Bioluminescence for USP Sterility Testing of Pharmaceutical .BIOLUMINESCENCE FOR USP STERILITY TESTING

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Vol. 51, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1986, p. 349-3550099-2240/861020349-07$02.0O/OCopyright 1986, American Society for Microbiology

Bioluminescence for USP Sterility Testing of PharmaceuticalSuspension Products

DIANE M. BUSSEY AND KIYOSHI TSUJI*Control Analytical Research and Development, The Upjohn Company, Kalamazoo, Michigan 49001

Received 19 June 1985/Accepted 22 November 1985

Bioluminescence measurement significantly improved the accuracy, sensitivity, precision, and reliability ofthe current visual endpoint determination for the USP sterility test and eliminated the day 7 transfer/dilutionstep required for testing suspension products. Thirteen strains of bacteria and fungi (representing potentialcontaminants in sterile products), three pharmaceutical suspensiori products, and four media were used in theexperiment. No interference from suspension products was encountered in the detection of microbial growth bythe bioluminescence measurement. The poor fungal growth encountered was attributed to insufficient diffusionof oxygen into the medium and was circumvented by use of a large tube size (38 by 200 mm) or by vortexingthe medium once during the 2-week incubation period. Bioluminescence measurement would facilitateautomated handling of the sterility test endpoint readout operation. The optimum parameters of biolumines-cence measurement for application in sterility testing were determined.

The firefly luciferin-luciferase bioluminescence measure-ment for detection of ATP has been used for the qualitativeand quantitative determination of microbial content in fluidsand solids (1, 3, 5-6, 7, 9-11, 14, 15). Applications haveincluded clinical screening for bacteriuria in urine and blood(1, 3, 7, 11, 15), antibiotic susceptibility testing (9, 10),microbiological potency assays of antibiotics and vitamins(9, 10), and determination of biomass in environmentalsamples (e.g., water, soil), foods, and beverages (9, 10, 14).The purpose of this paper was not to shorten the incubationtime of the sterility test required by the U.S. Pharmacopeia(16) but to provide objectivity to the visual endpoint readoutoperation currently in use and to eliminate the time-consuming day 7 transfer/dilution operation, which is apotential contamination source, for testing pharmaceuticalsuspension products.

MATERIALS AND METHODSReagents. (i) Bioluminescence. The Firelight ATP test kit

from Analytical Luminescence Laboratories (San Diego,Calif.) was used to perform the bioluminescence measure-ment. The reagents were stored in a refrigerator (4C) andallowed to reach room temperature before use. The test kitincluded HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.75), Extralight ATPreleasing reagent, Firelight luciferin-luciferase, and an ATPstandard. Reagents were prepared fresh each day accordingto the manufacturer's instructions. The ATP stock standardsolution was prepared by addition of 1.0 ml of sterilewater-for-injection (WFI; The Upjohn Co., Kalamazoo,Mich.) per ATP standard vial to provide 2 x 10-8 M ATP.ATP working standard solution was freshly prepared eachday by diluting the ATP stock solution. To determineday-to-day assay variation, several vials containing ATPstock solution were combined and frozen until used.

(ii) Media. Four media were used to conduct the simulatedsterility test: Trypticase soy broth (TSB), TSB withazolectin and Tween-80 (TSB-N), thioglycolate (Thio), andThio with azolectin and Tween-80 (Thio-N), all from BBLMicrobiology Systems, Cockeysville, Md. The media were

* Corresponding author.

prepared in Pyrex tubes (25 by 200 mm), 40 ml per tube,capped with Kap-uts (Bellco, Vineland, N.J.). For theoxygen diffusion study, the media (TSB and TSB-N) wereprepared in Pyrex tubes (38 by 200 mm) and autoclaved for15 min at 121C. For plate counts and streak plates, Trypti-case soy agar, antibiotic medium no. 1, Schaedler agar, andSabouraud dextrose agar (Difco Labs, Detroit, Mich.) wereused.

Test microorganisms. The following 13 strains, represent-ing potential environmental contaminants in sterile products,were inoculated in pharmaceutical suspension products tosimulate positive sterility samples: Aspergilluis niger ATCC16404, Bacillus subtilis ATCC 6633, Candida albicansATCC 10231, Clostridium sporogenes ATCC 11437, Cory-nebacterium sp. strain UC 9165, Escherichia coli UC 3114,Micrococcus sp. strain UC 9168, Penicilliim sp. strain UC7296, pink-pigmented bacterium strain UC 9166, Pseuidomo-nas aeruginosa UC 9170, Pseudomonas cepacia UC 9463,Staphylococcus epidermidis UC 719, and Streptococ cuspyogenes UC 3113. For microcount determinations andstreak plates, the following media were used: antibioticmedium no. 1 for P. aeruginosa and P. cepacia, Schaedleragar for C. sporogenes, Trypticase soy agar for all otherbacteria, and Sabouraud dextrose agar for fungi.

Bioluminiescence. Portions (0.1 ml) of sample or ATPreference standard were pipetted into plastic cuvettes (cat-alog no. 2000-10, Analytical Luminescence Laboratories).For the negative control, 0.1 ml of WFI or medium wassubstituted for the sample. A 0.1-ml volume each of HEPESbuffer and Extralight reagent were added to samples, and themixture was incubated for 7 min at room temperature torelease/extract ATP from microorganisms. The cuvette wasthen placed in a bioluminometer (Monolight 2001, AnalyticalLuminescence Laboratories), and 0.1 ml of Firelightluciferin-luciferase reagent was automatically pipetted intothe sample. The amount of bioluminescence emitted wasintegrated for 10 s and recorded in relative light units. Allsamples and standard solutions were tested in quadruplicate.The simulated positive samples, microbial suspensions, andATP standards in media were diluted 1:10 in WFI beforeassay. Gilson precision microliter pipettes (Rainin Instru-ment Co., Woburn, Mass.) equipped with sterile disposable


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pipette tips were used to prepare dilutions and to transfersamples into the cuvettes.Simulated positive sterility test sample. For each of the 13

organisms 1.0-ml volumes, containing less than 100 cells perml, were pipetted into 12 tubes, each containing 40 ml ofTSB, TSB-N, Thio, or Thio-N. A 1.0-ml volume of eachsuspension product (Depo-Medrol Sterile Aqueous Suspen-sion, Cortef Sterile Aqueous Suspension, and Depo-ProveraSterile Aqueous Suspension) was then aseptically added tothe tubes. Negative controls contained no microorganisms,and positive controls contained no product. The sampleswere then incubated for 2 weeks at room temperature forTSB and TSB-N media and at 35C for Thio and Thio-Nmedia to simulate sterility test condition. On days 3, 7, and14 of the incubation period, four tubes of the inoculatedsamples and one tube each of the positive and negativecontrols were tested for bioluminescence.For the bioluminescence assay, a sample was recorded as

positive if the relative light units value was greater than orequal to twice the background of the respective negativecontrol. On each of the three testing dates, all biolumines-cence-negative samples were streaked on agar plates toverify the lack of microbial growth or to confirm that thepopulation was below the detection limit of the biolumi-nescence measurement. Microorganisms in positive-biolu-minescence samples were identified to verify growth of theinoculated microorganism. On day 14, microcounts wereperformed to determine the sensitivity and detection limitsof the bioluminescence method.TSB and Thio were used for Depo-Provera Sterile Aque-

ous Suspension and Cortef Sterile Aqueous Suspension,while TSB-N and Thio-N were used to test Depo-MedrolSterile Aqueous Suspension.Oxygen diffusion study. To examine the oxygen content

and diffusion rate, TSB (40 ml per tube) was prepared in twodifferent size containers: a large tube (38 by 200 mm) and asmall tube (25 by 200 mm). The oxygen level of medium wasdetermined with an oxygen probe (model 97-08; OrionResearch, Inc., Cambridge, Mass.) coupled to a pH meter(model 130; Corning Glass Works, Coming, N.Y.). Resultswere expressed in parts per million (microliter per liter) ofoxygen.

RESULTS AND DISCUSSIONOptimization of bioluminescence assay. ATP standard

curves were constructed using the peak and the integrationmodes. Although the peak mode gave better precision (rel-ative standard deviation [RSD], ca. 5 to 8%), the integrationmode (RSD, ca. 4 to 15%) was chosen because of its superiorsensitivity (10-14 M ATP) over the peak mode (10-13 MATP).

(i) Integration time. An integration time of 10 s wasselected from a choice of 10, 30, or 60 s because of lowerbackground bioluminescence, superior linearity (r = 0.9992),sensitivity (10-11 to 10-14 M ATP), and shorter assay time.

(ii) Precision sensitivity. Results of ATP standard curvesperformed with 25- and 100-pdl samples indicated that the100-,ul volume provided slightly better sensitivity (2 x 10-14M ATP) than did 25 RI (5 x 10-14 M ATP). The linearity ofthe two standard curves was comparable, and the number ofreplicates (either three or four) had no significant effect (P