den,in terms ofits concentration in the formulation,must beevaluated to minimize the bacterial challenge to the sterilizingfilter and the endotoxin content ofthe product.Informationabout the properties and specifications ofpharmaceutical in-gredients is available in the
Handbook ofPharmaceutical Excipients
(4).The bioburden ofpackaging components must be evaluatedwith respect to the sterilization process that will be used in themanufacture ofsterile products.The bacterial endotoxin levelsand the potential populations ofgram-positive,spore-formingbacteria associated with stoppers and vials are a consideration.Vials must be inspected and packaged for shipment to the cus-tomer in a controlled environment.Individual vials must beseparated with non-shredding dividers and shrink-wrapped toprevent glass-to-glass contact and particulate contamination.Vials are washed to remove particulates,depyrogenated to re-move bacterial endotoxins,and sterilized before aseptic filling.The maximum temperature and belt speed are established fora depyrogenating tunnel that adequately depyrogenates andconcurrently sterilizes the vials as they move through the tun-nel into the aseptic filling area.Stopper preparation methods should physically remove bac-terial endotoxins and nonviable particulates before siliconiza-tion.The cleaning and siliconization process should not con-tribute to the bioburden.The sterilization cycle ensures that thestoppers are sterile and dry so they can be stored before theaseptic filling operation.It should be noted that steam steril-ization is not a depyrogenation step.Typically,stoppers aresteam sterilized in heat-sealed,nonwoven,high-density poly-ethylene bags that enable steam penetration and moisture re-moval during the sterilization,exhaust,and drying processeswithin a pass-through autoclave.Cleanroom personnel usuallysize the bags to replenish the stopper hopper with a single loadto reduce the potential for microbial contamination duringmultiple handling.Alternately,semi-automated stopper prepa-ration equipment can be used to prepare sterile siliconized stop-pers.Stoppers also can be purchased clean,siliconized,andsterile.Initial bioburden and endotoxin monitoring ofincom-ing packaging components should be conducted to establishwhether the challenge levels for the cleaning,depyrogenation,and sterilization processes are adequate.
Preservation system development.
Testing for antimicrobial ro-bustness is an important part ofa drug product’s developmentalphase.In general,the use ofa preservative in single-use prod-ucts to replace good manufacturing practices (GMPs) is notsupported by regulatory agencies.Multiple-use products thatare stored in stoppered vials can be contaminated during re-peated syringe needle entries.Thus,they are formulated withpreservative systems that are tested for preservative efficacy dur-ing development using
,“Antimi-crobial Effectiveness Test”(5).The use ofthis test,when ap-propriate,can generate a developmental history ofa formulationin terms ofits preservative effectiveness against a range ofmi-croorganisms.The test also can indicate whether a product ismicrobiologically stable even without the presence ofa preser-vative system (i.e.,self-preserving).During the developmentphase ofthe product life cycle,the lowest concentrations atwhich the preservative system is effective can be established.The proposed formulation should be tested with the antimi-crobial effectiveness test at 50,75,and 100% ofthe target preser-vative concentration to establish the shelfspecification for theproduct on the basis ofpreservative efficacy and stability.A typ-ical preservative specification for a pharmaceutical product maybe 80–120% ofthe label claim.Thus,the preservative systemmay be monitored in the research and development stage andin product stability programs using a stability-indicating chem-ical assay in place ofmore time-consuming and more-variableantimicrobial effectiveness tests.
The container–closure integrity of the packaging components also is addressed during productdevelopment using a sensitive and adequately validated test.Recommendations for various container–closure combinationsfrom packaging suppliers are usually helpful.A physicalcontainer–closure integrity test may be selected and validatedusing a bacterial liquid immersion or aerosolization test.In gen-eral,physical tests are more sensitive than bacterial challengetests.Therefore,the leakage observed during a physical test maynot be indicative ofsterility assurance loss.A comprehensivediscussion about leak testing ofpharmaceutical packaging sys-tems has been published (6).When selecting a test method,the container–closure typeshould be considered.Although stoppered vials are subjectedto a bacterial immersion test,prefilled syringes are subjected toa bacterial aerosolization test because the latter has a moretorturous path for container–closure integrity.Physical testmethods described in the literature include the bubble method,helium mass spectrometry,liquid tracer (dye),headspace analy-sis,vacuum and pressure decay,weight loss or gain,and high-voltage leak detection.There are two phases to the container–closure integrity assessment:the initial evaluation and selec-tion ofthe container–closure system and integrity testing withinthe premarketed stability program.Suitable testing intervals are0,3,6,9,12,18,and 24 months during the premarketed stabil-ity program and annually during the postmarketed stabilityprogram.The number ofsamples tested at each time intervalreflects the sampling requirements found in
,“Sterility Test”(7).Whenever possible,physicalcontainer–closure integrity tests for product monitoring shouldbe substituted for sterility testing.
Sterility test development.
The development ofa sterile prod-uct requires initial and ongoing consultation with an experi-enced pharmaceutical microbiologist.The application ofthesterility test is one indicator ofthe presence or absence ofcon-taminating microorganisms in a sterile batch,but the sterilityassurance is established by process design and validation,notsimply finished product testing.Validation ofthe sterility testincludes bacteriostasis and fungistasis testing and follows a pro-cedure that is defined in
,“SterilityTests.”Biological products marketed in the United States thatmust meet the 21
610.12 sterility testing requirements andproducts that differ in the subculturing requirements are an ex-ception.The development microbiologist uses results from ster-ilization process validation,aseptic process simulation usingmedia fills,and congruent environmental and personnel mon-