In the manufacture of biologicals single-use disposable flexible vessels are commonly used as bioreactors, mixers and sterile fill stations. To guarantee the vessels are of the quality needed to ensure process integrity, they must be proven free from biological contamination and free of leaks that could be an ingress route for contaminants such as bacteria. The traditional pressure decay method has significant limitations, so ATMI has created a reliable and repeatable method involving pressurising the vessel with helium instead of air. By using this method, defects as small as 10µm can be easily detected.
Tiny holes in the vessel walls or seals can provide an ingress route for contaminants
Proving that disposable plastic vessels are leak-free has become a critical requirement for those involved in the manufacture of biologicals. Engineers at ATMI LifeSciences have developed a method using helium to improve the integrity testing for such vessels.
There has been a trend in the past few years for the biopharmaceutical manufacturing sector to use disposable plastic vessels in place of traditional steel and glass containers. Employing single-use vessels can offer technical, operational and economic advantages by reducing the risk of cross-contamination and eliminating the need for time-consuming and expensive cleaning and validation procedures.
Single-use flexible vessels were first used to store GMP materials, but now they are commonly used as bioreactors, mixers and even sterile fill stations. The use of these vessels for storage applications was rapidly accepted in the industry over the past decade, but they have really gained significant market penetration in biomanufacturing only in the past three years or so.
They are now available in a wide range of sizes, from five litres up to 2,000 litres. Many companies have switched over to exclusive use of disposable technologies throughout their biomanufacturing processes, while others run parallel applications using a combination of traditional and single-use technologies.
This increasing demand for disposable reactors has been accompanied by a growing requirement from users for some form of guarantee that the vessels are standardised, and also of the quality they need to ensure process integrity. The vessels themselves are manufactured from multilayered plastic, with the product contact layer normally being a low-density polyethylene. This is laminated to layers of other polymers to give specific performance characteristics, including an ethylene vinyl alcohol polymer, which minimises gas diffusion and provides a degree of flex-crack resistance, and polyamide, which prevents punctures.
All of these properties must be fully validated by the vessel supplier. It is vital that these materials are neither leachable nor extractable, as extraneous chemicals in the biomanufacturing process cannot be tolerated.
Proving that the vessel is free from biological contamination before the manufacturing process starts is fairly straightforward, but proving that the vessel has no leaks is far more difficult. Tiny holes in the walls or seals of the vessel can provide an ingress route for contaminants from the environment, such as bacteria, and the joints and seals between the vessel and any connected tubes and valves are particularly vulnerable. It is rare for the vessels to have only one inlet, and the joint vulnerability problem increases with the complexity of the vessels and the number of connectors attached to it.
The traditional technique for leak detection – the pressure decay method – has significant limitations. Using this method, the vessel is first filled with air to a predetermined pressure, sealed, and then left to stabilise for anywhere between 30 seconds and 10 minutes. The pressure is measured again, and if it has decreased this shows that some of the air has escaped. The size of the pressure drop correlates to the size of the defect, and it is possible to detect holes of about 250–500µm in this way.
However, larger vessels are more difficult to test accurately as the vessel will sag, affecting the rate of air loss through the defects. To overcome this limitation, a modification to the testing method was introduced, in which the vessel is placed between two fixed plates and then pressurised. Constraining the vessel like this reduces its ability to deform under the influence of gravity, providing more accurate results. With this method the defect detection limit reduces to around 100µm.
However, this method still does not always ensure the level of integrity testing that is required by biomanufacturing companies. The problem is that 100µm holes are still quite large when compared with the size of the contaminants and bacteria that could potentially penetrate holes as small as 15µm. Ideally the detection limit should be about 10µm to give confidence that no bacteria would be able to enter the vessels. Yet this is well below the detection limit of even the modified version of the pressure decay technique.
The overly high detection limit of the constrained plate pressure decay method is not its only drawback, however. To test a vessel in this way, any connectors, valves and tubes must be removed before the vessel is placed between the plates, and the holes left behind sealed off. But in the real world, these connectors, valves and tubes are the most vulnerable parts of the vessel – the seals around them are precisely where any defects are most likely to appear.
Removing them for testing significantly increases the likelihood of any defects that could affect the biomanufacturing process.
Sealing the fittings and tubing onto the vessel after the test is performed means that those areas are being missed, as only the vessel itself is tested, not the parts where defects are most common.
A more sensitive method is thus essential, and a solution to the problem comes in the form of helium integrity testing. The technique is already tried and tested in other sectors, notably the aerospace, automotive and vacuum industries, but testing single-use bioprocess vessels is a new application. ATMI has created a reliable and repeatable method to address this.
The empty vessel is placed inside a rigid, well-sealed container, and a vacuum pulled to remove all the air
Although it still involves pressurising the vessel, there are several important differences. Most importantly, it is filled with helium instead of air. This allows the escape of gas to be measured directly by detecting helium, rather than indirectly by looking for a pressure drop. As air normally contains only about 0.0005% helium, any helium that is detected must have escaped from the vessel.
To carry out the test, first the empty vessel is placed inside a rigid, well-sealed container, and a vacuum pulled to remove all the air. An inlet valve is then attached to the collapsed vessel, and helium injected. The vacuum inside the container will cause helium to be pulled out through any small defects in the vessel, and any helium that has escaped in this way is detected using a mass spectrometer. If there is no helium, then the vessel has no defects. If some helium is detected, the amount can be quantified using a mass spectrometer, which allows for correlation to the defect size.
By using this method, defects as small as 10µm can be easily detected, which gives confidence that bacteria and other contaminants will not be able to enter the vessel when it is being used in a manufacturing process. No other method is currently known to be able to detect defects at this low level. Even smaller defects can be detected using this method, but it takes much longer and the cost is higher as all background helium must first be removed from the air. In any case, the 10µm level is satisfactory as it is below the penetration limit for bacteria.
The problems that are often seen with traditional pressure decay testing methods are circumvented with the helium integrity testing method. As the testing is done within a sealed container, there is no requirement for the fitments and connectors on the vessel to be removed. The vessel, including all attachments, is simply put inside the container for testing; the only additional requirement is that any open tubes need to be sealed. Thus it is possible to test the integrity of the whole system – vessel and attachments.
As manufacturers of biologicals are becoming increasingly reliant on single-use vessels, their market penetration has drastically increased. It is therefore vital that the integrity and sterility of these vessels can be proved and maintained, and materials testing is part of that process. Helium integrity testing is an important development in the arsenal of techniques that can be used to ensure that a vessel is sterile before it is used – and will remain that way while in use.
Vishwas Pethe, Research Engineer
Richard Bhella, Global Product Manager, Single-Use Storage and Delivery
Alex Terentiev, US R&D Director