Single-use vessels drastically reduce requirements for cleaning and validation compared to stainless steel or glass tools, which saves a great deal of time and resources, such as water. The vessel arrives clean and validated and stays that way until it is plumbed into the process train. As long as the vessel has no holes, sterility is assured. The downside of single-use vessels, however, is the possibility of defects in the plastic vessel (i.e., bag) or its seams.
As the biopharmaceutical industry has become more confident in using single-use technologies, the scale--and the value--of what is contained in single-use biovessels has grown. It is not uncommon to see single-use vessels on the market that accommodate up to 2000 L of product. In these cases, the value of what is in the vessel could be more than hundreds of thousands or even millions of dollars. In these instances, if a defect were to be present in the bag, the potential financial impact would be enormous, regardless of whether the vessel is being used for storage or as a bioreactor.
The rapid uptake of single-use vessels for use in bioprocessing applications has made assuring integrity that much more crucial. Although major failures in the seams or large punctures in a vessel should be visible to the naked eye, the real problem lies in the potential presence of microscopic holes. These smaller imperfections are often undetected and can cause not only leaks, but also the ingress of microbial or other contaminants into the vessel, thus ruining the entire batch.
It is crucial that each vessel is tested prior to use to assure that there are no holes or leaks in either the vessel’s walls, seals, or--importantly--the joints and seals between the valves and tubings that enter and leave the vessel. These joints are the most vulnerable points at which leaks can occur.
Historical integrity test methods
For many years, the only nondestructive technique available for detecting leaks was the pressure-decay method. To conduct this test, the vessel is filled with air to a predetermined pressure and left to stabilize for a set amount of time. The pressure is then remeasured; a drop in pressure indicates that some of the air has escaped from the vessel. The drop in pressure and the time elapsed can be used to calculate the total size of any defect(s) present in the vessel through which the air escaped. Typically, holes that are 250 µm (for a 200-L vessel) or 500 µm (for a 1000-L vessel) can be identified in this way.
Clearly, this level of accuracy is not sufficient to guarantee sterility in a vessel being used in bioprocessing applications. ATMI LifeSciences conducted studies to find a reasonable cut-off point for the bags used as bioprocessing vessels. The studies began with guidance from the food sector, where it has been shown that microbes can pass through holes as small as 15 µm. Using 15 µm as a marker, ATMI coordinated independent, aerosol microbial-challenge laboratory studies. These studies confirmed that microbes are unlikely to penetrate sterile vessels with hydrophobic film surfaces through any defects of 12 µm or smaller; microbes proved to be unable to penetrate a vessel defect of 10 µm or smaller (1).
A new solution was required to detect defects as small as 10 µm. ATMI developed a solution using helium tracer gas (HIT, ATMI). Unlike the pressure-decay method that measures pressure loss in a vessel, this method measures the amount of tracer gas leaking through a defect. The amount of leaking gas can be correlated to defect size. Although helium was well established in leak-testing protocols in the automotive, aerospace, and vacuum industries, ATMI’s method was the first use of helium in integrity testing of flexible vessels used in the bioprocessing industry.
Figure 1shows the ATMI HIT system.
To carry out HIT testing, the vessel is placed inside a well-sealed, rigid chamber and connected to a helium inlet valve. The seals ensure no air or other gas can enter or leave while the test is in progress. All the air is then pulled from the chamber with a vacuum until there is a negligible amount of helium in the chamber. A predetermined amount of helium is injected into the vessel. If there are no defects in the vessel, the helium will remain inside it. If there are any defects (e.g., holes or splits), the vacuum will cause helium to escape from the vessel into the chamber. If this happens, the helium is detected using mass spectrometry; the amount of helium detected correlates precisely to defect size.
Defects down to 10 µm can be identified in this manner. In the rare case that a lower detection limit is required, the HIT equipment can be designed to meet those requirements, although it would be more technically challenging to set up. For these types of studies, the user must account for the influence of the tiny amount of background helium in the air.
Detection limits of HIT, however, are not the only advantage over the constrained-plate method. In the real world, a single-use vessel is not just a vessel with a single inlet. It will have a number of different inlet ports, all attached to connectors and tubes, particularly if it is being used as a bioreactor instead of a product-storage container. These ports and connectors are the most vulnerable part of the vessel in terms of leaks, holes, and other defects. To run a constrained-plate pressure-decay test, however, all of these ports and inlets must be removed from the vessel before it can be placed between the plates. Therefore, not only does the constrained pressure-decay method offer lower sensitivity, it also omits testing of the ports and inlets that have shown to be most likely to fail. In contrast, the HIT method allows a fully assembled vessel to be tested. The entire vessel, including the tubing sets, is placed inside the test chamber. If end connectors have porous membranes on the tube ends, the outlets are blocked using a plug or sealing mechanism to allow testing of the joints between the tube and end connector.
In addition to being offered for the preshipping validation of vessels and manifolds manufactured by ATMI, HIT technology is now available to end-users for testing in their own facilities of single-use systems from any supplier. On-site testing enables processors to verify the integrity of vessels at the point-of-use, immediately prior to use, giving a further failsafe to ensure that expensive batches of biopharmaceutical products are not spoiled by microbial contamination or lost through a leak. HIT testing can currently be applied to vessels up to 200 L, with plans to extend this to 2000 L.
As the penetration of single-use systems in the marketplace continues to expand, the need to assure the integrity of those systems has become more important. Supply-chain security and product integrity are core components of biopharmaceutical manufacturing success. Innovations like the HIT platform are important to advancing the potential of the industry as a whole, regardless of single-use provider.
1. ATMI, Internal research report.