Microbial contamination is a

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1 B I O P R O C E S S TECHNICAL Barrier Vial Technology A Global Approach to the Aseptic Filling Process Diego López-Alvarez, Sergi Roura, and J.A. Garcia Microbial contamination is a concern and a constant challenge in research laboratories as well as in sterile medicine production plants. Yes, the distinguished scientist Alexander Fleming did discover penicillin, one of the most outstanding discoveries of modern medicine, as a consequence of an accidental contamination in a bacterial culture. But it is vital to prevent such contaminations in sterile formulas. To minimize the risk of contamination in sterile filling, the pharmaceutical industry has implemented more and more rigorous procedures and technologies. In the late 1980s, one company that manufactures plasma derivatives applied its many years of such experience to developing a new aseptic filling process: barrier vial technology (BVT) (1). This process has continued to be refined over the past 20 years. PRODUCT FOCUS: ALL BIOTECH PRODUCTS PROCESS FOCUS: MANUFACTURING (FILL AND FINISH) WHO SHOULD READ: FORMULATIONS AND PROCESS DEVELOPMENT, FACILITIES MANAGEMENT AND MANUFACTURING, AND ENGINEERING PERSONNEL KEYWORDS: CONTAINER CLOSURES, AUTOMATION, LYOPHILIZATION, LIQUID FORMULATIONS, ASEPTIC FILLING LEVEL: INTERMEDIATE An existing BVT filling room (human albumin production in progress) GRIFOLS ENGINEERING SA ( BVT does not simply cover aseptic filling alone, but rather every single step of aseptic processing, from preparation and sterilization of containers and closures to the laseretched identification of vials after dosing. Both liquid and freeze-dried products can be dosed using the BVT sterile filling process. The description of this process herein refers to a liquid filling plant. The set of practices and procedures described here demonstrate a unique approach used to minimize the risk of particulate and microbial contamination during every step of an aseptic process. The most important safety measures to take against particulate contamination include a highquality clean area and the use of physical barriers that protect sterile containers and stoppers. Among other relevant features, BVT vials are partially closed with physical barriers during handling. This means that contamination risk is very much minimized. However, the vials allow steam to enter for proper sterilization. CONTAINER DESCRIPTION Three elements comprise a typical container handled in a BVT aseptic filling process: a vial, a capsulestopper set, and a protector (Figure 1). The vial is a standard container 46 BioProcess International DECEMBER 2007

2 Figure 1: A typical container in the BVT aseptic filling process made of glass or plastic for pharmaceutical or medicinal use. The capsule (Figure 2) is also standard, but the stopper is specially designed for aseptic processing. The stopper has a stepped outside contour that allows it to stay in two different stable positions inside the neck of the vial: partially inserted to allow the container to be sterilized (Figure 2A), and fully inserted to seal the vial after filling (Figure 2D). A set of grooves in the body of the stopper (similar to the grooves of stoppers for lyophilized products) allows the sterilization steam pass into the container. And a flange shape provides a tight fit between the stopper and the capsule. The protector (Figure 1, Figure 2A) rests on the vial neck covering the capsule-stopper set. This piece has two functions: to create a labyrinthine path between the vial and the capsulestopper set that prevents particulates from entering washed and sterilized containers and to prevent the capsule stopper set from being fully inserted if improperly handled before sterilization (when being handled manually). The originality of this container closure system is not based on its use of standard vials or redesigned closures but on handling of the vial itself. The capsule stopper set and protector are in place on the vial from the earliest steps of this aseptic process, which creates a physical barrier against microbial contamination from the beginning. ASEPTIC PROCESS DESCRIPTION Figure 3 provides a BVT flow diagram, from container and closure preparation through component sterilization and aseptic filling to sealing and identification of vials. Container and Closure Preparation: Specially designed, prewashed, gamma-irradiated, and clean-packed stoppers are automatically inserted inside the capsule bodies. Product contact surfaces are rinsed with water for injection and blown with filtered air. Vials are thoroughly rinsed and blow-cleaned inside and out at different stations to meet pharmaceutical standards in conventional washing machinery. The capsule stopper sets are partially inserted into vials, and then a protector is simply placed over each vial. Once that is done, the partial closure creates a labyrinthine path to reduce the probability that particulates will enter while vials still can be sterilized. The containers will continue to have these labyrinthine seals until dosing. Component Sterilization: The containers are arranged on trays, and those trays are loaded on wheeled racks. The racks are conveyed into an autoclave, where containers (including capsule stopper sets and protectors) are sterilized by moist heat. The labyrinthine container seals permits air to be removed with a preliminary stage of vacuum pulses, then steam enters and sterilizes each vial. Just after that sterilization stage of the autoclave cycle, a drying stage prevents condensation from forming inside the vials. This intensive vial washing and long sterilization process guarantee endotoxin reduction by at least three logs for the containers, as specified in CGMPs (2). After sterilization, the wheeled racks remain under a laminar flow to cool the containers in the aseptic processing area. Aseptic Filling: Those wheeled racks are then brought near the filling room. An operator places the containers of each tray onto the infeed rotary table of the filling line. Dosing takes place in a Grade A (class 100) environment equipped with horizontal laminar flow. Inside this area, the protectors are discarded. As soon as vials reach the filling point, the capsule stopper sets are removed so the filling nozzle can dose the liquid pharmaceutical formulation. After dosing, each capsule stopper set is inserted completely into its vial. Thus, the amount of time during which vials remain open within the Grade A environment is reduced to just that required to unstopper, fill, and restopper the vial (full insertion). Unlike with conventional filling lines, no extra machinery is necessary to feed stoppers and capsules because each vial reaches the filling point with its stopper capsule set already mounted. The absence of stopper-feeder equipment Figure 2: A container for BVT aseptic processing (A) sterilization (blue arrow indicates air suction during preliminary vacuum pulses in an autoclave chamber, green arrow indicates steam from the autoclave chamber; (B) protection discarded before filling (under horizontal laminar flow); (C) capsule stopper set removed just before filling (under horizontal laminar flow); (D) capsule stopper set replaced after filling (under horizontal laminar flow); and (E) capsule crimping (under vertical laminar flow). 48 BioProcess International DECEMBER 2007

3 Figure 3: The BVT process illustrated in a filling area reduces particulate counts and obtains better overall particle results during monitoring. Sealing and Identification: After a video camera has recorded the whole filling process, stoppered vials are conveyed outside the filling room, where their capsules are crimped under a laminar flow by standard crimping machinery. The filling and crimping processes take place in different rooms with physical separation and different pressure levels to prevent pressure reversal (3). So no particulates generated during the crimping operation can reach the filling area. After crimping, a laser system marks the batch code, filling time, and vial number on each glass vial. This laser marking is durable and cannot be eliminated without damaging containers. In addition to anticounterfeiting benefits, such marking is also helpful for traceability. The filling process video recording and laser marking will be very useful in the event a quality investigation must be carried out. Both the filling time etched on every filled vial and the video of the whole filling operation allow complete tracking of the filled units. FILLING SUITE AND FILLING AREA The filling suite has four rooms (Figure 4): a vial-loading room, a filling room (where the filling area is located), a vial-finishing room, and a service room behind the filling room. An operator supplies vials to the filling line from the vial-loading room. Control and oversight of the filling line are done from the same room. This design feature makes it possible to minimize the presence of operators inside the filling room. Operator tasks inside the filling room are limited to preparing for vial filling (set-up of a nonviable particulates monitoring system and sterilized filling equipment including tubing, filling nozzles, containers, and so on), troubleshooting, and environmental control for viable particulates. This equipment is designed to operate in an at-rest occupancy state: At rest is when the equipment is installed and operating, but no operating personnel are present (4). Because the containers used in a BVT aseptic filling process include a preassembled capsule stopper set (Figure 1), the stopper feeding system in conventional filling rooms is eliminated, so operators need not enter the filling room to load stoppers into a feeding system. There are fewer particles (because any feeding device generates particles), and the filling room is smaller. In fact, the filling area (blue area in Figure 4) is a tiny space protected with a horizontal laminar flow where the following steps take place (Figure 5): A vial arrives with its protector and capsule stopper set already mounted (step 1); the protector is discarded (step 2); the vial moves into filling position (step 3); the vial is unstoppered (step 4); a filling nozzle fills the vial (step 5); and the vial is fully stoppered (step 6). Distinctive features of such a small filling area are its size, horizontal laminar flow, proximity of the vials being filled to a HEPA filter, location of equipment within the filling area, vial handling, and restricted access. The height and the length of the filling area are of the same dimensions as the HEPA filter of the laminar flow hood. A horizontal air flow reduces the risk of particulates entering vials. Proximity of the vials to the HEPA filter (150 mm) reduces the potential for contamination of air flow that reaches each vial. Because of the location of equipment within the filling area, moveable parts are placed downwind of the filling point and are carefully designed to maintain the characteristics of laminar air flow. A device located outside the filling room pushes vials into the filling area, which eliminates belts, chains, and similar conveying systems that can be difficult to clean. A sensor system detects the proper positioning of each vial under a filling nozzle. And safety barriers are installed in the filling area to protect the sterility of the process. If the light barrier detects a breach, the filling machine automatically stops, and all vials are immediately stoppered. If the process is then restarted, the machine will run some cycles without filling to reduce the possibility of contamination from intrusions into the filling area. Once all units are filled and stoppered, the vials reach a vial discharge room, where they are crimped (yellow area in Figure 4) and laser marked. Maintenance is performed from outside the filling room. Because the filling line is integrated into the wall panel, the inside workings of the machine are accessible for maintenance from the service room, in accordance with GMP equipment design recommendations (4). The design of the filling machine installed

4 in the filling room is compatible with any filling system: piston pump, diaphragm pump, time-pressure, weight control, disposable filling, peristaltic pump, and so on. Selection of the most suitable system depends on accuracy (the more expensive the product, the greater the necessary accuracy), volume adjustment (fixed volumes or variable volumes, depending on product activity), amount of liquid to be filled (from small to large volumes), filling time, batch size, and so on. VALIDATION AND PRODUCTION EXPERIENCES Extensive validation work has been carried out to test the protective qualities of physical barriers on sterile containers and stoppers in preventing contamination. Two studies tested the effectiveness of sterile containers with a physical barrier used in a BVT aseptic filling process (Figure 1): exposure to different microbial environments and an airborne microbial challenge. The first study compared sterile open vials and sterile containers with a physical barrier containing sterile culture medium (aseptically filled) Figure 4: A BVT filling room and its filling area when exposed to different environments (5). Specifically, those were grade A (class 100), grade B (class 1,000), and nonfiltered air for a period of seven days. Results of this study demonstrate that no single sterile container with a physical barrier was found to have microbial contamination after seven days exposure to any environment. Every sterile open vial was found to have microbial contamination in the case of nonfiltered air, and 1.4% of such vials were found to have microbial contamination after seven days exposure to both grade A and grade B environments. The second study presented an airborne microbial challenge of sterile containers with a physical barrier containing aspetically filled, sterile culture medium (6). A microbial suspension of bacillus bacteria (Bacillus atrophaeus) was aerosolized over the containers (inside a sealed chamber) at a final concentration of the maximum microbial level accepted for a grade D (class 100,000) area. After 60 minutes of exposure, the containers were fully stoppered, crimped, and incubated for 14 days at C. Results of this study show that in both concentration cases, not a single container with a physical barrier had microbial contamination. The results of both studies demonstrate that the labyrinthine seal created by a vial and its physical barrier increases the safety against microbes of aseptically filled containers. And if bacteria-carrying particles in room air are large, and gravitational settling is the most important way they are deposited (7), then it can be asserted that containers with a physical barrier help minimize the risk of vial contamination because potential microbe-carrying particulates should not be able to overcome the labyrinthine seal against gravity. Taking into account that personnel are the primary source of bacterial contamination in an aseptic cleanroom, the two key factors that increase the confidence of sterility of filled units in a BVT process are the at-rest occupancy state of the filling room and the physical barrier of the container. This process is used for the manufacture of injectable products derived from human plasma approved by the US FDA and European authorities. BVT was developed over the course of the past 20 years, during which time improvements were added and the latest technology was integrated (e.g., filling techniques, microbial control, and machine automation). Media fill simulations have been performed extensively following BVT procedures and practices at existing production facilities. Since 2002, our company has filled more than 350,000 vials with media using this technology, and no revalidation was necessary for any batch. ADVANTAGES AND DISADVANTAGES Advantages: As described above, BVT offers four advantages over conventional aseptic filling processes: particulate and microbial safety, environmental control, traceability of filling operations, and installation cost and size. 52 BioProcess International DECEMBER 2007

5 Particulates and Microbial Safety: Vials are kept closed, though not hermetically, because of the labyrinthine seal that exists between washing and filling. So the time each sterilised unit is exposed to the environment is minimized. No capsule- or stopper-feeding equipment (both sources of particles) is needed within the filling room. Horizontal laminar flow reduces potential risk of particulate entry into the vials. Vials stay very near the laminar flow during filling. Equipment inside the filling area is placed downwind of the filling point and was carefully designed to prevent air disturbances. There is no need for an operator to work inside the filling room, which limits personnel intervention in critical areas (e.g., only for troubleshooting and environmental control). Maintenance is performed from outside the filling room because the filling line is embedded in the wall. Environmental Control: A small filling area facilitates monitoring of both viable and nonviable particulates. Its integrity can be better guaranteed than with a larger area. Traceability of Filling Operations: Video recording and laser marking makes the dosing process trackable. Cost and Size: This technology is less machine-intensive than convential aseptic processing modes that use depyrogenation tunnels rather than an autoclave. The overall size of facility required for BVT filling is much smaller than for conventional filling. Therefore, it will be easier to maintain and the process more readily validated. Disadvantages: Two disadvantages make BVT aseptic processing most suitable for small to medium batch sizes (20 11,000 vials). Most biomanufacturing processes operate in batch mode. Use of an autoclave for sterilization/depyrogenation of containers (with intensive prewashing of the vials) limits BVT aseptic processing to batch production only (continuous production). Autoclaves must be designed according to an expected maximum batch size. To minimize the time during which vials are open and exposed to the environment, unstoppering, filling, and stoppering must be performed sequentially (sections A- A, B-B, and C-C in Figure 4). This means that unstoppering a vial for filling and stoppering it for sealing increases the overall cycle time. Therefore, throughput of the machinery designed for BVT aseptic filling will be slower than for conventional filling lines. FURTHER DEVELOPMENTS Experimental studies and tests are being carried out to integrate the protector element into the capsule design. As described above, containers used in a BVT aseptic filling process are each made up of a protector, a capsule stopper set, and a vial. Currently the capsules are standard, and vials are crimped by means of standard machinery. But a plastic capsule that clips to a vial has been designed (8). This plastic capsule is designed to be long enough to play the role of a protector forming the labyrinthine seal. The three main advantages of such a protector capsule will be reduction in the number of components being handled, substitution of the crimping machine with a simple press that clips the capsule stopper set into each vial (eliminate particles generated during crimping), and complete sealing vials in front of a class 100 horizontal laminar flow immediately after filling. With that adaptaion, BVT is also applicable to the sterile filling of freeze-dried products. After a vial is filled with a liquid formulation, a protector capsule (including the stopper) partially stoppers the vial, allowing the lyophilization process to proceed. The freeze-dryer shelves then clip the protector capsule, securely closing the vials. Use of the protector capsule thus eliminates the need for crimping found in a conventional manufacturing process. These developments meet the requirements stated in clause 93 of a recently proposed revision to Annex 1 of the EC guide to good manufacturing practice: The container Figure 5: Step-by-step filling process inside the filling area

6 closure system for aseptically filled vials is not fully integral until the aluminium cap has been crimped into place. Vials should be maintained in a Grade A environment until the cap has been crimped (9). There is also a BVT specifically adapted for aseptic filling of extremely small batch sizes, such as for personalized medicines. In such cases, drug product losses are minimized because of the filling system s full drainability. Furthermore, the system can be mounted and installed in a modular cleanroom that is delivered and prevalidated before factory acceptance testing. A PROVEN ALTERNATIVE Barrier vial technology is an aseptic processing approach for high valueadded pharmaceutical products such as biotech medicines, plasma derivatives, and others that are not stable enough to undergo final product sterilization by heat. BVT is applicable both to liquid and freeze-dried products. Although it is in some ways similar to conventional aseptic processing (e.g., vial washing process, integration of any filling systems), BVT increases microbial safety for aseptically prepared products and maximizes the exclusion of particulates from all phases of aseptic processing. It offers other advantages such as environmental control, traceability, anticounterfeiting, and lowered facility costs when compared to conventional procedures. BVT has been used for the past 20 years in a plasma-derivatives factory approved by US and European authorities for the production of medicines. ACKNOWLEDGMENTS The authors thank Dr. Victor Grifols (inventor of the barrier vial technology) for his comments and suggestions during preparation of this paper as well as Instituto Grifols for information provided on the validation of this technology. REFERENCES 1 Grifols, Victor. Method for the Sterile Dosing of Vials. US patent 6,832,463: January CBER/CDER/ORA.Guidance for Industry: Sterile Drug Products Produced By Aseptic Processing Current Good Manufacturing Practice. US Food and Drug Administration: Rockville, MD, September 2004; 17; fnl.htm. 3 ISPE. Sterile Manufacturing Facilities. Pharmaceutical Engineering Guides for New and Renovated Facilities, ISPE Baseline Pharmaceutical Engineering Guide, First Edition, Vol. 3. ISPE: Tampa, FL, January 1999; Ad Hoc GMP Inspections Services Group (European Commission). Manufacture of Sterile Medicinal Products: Medicinal Products for Human and Veterinary Use. EU Guidelines to Good Manufacturing Practice, Vol. 4, Annex 1, clause 3, clause 33, May Evaluation of the Contamination of Sterile Vials to Different Exposure Conditions. IG_ITEC _ING, internal report. Insituto Grifols: Barcelona, Spain, Demonstrating the Effectiveness of Physical Barriers in Sterility Assurance of Vials Subjected to Airborne Microbial Challenge Test. IG_ITEC _ING, internal report. Instituto Grifols: Barcelona, Spain, Whyte W. Sterility Assurance and Models for the Assessing Airborne Bacterial Contamination. J. Parenteral Sci. Technol. 40(5) 1986: Grifols Company. Stopper for Flasks of Sterile Products and Use of Said Stopper in Sterile Measured Filling. US Patent Application US11/759,578: June Clause 93 of GMP Annex 1: Proposals for Amendment to the Environmental Classification Table for Particles and Associated Text, Amendment to Section 42 Concerning Acceptance Criteria for Media Simulations, Amendment to Section 52 Concerning Bioburden Monitoring, and Additional Guidance in Section 88 on the Sealing of Vials. European Medicines Agency (Inspections), September 2005: 4. Corresponding author Diego López- Alvarez is applied engineering department manager at Grifols Engineering, Can Guasch 2, PI Levante, Parets del Valles, Barcelona (Spain); , fax ; diego.lopez@grifols.com, Sergi Roura is general manager of Grifols Engineering, and J.A. Garcia is vice president of manufacturing at Grifols Biologicals. 54 BioProcess International DECEMBER 2007