaluated

may just require requalification
any c"an$es to loadin$ #atterns, new containerHclosure
systems or cycle #arameters require full validation
39
Routine 'roduction
Issues considered for routine #roduction
Manufacturin$ environment s"ould be controlled
'rocedures in #lace to minimize t"e #re-

sterilization bioburden
bioburden limits s#ecified *alt"ou$" not so im#ortant
w"en over4ill cycle used+
)ime between fillin$ and sterilization s"ould be

s#ecified
Inte$rity of containerHclosure system s"ould be

#eriodically verified
'eriodic lea4 testin$ of c"amber *if vacuum is #art

of cycle+
40
Routine 'roduction
(oolin$ water s"ould be sterile
Differentiation between sterilized and not-yet

sterilized #roduct
'"ysical se#aration *double ended autoclave+
3abellin$ and use of visual indicators *e=$

autoclave ta#e+
'eriodic testin$ of containers to verify inte$rity of

containerHclosure system
Nuality of steam s"ould be defined and

#eriodically tested for contaminants
41
Routine 'roduction
:ac" sterilization cycle must be monitored
tem#erature, time and #ressure recorded
tem#erature recorder inde#endent from cycle

controller
second inde#endent tem#erature recorder
drain tem#erature s"ould be recorded
c"emical and biolol$ical indicators *if

a##licable+
Sterilisation cycle records s"ould form

#art of batc" records
42
Ot"er Sterilization 'rocesses
Sterilization usin$ ot"er #rocesses s"ould
follow a similar a##roac" as t"at
described for moist "eat
6alidation #rotocol
:qui#ment calibration
Determinin$ t"e #rocess t"at will deliver t"e

desired S23 */0
-1
+
IN, ON, 'N
Requirements for routine monitorin$ and

control
43
Dry %eat
S"ould "ave air circulation in t"e c"amber
'ositive #ressure in t"e c"amber to #revent

entry of non-sterile air
%:'2 filtered air su##lied
!iolo$ical indicators containin$ Bacillus

atropheus *if used+
removal of endoto&in is usually sufficient
,"en removin$ #yro$ens need to validate

#rocess usin$ c"allen$e tests
44
Radiation
Asually #erformed by contractin$ service

*need to ensure validation status,
res#onsibilities+
!ased on bioburden of #roduct bein$

sterilised
!iolo$ical indicators may be used as additional

control but may not be as resistant as naturally
occurin$ bioburden
Met"od defined in International Standard ISO

///EI
45
Radiaton *;+
(orrect dose of radiation *Q;D 4@y+ received

by all #roducts *measured wit" dosimeters+
quantitative measurement
number, location, wit"in calibration time-limit
Radiation sensitive colour discs a##lied to

#ac4a$in$
#rocedures to distin$uis" irradiated and non-

irradiated materials
6ariation in density of #ac4a$in$ s"ould be

addressed durin$ validation
46
@asses and .umi$ants
e=$= et"ylene o&ide, "ydro$en #ero&ide va#our
Only w"en no ot"er met"od is suitable
Must demonstrate t"at #rocess does not

adversely affect #roduct
'ac4a$in$ must be able to #ermit in$ress of

$as and "umidity
:nsure #roduct load is adequately "eated and

"umidified #rior to sterilization *called
conditionnin$+
need to ta4e into account validation #erformed in
summer or winter
47
@asses and .umi$ants *;+
)em#erature distribution is acce#table
(oncentration of sterilant $as is sufficient
Ase of biolo$ical indicators is im#ortant

*Bacillus atropheus+
%alf cycles
If cycle of "alf normal time destroys biolo$ical indicators
*/0
1
or$anisms+, double time will ac"ieve S23 of /0
-1
2eration
6entilated conditions
Defined limits of residuals
'rocess included in validation
Safety and to&icity issues considered

48
Aseful 'ublications
ISOH:5 /I11D - Sterilization of health care

products - Moist Heat *'arts / and ;+
ISOH:5 ///ED - Sterilization of health care

products - Ethylene Oxide *'arts / and ;+
ISOH:5 ///EI - Sterilization of health care

products - adiation *'arts /,; and E+
!"alidation of Moist Heat Sterilization #rocesses$

Cycle %esign& %e'elopment& (ualification and
ongoing Control) 'D2 )ec"nical Re#ort 5o= /
Revised ;00I
49
Nuestions-

Sterilization - Validation, Qualification Requirements: Mrs Robyn Isaacson

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Manufacture of sterile medicines Advanced workshop for SFDA GMP

inspectors - Nanjin! Novem"er #$$%

Discuss definition of Sterile

!riefly describe sterilization met"ods

Describe a##roac"es to be used for t"e

Describe requirements for routine monitorin$

Review issues t"at are s#ecific to ot"er

(ertain #"armaceutical #roducts must be sterile

)wo cate$ories of sterile #roducts

t"ose t"at can be sterilised in final container *terminally

t"ose t"at cannot be terminally sterilised and must be

.ree from microor$anisms

Defined as t"e #robability of / in a million of a

)"is referred to as t"e Sterility 2ssurance 3evel

Or$anisms are 4illed in an e&#onential fas"ion

2 sterilization #rocess must deliver a

It is not #ossible to measure /0

)"e required S23 can be ac"ieved by

@as *:t"ylene o&ide+

Radiation *@amma or :lectron+

Ot"ers - A6, Steam and formalde"yde,

/;/C( for /D minutes

/EFC( for E minutes

Ot"er cycles of lower tem#erature and lon$er

Ased for sterilization ofB

terminal sterilization of aqueous injections,

not suitable for non-aqueousHdry

#referred met"od of sterilization

3et"ality due to o&idative #rocesses

%i$"er tem#eratures and lon$er e&#osure

/10C( for /;0 minutes

/I0C( for 10 minutes

/<0C( for E0 minutes

tunnels used for t"e sterilisation of $lass vials

$lassware and #roduct containers used in

also used for de#yro$enation of $lassware

*'yro$ens - substances found in cell wall of

:it"er #ure or in mi&tures wit" ot"er inert

Requires #resence of moisture

;D-/;00 m$H3 $as

E0-<D9 relative "umidity

"eat labile #roduct containers

surface sterilization of #owders

2dequate aeration to reduce to&ic

@amma rays $enerated by (obalt 10 or (aesium

2ccelerated electrons from an electron $enerator

;D 4ilo$rays *4@y+ usual dose

#rocess must be #ro#erly validated

can cause unacce#table c"an$es

Removes or$anisms from liquids and

0=; - 0=;; micron for sterilization

com#osed of cellulose esters or ot"er

filter material must be com#atible wit"

used for bul4 liquids, $asses and vent

Selection of sterilzation #rocess must be

terminal sterilization is t"e met"od of c"oice

moist "eat *autoclavin$+ is t"e most common

#roduct must not be affected by "eat

containerHclosure inte$rity must be establis"ed

items bein$ sterilised must contain water *if

6alidation #rotocol s"ould include t"e

#rocess objectives in terms of #roduct ty#e,

s#ecifications for time, tem#erature, #ressure