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sterilization

sterilization

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his article provides an update ofthe validation ofmoistheat sterilization.It brings together practical informationone needs when validating an autoclave,from procure-ment through routine use.In Part I ofthis article,thesterility concept,sterilization principles,development ofsterili-zation cycles,and the measurement ofsterilization efficiency arediscussed.Part II will be published in
Pharmaceutical Technol-ogy 
’s October issue and will review the qualification–validationprocedure and the probability ofnonsterility ofa load duringthe validation ofthe steam sterilization process.
62 
Pharmaceutical Technology 
SEPTEMBER 2002 
www.pharmtech.com
An Overview of theValidation Approach forMoist Heat Sterilization,Part I
B.M.Boca,E.Pretorius*,R.Gochin,R.Chapoullie,and Z.Apostolides
B.M. Boca
is a doctoral student at the University of Pretoriain South Africa;
E. Pretorius, PhD,
is a senior lecturer in theDepartment of Anatomy, Faculty of Medicine, University ofPretoria 0002, Pretoria, South Africa, tel.
27 12 319 2533, fax
27 12 319 2240, rpretori@medic.up.ac.za;
R. Gochin
is thequality assurance manager of the Pharma Division at RocheProducts Pty Ltd.;
R. Chapoullie
is the quality manager atSteren Support Systems CC; and
Z. Apostolides, DSc,
issenior lecturer in the Department of Biochemistry, University ofPretoria.
*To whom all correspondence should be addressed.
 
T
This article illustrates aqualification–validation strategy for moist heatsterilizationand briefly discusses the sterilityconcept and common sterilization principles.In Part I,the authors present examples forcycle types,parameter requirements for astandard cycle as defined by pharmacopeias,methods used to design sterilization cycles,and various approaches used to measure theefficiency of the sterilization process.
The sterility concept and sterilization principles
Sterility 
is defined as the absence ofany microorganism capa-ble ofreproduction (1).Sterility can be achieved through thedestruction ofall viable life forms by applying a lethal agent tothe item that must be sterilized.Several sterilization principlesexist according to the lethal agent used (2).Each treatment de-stroys microorganisms in a unique manner and with a differ-ent degree ofeffectiveness.A few examples ofthese treatmentsare
G
heat sterilization with dry or moist heat
G
radiation sterilization with gamma and X-rays,which pro-duce positively charged ions in their passage through the mat-ter,thereby further generating free radicals with lethal effectsin the cells
G
chemical sterilization with glutaraldehyde,chlorine,iodine,hydrogen peroxide,quaternary ammonium salts,ozone,pera-cetic acid,and phenolic compounds
G
gaseous sterilization with chemical agents such as ethyleneoxide,propylene oxide,and formaldehyde,all ofwhich havea lethal effect that consists ofthe alkylation ofproteins,DNA,and RNA
G
filtration sterilization,which physically removes micro-organisms from the product by means ofretentive filters.New technologies also have been developed,including sterili-zation with free radicals generated by the combination ofUVirradiation with hydrogen peroxide and sterilization with chlo-rine dioxide.The sterility criterion is a mandatory requirement for paren-teral products,ophthalmic preparations,and products appliedto injured skin or used to irrigate the body cavities.All prod-ucts that must be manufactured as sterile should pass the steril-ity test described by the pharmacopeias.When exposing homogenous populations ofmicroorgan-isms to a sterilizing agent,the microbial inactivation followsan exponential decrease curve (see Equation 1).Mathemati-cally,the inactivation ofmicroorganisms can be expressed asa first-order kinetic process.A finite probability ofsurvivingorganisms,independent from the magnitude ofthe deliveredsterilizing agent,can be expressed as:
 
64 
Pharmaceutical Technology 
SEPTEMBER 2002 
www.pharmtech.com
[1]in which N
is the number ofsurviving organisms after time
,N
0
is the number ofmicroorganisms at time zero (i.e.,thebioburden),
is the exposure time,and
k
is a microbial inacti-vation rate constant.For a given process,the probability ofsurvival is determinedby the number,type,and resistance ofthe microorganisms pre-sent and by the environment in which the organisms exist dur-ing the treatment (e.g.,moisture content,thermal energy,andtime for steam sterilization) (3).For pharmaceutical products,the term
sterile
is applied to products that have been treated insuch a manner that,on completion ofthe process,individualitems have a probability ofbeing nonsterile or have a sterility assurance level (SAL) equal to 1
10
6
or more (4).Such a levelofsterility assurance is required for terminally sterilized injectableproducts.This definition ofsterility as a probability functiondoes not assume that one in a million products is allowed to benonsterile but admits a finite statistical probability that a micro-organism may survive the sterilizing process (3).
Sterilization by saturated steam in the autoclave
When heat is used as a sterilizing agent,the vibratory motionofevery molecule ofa microorganism is increased to levels thatinduce the cleavage ofintramolecular hydrogen bonds betweenproteins.Death is therefore caused by an accumulation ofirre-versible damage to all metabolic functions ofthe organism (5).N
N
0
e
kt 
Heat can be used either alone or mixed with steam.In theautoclave chamber,terminal sterilization is based on highly ef-ficient heat transfer from the saturated steam to the autoclaveload (6).Heat transfer occurs by the release ofthe latent heatfrom saturated steam under pressure as it condenses.Heat transfer has maximum efficiency ifthe steam is keptalong the phase separation line ofthe water
steam phase dia-gram.Heat transfer from saturated steam to the chamber envi-ronment is much more effective and timely for the coagulationand denaturation ofnucleic acids and proteins than from dry heat or superheated heat (7).Moist heat in the form ofsatu-rated steam under pressure is therefore the most reliable steri-lizing agent and is the method ofchoice whenever it can beused,especially for aqueous preparations (2,8).
Types of cycles used in moist heat sterilization
For saturated steam sterilizers,the physical parameters gov-erning the efficiency ofthe sterilization process are exposuretime,temperature,and pressure.The last two parameters vary in a direct proportional relationship to each other.Generally,a cycle comprises heating,sterilizing,and coolingphases (9).The choice ofa cycle for a particular load depends onthe heat sensitivity ofthe load material and on the knowledge of the heat penetration in the articles.Figures 1 and 2illus-trate the cycles typically used in the moist heat sterilization process.For a saturated steam-vented cycle (see Figure 1) the steamis injected at the top ofthe chamber and displaces the cold air,which exits through a trap.This cycle is recommended for con-tainers filled with aqueous preparations (i.e.,parenterals,oph-thalmics,media,and buffers).The prevacuum cycle (see Fig-ure 2) ensures a more effective penetration ofthe load by saturated steam and is generally used for porous loads (e.g.,surgical dressings and wrapped materials).For moist heat sterilization,the accepted range ofsterilizingtemperatures is 118
134
C.The
US Pharmacopeia (USP)
ex-plains in a footnote that
an autoclave cycle,where specified inthe compendia for media or reagents,is a period of15 min at121
C,unless otherwise indicated
(10).The
European Phar-macopoeia
(
EP 
) and the
British Pharmacopoeia
(
BP 
) recom-mend a heating process at a minimum of121
C for 15 min asa reference condition for aqueous preparations (3
4).The textofboth books states that other conditions oftime and tempera-ture may be used provided that the process chosen has been sat-isfactorily demonstrated to deliver an adequate and reproduciblelevel oflethality when operating routinely within the estab-lished tolerances (3
4).
Development of sterilization cycles
Theoretically,the timing ofthe exposure phase begins when thetemperature sensor placed in the chamber drain (i.e.,the cold-est spot,theoretically) reaches the set sterilizing temperature.Experiments using thermocouples have shown that the tem-perature profile ofthe chamber under loaded conditions is dif-ferent from the one obtained for the empty chamber.The rateofheating and cooling a product in a container is a function of the container type and size,the viscosity ofliquids,and the sizeand arrangement ofthe load (2).Therefore,to ensure sterili-
Heatingphase
   T  e  m  p  e  r  a   t  u  r  e   (
              
   C   )
Exposure phaseSterilizing temperatureCoolingphaseVent closureTime (s)T
0
T
100
C
1
0
1
2
3
4
1
2
3
4
1
Figure 1:
The temperature profile for a saturated steam–vented cycle.
0
to
is the time interval in the computation of the
value.
1
to
areas represent the lethal rates in the product calculated for eachdiscrete time interval. The shaded area under the curve obtainedthrough graphical summation of
1
to
values represents the totalcalculated
value per cycle. In the heating phase,saturated steam isadmitted into the chamber,displacing the cold air until the exposuretemperature and corresponding saturated steam pressure are attained.In the exposure phase,the sterilizing temperature is maintained in thechamber by saturated steam for the prescribed exposure time. Thecooling phase can be achieved by slow exhaust (for containers filledwith liquids) or fast exhaust (for goods required to be dry aftersterilization). This phase is completed when the pressure in thechamber reaches atmospheric pressure.
 
www.pharmtech.com
66 
Pharmaceutical Technology 
SEPTEMBER 2002 
zation efficiency,sterilization must begin after a certain equili-bration time when the temperature ofthe material being steri-lized has reached the set sterilizing temperature.This meansthat a lag time must be added to the cycle exposure time thatrepresents the time necessary for the coldest spot inside the loadto reach the sterilizing temperature.The lag time must be de-termined experimentally and validated with each loading con-figuration that will be used in the autoclave for any further rou-tine sterilization.The larger the containers and the volumes tobe sterilized are,the longer the lag time is.The modern design ofa sterilization cycle can be based oneither the initial population ofmicroorganisms in the productthat is examined during a suitable time period or on the mea-surement ofthe physical parameters attained in the load dur-ing the sterilization process.
BP 
indicates that
sterilization meth-ods must be validated with respect to both the assurance of sterility and integrity ofthe product and to ensure that the finalproduct complies with the requirements ofthe monograph
(4).
USP 
states that
the design or choice ofa cycle for givenproducts or components depends on a number offactors,in-cluding the heat lability ofthe material,knowledge ofheat pene-tration into the articles,and other factors described under thevalidation program
(10).Three approaches are used in de-signing sterilization cycles:the bioburden approach,the overkillapproach,and a combination ofthe two approaches.The bioburden approach is based on determination ofthenumber,type,and resistance characteristics ofthe organismscontained in the bioburden (11).For heat-sensitive products,the heat exposure must be minimized in such a way to reach anoptimum balance between an acceptable degree ofsterilizationand an acceptable stability ofthe product after its sterilization(12).In the bioburden approach,the process will provide lessthan 1
10
6
probability ofmicroorganism survival.The steri-lizing conditions used for the bioburden approach are less ex-treme than the conditions used for an overkill approach.The design ofa sterilization cycle also can be based on theheating characteristics ofthe containers located in the slowestheating zone ofthe load.The overkill approach stems from theconcept that the sterilization process will inactivate a high micro-biological challenge with an additional safety factor.The micro-biological challenge will consist ofa biological indicator witha specific number ofmicroorganisms (10
3
10
6
),and a worst-case assumption is made that the heat resistance ofthe biobur-den is equivalent to that ofthe biological indicator.Therefore,the cycle conditions established are more severe than those re-quired to inactivate the real product bioburden,and a theoreti-cal spore reduction of10
12
is expected to prove the overkillassurance.Considerably exceeding the sterilization time is rec-ommended for heat-resistant products (10).Therefore,
0
val-ues (see definition in the
Mathematical approach
section) inthe range of30
60 are expected.The bioburden approach emphasizes the stability ofthe finalproduct while the optimum sterility is achieved by knowing thecharacteristics ofthe bioburden;however,the overkill approachplaces the emphasis on sterility.A combination ofthe two ap-proaches is sometimes useful to reach a balance between themaximum acceptable SAL and the maximum allowable con-comitant adverse effect upon the material to be sterilized.
Measuring the efficiency of the sterilization process
Assessment ofsterility is based on the demonstration ofthe ab-sence ofgrowth following the sterility test.Performance ofthesterility test is not always a guarantee ofsterility for a product(13).Therefore,two methods that incorporate microbial inac-tivation data have been developed to measure,control,anddemonstrate the efficiency ofa sterilization process.
Biological approach.
In the biological approach,known alsoas the
Latin approach
,one determines the lowest probability todetect a nonsterile unit in a sterile load and uses biological indi-cators (BIs) (14).BIs are standardized preparations ofmicro-organisms specific to the type ofsterilization process being stud-ied.These BIs are used to show a reproducible logarithmicinactivation.Many types ofBIs consist ofa given populationofbacterial spores considered to be the most resistant formsagainst a particular sterilization principle.In sterilization microbiology the
D
value is frequently usedinstead of 
k
as a measure ofthe rate ofmicrobial death.Equa-tion 1 can be formulated in a logarithmic manner as follows:[2]The
D
value represents the temperature coefficient for thelethal process and is the exposure time in minutes required tolog
0
 N 
kt 
2.303
 D
   A   i  r  r  e  m  o  v  a   l   T  e  m  p  e  r  a   t  u  r  e   (
              
   C   )   H  e  a   t   i  n  g
Exposure
   E  x   h  a  u  s   t   D  r  y   i  n  g   V  a  c  u  u  m   r  e   l   i  e   f
Time (s)T
0
T
100
C
1
0
1
2
3
4
1
2
3
4
1
Sterilizingtemperature
Figure 2:
The temperature profile for a saturated-steam with forcedair
removal cycle.
0
to
is the time interval in the computation of the
value.
1
to
areas represent the lethal rates in the product calcu-lated for each discrete time interval. The shaded area under the curveobtained through graphical summation of
1
to
values representsthe total calculated
value per cycle. In the air-removal phase,air isremoved from the chamber and load by several vacuum pulses. In theexhaust phase,steam is exhausted from the chamber until theatmospheric pressure is reached. In the drying phase,the temperaturein the jacket and the vacuum in the chamber are maintained for apredetermined time period for products required to be dry. In thevacuum-relief phase,air is admitted to the chamber through amicrobiologically retentive filter until atmospheric pressure is reached.

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