n°6 November 2009

bi Pharmabi Pharma

Welcome to the sixth issue ofbioPharma, focused on SterilityControl, a critical activity for thebiopharmaceutical industry. As inprevious issues, a significant part ofbioPharma looks at innovation andI am particularly pleased to present

a number of contributions from experts.

At a time of increasing demand in the areas of quality control and risk prevention, our long-standing expertise inmicrobiological control allows us to reinvent traditionalmethods to meet your changing needs. bioMérieux hasdeveloped two groundbreaking ranges of culture mediaspecifically designed for Media Fill Tests, one of this issue’smain topics. Susanne Edström, Head of QC SectionMicrobiology at Octapharma (Sweden) has recentlyswitched from home-made to bioMérieux’s ready-to-useClean Room range media and shares her experience.

Automated methods provide rapid and standardizedresults as well as traceability, while fulfilling regulatoryrequirements. As Dawn McIver (Microworks, Inc. – USA)puts it, “rapid methods are the future in the pharmaceuticalindustry” and this is the reason why she purchased theBacT/ALERT® 3D system for her company’s facility. PaulStephney, Head of Technical Services Team at PharmatelFresenius Kabi Pty Ltd (Australia) explains how his companyuses BacT/ALERT to enhance the in-process microbialdetection method used during aseptic manufacturing ofTotal Parenteral Nutrition (TPN) IV infusions.

I would like to thank you for your continuing contributionto this newsletter. This issue is no exception with morethan 10 different readers providing articles or testimonials.As you look through these pages, think of what you wouldlike to see developed in future editions and send it alongto us. Your feedback will continue to make bioPharma a valuable resource for you and your peers throughoutthe world.

editorial at stake

AUTHOR: O. CHANCELTITLE: TECHNICAL PROJECT

MANAGERCOMPANY: MERIALCOUNTRY: FRANCE

Originally, aseptic process simulation alsoknown as media fill test, is associated withaseptic production and filling, and their corresponding direct environment. Asepticprocess simulation subsequently came toplay a new role in generating information onthe rim of the process, mainly upstream, orfor which no other approach exists. In myopinion, aseptic process simulation is onlyone part of demonstrating sterility assurance,and is regularly discussed at excessive lengthwith the risk of losing focus on its primaryobjective.Why simulate aseptic processes? This questionmay appear incongruous insofar as the subjecthas already been widely addressed in differentguidelines, at conferences dealing with asepticprocesses or in specific professional literature…Furthermore, no-one would dare challengethe purpose of this kind of simulation asmuch as the tool has already proved its

worth in identifying weaknesses of asepticprocesses.However, the relationship which we aredeveloping with this test is a very specialone, and sometimes leads us to forget morespecific tests which are probably more “factual”.Which one of us has not anxiously waited for the end of the incubation period of theMFT, just much like a student awaiting his orher exam results? Does the absence ofcontamination following the test not inducea sensation of satisfaction, a feeling of dutyaccomplished, even for the most experiencedof us?Apart from the fear that it induces, and theconsequences if it fails to conform, asepticprocess simulation unquestionably enjoys anunrivalled aura among the aseptic validationspecialists. By definition, it is required tosimulate, as closely as possible, routine andindeed worst case production conditions,thereby achieving the force of global proofde facto. But the major question is “does theMFT ensure aseptic control of our processes?”Perhaps this is too much to expect, insofar asan aseptic process is multifaceted and itscontrol difficult to prove? Considering anaseptic process as validated when this is not

the case is a major risk. I thus sought to question the assurance provided by a conformingaseptic process simulation regarding the sterility of products and the induced risk of an “aseptic mirage or illusion”. The ultimatechallenge is not one of voicing an opinion onrequalification of an aseptic process followingdiscovery of one or two contaminated units,but rather the attitude to adopt in a situationof zero contaminated units! The “asepticmirage” risk is a real problem. To say that contamination of an aseptic process simulationis a “true opportunity to revisit the way ofcontrolling aseptic processes is a justifiedreaction, if not somewhat masochistic.However, this is the reality: a conformingaseptic process simulation is much less of aninvitation to challenge aseptic practices! In any case, can one really expect an asepticprocess simulation to validate the completeaseptic process? Having no intention of discrediting aseptic process simulation, anumber of limits could be discussed toimplement new alternative solutions, suchas more sensitive microbiological or nonmicrobiological in process testing whichcould advisedly precede implementation of aseptic process simulations.

A Philosophical Approach to the Aseptic process simulation.

1

Alexandre MérieuxCorporate Vice-President

Industrial Microbiology

2

innovation

Production of parenteral drugs has always beenconsidered as the most critical process in the pharmaceutical industry. To ensure sterility ofthese products, manufacturers use an asepticfilling process and must prove that this processis under control. Media fills have become thestandard approach to validate the protectionfrom microbiological contamination providedby the aseptic filling procedure.

According to the European and US regulatoryagencies, validation of an aseptic line mustbe realized with three successive successfulMedia Fill Tests (MFT), and each validatedline has to be re-evaluated at least twice ayear. MFT are critical for pharmaceuticalcompanies because they have to be performed in the worst-case productionconditions and each single unit has to be visually controlled after incubation. The culturemedium that replaces the pharmaceuticalproduct has to be carefully selected, it mustshow excellent growth performances and hasto be safe and easy to use.

bioMérieux developed two groundbreakingranges of culture media specifically designed forMFT. Both ranges comply with pharmacopoeiasrequirements for the growth of non-fastidiousmicro-organisms, including some anaerobicstrains and, are irradiated at least at 25kGy to

ensure the absence of viable micro-organismsincluding mycoplasmas. These products are coldfilterable.

One of the products contains vegetable pep-tones instead of animal-derived peptones for“animal-free” facilities. In addition, conside-ring the importance and the sensitivity of theMFT, bioMérieux has innovated and included,in this vegetable formulation, a growth-based proprietary color indicator to help reading of contaminated units. Contamination and turbidity can easily be revealed with an irreversible color change from red to yellowif growth occurs after incubation.

bioMérieux’new dehydrated culture mediaTSB 3P™ AP and TSB 3P VP dedicated to Media Fill Test are available in two presen-tations: 500 g powder in a plastic flask and5 kg double wrapped powder within a plasticbucket. Packaging for both are tamper proofwith an individual seal and the dehydratedmedia in their final packaging are irradiated,cold filterable and available with animal or vegetable peptones. In the vegetable formulation, a red proprietary color indicatorspecific for microbial growth was added foreasier contamination detection. Evaluationresults are presented in the last page of thisnewsletter.

bioMérieux solutions for Media Fill TestExample of Media Fill Test (Courtesy of Merial)

Pharmacopeial sterility tests as described byUSP <71>, EP 2.6.1 and JP 54 are of criticalimportance in the bioPharmaceutical industrydue to the nature of the products that areproduced. Two well-understood drawbacksof the Pharmacopeial method for sterility testingare the 14-day incubation period and the necessity for trained personnel to subjectively determine if visible micro-bial growth has occurred. This prolonged

14-day incubation period results in signifi-cant inventory holding costs during in-processquality control and false positives are alwaysa concern due to the subjectivity of thePharmacopeial sterility test method. Inaddition, the bioPharmaceutical industrycontinues to develop novel cellular therapieswith short-shelf lives rendering the 14-dayincubation period especially inadequate.

To address the d rawbacks o f thePharmacopeial ster i l i ty test methodbioMérieux has developed the BacT/ALERT®

3D Dual-T. The BacT/ALERT® 3D Dual-Tis a fully automated, growth based,dual temperature, microbial detectionsystem capable of incubating BacT/ALERT®

culture media bottles at 32.5°C and22.5°C. As a growth based method capableof incubation at 32.5°C and 22.5°C theBacT/ALERT® 3D Dual-T is a true alter-native to the traditional Pharmacopeialsterility test method. The BacT/ALERT® 3DDual-T may be readily utilized for sterility testing in a wide variety of sample matricesincluding but not limited to: mammalian cellculture, vaccine production, culture mediaand, in the United States, autologous cell therapy and tissue banks.

The BacT/ALERT® 3D Dual-T is comprised ofthree components: a BacT/ALERT® 3DController Module, a BacT/ALERT® 3DIncubator Module (32.5°C) and aBacT/ALERT® 3D Low Temperature (LT)Module (22.5°C). bioMérieux specifically designed and validated the LT Module to

incubate BacT/ALERT® culture bottles at 22.5°Cfor the application of sterility testing. Datagenerated during the verification and validationprocess of the LT Module demonstrates theability of the LT Module to detect relevantmicroorganisms within the acceptedPharmacopeia standards for t ime-to-detection.

Each sample that’s incubated in theBacT/ALERT® 3D Dual-T is continuously readevery 10-minutes for CO2 production, thisindicates microbial growth. Proprietary algorithms within the BacT/ALERT® 3DController Module automatically evaluate datagenerated from each sample, this enablespositive samples to be immediately detected.As a non-destructive method when theBacT/ALERT® 3D Dual-T detects a positivesample subcultures may be taken directlyfrom the culture bottle for subsequent analysis and/or identification.

Implementation of the BacT/ALERT® 3D Dual-Tenables a much more rapid and objectivemethod to conduct sterility testing forbioPharmaceutical products. BacT/ALERT®

customers have demonstrated the ability todetect sample contamination within 24-48 hours; this is a clear advantage relative tothe traditional 14-day test. Also, theBacT/ALERT® 3D Dual-T uses an objectivemethod, production of CO2, to determine ifmicrobial growth has occurred. This eliminatesthe need for trained personnel to monitorindividual samples on a daily basis reducingthe overall labor costs to conduct sterility tests.

In addition, BacT/ALERT® customers havedemonstrated a reduction in the false positiverate relative to the Pharmacopeial sterilitytest method.

bioMérieux has validated its line of IndustryBacT/ALERT® culture bottles for use in the BacT/ALERT® 3D Dual-T, each bottle hasan Intended Use specific for Industrial applications. In addition, all IndustryBacT/ALERT® culture media bottles have performance characteristics in theirInstructions for Use with microorganisms usedfor growth promotion tests as described inthe Pharmacopeial chapters for sterility testing.bioMérieux also conducts quality control oneach lot of Industry BacT/ALERT® culturemedia bottles with the appropriate growthpromotion microorganisms as suggested inthe Pharmacopeias.

Regulatory agencies continue to support theuse of alternative methods for microbial testing; USP <1223>, EP 5.1.6 and EP 2.6.27all contain information on the validation ofalternative methods. As a growth based, dualtemperature method for sterility testing vali-dation of the BacT/ALERT® 3D Dual-T isstraightforward since it mimics the conditionsfound within the traditional sterility testmethod. This, in addition to the performancebenefits provided by the BacT/ALERT®

3D Dual-T, make it a system uniquely capableand well adapted for use as an alternativeto the steril ity test method found inPharmacopeial chapters USP <71>, EP 2.6.1and JP 54.

AUTOMATED MICROBIAL DETECTION AT TWO TEMPERATURES*

BacT/ALERT® 3D LT Module

B A C T / A L E R T 3 DDual-T

Manufacturing tankLot of 4,000 liters

Visual InspectionAt 7 and 14 Days

Incubation at 32.5°CDuring 7 Days

Incubation at 22.5°CDuring 7 Days

Aseptic Filling: 250 mL polypropylen Bottles

6,000 Bottles

Transfer Bag1,000 liters

Transfer to the filling line

Pre-Filter Sterilizing Filter 0.2 µm

Growth Promotion

Test

?or

Sterilizing Filter 0.2 µm

Storage Tank

*Please, consult your bioMérieux representative for availibity in your country.

Addressed with a focus on the Identifiation-Typing-combination of Vitek® 2 Compact and Diversilab®,bioMérieux Germany took part at the 2nd EuropeanMicrobiology Conference of European ComplianceAcademy (ECA) in Munich in May 2009 BioBall®

and the new Media Fill Tests were also presented.Both products generated great interest among high-level representatives of the European pharmaceutical industry. The first day of the meetingwas dedicated to “Regulatory Requirements andNew Developments”, the second day to “Facilitiesand Utilities” and “Biotechnology”. Speakers camefrom international companies such as Novartis,Roche Diagnostics, Baxter, Vectech and manyothers. Richard Lilischkis (bioMérieux, Australia)spoke on “Equivalence of Bacterial Strains fromDifferent Reference Stocks”.

expert opinion

INTERVIEW

INTERVIEWEE: DAWN McIVER

FUNCTION: PRESIDENT

COMPANY: MICROWORKS, INC

COUNTRY: USA

bioPharma: Would you please tell our readers about you and your company?

Dawn McIver: I am a Microbiologist withtwenty-two years of experience. I have workedboth in the food industry and the pharma-ceutical industry. I founded MicroWorks, Inc. in 1996. For thefirst 12 years MicroWorks specialized inconsulting and training for the pharmaceuticalindustry. We have been in many large andsmall companies throughout the industryand performed almost every type of Microtest and validation out there. We havealways been a hands-on company which I believe is what has made us successful. In 2008, we bought a 10,000 square feetfacility in Crown Point, IN and have establisheda cGMP compliantMicrobiologicaltesting laboratory.We perform tradi-tional Microbiologytesting as well asspecial projectssuch as disinfectantstudies, new equip-ment qualification

and environmental monitoring studies. We continue to consult as well and offer on-site and off-site training programs.

bioPharma: What types of products are tested in your facility and how did you historically perform sterility testing for theseproducts?

D.M.I.: All types of pharmaceuticals, medicaldevices, raw materials, EM samples.I have experience with the USP <71> membrane filtration and direct inoculationmethods.

bioPharma: What are some of the key reasons you decided to implement analternative method for your sterility testing?

D.M.I.: I believe that the rapid methods arethe future in our industry. We purchasedthe BacT/ALERT® 3D system for our facilityin order to bring the possibility of rapid tes-ting to the contract laboratory. This will allowus to assist companies that are thinking ofmoving to this technology with method validations and feasibility studies. Smallercompanies that do not have the resourcesto implement this technology will haveaccess to rapid methods performed hereat MicroWorks.

bioPharma: As an automated systemfor sterility testing that tests at 32.5°C

and 22.5°C, how doyou anticipate theBacT/ALERT® 3DDual-T to performrelat ive to your historic method oftesting?

D.M.I.: I am quiteconfident that theDual-T will perform

as well or better than thetraditional method. I thinkhaving both temperatureswill align this system betterwith the traditional methodand eliminate the possibilitythat organisms which grow atlower temperature will be missed.Since this system is non-destructiveit will allow for identification of positiveresults. I see this as a major advantageof this system.

bioPharma: Being a system that incubatesat 32.5°C and 22.5°C how would youexpect val idat ion of the Dual-T and acceptance by competent authorities toprogress?

D.M.I.: I expect the val idat ion to gosmoothly since the system now so closelyrelates to the traditional methods. We havealready performed our equipment IQ andOQ on the instrument and had successwith all the testing that we have done withthe system so far. This makes me optimisticthat the addition of the low temperatureincubator will only enhance the system.I think the current regulatory environmentis open to alternative methods as long asthe science behind them is strong.

bioPharma: Please tell us how you wouldenvision utilization of the BacT/ALERT® 3DDual-T for sterility testing at your facility.

D.M.I.: This will allow us to expand our current capabilities by offering rapid sterilitytesting to our clients. We also plan to workwith clients who are thinking of buying theirown systems to assess the feasibility of thissystem with their products and to assistthem with validation and training.

BacT/ALERT drawer

BacT/ALERT culture bottles

3

bioMérieux Germanyat the 2nd EuropeanMicrobiologyConference of ECA

news

For the official launch of the new media rangeTSB 3P™ vegetal peptones and color indicator,the program of the fifth Symposium was natu-rally focused on the management of the MediaFill Test in the pharmaceutical industry. The wholeprocess was discussed from implementation to the management of an investigation.

Olivier Chancel from Merial presented the evaluation conducted on an industrial scale withthe new TSB 3P bioMérieux’ media for MFT…Such a nice opportunity to bring a very coloredtouch to this day!

Furthermore, with two subjects (the bioburdenbefore sterilisation and the establishment of an environment control plan) annex 1 of cGMP was widely discussed, confirming that it is more than ever at the heart of

concerns in the pharmaceutical industry.

Two more technical presentations completed the program:

• The first by Arnaud Carlotti from IDMYK, wasan overview on contaminants. Then RolandGuinet from AFSSAPS elaborated on theimportance of typing for investigations, recal-ling the difference between identificationmethods and typing methods.

• The final presentation given by Vincent Boudyfrom APHP, a specialist in risk management, succeeded in getting the meeting with his passion for metrology, a complex science present everywhere in industry.

Finally, the now traditional forum concluded the day giving participants the opportunity to exchange each other.

Around 150 persons attended to this eventand left the meeting with the firm wish tocome back in 2010. The success in partici-pat ion demonstrates, once again, theconfidence of pharmaceutical industry in

bioMérieux and widely comfort us that thebest partnerships are based on confidence,scientific exchanges and obviously the deve-lopment of innovating technologies, adaptedto our customer needs.

bioMérieux Symposium 2009News and prospects in pharmaceutical microbiology

Lecturers of this 2009 Symposium

news

User’s contribution to bioPharma

INTERVIEW

INTERVIEWEE: PAUL STEPHNEY

FUNCTION: HEAD OF TECHNICAL SERVICES TEAM

COMPANY: PHARMATEL FRESENIUS KABI PTY LTD

COUNTRY: AUSTRALIA

bioPharma: Please briefly present the profile of the PFKOrganisation and tell our readers about your role and activityin the laboratory and production facility?Paul STEPHNEY: Pharmatel Fresenius Kabi Pty Ltd (PFK) isa Pharmaceutical Healthcare company that formed in 2004as a result of a joint venture between a family owned AustralianHealthcare company, Pharmatel, and a German based multinational, Fresenius Kabi. PFK specialises in treatment ofcritically ill patients in key areas such as, Anaesthesia, BloodVolume Substitution, Fluid Management, Gastroenterology,Nutrition and Oncology. Our customers are public and private hospitals and healthcare professionals.

At our compounding facilities in Sydney and Melbourne we manufacture Oncology and Total Parenteral Nutrition (TPN)IV infusions under TGA license in ISO class 7 certified cleanrooms.

Jerome and I head up the Technical Services team at theSydney facility. We’re fortunate enough to have a wide rangeof day to day responsibilities, however I would consider ourprinciple role is to ensure the facility operates within specifi-cation from a mechanical and environmental perspective.We’re also responsible for implementing all microbiologicalprocedures in the facility. The other significant part of our jobis the more scientific, ongoing project based work that weundertake for other teams.

bioPharma: How did you find out about BacT/ALERT® system?

P.S.: Last year we appointed a new Operations Director. Hebrought with him over 25 years of aseptic manufacturingexperience from compounding facilities in South Africa andthe United Kingdom.

As our colleagues in South Africa have been using theBacT/ALERT as part of their QC protocols for aseptic manu-facture it seemed logical for us to adopt this process as well.

bioPharma: What methods and processes were you tryingto improve for the PFK Organisation?

P.S.: We wanted to enhance the in-process microbial detectionmethod used during the aseptic manufacture of TotalParenteral Nutrition (TPN) IV infusions.

TPN is a complex nutritional infusion admixture that’s admi-nistered via the parenteral route to patients who have impaireduse of their gastrointestinal tract due to disease or surgery.

TPN infusions typically contain large volumes of glucose, aminoacids and lipids. This makes TPN a particularly suitable growthmedium for microbes. A TPN infusion contaminated with justa few colony forming units would provide a suitable environmentfor exponential microbial growth in a relatively short periodof time, even if the product is refrigerated.

TPN therefore requires a high level of quality control due to the nature of the solution and because it’s administeredintravenously.

Obviously maintaining asepsis is paramount when compoundingTPN so it was imperative to incorporate an in-process testthat was accurate and representative of the manufacturingprocess. The original procedure had several limitations in sampling,result detection and extended incubation time (14 days asper EU Pharmacopeia method). The reduced incubationtime has allowed us to have improved batch to batch controlsin place.We required a microbial detection system that was simpler,more specific to the batch and provided quicker results, withoutthe subjectivity associated with visual detection.

bioPharma: What are the applications the BacT/ALERT system is currently being used for? Have you consideredother applications since you have started using theBacT/ALERT for the future?

P.S.: Currently the BacT/ALERT system is now used for in-process testing of all aseptically prepared TPN IV infusions.

I envisage incorporating BacT/ALERT into other areas of asepticmanufacture and validation. Implementing the system to TPNwas the priority given the nature of the product. I see no reason why we can’t incorporate the BacT/ALERT system forother projects both for in-process testing and part of releasecriteria for batches.

bioPharma: What was your experience with validatingthe BacT/ALERT for your laboratory?

P.S.: The validation was generally straightforward. The protocolprovided by bioMérieux was comprehensive and relativelysimple for us to apply the validation to our own product. Ourmain aim was to challenge the system’s specificity, sensitivityand speed of detection. The protocol fulfilled these criteriawith only minor amendments required.

The Installation Qualification portion was completed mostlyby bioMérieux’s Field Service Engineer and ApplicationsSpecialist who also provided us with on site training. Since the BacT/ALERT 3D user interface is so simple to useJerome completed the Operational Qualification portion ofthe validation without any setbacks.

The validation was a success – all seeded media bottlesrecorded positive results within 48 hours. Subsequent sub-culturing of these samples confirmed the positive result,as well as recovery and identity of the original challengemicroorganisms.

bioPharma: What are the mains benefits of incorporating theBacT/ALERT system into your lab and into the productionfacility? What are the main benefits to your business overall?

P.S.: Early microbial detection as well as having an automated microbial detection system drastically reducesthe need for “hands on” time for operators for sample preparation and inspection.

We expect to make significant savings in the short term sincethe previous system used multiple bottles of media perbatch as a opposed to a single iAST bottle required for the BacT/ALERT.

Implementing the BacT/ALERT system has made our overalloperations more efficient; microbiological data is given inreal-time with reduced time constraints. We have a moreresponsive, accurate and efficient in-process quality controlmethod with improved overall quality assurance of our products.

bioPharma: Do you have any other comments or suggestionsfor the readers?

P.S. : I f your products demand fast results without compromising quality control, then I would definitelyrecommend considering the BacT/ALERT system as methodfor microbial detection.

news

In September 2009, bioMérieux has stepped up its production capacity for custom culture media bybuilding a dedicated production unit in Craponne(France), where the company’s main culture mediaproduction center is located.

The new facility will bolster bioMérieux’s product offering and ability to satisfy the biopharmaceutical and agri-food industries’ needs for custom manufacturingto meet their demanding quality requirements. Custommanufacturing is already possible at several bioMérieuxsites. The creation of this new production unit heraldsan important new step in the company’s commitmentto improving customer satisfaction.

A high degree of flexibility is necessary (in terms ofpackaging, volume and media formulation) to satisfya wide variety of industrial requirements, andstandard products cannot always offer such flexibility. The new production unit will make it possible to supply very specific culture media, regardless of thebatch size to be produced. The solutions provided by bioMérieux are validatedand produced in compliance with ISO 9001 and GMPstandards.

In addition, a dedicated scientific team is available toadvise customers in selecting the most appropriatemicrobiological solution. This team also offers scientificsupport to facil itate product validation on the customers’ premises.

bioMérieux Creates New Facility Specializedin the Production ofCustom Culture Mediafor use in IndustrialMicrobiology Laboratories

4

From left to right: Melissa J BOURKE (bioMérieux), Jerome CHEUNG and Paul STEPHNEY

User’s contribution to bioPharma

INTERVIEW

INTERVIEWEES: SUSANNE EDSTRÖM, HEAD OF QC SECTION MICROBIOLOGY.

KERSTIN MOËLL, LABORATORY ENGINEER

KARIN HULTMAN, LABORATORY ENGINEER

COMPANY: OCTAPHARMA, STOCKHOLM

COUNTRY: SWEDEN

bioPharma: How would you presentOctapharma to our readers?

Susanne Edström: Octapharma is a privately owned company with internationalrelations and is the manufacturer of essentialprotein medicinal products derived fromblood plasma or by means of gene technology.Octapharma’s medical products are usedin haemophiliac therapy, immuno therapyand intensive care. Octapharma has approx.3,000 employees in more than 30 countries.One of the company’s production sites islocated in Stockholm.

bioPharma: What are your quality objectives?

S.E.: Octapharma guarantees that their products meet regulatory requirements andare suitable for intended use with respectto safety and product quality. StandardOperating Procedures, SOPs, are used to guide management and personnel indecisions and actions for any operation orsystem, to assure first-rate product quality.

bioPharma: What are your sterility controlneeds?

S.E.: It is one of the tests we have to per-form on our finished product according to pharmacopœia.

bioPharma: What solution do you use forsterility control?

Kerstin Moëll: We perform sterility testingin accordance with pharmacopoeia andhave used the same method for more than 20 years. Until a year ago we prepared TSB and Thioglycollate ourselves, but we have now switched to bioMérieuxready-to-use media.

bioPharma: What was the reason for the switch?

S.E.: We made a long-term plan to replaceour home-made media with ready-to-usemedia in order to minimize the substrateproduction to be able to utilize resourcesfor sample testing and other laboratory tasksrequiring qualified personnel. bioMérieuxsupplies ready made substrates, whichsatisfy our demands and objectives.

bioPharma: Why did you choosebioMérieux as supplier?

S.E.: We tested different suppliers simulta-neously, and media from bioMérieux werefound to be the best suited for our company.

K.M.: Sterile triple-wrapping was veryimportant for us. In the beginning we hadongoing discussions about the packing, andthe fact that there were no plastic caps tocover the membrane. We received goodresponse, and all our questions were answered. The media is very clear and it iseasy to interpret.

bioPharma: Please inform us about thevalidation you performed on of the bottles?

S.E.: The validation was part of our routine.We checked the growth enhancing abilities

of the media in the same way as wechecked our home-made batches. Wecontrolled three different batches with themicroorganisms listed in the pharmacopœia,and were able to conclude that thebioMérieux media were equal to the mediawe used previously.

bioPharma: How do you regard bioMérieuxas a supplier?

S.E.: bioMérieux is an important supplierfor us, and I believe we have good communications. You always try to findsolutions for us whenever required. Karin Hultman: We have experienced that logistics have improved during therecent years and now we receive detailedinformation of all deliveries.

bioPharma: bioMérieux aims at supplyingcomplete solutions to our customers. Whatother products/solutions would you find tobe of use?

S.E.: We have reduced the number of suppliers during the recent years. We haveno specific wishes right now, but your newBioBall® product looks very interesting.

The Octapharma quality policy

5

News

In July 2009, bioMérieux announced its productionincrease to support the global effort to fight the Influenzapandemic. The company’s culture media for sterility testingis used by vaccine manufacturers during the qualitycontrol of their production process to help ensure productsafety. Sterility testing is critical for the control and releaseof vaccines on the market.

bioMérieux’s main culture media production site in Craponne (France) is mobilized to meet the growing needfor sterility testing media. The company increased its safety stock at the first signs of a potential pandemic, enabling it to meet the extra demand from global vaccinemanufacturers in recent weeks. It is preparing for an expected rise in orders over the coming months, whenthe first batches of Influenza A/H1N1 vaccine will be produced and released. A global continuity plan is underway to ensure consistent service and a steady supplyof priority products for the pandemic.

“bioMérieux has been in close communication with its pharmaceutical customers globally to support increasedproduction capacity efforts for the influenza vaccine,” said Alexandre Mérieux, Corporate Vice President, IndustrialMicrobiology. “We are taking the necessary measures toready our teams and ensure a secure supply of productsat a critical time for public health worldwide”.

Together with the recently launched Media Fill line, bioMérieux provides a complete solution for the control ofprocess and product sterility to ensure patient safety.

In addition to its solutions for industrial microbiologicalcontrol, bioMérieux is contributing to the fight against Influenza A/H1N1 in the area of clinical diagnostics. To help physicians rapidly assess their patients and take the necessary isolation steps, the company is adapting its NucliSENS EasyQ® Influenza A/B test to include this newstrain. bioMérieux is currently building stock of specific reagents for its NucliSENS® easyMAG® molecular diagnostics system, which is being used for extraction ofinfluenza viral nucleic acids, a preliminary step in testing, atseveral public health laboratories in the world. bioMérieux also distributes Quidel’s QuickVue® InfluenzaA+B and QuickVue Influenza A/B rapid tests. However, theeffectiveness of these tests to detect the H1N1 strain iscurrently being evaluated.

Quality means safety We ensure that our products meet regulatory requirementsand that they are suitable for their intended use with respect to safety, quality and efficacy.

Quality claims trust We strive for a fair, open and long lasting co-operationwith our internal and external customers, partners andsuppliers.

Quality safeguard, Quality Assurance activities directly contribute to our future the performance and result of our company.

Quality encourages Our management culture is based on the responsible caredevelopment of all our employees and focuses on the quality of our work. of our employees We contribute in this manner to the development of

employees’ skills and generate a motivating workingenvironment.

Quality asks for It is our aim to achieve quality by continually improving ourcontinuous improvement products and processes. Every employee is encouraged

to contribute to the process of continuous improvement.

bioMérieux is Adapting Production to Support Global VaccineProduction Effort in the Fight Against Influenza A/H1N1

science & technology

bioPharma is an International publicationof bioMérieux Industry.• Publishing director: Alexandre Mérieux• Editor: Pascal CruveillerHave contributed to this issue: Philippe Bechaud,Fabienne Cassagne, Michele Storrs-Mabilat,

Koren Wolman-Tardy, Renaud Jonquières, Arnaud Paris, Laurent Leblanc, Keith Hansen,Sandy Hume, Alexander Pfülb.

Special thanks to: Olivier Chancel (Merial), DawnMcIver (Microworks, inc), Paul Stephney

(Pharmatel Fresenius Kabi Pty Ltd), SuzanEdström (Octapharma), Kertsin Moëll(Octapharma), Karin Hultman (Octapharma).Layout and design :ISSN: 1777-6856

bioMérieux S.A69280 Marcy l’Etoile - FranceTél. 33 (0) 4 78 87 20 00Fax. 33 (0) 4 78 87 20 90www.biomerieux.comwww.biomerieux-industry.com

We want to hear from you! Send us comments and suggestions or submit articles to be published:biopharma@eu.biomerieux.com

bioMérieux applications

As the world leader in microbiological control, bioMérieux provides a unique range of microbiology quality assurance testing solutions, coveringenvironmental monitoring, product sterility testing and microbial identification. We are committed to helping pharmaceutical and cosmetic industrypartners as well as blood banks, tissue banks and biotechs provide safe products in total confidence.

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ABSTRACTMedia Fill Tests (MFT) are critical microbiologicaltests carried out to simulate the normal manu-facturing conditions by replacing thepharmaceutical product by culture media.Manual reading of several thousands pharmaceutical containers filled with culturemedia is the current practice. Since eachcontainer must be individually checked, thisstep is extremely time-consuming and can bea source of potential errors: it requires highlyqualified technicians to be performed inappropriate conditions.bioMérieux has developed two groundbrea-king ranges of culture media specificallydesigned for MFT. This study describes howboth those new ranges of TSB 3P™(Pharmaceutical Proven Performances) culturemedia with animal and vegetable peptoneswere designed and optimized to meet thehighest requirements from the pharmaceuticalindustry.

MATERIAL AND METHODSThe growth performance analysis was performedon the following bioMerieux references:

• Irradiated TSB 3P™ Animal Peptones, ref. 51101/51102 (TSB 3P AP)

• Irradiated TSB 3P Vegetable Peptoneswith color indicator, ref. 51103/51104(TSB 3P VP)

• Trypcase Soy Broth, ref. 51019 (TSB) wasused as a positive growth control.

Performances of two MFT media from othersuppliers were compared to bioMérieux MFT media for the growth of anaerobic ormicro-aerophilic micro-organisms:

Growth Performance StudyThe growth promotion performances werechecked on Pharmacopoeias strains and on cleanroom isolates. One hundred and one (101)micro-organisms were tested in this study.

Inocula were prepared from fresh micro-orga-nisms culture by serial dilutions; their finalconcentration was between 10 and 100 CFU.The inoculum cells was enumerated on anappropriate Petri dish. Culture media were inoculated with 50 µl ofeach strain (4 tubes for each medium), then2 tubes were incubated at 20-25°C and 2 tubes at 30-35°C during 14 days in aerobicor anaerobic conditions. Reading of the media was performed for dailyand growth was measured according to aninternal turbidity scale.

Color Indicator StudyThe evaluation of the TSB 3P VP media colorchange was performed on aerobic strains.Tubes were inoculated and incubated; turbiditywas read according to the same protocol asdescribed for the Growth Promotion Study.At the same time, the color change of theindicator was also evaluated and the disco-loration was rated under three conditions:total, partial or no discoloration.

Filterability Study - Vmax Determination Vmax is the theoretical maximum volumetricthroughput for a filter under a constant pressure. Vmax was determined on 3 differentfiltering membranes:- Polyvinylidene (PVDF), 0.22 µm,- Polyethersulfone (PES), 0.20 µm,- Nylon (NR), 0.22 µm.The test was repeated 10 times on eachmembrane.

Irradiation Cycle ValidationThe killing validation was made using Bacilluspumilus ATCC 27142 biological indicators calibrated at 1.5x108 CFU on strips. Stripswere introduced in 500 g and 5 kg bucketsof dehydrated culture media and were irradiatedwith a worst case condition of 25 kGy dose(the lowest dose). Irradiated strips were thenincubated in TSB culture media bioMérieuxref. 44011 and growth was monitored by turbidity reading.In addition, growth performances of the mediawere controlled after irradiation. 500 g and 5 kgbuckets of dehydrated media were irradiatedwith a worst case condition of 40 kGy dose(the highest dose) and growth performanceswere then evaluated on a specific samplegroup of micro-organisms.Mycoplasma AbsenceThe “over-kill” effect of irradiation for myco-plasmas was validated by verifying theabsence of mycoplasmas before and afterirradiation at 25 kGy. This was tested by anexternal laboratory who analyzed samples tocheck for the absence of the mycoplasmasdescribed in the pharmacopœia.Two methods were used to detect mycoplasmasin the dehydrated media: - An indirect culture growth method with

indicator cells and a PCR-based method witha genetic amplification.

- A spiking method with mycoplasmas biolo-g ical indicators and a pharmacopœiacompliant growth method on culture media.

RESULTS AND DISCUSSIONRaw Material SelectionSeveral conditions were used for the rawmaterial selection:- Absence of mycoplasma,- Limited bioburden,- Good filterability of the peptones,- High growth performances.The bioMérieux TSB 3P AP and TSB AP VPmedia were manufactured with TSE-free peptones and all the raw materials enteringin the composition of the final MFT mediawere tested in pilot batches. Growth PerformancesGrowth promotion test was per formed on bioMérieux TSB 3P media with 101 strains, either ATCC strains or isolatesfrom the production environment (see Figure 1and Figure 2).

Optimized Performances For QuickAerobes Detection

TSB 3P AP and TSB 3P VP are equivalent tothe non irradiated TSB control in terms ofbroad range detection.

As shown in the above figure, all data obtai-ned during the growth promotion test supportthe fact that the irradiation process doesn’thave any effect on the performances of the TSB 3P media for MFT, and that the rapiddetection performances were optimized compared to the reference.

Optimized Performances For QuickAnaerobes DetectionBoth TSB 3P AP and TSB 3P VP were speciallydesigned and optimized to grow anaerobicbacteria as shown in Figure 3.

TSB 3P media have higher performances thanthe non-irradiated TSB control and the othermanufacturers. 100% of the anaerobic strainsgrew in TSB 3P AP and TSB 3P VP after 8 days of incubation at 20-25°C.

These results clearly illustrate that TSB 3Pmedia were optimized to be adapted for efficient and rapid anaerobe growth: an earlydetection of anaerobic micro-organisms underanaerobic conditions occurs during the firstpart of the incubation at 20-25°C.

Color Indicator Study

bioMérieux MFT culture media references51103 and 51104 (with vegetable peptones)contain a Redox color indicator. The recons-tituted culture medium has a red color thatis discolored with microbial growth. This indicator was selected because of its ability to be reduced by a very broad range of micro-organisms metabolism. We demonstrated on 92 aerobic strains the correct discoloration of the TSB 3PTM

with vegetable peptones medium.The results of this study i l lustrate that the chosen color indicator is well adapted foruse as a growth indicator. After a 14 daysincubation, a wide panel of microorganismsshowed an efficient discoloration.

Irradiation Cycle Validation

A 25 kGy irradiation establishes a 8 log killingcondition. In accordance with the raw materialbioburden analysis (data not shown), the irradiation process ensures a dehydrated culture media for Media Fill Tests free of anyviable micro-organisms. Complementary growth promotion testsdemonstrated that no performance differenceswere observed between the irradiated andthe non-irradiated media.

Mycoplasma AbsenceNo mycoplasma were found in the powderwhich clearly establishes the high quality of the raw material used in the MFT mediamanufacturing.The absence of mycoplasma was also confirmedafter a 25 kGy irradiation cycle. The irradiationestablishes a 8 log killing condition.

Filterability StudyThe animal peptone formulation showed asimilar range of filterability with the differenttypes of membranes. The animal formulationis more filterable due to the fact that animalpeptones are more fluid.However, it was demonstrated by a real casestudy in the pharmaceutical industry that a4,000 liters TSB 3P VP media preparation wasfinally filtered on 0.22 µm without any cloggingproblem. The conditions were with a pre-filtrationon 0.2 µm and a 2.5 Bar pressure.

6

0

20

40

60

80

100

% of micro-organisms after 7 days at 20-25 °C

TSB control TSB 3P AP TSB 3P VP

CONCLUSIONIn this study, bioMérieux presented a breakthrough improvement for the MFT reading accuracywith a new irradiated cold filterable TSB medium, containing vegetable peptones and a uniquecolor indicator. This product was developed with selected raw materials to guarantee the culture medium tobe TSE-free and to be free from any viable micro-organism and mycoplasma.The presence of the color indicator facilitates the visualization of the microbial growth.The study also illustrates the optimization and the ability of the TSB 3P culture media to recoveranaerobic strains from environment.

A Groundbreaking Culture Media RangeTo Simplify And Enhance Media Fill Tests Accuracy

Fig 1: Proportion of all the strains that grew after 7 days of incubation at 20-25°C.

250

260

270

280

290

300

Intensity of the turbidityafter 7 days at 20-25 °C

TSB control TSB 3P AP TSB 3P VP

Fig 2: Growth promotion results on allstrains after 7 days at 20-25°C.Turbidity values were summed.

Intensity of the turbidityafter 8 days at 20-25 °C

5.000

TSB control TSB 3P AP TSB 3P VP ManufacturerA

ManufacturerB

10.000

15.000

20.000

25.000

0

Fig 3: Growth promotion results on all anaerobic strains after 8 days ofincubation at 20-25°C. Turbidity values were summed.