Quality is the word on everyone’s lips in the biopharmaceutical market. The outcry from end users (patients) regarding highly publicized contamination events has effected a great amount of transformation in the industry. New regulations have been — and will continue to be — brought to the market in an effort to effectively monitor and increase quality for the industry. With these new regulations, the structure of accountability has changed and drug manufacturers are now more accountable than ever to meet and exceed regulations, from sourcing of raw materials through to putting the product “on the shelf.”

This changing regulatory landscape means that every factor of the manufacturing process is under scrutiny, and as new technologies are introduced to advance the state of the art, they must also be validated to meet these upgraded standards. The application of single-use technologies in bioprocessing is no exception.

What was looked at just a decade ago as a promising-but-questionable offering is now becoming commonplace. The rise of the flexible facility offers a new avenue for manufacturers to combine the benefits of single-use bioprocessing technologies with traditional stainless steel and glass technologies to produce high quality products, while also controlling costs and eliminating the need for excessive validation and exceedingly large facilities, with their accompanying capital costs.

Though single-use technologies offer an exceptional solution for biopharma manufacturers, they also give rise to new considerations. After all, whether it is a bioreactor or a mixer, the core component of the machine is, at the end of the day, a plastic bag (a.k.a. a bioprocess vessel). The further downstream you get in the manufacturing process, the greater risk any type of failure presents. A failure at this point could conceivably run into the millions of dollars if a whole batch has to be thrown away due to bacterial contamination in the vessel. We shall provide an overview of aseptic validation for single-use technologies, including the type of testing available to the market and a new approach to ensuring aseptic validation of bioprocess vessels once they arrive at the customer’s facility for use.

Cause for Concern
It is a common misconception that the bag is the only component of a single-use technology that causes contamination concerns; each bag has many different devices that must be in sync for it to operate appropriately. For instance, there will be a closure device, tube-fitments, and connectors, all of which must be sealed to create integrity of the bag. Any time a closure device is missing, or a seal is incomplete or weak, it is a defect that creates a greater possibility of contamination.

A major culprit for defects is the shipping process. Shipping validation is a complicated art, but no matter how carefully a bag is packed and however effectively the packaging of the shipment itself is validated, once it is in the hands of the shipping company, a manufacturer has limited (if any) control of what happens along the way. If the vessel is dropped at any point, this might cause a T-fitting to break — a particular problem for bags with complicated manifolds that have many connection points. The shipping box might also be damaged during shipping, exposing the vessels to risk.

Extremes of temperature can affect the integrity of plastics. The polymers used in the silicone tubing, rigid plastic connectors and flexible film bag all expand and contract in response to temperature at different rates. If the shipment sits in the hold of a plane on a hot runway for some time, and then gets very cold at 40,000 feet during the flight, the temperature difference it experiences can easily exceed 100°C, offering a lot of potential for the joints between different types of plastic to split.

Problems can also occur within the customer’s own facility. It is all too easy to inadvertently overstress the joints or connections — perhaps too much lateral pressure is applied on a fitting during use, which causes it to break. Altering the process or the material out of which the fitting is manufactured can solve this; however, being able to verify integrity of the vessel on the customer’s end is important.

Testing for Integrity
Prior to any bioprocess vessel or other component leaving the manufacturer, it is subjected to a battery of quality checks in order to validate aseptic integrity. This can include visual inspections and statistical burst sampling, both of which do not provide a very high level of accuracy in application. One other, and more widely used method is pressure decay testing; this is often considered to be the industry standard for aseptic validation.

There are two ways to perform pressure decay testing: either through an inflation and hold test (also referred to as free-inflation), or through a constrained test. In the free-inflation test, the bag is filled with air to create pressure and is left to stabilize over time. The pressure in the bag will then be measured to see whether air is escaping or not and is accurate down to 500 µm to 100 µm, depending on the size of the vessel. The constrained test is the same approach, but in this case the vessel is placed and constrained between two plates to put extra pressure on the bag. Adding in constraint increases the testing accuracy to 20µm.

Drawbacks of Pressure Decay Testing
When looking at pressure decay testing, it is important to consider the rate of pressure decay testing a manufacturer completes. We perform pressure decay testing on 100% of bags, but not all manufacturers operate in this manner. Additionally, while pressure decay testing is useful, it cannot totally eliminate risk and brings a few drawbacks along with it.

One of the greatest drawbacks is that smaller bags are more sensitive to pressure decay testing than larger bags are, which means that defects are harder to isolate in the larger bags. For any given defect size, the amount of gas escaping will be nearly identical irrespective of the bag size. However, the amount of gas present in a bag during pressure decay testing is directly proportional to the bag’s rated volume. So, the proportion of gas escaping through a defect (in comparison to the gas in the bag) is much higher in smaller bags versus larger bags. This makes the signal for defects in smaller bags more noticeable as compared to larger bags for the same defect size.

Another drawback of pressure decay testing (in constrained application) is not being able to test the full manifold. When using this method, only the bag can be tested, and there is no opportunity to perform the testing with any of the tubing or fitments attached.

Imitating Real-Life Applications
Studies carried out within the food sector suggest that microbial contaminants can migrate through holes as small as 10µm in size, but can rarely migrate through anything smaller. We conducted further studies with aerosol testing to determine when microbial ingress happens, and at what level. The tests were designed to simulate real-life situations (i.e. a sneeze in the range of the bag) and to provide greater clarity about when bacterial ingress happens and how likely it is.

In the aerosol trials performed in 2009, the single-use manifold was subjected to a 10-minute exposure of contaminant in aerosol format. In these trials, the lowest point of microbial ingress was 15 µm.

A Solution with Integrity
In March of 2011, we began to commercially perform helium integrity testing (HIT), a new type of testing to validate single-use bags for the biopharma industry. The technology is able to isolate defects down to 10µm.

To execute the HIT process, a vessel is placed within a sealed container, connected to an inlet hole on the side of the container, and a vacuum is pulled to remove all air in it. A measured quantity of helium is then injected through the inlet. If there are any defects, the helium will escape through them into the container. Any escaped helium can be detected and quantified using mass spectrometry; the amount of helium detected correlates to the size of the defect.

In addition to being the only type of testing in the industry capable of finding defects at this level, HIT technology also has another large advantage. Instead of just being applicable to the bags, HIT technology allows testing to be carried out with all of the connectors and valves in place. This is extremely important because, as noted earlier, defects often occur at the joints of the connectors and valves. HIT technology can detect missing or half-closed connectors in a way that pressure decay testing cannot.

Focused on the Industry
HIT was initially offered as an add-on service for 2-D vessels, but market feedback made it clear that the majority of opportunities for defects arise after a bag leaves the manufacturer’s facility. We have also been investigating the licensing of HIT technology for use at biopharma manufacturing facilities.

Before it is up and running, the HIT system itself will be validated on site, with many different bag assemblies being tested,including some with deliberately introduced defects to ensure they are detected. Using this setup means that bags from any supplier can be tested and validated prior to use, so users are not tied to one supplier.

Technologies like HIT start new conversations in the industry and are helping to make strides toward reduction of risk for drug manufacturers and patients. Regardless of what type of bag is used, the bottom line is that aseptic validation is imperative to quality. The smaller the defect that can be detected, the higher the assurance of sterility maintenance for drug manufacturers.