Qualification Results of a New System for Rapid Transfer of Sterile Liquid through a Containment Wall

The transfer of sterile liquid is an operation performed frequently during the development of sterile-liquid drugs such as injectables or ophthalmic drops. To achieve such transfers, operators should set up a connection after preparing the sterile liquid. Several types of transfer can be performed, each requiring a validated technology to ensure the sterility-assurance level.

Transfer between two containers usually is achieved with a valve-to-valve connection, followed by steam sterilization of the space between the two valves. This procedure is well established, mostly in the context of large stainless steel equipment, and takes at least two hours, including a cooling down period. In addition, it requires clean steam, the evacuation of condensates, and a system of recording the sensed parameters.

This technique is not especially attractive for applications that involve disposable equipment or more-frequent connections of multiple, smaller containers. Blood banks typically perform these types of transfers. In the early 1980s, the technique of flame-sterilizing the tubing ends under laminar flow was replaced by hot-plate welding (also called "tube fusion"). This technique is suitable for small-diameter thermoplastic tubing made of polyvinyl chloride, thermoplastic elastomer, or other materials.

Disposable connection devices have been introduced for applications requiring connections between two tubes. Kleenpak from Pall (East Hills, NY), DAC from BioQuate (Clearwater, FL), and Lynx S2S from Millipore (Billerica, MA) are presterilized disposable systems that reportedly are safe for low-classification or unclassified environments.

The addition of barriers and isolators around critical aseptic processes created a new environment for aseptic transfer. In such systems, it clearly is preferable to leave containers such as bulk containers outside the protected environment. Sterile liquid must then cross the wall through a connection mechanism. It is quite difficult to introduce the container in the protected environment because the container's exterior cannot be thoroughly sterilized.

The first transfer system also originated in the context of large, infrequent transfers involving the well known rapid-transfer port (RTP) transfer container from La Calhène (Vendôme, France). It consists of steam-sterilizing tubing in a stainless steel transfer container docked to the barrier wall, unfolding the tubing, and introducing it into the barrier or isolator. In some cases, it is possible to sterilize the preassembled system (e.g., the RTP container and the empty tank), but in many cases, the classic valve-to-valve steam sterilization is still necessary.

Because of the increased use of flexible pouches, Stedim (Aubagne, France) introduced its rapid aseptic fluid transfer (RAFT) system, which connects to pouches using a Biosafe port. The stainless steel container described previously is replaced by a plastic bag equipped with a disposable flange that is docked to an RTP port. The disposable part is attached beforehand to the flexible pouch and gamma sterilized with it as a closed system. Unfortunately, the docking system does not allow multiple uses because reclosing and reopening is not feasible unless multiple flanges are attached to the bag.

This article describes a new approach for aseptic fluid transfer: the Sartorius Aseptic Rapid Transfer (SART) connection technology. The system incorporates a SART port and a disposable Gammasart aseptic transfer device (ATD) connector and was developed to provide a high-quality aseptic connection and simplify passage through a wall. The authors will discuss SART's product-qualification results to show that this technology and its various components meet the stringent pharmacopeial product-qualification requirements.

Principle of the technology

The SART technology was developed for the aseptic transfer of liquid through a barrier or any separating wall. The system has the following features:

• a port of minimal size that is fixed on the separation (in many isolators, wall space is restricted);

• an RTP system comprising four matching "V-shaped profiles," two of which have seals at the tip. The four components' small size enables accurate matching of the shaped profiles.

• an incoming-liquid tube that does not rotate or twist during docking in the port;

• a mechanical-interlock system that prevents accidental opening of the port when the connector is absent;

• a disposable connector to be attached to the external container's tubing. The connector should be small to accommodate small containers.

• rigid plastic not subject to pinholes that provides sterility protection for the connecting inner tubing;

• a leak-test limit that meets standards comparable with those in place for the closure integrity of the attached container. All connectors are tested individually during the manufacturing process.

• a maximum of five reclosing and reopening cycles to withstand the partial emptying of a container and to provide flexibility if operation is interrupted;

• compatibility with rigid liquid containers (e.g., stainless steel, glass, plastic) and flexible container (e.g., pouches);

• simplicity and low cost that enable multiple volumes or a small volume of liquid to be transferred.

The Gammasart ATD connector consists of two parts: the connector body that contains the rigid tubing and the connector cover (see Figure 1). To open the docked connector, an operator locks the cover into the SART port and removes it by unscrewing it. Thus, the rigid tubing is released into the aseptic area.

Figure 1

Both the connector body and the connector cover are made of polybutylene terephthalate and molded in a classified environment. A seal made of Santoprene (Advanced Elastomer System, Newport, UK) thermoplastic elastomer is overmolded on the connector cover. This seal ensures the quality of the system's closure integrity.

The principle of the SART connection is based on the alpha-beta concept of four V-shaped profiles that are in contact at the tips. This alpha-beta concept initially was developed for the nuclear industry in the 1960s. The concept has been approved by pharmaceutical industry authorities and used widely since La Calhène introduced it in the 1970s. As illustrated in Figure 2, the four V-shaped profiles of the SART system are the connector body, the connector cover, the internal part of the port, and the seal of the port. Two of them are rigid (the connector body is made of polybutylene terephthalate, and the internal port is made of stainless steel) and two are flexible (the connector-cover seal is made of Santoprene, and the external port seal is made of silicone).

Figure 2

The connector device must be subjected to at least 25 kGy of gamma irradiation to ensure its sterility. The device was validated at 45 kGy to ensure that the entire radiation range was validated. The connector should be irradiated alone if it will be attached to a nonirradiated container such as a stainless steel vessel. If it will be used with a stainless steel vessel, the connector can be sterilized by autoclaving. High- and low-pressure cycles should be used to push water vapor into the connector and dry it. Preliminary tests performed with 10⁶ biological indicators located in the assembled connector have shown total kill. If the connector will be used with containers such as plastic pouches, it should be irradiated after assembly.

The connection process includes the following steps (see Figure 3):

• The Gammasart ATD disposable connector is introduced into the port and secured with two clamping devices on the external port (see Figure 3a).

• The internal port's clamping system firmly holds the connector cover inside. The objective is to contain all the connector cover's exposed outer parts inside the port (see Figure 3b). The internal port is sanitized in a clean environment in parallel with the rest of the main equipment (e.g., with a vaporized hydrogen peroxide cycle).

• The port is opened by rotating the internal port (see Figure 3c).

• The tubing (e.g., silicone) is placed in the contained area on the connector's sterile tubing (see Figure 3d).

Figure 3

The connector can be closed and reused as many as five times within one week. This feature is useful for multiple transfers (e.g., a single bag containing five volumes of a formulation component to be transferred in five different batches) or in the exceptional cases of major operational troubles. For example, a major breakdown on a liquid-filling line would risk the destruction of connected bulk material. The Gammasart ATD connector safely allows the bulk to be removed from the line, stored, and reused within a few days. When the connector cover is screwed an additional 9 ° beyond its initial position, the seal is recompressed, and good closure integrity is maintained.

Application of Gammasart ATD

The connector can be used to cross any type of separation between two areas with different containments. The most common application in the pharmaceutical industry is the transfer of formulated bulk product to the aseptic filling line. The transfer may be from a cleanroom to a protected environment (e.g., an isolator or restricted-access barrier system [RABS]) or from a corridor to the class A–ISO 5 cleanroom.

Other possible applications include transfer during formulation, which can be accomplished in two ways:

• by transfering the different components to the formulation container;

• by transfering the formulated bulk product to the different storage units.

In the latter application, Gammasart ATD will be used in a reverse direction. The flow will move from inside the contained area to the sterile container.

Because the connector resists both gamma-irradiation and steam sterilization, various kinds of containers may be selected. Both sterilization procedures can be combined. Therefore, two possibilities for the sterilization of the container–connector assembly can be envisaged:

• In containers that can be sterilized by gamma irradiation (e.g., flexible pouches), the connector should be preassembled, and the full assembly should be irradiated.

• For containers that can only be steam-sterilized (e.g., stainless steel containers), the preirradiated connector should be assembled before steam sterilization.

Qualification of the Gammasart ATD connector

The connector was validated completely (see Table I). The tests were conducted on connectors subjected to 45 kGy of irradiation and a cycle of steam sterilization.

Table I: Overview of tests performed on connector and results obtained.

Leak test. The leak test is critical to ensure that the seal effectively preserves the sterile environment of the Gammasart ATD connector and to eliminate the risk of viable-particle penetration inside the connector during storage. The leak limit was fixed at 0.9 cm³ /min at 350 mbar or 35,000 Pa, corresponding to the closure integrity of the attached container.

Leak test after multiple opening–closure cycles. This test was performed to validate the fact that a connector can be opened and reclosed as many as five times within one week. The relatively short period of time results from the fact that the connector seal is overcompressed upon its first reclosure to obtain new closure integrity. Nevertheless, because over-compression can occur only once, the closure integrity cannot be maintained for a long time period thereafter, especially after subsequent openings.

Aging conditions. The connector was tested with various aging times to verify that the connector specifications such as closure integrity are maintained. The various aging periods were as follows:

• one month aging at room temperature;

• accelerated aging for 74 days at 60 °C, corresponding to roughly two years of aging in normal conditions;

• one year of aging at room temperature.

Low temperature. The connectors were stored at –20 °C and –60 °C for 15 days before use. After being unfrozen, the connectors showed a behavior similar to that of connectors that were not frozen.

Fitting and traction test. This tested the ease of installing the tubing when the connector was open and verified the connection's resistance to rapid flow.

Burst-pressure test. The connector's resistance to high pressure was tested to eliminate the risk of operator injury if the connector accidentally came unscrewed after pressure increases occurred inside the container attached to the connector (e.g., in the case of liquid transfer inside a container with a defective vent filter). The test was conducted at 6.5 bar for 30 s. Roughly half of the connectors showed seal deformation, but none of them became disassembled.

Particle test. Because the pharmacopeia allows the presence of a limited amount of particle from the container and filling process, particle presence was recorded in various volumes collected over time. The objective was not to exceed a count of 600 particles bigger than 10 μm and 60 particles bigger than 25 μm per 50-mL volume. These data correspond to 10% of the pharmacopoeia limits for a single injection, independent of volume.

USP cytotoxicity and biocompatibility. These tests confirmed that the polybutylene terephthalate material meets the most stringent safety criteria (USP class VI). No sign of toxicity was recorded on the cell or when the material was administered to animals using three different methods.

Extractable profiles. Connector bodies were placed in a worst case-situation. They were immersed in water for injection for 24 h at 80 °C or in ethanol for 24 h at 50 °C. Analyses showed a release of material that always was less than 50 μg/connector and usually less than a few μg/connector. To extrapolate the amount of release from the internal tubing surface, the data must be divided by a factor of 10.

Endotoxin detection. Preliminary tests showed an undetectable level of endotoxin inside the connector tubing where the liquid passes through. This test will be repeated when the final connector packaging is available.

Media-transfer simulation. Media-fill simulations were performed in a workshop using closed-vial technology to validate the quality of the connection. Three lots of ~6300 vials were filled in the assembly workshop (i.e., a nonclassified area). Two connectors were used for each lot to create a circulation loop. Seven, 1-m³ samples of air were collected inside the RABS to record the presence of colony-forming units (see Table II). The recorded samples show no presence of contaminant in the vial or in the environment.

Table II: Media fill results.

Conclusions

The SART connection technology performs aseptic transfers through any type of separation between areas with different containments. The Gammasart ATD connector provides several advantages for users. The major advantage is the robustness of the connection system that prevents contamination of the area and provides the highest sterility assurance level. These results are achieved through the use of the precise alpha-beta system that has been used extensively for several models of connections (such as the RTP container or beta-bag) to bring autoclaved material inside the protected environment.

The second key advantage is the technology's ease of use, which includes simple manipulation and a setup time of less than one minute. In addition, the container is kept outside the critical area. Ease of use combined with security features such as interlock prevents operator mistakes and, therefore, reduces the risk of contaminating the transferred product.

The third advantage is the possibility of reversing the connection in the event of problems during the process. Most systems do not allow reversing the connection and saving the transferred product. This characteristic creates the potential risk of product loss. It also may force operators to fix problems in suboptimal conditions, leading to an increased contamination risk.

Finally, the solution is inexpensive and flexible, allowing users to adapt it to various situations.

The connector's qualification plan confirmed that the materials used, the port design, and the connector device meet the most stringent criteria. Therefore, the SART connection system is a suitable candidate for multiple-transfer applications in the pharmaceutical industry.

Acknowledgments

The authors would like to thank Thomas Paust, Paul Priebe, and Patrick Balériaux for their recommendations on this article. Aseptic Technologies benefits from grants from the Walloon Region and from the Agence Wallone à Exportation.

Benoît Verjans* is the commercial director, Jacques Thilly is the technology director, and Christian Vandecasserie is a consultant at Aseptic Technologies, tel. +32 81 409 417, fax +32 81 409 411, benoit.verjans@aseptictech.comHartmut Hennig is the head of new technologies in the quality assurance and research and development departments at Sartorius. Patrick Evrard is the manager of TS biotech devices at GSK Biologicals.

*To whom all correspondence should be addressed.

Submitted: Feb. 18, 2007. Accepted: Mar. 6, 2007.

Keywords: aseptic transfer, barrier–isolator, containment, sterile connections