The Introduction of Qualitative Rapid Microbiological Methods

Transcription

1 The Introduction of Qualitative Rapid Microbiological Methods for Drug-Product Testing Paul Newby, Gilberto Dalmaso, Silvano Lonardi, Bryan Riley, Peter Cooney, and Kim Tyndall* FDA recently approved the first PAT applications for the introduction of rapid microbial testing of drug products and pharmaceutical-grade waters. Officials from FDA and GlaxoSmithKline worked together to ensure the appropriate scientific evaluation of the methods. Team members report on the successful validation approach and identify technical issues to be considered for the future. Paul Newby, PhD, is the team leader of pharmaceutical microbiology, at GlaxoSmithKline (Ware, Hertfordshire, England). Gilberto Dalmaso, PhD, is the microbiological laboratory head and Silvano Lonardi is a manufacturing technology/pat champion,at GlaxoSmithKline (Parma, Italy). Bryan Riley, PhD, is a senior review microbiologist and Peter Cooney, PhD, is the associate director for new drug microbiology at the US Food and Drug Administration, Center for Drug Evaluation and Research, Office of Pharmaceutical Science (Rockville, MD). Kim Tyndall is an associate director, CMC regulatory affairs, at GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, tel , fax , kim.m.tyndall@gsk.com *To whom all correspondence should be addressed. Conven ti onal microbi o l ogical test met h od s, b a s ed on n i n eteen t h -cen tu ry tech n i qu e s, a re ti m e - con suming and l a bor- i n ten s ive, l ack sen s i tivi ty, and are su bj ective and poorly va l i d a ted. Because microbi o l ogical te s ting is a bo t t l en eck in produ ct rel e a s e, the ph a rm aceutical indu s try s interest in rapid microbi o l ogical met h ods (RMMs) has grown considerably during the past ten years. RMMs su ch as adenosine tri ph o s ph a te (ATP) bi o lu m i n e s- cen ce and solid-phase laser cytom etry are being inve s ti ga ted for the adva n t a ges they of fer in speed, s en s i tivi ty, and acc u rac y. Despite significant progress in the development of such method s, h owever, the ph a rm aceutical indu s try has been hesitant to i m p l em ent them for produ ct te s ting because of con cerns abo ut reg u l a tory accept a n ce and technical barri ers. Technical and regu l a tory barri ers inclu de the com p l ex ch emical natu re of t h e tech n o l ogy, the ch a ll en ge of technical tra n s fer of these sys tem s f rom the food, bevera ge, and co s m etic indu s tries (wh ere they have been used for several decades) to the pharmaceutical sector, and the lack of a ppropri a te guidance for va l i d a ti on and implementation. Va l i d a ti on and reg u l a tory requ i rem ents for RMM tech n o l o- gies are beginning to em er ge fo ll owing FDA s en dors em ent of RMMs as a process analytical technology (PAT), as part of the a gen c y s qu a l i ty initi a tive, P h a rm aceutical CGMPs for the 21st Century: A Risk-Based Approach. PAT is a framework for scien ti f i c, ri s k - m a n a ged ph a rm aceutical devel opm ent that requ i re s ti m ely, i n - process measu rem ent of m a terials and processes to en su re the qu a l i ty of final produ ct s. Re a l - time or near- re a l - ti m e microbiological testing can be conducted as part of manufacturing process control only through the use of RMMs. G l a xo Sm i t h Kline has taken the opportu n i ty provi ded by the PAT initi a tive to establish rapid microbi o l ogical met h ods for m i c robial-limit te s ting of ph a rm aceuti c a l - grade waters and produ ct release of s el ected do s a ge form s. F DA s approval of t h e s e m et h ods marks the agen c y s first approval of a PAT met h od. This arti cle ad d resses some of the technical qu e s ti ons assoc i- a ted with introducing qu a l i t a tive RMMs for dru g - produ ct te s t- ing such as instrument qualification and microbiological performance testing. 6 Pharmaceutical Technology PROCESS ANALYTICAL TECHNOLOGY 2004 w w w. p h a r m t e c h. c o m

2 Figure 1: Qualitative testing strategy for two-tiered testing, using the ATP bioluminescence method and a conventional MLT method (RLU denotes relative light units). Background The current com pendial met h ods for microbial te s ting of f i n i s h ed d rug produ cts or ph a rm aceuti c a l - grade waters gen era lly use ei t h er a mem brane filtra ti on - b a s ed or po u rp l a te / s pre ad p l a te - b a s ed microbial-limit test (MLT). These growt h - b a s ed test met h- ods typ i c a lly take four to seven days to com p l ete and requ i re visual examination and identification. The MLT defined by the current US Pharmacopeia is a threefold test, entailing enumeration of microorganisms (quantitative) determ i n a ti on of the pre s en ce or absen ce of ph a rm acopei a l indicator microorganisms (qualitative), and identification of recovered microorganisms (qualitative). Two new tech n o l ogies for microbial determ i n a ti on, ATP bi o - lu m i n e s cen ce and laser cytom etry, a re su peri or to trad i ti on a l tech n i ques in many re s pect s, i n cluding sen s i tivi ty, s el ectivi ty, and speed. The ATP bi o lu m i n e s cen ce assay qu i ck ly and ef fectively determines the pre s en ce of vi a ble microor ga n i s m s. Bi o- lu m i n e s cen ce occ u rs natu ra lly in the firef ly (Ph oti nus pyra l i s ), wh en the en z yme lu c i ferase cataly zes the re acti on of lu c i feri n and the nu cl eo ti de ATP to produ ce ligh t. By producing ligh t, lu c i ferase thus can qu i ck ly and acc u ra tely detect the pre s en ce of ATP (found in all living cell s, i n cluding microbial cell s ). In l a s er cytom etry, a flu ore s cent stain signals vi a ble microbial cell s. The stain is converted to a flu orescing state on ly wh en estera s e en z yme activi ty and an intact cell mem brane are pre s en t ; n onvi a ble cells thus are not detected. Va l i d a ti on requ i rem ents for microbial-limit te s ting wi t h RMMs are def i n ed in PDA s Technical Report 33, Eva lu a ti on, Va l i d a ti on and Im p l em en t a ti on of New Mi c robi o l ogical Te s t- ing Met h od s ( 1 ). The va l i d a ti on cri teri a, l i s ted in Ta ble I for both qu a l i t a tive and qu a n ti t a tive met h od s, a re indepen dent of the tech n o l ogical platform. The equ iva l en ce of RMMs to conven ti onal met h ods can be dem on s tra ted thro u gh a series of experi m ents de s i gn ed to assess the va l i d a ti on cri teri a, wh i ch can be perform ed by the pro s pective user or by others (i. e., l i teratu re referen ces can be used to ad d ress va l i d a ti on cri teri a ; s ee the bi bl i ogra phy at the end of this arti cl e ). Validation of RMMs: a case study G l a xo Sm i t h Kl i n e s microbi o l ogists needed to determine the appropri a te va l i d a ti on stra tegy and app l i c a ti on of qu a l i t a tive MLT methods for drug products and pharmaceutical-grade waters. Because of the divers i ty of requ i rem ents for produ ct te s ting and monitoring, no single technology platform has the capacity to satisfy all testing requirements. Therefore, a two-stage method was sel ected, com bining a rapid microbi o l ogical met h od for qu a l i t a tive te s ting and conven ti onal MLTs. In this approach, t h e RMM is used as a screening te s t ; b a tches that pass need not under go conven ti onal te s ti n g. This type of s c reening is app l i c a bl e for high - vo lume produ cts wh ere historical microbial data ind i c a te that the produ ct typ i c a lly is free of bi obu rden. R M M s c reening all ows rapid produ ct rel e a s e, in hours inste ad of d ays or wee k s, for batches that dem on s tra te the absen ce of m i c ro - organisms. The ATP bi o lu m i n e s cen ce met h od was va l i d a ted as a microbial-limit testing screen, and laser cytometry was validated for qu a n ti t a tive microbial analysis of ph a rm aceuti c a l - grade w a ter. The enu m era ti on and iden ti f i c a ti on aspects of the produ ct s accept a n ce cri teria rem a i n ed unch a n ged. Va l i d a ti on and i m p l em en t a ti on were no different for RMMs than for any other test, with the following exceptions: MLT acceptance criteria were revised for use with RMMs a strategy was developed for handling batches that gave positive results in the screening test. Conven ti onal accept a n ce cri teria for microbial limits are not a pp l i c a ble to RMMs because RMMs are mu ch more sen s i tive than conven ti onal met h od s. Th erefore, we adopted the fo ll owing two-tiered specification: Tier 1 (RMM): negative (the result is below the set limit) Ti er 2 (conven ti onal met h od ) : The total aerobic microbi a l count is not gre a ter than x co l ony - forming units per ml ( c f u / m L ), and St a p hyl o co ccus aureu s, Pseudomonas aeru ginosa, Salmonella species, Escherichia coli, and Enterobacteriaceae are absent in x ml of sample. A stra tegy was devel oped that all ows for rapid release of bi obu r- den - f ree produ cts wh en the RMM yi elds nega tive re sults for the pre s en ce of AT P. Wh en the screen ind i c a tes the pre s en ce of m i- Table I: Microbial-limit test validation criteria for rapid microbiological methods. Qualitative: ATP Quantitative: Parameter bioluminescence laser cytometry Accuracy Precision Specificity Equivalence Limit of detection Limit of quantitation Linearity Range Repeatability 8 Pharmaceutical Technology PROCESS ANALYTICAL TECHNOLOGY 2004 w w w. p h a r m t e c h. c o m

3 Table II: Experiments for validation of the microbiallimit test using rapid microbiological methods. Experiment Absence of interference (TLV) Specificity Limit of detection Ruggedness and repeatability Robustness Acceptance criterion The product formulation does not inhibit or enhance method performance, and a threshold limit value (TLV) has been established. The method has an acceptable ability to detect all the microorganisms with which it is challenged. The method has an acceptable ability to detect microorganisms in samples spiked with 1 to 10 cfu. The method demonstrates acceptable ruggedness and repeatability in analyses with various analysts, various instruments, and various reagent batches. The method demonstrates acceptable robustness in analyses with deliberate variations of incubation time, shaking speed, incubation temperature, microbiological physiological conditions, and compositions of mixed cultures. Figure 2: The V-model for system qualification. c robial AT P, the conven ti onal MLT is perform ed to determine the nu m bers and types of m i c roor ganisms pre s ent and to dem on s tra te com p l i a n ce or non com p l i a n ce with the microbi o l ogical spec i f i- c a ti on. F i g u re 1 illu s tra tes this proce s s. Va l i d a ti on of the RMMs foc u s ed on both microbi o l ogi c a l testing performance and technology platform qualification. A va l i d a ti on model that Glaxo Sm i t h Kline has used su cce s s f u lly with RMMs is a V- m odel, wh i ch is wi dely used in com p uter s of t w a re devel opm en t. The model was mod i f i ed to inclu de microbiological performance qualification attributes (see Figure 2). Other validation models exist, and the use of the modified V-model in this example is not meant to suggest that it is requ i red. However, the basic el em ents of de s i gn qu a l i f i c a ti on ( D Q ), i n s t a ll a ti on qu a l i f i c a ti on (IQ), opera ti onal qu a l i f i c a ti on ( OQ ), and perform a n ce qu a l i f i c a ti on (PQ), as illu s tra ted in this m odel, a re important parts of the va l i d a ti on of a ny new analytical met h od. The mod i f i ed V- m odel also provi des a conven i ent way to com bine equ i pm ent qu a l i f i c a ti on requ i rem en t s with a microbiological performance evaluation. Design qualificat i o n. G l a xo Sm i t h Kl i n e s microbi o l ogists developed a va l i d a ti on master plan for RMM eva lu a ti on. The plan inclu ded both equ i pm ent and microbi o l ogi c a l - perform a n ce eva l- u a ti on details su ch as user requ i rem en t s, perform a n ce capabi l i ti e s, com p uter hardw a re and sof t w a re requ i rem en t s, su pp l i er audits, co s t s, and ben ef i t s. The flow diagram shown in Figure 3 illu s tra te s a process for assessing the fe a s i bi l i ty of a ny rapid microbi o l ogi c a l tech n o l ogy, using ATP bi o lu m i n e s cen ce as an ex a m p l e. I n s t a l l ation qualificat i o n. The IQ stage veri f i ed and doc u m en ted that the RMM instru m en t a ti on had been su pp l i ed, i n s t a ll ed, and te s ted according to the manu f actu rer s spec i f i c a ti on s. Th e i n s t a ll a ti on qu a l i f i c a ti on pack a ge inclu ded doc u m en t a ti on of a vi sual inspecti on of a ll equ i pm en t, copies of a ll opera ti on manu a l s, and con f i rm a ti on that all requ i red uti l i ties (e. g., el ectri c i ty, vac u u m, and laminar flow hoods) were install ed properly. Cop i e s of pro tocols and re sults for all tests perform ed by the ven dor and on site are maintained in the event of an inspecti on. Operational qualification. The manufacturers of rapid microbi o l ogical tech n o l ogies su pp ly the pro tocols for the OQ of t h ei r instruments. These protocols may be used as is or modified by the end user as appropriate. Typical tests include verifying the i n terf ace bet ween the sof t w a re and the instru m en t, veri f yi n g u s er access to each input message or command proce s s ed by the sof t w a re, c ro s s - ch ecking each ex ternal file or data record referenced by the supplier, and verifying output messages, displays, and recorded data generated by the software. These tests were perform ed and doc u m en ted with both com pendial micro - or ganisms and site - s pecific envi ron m ental microor ga n i s m s. This OQ was cri tical at this stage, because it en su red correct operation of the method under working conditions. Pe r fo rm a n ce qualificat i o n. PQ dem on s tra ted the su i t a bi l i ty of the RMMs for the purpose of m i c robial-limit te s ti n g. Va l i d a- ti on ex peri m ents were de s i gn ed to dem on s tra te and ju s tify the use of the RMMs for te s ting specific drug produ cts and ph a rm aceuti c a l - grade waters. P DA Technical Report 33 was fo l- l owed in performing the va l i d a ti on ex peri m ents listed in Ta bl e I I. Te s ting for each cri teri on (absen ce of i n terferen ce, s pec i f i c i ty, limit of detecti on, ru ggedness and repe a t a bi l i ty, and robu s t- ness) was out l i n ed in pro tocols with spec i f i ed accept a n ce criteri a. The perform a n ce tests used for the ATP bi o lu m i n e s cen ce method are summarized below. Absence of interference. Interaction of the drug product or sample matrix with the luciferase ATP enzymatic reaction (i.e., inhibition or enhancement) was evaluated through tests of noncon t a m i n a ted samples and samples spiked with the fo ll owi n g contaminants: Gram-positive bacterial species: S. aureus Gram-negative bacterial species: E. coli Spore-forming bacterial species: Bacillus subtilis Fungus: Aspergillus niger 10 Pharmaceutical Technology PROCESS ANALYTICAL TECHNOLOGY 2004 w w w. p h a r m t e c h. c o m

4 The re sults of these tests (mu l tiple data points) were used to s et the threshold limit va lue (T LV), a bove wh i ch samples are cons i dered con t a m i n a ted and bel ow wh i ch they are con s i dered not con t a m i n a ted. The accepted prob a bi l i ty of a false-nega tive re su l t ( i den ti f ying a con t a m i n a ted sample as non con t a m i n a ted) was %. This va lue is equal to the prob a bi l i ty of a false-negative re sult accepted for the media fill test of s terile drug produ ct s. Spe c i f i c i ty. To dem on s tra te the te s t s abi l i ty to detect a ra n ge of microorganisms, at least six replicate samples of drug product were spiked with 10 to 100 cfu of m i c roor ganisms from site - s pecific isolates and the fo ll owing com pendial microor ga n i s m s (strains of the American Type Culture Collection, ATCC): E. coli (ATCC 8739) S. aureus (ATCC 6538) P. aeruginosa (ATCC 9027) B. subtilis (ATCC 6633) Salmonella abony (ATCC 6017) Candida albicans (ATCC 10231) A. niger (ATCC 16404) The te s t s abi l i ty to differen ti a te bet ween non con t a m i n a ted samples and samples containing diverse microor ganisms was dem on s tra ted by a two - w ay analysis of va ri a n ce, using five cont a m i n a ti on factors (thre s h o l d, f u n g u s, and gra m - n ega tive, gra m - positive, and spore-forming bacteria). Limit of dete ct i o n. The te s t s abi l i ty to detect low con cen tra ti on s of m i c roor ganisms was dem on s tra ted using samples of d ru g produ ct spiked with 1 to 10 cfu of the same microor ga n i s m s used to test specificity. Ruggedness and re pe at a b i l i ty. The te s t s prec i s i on was dem on s tra ted by analyzing the same bioburden-free samples under a variety of n ormal test con d i ti on s ; t wo different analysts carri ed out the tests with two different instru m en t s, re a gent kit lots, and membrane lots, and results were compared. Ro b u s t n e s s. The te s t s abi l i ty to yi eld re sults unaffected by small but del i bera te va ri a ti ons in met h od para m eters was dem onstrated by varying the following parameters: i n c u b a ti on ti m e : f rom 0 to 24 h, using a gra m - po s i tive bacterial species and a fungus, both chosen for slow growth. shaking speed : 200 or 300 rpm, b a s ed on 250 rpm as establ i s h ed by current good manu f actu ring practi ce s, using a gra m - positive bacterial species and a fungus, both chosen for high oxygen requirements. i n c u b a ti on tem pera tu re : 30 8C or 35 8C, using a gra m - po s i tive bacterial spec i e s, ch o s en as a repre s en t a tive bacteri a l species, and a fungus chosen for its temperature sensitivity. m i c robial phys i o l ogical con d i ti on s : s oybe a n - c a s ein dige s t medium at ph levels of 7.3, 5.5, or 3 and a sodium chloride con cen tra ti on of 0. 5, 1. 5, or 5%, using gra m - po s i tive and gra m - n ega tive bacteri a. The con d i ti ons were ch o s en to ch a l- l en ge the test sys tem, because the bi o lu m i n e s cen ce en z ym e com p l ex is sen s i tive to high and low ph as well as to high salt concentration. m i xed cultu re s : (a) gra m - po s i tive and gra m - n ega tive bacteri a and a fungus or (b) a spore - forming bacterium and a fungus. Eq u i va l e n ce. The cri teri on for equ iva l en ce is that the new method be at least as good as the current method. In conventi onal MLT met h od s, the limit of detecti on is approx i m a tely 10 Figure 3: Process for design qualification of rapid microbiological technology, as applied to ATP bioluminescence. Figure 4: MLT results for purified water tested by a conventional pourplate method and by laser cytometry. c f u / m L, wh ereas RMMs can easily detect microbes at the s i n gl e - cell level. Taking into account this significant increase in s en s i tivi ty, a mu l ti f aceted approach was used to establish met h od equ iva l en ce, com bining a revi ew of the publ i s h ed litera tu re, laboratory experiments with spiked samples, and parallel testing of ph a rm aceuti c a l - grade water samples with rapid and conven ti onal met h od s. In the case of the ATP bi o lu m i n e s cen ce m et h od, l i tera tu re citati ons were su f f i c i ent to establish equ iva l en ce. F i g u re 4 shows an example of the re sults obt a i n ed in p a ra ll el te s ting of ph a rm aceuti c a l - grade water with a po u rp l a te Pharmaceutical Technology PROCESS ANALYTICAL TECHNOLOGY

5 m et h od and with laser cytom etry over a 20-day peri od. The results show similar tren d s ; h owever, the conven ti onal met h od s took several days, while the laser cytom etry re sults were obtained within hours, without the need for pre-enrichment. Moving from data to knowledge Broad acceptance and implementation of rapid microbiological met h ods by the ph a rm aceutical indu s try wi ll be a slow process. Given the range of skills required, equipment qualific a ti on and microbi o l ogical perform a n ce va l i d a ti on are be s t tackled through a team approach involving the system manufacturer to ensure that expertise is effectively transferred from the manufacturer to the operators using the equipment. It is essen tial that du ring the va rious stages of eva lu a ti on, i m- p l em en t a ti on, and va l i d a ti on, a thoro u gh understanding of t h e n ew te s ting sys tem and a true pictu re of its capabi l i ties be ga i n ed. Data gen era ted must be tra n s l a ted into knowl ed ge, wh i ch mu s t be ef fectively doc u m en ted thro u gh a com preh en s ive va l i d a ti on process and passed on through a training program. Future direction In terest in rapid microbi o l ogical met h ods in the ph a rm aceutical indu s try is high and is ex pected to increase fo ll owing reports of su ccessful implem en t a ti on. Adopti on of RMMs is warra n ted by significant adva n t a ges in speed of re su l t s, proce s s efficiency savings, sensitivity, and business benefits. The advent of PAT has given a new impetus to the introdu c- ti on of R M M s. Conven ti onal microbi o l ogical test met h ods are not capable of delivering real-time or near-real-time results, a prerequ i s i te for su ccessful ex p l oi t a ti on of PAT ben ef i t s. Bec a u s e RMMs do have this capabi l i ty, t h ey wi ll be an inva lu a ble aid to successfully realizing the objectives of PAT increased quality and efficiency. The current reg u l a tory envi ron m ent for implem en ting ra p i d m i c robi o l ogical met h ods also is en co u ra gi n g. The reg u l a tory a ut h ori ties in the Un i ted States and Eu rope have approved RMM tech n o l ogies for produ ct te s ting and ph a rm aceuti c a l - grade water testing. The inclusion of these methods as part of the PAT initiative is also indicative of FDA s support for RMMs. Reference 1. PDA, Technical Report 33, Evaluation, Validation and Implementati on of New Mi c robi o l ogical Te s ting Met h od s, J. Ph a rm. S ci. Te ch n ol. 54 (3), Suppl. TR33 (May June 2000). Bibliography (references and guidances) F DA, Gu i d a n ce for In du s try: PAT A Fra m ework for In n ova tive Pharm aceutical Ma nu f actu ring and Quality As su ra n ce, D raft Gu i d a n ce, (August 2003). F DA, Gu i d a n ce for In du s try: Com p a ra bi l i ty Pro toco l s Ch em i s try, Ma n- ufacturing, and Controls Information, Draft Guidance, fda.gov/cder/gdlns/cmprprot.pdf (February 2003). G en eral Ch a pter,6 1., Mi c robial Limit Te s t s, USP 27 NF 22 (US Pharmacopeial Convention, Rockville, MD, 2004). G en eral Ch a pter, , Va l i d a ti on of Com pendial Met h od s, U S P 27 NF 22 (US Pharmacopeial Convention, Rockville, MD, 2004). General Chapter,1227., Validation of Microbial Recovery from Pharmacopeial Articles, USP 27 NF 22 (US Pharmacopeial Convention, Rockville, MD, 2003). G en eral Ch a pter, , Va l i d a ti on of Al tern a tive Mi c robi o l ogical Met h- ods, USP 27 NF 22 (US Pharmacopeial Convention, Rockville, MD, 2003). M.A. Brailsford, A Two-Hour Method for the Enumeration of Microorganisms in Envi ron m ental Sa m p l e s, in Pro ce ed i n gs from PDA An nu a l Meeting (PDA, Bethesda, MD, 1998). K. Ha berer and G. Wa ll n er, Eva lu a ti on of the Ch emscan sys tem for Ra p i d Mi c robi o l ogical An a lysis of P h a rm aceutical Wa ter, in Pro ce ed i n gs fro m P DA In tern a tional Co n gre s s Adva n ces in Ph a rm a ceu tical Ma nu f a c- turing, (PDA, Bethesda, MD, 1998). D. Jon e s, M. Bra i l s ford, and J. - L. D roco u rt, Solid Phase, L a s er Scanning Cytom etry: A New Two - Hour Met h od for the Enu m era ti on of Mic roor ganisms in Pharm aceutical Wa ter, Ph a rm a copeial Fo ru m 2 5 ( 1 ) (1999). R. L. Kepn er and J. R. Pra t t, Use of F lu oroch romes for Di rect Enu m eration of Viable Bacteria in Environmental Samples: Past and Present, Microbiol. Rev. 60 (4), (1994). C. L a P l ace - Bu i l h e, et al., App l i c a ti on of F l ow Cytom etry to Rapid Mi c robial An a lysis in Food and Drink In du s tri e s, B i ol. Cell 78 ( 1 2 ), (1993). W. D. Mc E l roy, The Ener gy So u rce for Bi o lu m i n e s cen ce in an Is o l a ted System, Proc. Nat. Acad. Sci. USA 33, (1947). P. J. Newby, Rapid Met h ods for Enu m era ti on and Iden ti f i c a ti on in Mic robi o l ogy, in Ha n d b ook of Mi crobi ol o gical Quality Co n trol, R. M. Baird, et al., Eds. (Taylor & Francis, New York, 2000), pp K. Wi ll s, et al., Mu l ti - Cen tre Va l i d a ti on of Rapid Bi o lu m i n e s cent Techn i que for the Enu m era ti on of Mi c ro - or ganisms in Pharm aceuti c a l Process Water, in Abstracts of the 97th General Meeting of the American Soci ety for Mi crobi ol o gy ( Am erican Soc i ety for Mi c robi o l ogy, Wa s h- ington, DC, 1997).P T 12 Pharmaceutical Technology PROCESS ANALYTICAL TECHNOLOGY 2004 w w w. p h a r m t e c h. c o m