Sterilization

Acrucial step in pharmaceu-tical production is steril-ization. There are manysterilization methods tochoose from, such as

steam, sterile filtration, ethylene oxidegas (EtO), electron beam (E-beam),and gamma radiation. Each techniquehas aspects that make it suitable orunsuitable for the sterilization of aparticular product.

For example, EtO, while being ahighly effective method, leaves behindpotentially hazardous residuals and can-not reach products in airtight packages.E-beam, while being one of the fastestmethods of sterilization, cannot pene-trate well into dense product or bulkpackaging of some products. In addi-tion, the product complexities of hetero-geneous components often requireextensive product qualification. Gammaradiation can cause certain product andpackage materials to degrade.

GAMMA BENEFITSGamma radiation does have some

significant advantages over othermethods of producing sterile product.These benefits include:

• Better assurance of product sterilitythan filtration and aseptic processing.

• No residue like EtO leaves behind.• More penetrating than E-beam.• Low-temperature process.• Simple validation process.

The first aspect to consider whensterilizing with gamma is product tol-erance to the radiation. During use ofthis type of radiation, high-energyphotons bombard the product, caus-ing electron displacement within.These reactions, in turn, generatefree radicals, which aid in breakingchemical bonds. Disrupting microbialDNA renders any organisms that sur-vive the process nonviable or unableto reproduce.

However, these high-energy reac-tions also have the potential to disruptbonds within the pharmaceutical for-mulation, to weaken the strength ofpackaging materials, and to causechanges in color or odor in somematerials. For these reasons, drugmanufacturers should perform pre-qualification Dmax (maximum dose)testing, whereby the drug and its pack-aging are subjected to a high dose ofgamma radiation and then evaluatedfor stability and functionality.

Usually, the manufacturer will bethe party responsible for drug testing.Parameters to characterize typically

include potency, efficacy, stability, bio-compatibility, and chemical accept-ability. Per guidelines under theInternational Conference on Harmo-nization (ICH), known as TechnicalRequirements for Registration ofPharmaceuticals for Human Use, it isrecommended to use high-performanceliquid chromatography (HPLC), massspectrometry, or gas chromatographyto characterize and compare differentanalytical aspects of irradiated prod-uct versus nonirradiated product.

A qualified laboratory should per-form package testing. It is often recom-mended to have an aerosol challengeperformed on the product and pack-aging. This test entails placing thepackaged product inside an aerosolchamber and exposing it to high levelsof bacterial spores. The product isthen subjected to a sterility test, whichshows whether or not the packagingmaintains a sufficient barrier.

In addition, at least one physicalchallenge should be performed on thepackaging, if applicable. These in-clude the peel test to determine theamount of pressure needed to openthe seal; the burst test to determine theamount of pressure needed to burstthe package and to locate areas ofweakness in the package; and the dye

Strategies forGamma Sterilization of Pharmaceuticalsby Ruth Garcia, Betty Howard, Rose LaRue, Glenn Parton, and John WalkerSteris Isomedix Services (Mentor, OH)

Sterility is desirable not only for medical devices, but also to ensure the safety ofparenterals or injectable drugs. Various methods of reducing microbial load

in drugs and parenterals are available.

PH

OTO

CO

UR

TES

Y S

TER

IS I

SO

ME

DIX

SE

RV

ICE

S (

ME

NTO

R,

OH

)

migration test, which determineswhether dye travels through the sealsof the package. If a shelf-life claim isdesired, most labs will perform accel-erated aging. Typically, incubation at55°C for 6.5 weeks equals one year ona shelf (this may vary depending onthe drug formulation). These tests areperformed on aged products.

Performing a fraction of or all ofthese tests following a high dose ofgamma radiation will give the manu-facturer a good idea of product andpackaging suitability for gamma radia-tion. (A high dose is usually consideredto be in the 50–60-kGy range or high-er, preferably twice the minimum.)Many materials are highly resistant toradiation. If possible, the manufactur-er should choose materials that areresistant to the effects of gamma priorto the initial production phases.

HANDLING DEGRADATIONIf a drug experiences degradation,

discoloration, or any other physicalmalady due to the high dose of 50–60kGy, the manufacturer can begin test-ing at lower doses. One methodinvolves testing at particular intervals,such as at 5 or 10 kGy. For example, adrug that fails at 50 kGy may be stableat 40 kGy.

However, some drugs may continueto exhibit effects from the radiation atextremely low doses. Another testentails dropping the dose to half ofthe original high dose. This would cutthe range of possible maximum dosesin half. If the product is stable at thenew dose, then the max dose will fallsomewhere within the top half of theoriginal high dose. If the product isstill showing instability, the max dosemust fall in the lower half of the origi-nal high dose tested. This method mayreduce the number of irradiationsnecessary for establishing this infor-mation. All in all, the end product ofthis testing should be a solid maximumtolerated dose for the particular drugproduct.

Many pharmaceutical products,including parenterals and orally ingest-

ed drug products, are composed largelyof water. Water dissociates as a resultof exposure to radiation and is a majorsource of free radicals. These free radi-cals can cause chemical compromise,so drugs with high water content oftenrespond poorly to irradiation.

Performing irradiation on productin a frozen state can mitigate theseeffects. If the product can be safelyfrozen and thawed, the potential existsto irradiate it without, or with less,product degradation. Freezing thedrug traps free radicals in the ice crys-tals, reducing their freedom to moveabout. This may induce them torecombine with each other, ratherthen disrupt molecules in the productitself. This would possibly improvedrug resistance to degradation duringgamma irradiation. Other optionssuch as freeze-drying and/or usingfree-radical scavengers may also alle-viate the degradation effects seen insome products.

FINDING THE RIGHT DOSEThe next step is to set the minimum

sterilization dose, which will providethe desired sterility assurance level(SAL). There exist two commonlyused, industry accepted, validationtechniques, with several variationsfor special circumstances. The firsttechnique for discussion, Method 1,is found in AAMI/ANSI/ISO11137:1994, “Sterilization of HealthCare Products: Requirements forValidation and Routine Control—

Radiation Sterilization.” Method 1 encompasses product

with bioburden up to 1 million colony-forming units (CFUs). It allows forextremely low and high doses and iswell known throughout the gammasterilization industry. The steps aresimple and straightforward. First ofall, 10 product samples from each ofthree separate production batchesmust have bioburden testing per-formed on them. This quantitativemeasure, or count, of the number oforganisms on the unsterilized productprovides an excellent tool for deter-mining the minimum dose necessaryfor sterilization.

Bioburden tests should be accompa-nied by a determination of recoveryefficiency. This allows the laboratoryto calculate a more accurate biobur-den number. The average bioburdenof each batch and the overall averageof all product units should be deter-mined. If any single-batch bioburdenlevel is more than twice that of theoverall bioburden, that batch averageshould be used. Otherwise, the overallaverage should be used.

Afterward, the verification or sub-lethal dose must be set. UsingAAMI/ANSI/ISO 11137 Table B.1,find the bioburden number equal to orjust higher than that of the product.Follow the row to the column labeledSAL 10–2, where the verification dosewill be found.

The final phase includes testing forBacteriostasis/Fungistasis (B/F) andsetting the verification dose. The B/Ftest validates the sterility test by deter-mining whether the product formula-tion inhibits bacterial or fungalgrowth. If inhibition is seen, stepsmust be taken to neutralize it. The testis required only once in the lifetimeof a product, but it is recommendedannually. Without such a test, sterility-testing results are meaningless.

To begin the verification dose exper-iment, send 103 product units (100 forsterility testing and 3 for B/F) to thesterilization provider for irradiation atthe verification dose ± 10%. If the

Sterilization

Steris’ new JS 10,000 continuous andincremental Cobalt-60 irradiator is ready toprocess customers’ products.

dose exceeds the prescribed verifica-tion dose by more than 10%, then theproduct must be sacrificed and newproduct irradiated. If the dose is lowerthan 90% of the prescribed dose, theremainder of the testing may be per-formed and a failing test would allowfor a retest.

The product should then be sent tothe laboratory for sterility testing andB/F testing. If two or fewer sterilitytests turn positive, the product haspassed the validation, and the nextstep is to find the sterilization dose.Manufacturers should follow the samerow in Table B.1 from which the verifi-cation dose was taken, to the columnmarked SAL 10–6. This is the mini-mum sterilization dose. The productnow qualifies to be irradiated at arange from the minimum dose to themaximum dose determined during thehigh-dose materials testing.

The second type of validation iscommonly known as VDmax. Found inAAMI TIR 27:2001, “Radiation Steril-ization, Substantiation of 25 kGy,” thismethod requires fewer products andresults in a minimum sterilization doseof 25 kGy. However, only products with1000 CFU or less qualify.

The first step of this process is iden-tical to that of Method 1. Bioburdendata from 10 products from each ofthree separate production batchesshould be collected. Using Table 2 ofthe TIR, the bioburden number equalto or just greater than the product’saverage bioburden is found. The sub-lethal dose is found by following therow to the column labeled “Verifica-tion dose” (SAL 10–1). Send 13 productunits (10 for sterility testing and 3 forB/F) to the sterilizer for irradiation atthe verification dose ± 10%. Once theirradiation is complete, send the prod-ucts to the laboratory for sterility test-ing. If one or fewer sterility tests turnpositive, the product can be irradiatedat a minimum dose of 25 kGy. If twopositive sterility tests occur, a retestshould be performed on 10 additionalproducts. This time, no positives areallowed for substantiation of 25 kGy.

Should positives occur, another dose-setting method must be used.

Also contained in AAMI 11137 isan alternative validation procedurereferred to as Method 2. Method 2provides for dose setting based onthe actual radiation resistance ofmicroorganisms as they naturallyoccur on a product. Of the methodscited, it can provide the lowest possi-ble minimum dose. It is not used asfrequently as Method 1 or VDmax,due to more sample requirements andassociated costs.

Method 2 uses incremental dosedata to select a verification dose.Groups of samples from three produc-tion batches are irradiated in doseincrements up to the point where anSAL of 10–2 can be determined. AMethod 2 validation starts with therandom selection of 280 samples(Method 2A) or 260 samples (Method2B) from each of three productionbatches of product. Samples are thendesignated in groups of 20 samples foreach dose increment. Method 2A usesnine increments in 2-kGy increments,and 2B uses eight doses at 1-kGy incre-ments. All samples are tested for sterili-ty. After the results of sterility testsare known, a series of calculationsdescribed in AAMI 11137 (sectionB3.4.2) a verification dose (D*kGy) isdetermined.

An additional 100 samples from thebatch designated from the initial sterili-ty tests are irradiated at the verificationdose and tested to confirm sterility. Fol-lowing these sterility tests, a steriliza-tion dose is calculated using theequation appropriate to the specificmethod chosen (2A or 2B).

In extreme circumstances in whichall efforts to neutralize bacteriostaticagents have been exhausted and othersterilization methods are unsuitable,dose setting can be done with inocula-tion of the product. The practice ofinoculation, commonly used in thepast, is not currently recommendedunless it is impossible to collect naturalbioburden data from the product. For-tunately, in most cases, product inocu-

lation is not necessary. The organism most commonly used

for radiation challenge is Bacillus

pumilis. It was once believed that thisorganism was highly resistant to gam-ma. However, many organisms natu-rally occurring in medical products aremore resistant to radiation than B. pumilis, rendering this a poor surro-gate organism. If no alternative exists,however, this method may be accept-able. A D10 value (D value) of anorganism, in this case, is the amount ofradiation (quantity of kGy) necessaryto reduce the bioburden level by 1 log.

An example of a published D valuefor B. pumilis is 1.7 kGy. Some cautionshould be taken in using a published Dvalue, as D values can vary dependingon the technique used to determinethem and/or the inoculation substrate.Also, D values, or the resistance of anorganism to gamma radiation, canchange over time, analogous to antibi-otic resistance in microorganisms.However, if this is the method to beused, the following is an example ofthe calculation for determining mini-mum sterilization dose.

Inoculation with 106 (1,000,000organisms):

SAL = 10–6

106 to 10–6 = 12 log reductionD value 1.7 kGy × 12 log reduction= 20.4 kGy20.4 kGy = 10–6 SAL dose

The following calculation deter-mines the necessary verification dosefor 10 products to show the efficacyof the above 20.4-kGy sterilizationdose:

Log bioburden – log (1/#samples)]× d-value = verification doseLog 1,000,000 – log (1/10)] × 1.7 =verification dose[6 – (–1)] × 1.7 = verification dose[6 + 1] × 1.7 = verification dose7 × 1.7 = verification dose11.9 kGy = verification dose

Sterilization

A radiation dose of 11.9 kGy ±10% is applied to 10 product units,which are then sent to a lab for sterilitytesting. If no more than one test out of10 turns positive, the sterilization dose,in this example, 20.4 kGy, is validated.

Finally, whichever method is used,the manufacturer must verify the doseevery 3 months in an experimentknown as a Quarterly Dose Audit. Todo this, 10 samples must be sent to thelaboratory for bioburden testing.

Furthermore, every organism cul-tured during the bioburden testshould be identified, at minimumwith a colony morphology and gramstain. Simultaneously, repeat the orig-inal verification dose experiment forwhichever method was used duringthe original validation. For example,if Method 1 determined the originalsterilization dose, then the Method 1verif ication experiment must berepeated. The original verificationdose, or a dose augmented from apast dose audit, is the dose that mustbe used.

The quarterly bioburden samplesserve as a trend-analysis tool. A newverification dose should not be deter-mined from new bioburden data.Should a product fail a dose audit, thebioburden data may hold valuableclues as to why the failure occurred,

e.g., a spike in bioburden number orshift in organism types. If neither ofthese is the case, there is possibly anincrease in the radiation resistance ofthe organisms.

A dose audit failure requires a doseaugmentation. The augmentationamount is found in the dose-settingtable used in the original validation.Beyond all of this, the dose auditshould also include manufacturingenvironment monitoring, such aswater testing, air sampling, and con-tact agar plates. Although regularenvironmental monitoring is recom-mended at shorter intervals, such test-ing quarterly meets the minimumrequirements.

The AAMI dose-setting methodsdescribed here are only recommenda-tions and do not exclude other dose-setting procedures that may bedeemed more appropriate by theirusers. The AAMI methods are widelyaccepted in North America. Whenproperly applied, they have beenaccepted by regulatory groups as validdose-setting procedures.

AAMI guidelines are regularlyreviewed and updated through collab-oration by industry experts (the latestdrafts under consideration are 11137-01, 02, and 03, which will encompassthe methods cited here in 11137:1994

and TIR 27) and are designed to pro-vide a guideline that encompassesthe latest in industry knowledge andrequirements.

Each method has advantages anddisadvantages, and care must be usedin selecting a method that best fitsthe needs and l imitations of theproduct being evaluated. Thesemethods can provide an acceptableand straightforward means of sub-stantiating dose selection for pharma-ceutical products.

Following this guidance will aid inthe successful validation of any radia-tion-stable pharmaceutical product forgamma radiation sterilization. Theideal time for considering the methodof sterilization is at the concept stage,so that gamma-compatible materialscan be chosen and the effects on prod-uct safety and efficacy can be consid-ered. With the variety of materialscurrently available, many pharmaceu-ticals and most packaging materialscan satisfactorily withstand the rigorsof gamma processing.

Steris Isomedix Services provides technical

support during all of these processes, including

research, turnkey validations, special projects,

and technical information. ■

Sterilization

Reprinted from Pharmaceutical & Medical Packaging News, May 2004 • Copyright © 2004 Canon Communications LLC

STERIS Isomedix Services provides contract sterilization, microbial reduc-tion, and materials modification services to medical device manufacturers, pharma-ceutical, biotechnology, and industrial customers. Through a network of NorthAmerican facilities, we deliver state-of-the-art Gamma, EtO, and E-beam processesas part of a complete managed program that emphasizes exceptional process quali-ty, efficient turnaround, and optimum cost containment. For more informationabout STERIS Isomedix Services please call (877) 783-7470 or log ontowww.Isomedix.com.