The result of eye drops beingcontaminated with Pseudomonas aeruginosacan be a considerable loss of vision or evencomplete blindness, as happened to someusers in Sweden in 1968. Infectiouspathogens – bacteria, viruses, fungi,parasites – that find their way into theblood stream can multiply and cause bloodpoisoning, called sepsis, a severe immuneresponse to pathogens or their toxins.

Minimising microbial contaminationduring the production of pharmaceuticalsis an absolute necessity. The GMPGuidelines therefore impose a steadilygrowing number of requirements thatoblige manufacturers to guarantee drugsafety by following appropriate qualityassurance and quality managementconcepts.

These GMP Guidelines deal not onlywith the pharmaceuticals themselves, i.e.with their production, packaging andtesting, but also with the supply of allstarting materials, including anymachinery and equipment required.Personnel hygiene and personnel trainingare also core fundamentals covered byGMP Guidelines.

Another essential component of GMPrequirements is to implement in-process

control and final product quality controltesting to detect any microbiologicalcontamination and to monitor thebioburden.

Sterility testing for batch releaseIn particular, strict requirements apply tosterile formulations, i.e. parenteralsintended for injection, infusion ortransplantation into the human or animalbody. Testing the sterility of the filledfinal product is an essential and a decisivecriterion for approving the release of acomplete batch.

Sterility testing of the final product canbe carried out either by the drugmanufacturer or by a certified contract

lab. According to the internationalpharmacopeias (USP <71>, EP 2.6.1, JP 4:06, WHO (QAS / 11,413 final),sterility testing is not only to be carriedout on parenterals, but is also mandatoryfor the release of eye drops, ointments,creams, aerosols and solids, such assurgical sutures.

The method for culturing micro-organisms to test for product sterility hasexisted since about 1930. It involves eitherfiltration of the pharmaceutical product tobe examined – the currently preferredmethod according to USP <71> or EP

September 2015 41PHARMACEUTICALS

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Sterility testing of parenterals is a decisive criterion contributing todrug safety. Without this test, no sterile medical preparation maybe released onto the market. Marcus Schütte, Product Manager,Microbiology, Sartorius Stedim Biotech, Goettingen, Germany,looks at some of the crucial aspects of sterility testing

STERILITY TESTING OFPARENTERAL DRUGS

Above: Typical sterility test procedure performedin an isolator

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2.6.1 – alternatively, if this method is notpossible, direct transfer of the product toa liquid culture medium. The sterility testis performed in a controlled environmentunder aseptic conditions, either in anisolator that meets the requirements of aEU cGMP Grade A cleanroom, or in alaboratory Grade A cleanroom (i.e. cleanbench, surrounded by a Grade Bcleanroom).

Measures taken to preventcontamination of the critical area may notaffect the growth of any micro-organismspresent in samples undergoing sterilitytesting. This needs to be ensured byappropriate validation procedures, forexample, bacteriostasis/fungistasis tests.To monitor the environmental conditions,methods that do not interfere with theprotection of the cleanroom area are used,such as active air sampling with the MD8AirScan. In especially critical areas, e.g.filling lines, isolators or blow-fill-sealequipment, users rely on the proven highretention and recovery rates of gelatinmembrane filters to detect viruses andmicro-organisms during air monitoring.

The demands on the sterility testingsystem, materials and on the personnelperforming this test are very high. Afterall, both false-negative and false-positiveresults must be avoided (US Departmentof Health and Human Services, Food andDrug Administration, [CBER], Office ofRegulatory Affairs).

According to 21 CFR 211.25, well-trained personnel are important in thisregard. Therefore, lab personnel shouldgive thought early on to the applicationitself as well as to its requirements andprepare a checklist or User RequirementSpecification (URS). In selecting asterility testing system, they also need toweigh process risks against the costbenefits.

Rules applying to sterility testingThe countries in which a pharmaceuticalproduct will be sold must first be defined,since knowledge of GMP Guidelines is afundamental requirement. For example,in almost all countries, sterility testing isperformed with two culture media andtwo test containers. For parenterals soldin China, a three-container variant ismandatory, whereas in South America andAfrica, because of the low labour costs,individual components assembled into asystem part by part are more commonlyused.

This individually assembled equipment,known in Germany as the ‘Schillersystem’, was widespread in Europe untilabout 1975. Due to the time required for

assembly and the risk of false-positiveresults, however, it is no longer used inEurope, the US or Japan.

The procedure for sterility testing isclearly regulated: it requires a cleanroom-suitable pressure supply, e.g. a peristalticpump that conveys the liquid to beanalysed into the sterility test units thatfilter it through an incorporatedmembrane. Having a nominal pore size of0.45µm, this low-adsorption membrane ineach test unit reliably retains micro-organisms.

Antibiotics and other growth-inhibitingsubstances are washed out by pumpingrinsing fluid through the sterility testingsystem. If the product to be tested is inpowder form, it must be made soluble byappropriate means. At the end of theprocess, the two test containers are sealedoff on the bottom with a plug.

Subsequently, the containers are eachfilled with the appropriate culturemedium: CASO (Casein Soybean Digest)for detecting aerobic bacteria and FluidThioglycollate Medium for detectinganaerobic/aerobic bacteria. Thepermissible rinsing fluids are specified inUSP <71>. Because the septa on therinsing fluid containers or product areusually pierced more than once, the usermust ensure that the needles are sharpenough to allow smooth and easy multiplepuncturing.

To enable GMP-compliant work, batchtraceability must be in place for allproducts used, including the sterility testsystem; preferably, however, it should bepossible to scan the barcode on eachpackage and save the data as a PDF file in

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The Sterisart Universal pump is availablefor liquid transfer of equal quantities of apharmaceutical product to the sterilitytest units. An integrated barcode scanneron the pump permits user-friendlytraceability of the products employed insterility testing, such as media andSterisart NF units. Special focus wasplaced on designing the pump to ensurecleanroom suitability. Its smooth surfacefinish is made of AISI 316 L stainlesssteel, and the pump can either beoperated inside a clean bench orintegrated into an isolator. In addition totheir H2O2 compatibility, the individualpump components are resistant to UVand are autoclavable. These two aspectsare particularly important for operationinside a clean bench.

The GMP-compliant pump is acompletely closed unit – without anyventilation slits.

Sterisart Universal pump for liquid transfer

Above: SterisartUniversal pump,including displayand barcodescanner, used totransfer media andliquids for sterilitytesting. Left: theintegrated scannerreads all standardbarcode labels forbatch traceabilityand saves them asproof of GMPcompliance foraudits

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September 2015 43PHARMACEUTICALS

CONTACTSartorius Stedim BiotechT +49 551 308 3522www.sartorius.de/de/bioprocess-solutions

accordance with 21 CFR Part 11.For final batch release testing of sterile-

labelled pharmaceuticals, Sartoriussupplies Sterisart NF single-use units.These systems feature a closed design fortesting bags, syringes, vials, ampoules andpowders, and function according to themembrane filtration method. Theobjective of using Sterisart NF units is toenable reproducible, reliable sterilitytesting that leaves no room for false-positive or -negative results.

During the 14 days of incubation toidentify any contamination or to validatea pharmaceutical product, it may benecessary to remove samples fromcontainers aseptically or to inject fluids,e.g. penicillinase, to deactivate antibioticswith a β-lactam ring system. Today,sampling and injection are generallycarried out by piercing the tube with aspike or by cutting the tubing andsubsequently pipetting in the solution.This increases considerably both the riskof contamination and the risk of puncture-related injury for the user.

Risks can be avoided and samplingmade much easier by using septumversions of Sterisart NF units. Recognisedby the medical sector, septum technology

has proven its efficacy. The septum iscapped, and can be disinfected with a wipewetted with isopropanol, if necessary. Ifthe pharmaceutical product itself rendersthe medium turbid, USP <71>recommends: ‘…14 days after thebeginning of incubation, transfer portions(each not less than 1mL of the medium) tofresh vessels of the same medium...’

Septum versions of sterility test unitsenable safe and reliable fluid transfer. Theconcept of liquid transfer involvingsimultaneous sterile venting of thecontainer can be implemented using apatented dual-needle spike. First, stainlesssteel dual-needle spikes are exceptionallysharp, which considerably facilitatespiercing septa; second, these needles areeasier to handle compared with individualneedles, as no separate vent filter isrequired.

Sterisart NF is a registered trademark ofSartorius Stedim Biotech

Left: Example of a Sterisart NF unit as a septumversion for sterility testing. This unit has a short,sharp, automatically sterile-vented dual-needlespike and a safety platform

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