by Thomas P. Burns
It takes one hole to cause a sterility breach.
The extra effort to find or prevent it is justified.
With the increased use of isolators in manufacturing operations and sterility testing laboratories, there is an urgency to ensure integrity of these units and their components. While there has been an effort to improve the methods used to test the integrity of isolator canopies and gloves, there has been minimal noted work on the testing of half-suits and waste containers.
Current leak-testing methods
Historically, leak testing these two components has been inconvenient at best. The current standard methods for testing half-suits and waste containers are as follows:
 Figure 1: Inflation plate for half-suits. Note inlet valve on right with 'quick disconnect' connector. |
For half-suits, one suit is installed in the isolator upside down (head on the floor), then a bottle of ammonium hydroxide is opened inside the isolator. When the isolator is saturated with the ammonia vapor, the inverted suit is checked with a yellow 'ammonia test' cloth for leaks. The cloth changes color from yellow to green when exposed to ammonia vapor.
There are many problems with this method. The suit to be tested is in an inconvenient location; the suit is not completely extended; the inverted suit is difficult to purge of ammonia vapor; only one suit at a time can be tested; and the workstation is unavailable for cleaning while testing is being performed.
To test a second suit, the ammonia vapor must be purged from the isolator, the inverted suit placed into the correct position, the second suit inverted and the ammonia steps repeated. This process is time consuming, as it takes an hour or more to purge the ammonia from the isolator and the half-suit.
For waste containers, a bottle of ammonium hydroxide is opened inside the workstation isolator. When the isolator is saturated with ammonia vapor, a waste container is connected to the isolator through a sterile isolator door, or double port transfer entry (DPTE), and the waste container lid removed. The container is then checked with a yellow test cloth for leaks. The problems here are that the waste container is in an inconvenient location under the isolator; it is not completely extended; the container is difficult to purge of ammonia vapor; and the workstation is unavailable during leak testing.
Process improvement
The first attempt to improve these processes was to find a way to fully inflate the half-suits and the waste containers.
An inflation plate was created for the half-suits, which included an attachment ring similar to the one in the isolator and an inlet valve (see Figure 1). A half-suit is suspended by the shoulder hooks above the inflation plate, and then attached to the plate with a standard half-suit rubber band and clamp. Because leaks are frequently discovered near the base of the suit skirt, the suit should be clamped to the plate exposing as much of the skirt as possible. In the isolator, the suit is slid to the bottom of the clamping ring before attaching the rubber band and clamping band.
On the inflation plate, the suit is slid on, the rubber band attached, and the suit slid up to the top of the clamping ring before attaching the clamping band. An air hose connects the benchtop air inlet to the inflation plate (for ease of use, 'quick disconnects' are recommended for all connections). The air valve on the benchtop is opened (30-40 psi, 2-3 kg/cm2), and the valve opened on the inflation plate. The suit is fully inflated, but not overly stressed, and all valves are closed. This allows thorough visual inspection of the entire suit.
Originally, a pressure gauge was to be used to monitor pressure decay, but because the relative pressure inside the suit is quite small, the pressure was simply gauged by the height of the suit arms. Drooping arms indicated a leak in the suit.
Inflating the suits also facilitates cleaning. Before using the inflation plate, the suits were cleaned by laying them across a table or hanging them by the shoulder hooks and wiping them down with cloths. An inflated suit is much easier to clean and dry, as folds and creases are minimized. Using a mild soap solution to wash the suits can also reveal leaks because holes in the suit will cause bubbles as the soap solution is passed over them.
Deflating the suit is achieved by simply unhooking the clamping band.
To test waste containers, an air inlet connector was created using a sterilization inlet cap and a short piece of pressure hose (see Figure 2). This air inlet connector is attached to the sterilization inlet port of a clean, empty waste container. An air hose with 'quick disconnect' connectors is then used to attach the benchtop air inlet to the air inlet connector. The waste container is inflated, being careful not to overinflate. The fully extended waste container is now easy to visually inspect for holes.
The pressure decay in the waste container can be measured by watching the air inlet connector: If there is a hole in the container, the connector will droop.
To deflate the waste container, the air hose is disconnected from the air inlet connector.
Ammonia leak testing
Although utilizing these inflation methods for visual leak testing greatly increased the chances of finding holes, there was still a desire to find a way to ammonia test the half-suits and waste containers. The first step in the process was modification of a pressure can. The intent of this process is to transfer ammonium hydroxide vapor, not liquid, so the dip tube was removed. Next, 'quick disconnect' connections were added so the pressure can could be connected and disconnected easily from the hoses (see Figure 3).
A second air hose is also required, and it should also contain the 'quick disconnect' connections.
 Figure 2: Air inlet connector for waste containers. |
The biggest concern with ammonia testing the half-suits was being able to exhaust the ammonia so an analyst could climb into the suit and hook it back into the isolator. An attempt was made to exhaust the ammonia through another valve in the base of the inflation plate, but the suit did not deflate efficiently (this extra 'outlet' valve may be seen on the left side of the inflation plate in Figure 1). This problem was solved by cutting the fingers off a suit glove and, using a PVC pipe connector and hose clamps, attaching it to a piece of 2-inch (50 mm) general-use 'flat' rubber hose (see Figure 4). The other end of the rubber hose is attached to the isolator exhaust manifold via a valve.
 Figure 3: Pressure can with dip tube removed and ‘quick disconnect’ connections added. |
Additionally, to ensure that the ammonia vapor circulated throughout the suit, a piece of Tygon tubing was attached to the inside of the inflation plate inlet. A four-way tubing connector was used, allowing the insertion of one piece of tubing down the non-exhaust arm, one in the helmet and one in the bottom of the inflation plate.
To ammonia test a half-suit, a suit was suspended from the shoulder hooks above the inflation plate. A small piece of yellow test cloth was tied to the inside framework of the helmet (so the color change can be observed). One of the ends of the circulating tubing was secured in the helmet (it was tied to the helmet framework with the yellow test cloth), a second piece of tubing was placed into the non-exhaust arm of the suit, and the third piece of tubing was allowed to dangle in the bottom of the inflation plate. The suit was attached to the inflation plate as stated above to allow maximum exposure of the skirt. The 'other' glove was removed from the suit, and replaced with the exhaust glove/tubing.
The pressure can was placed into a fume hood and the lid removed. A small amount of fresh ammonium hydroxide (at room temperature) was poured into a small bottle. This container was placed into the pressure can, and the lid replaced. It should be noted that originally the ammonium hydroxide was poured directly into the pressure can, but the resulting post-leak-testing cleanup was difficult. Placing the liquid into a removable bottle made cleanup and disposal much easier. One hose connected the benchtop air inlet to the inlet side of the pressure can.
A second hose connected the outlet side of the pressure can to the inlet valve of the inflation plate. The exhaust manifold valve (where the glove/tubing is attached to the manifold) was opened. The benchtop air inlet and the inflation plate inlet were both opened. Air now circulated through the pressure can, past the ammonium hydroxide and entered the suit. When the yellow test cloth began to turn green, the exhaust manifold valve was closed so the pressure could build up in the suit. When the suit was at the desired inflation, the inflation plate valve was closed (the benchtop air inlet valve would likewise have accomplished the same effect). The inflated suit is shown in Figure 4. The suit was washed with a mild soap solution, rinsed and dried thoroughly. A yellow test cloth was used to inspect the suit for leaks, and holes were patched as necessary. Patching a leak is performed easily with the suit inflated, as the pressure inside the suit exerts resistance against the patch; the small amount of air coming out the hole usually does not interfere with the adhesion of the glue.
 Figure 5: Inflated waste container. Air inlet connector is on the left and the sterilization exhaust is on the right. |
Now, the ammonia vapor had to be cleared out of the suit. First, the exhaust manifold valve was opened to release the pressure from the suit. The benchtop air inlet valve was confirmed closed, and any residual pressure was relieved from the pressure can by using the pressure relief valve. The air line was disconnected from the benchtop inlet line, and the air line from the outlet of the pressure can was removed and attached to the benchtop air inlet. To contain any residual vapor, the free end of the air hose was attached to the outlet of the pressure can to make a closed loop.
Now the benchtop air valve and the inflation plate inlet valve were opened, and the exhaust manifold valve was confirmed to still be open. The air flow was adjusted at the benchtop air inlet (the inflation plate valve would accomplish the same task) to keep the suit approximately halfway inflated, and air was allowed to purge the suit until the test cloth in the helmet changed from dark green to light yellow. When this occurred (approximately 30-60 minutes), the air inlet valve was closed and the suit was allowed to deflate.
The exhaust manifold valve was closed, the glove/tubing was removed and the suit glove was replaced.
The half-suit was removed from the inflation plate, and the circulation tubing and the small piece of test cloth were removed. By holding the shoulder hooks, the suit was transferred to the isolator, and the suit was hung in the isolator (because of the residual ammonium hydroxide vapors, it is best not to enter the suit at this time). The half-suit air supply line was attached to the suit, and the air was turned on 100 percent. The air was permitted to circulate for at least 30 minutes before connecting the half-suit to the isolator attachment ring. By using this method, virtually no residual ammonia vapor has been found in the suit.
A similar method is employed in ammonia testing of waste containers. A clean waste container was obtained and connected to the sterilization outlet line. The sterilization outlet valve was opened. The air inlet connector was attached to the inlet to the waste container. The top plate of the waste container was removed, and a small piece of yellow test cloth was tied to the inlet tube near the top (so it could be seen through the plastic sleeve), and the plate replaced. The pressure can (with ammonium hydroxide and associated air lines) was prepared as mentioned above, and the air line from the outlet of the pressure can was attached to the waste container air inlet connector.
 Figure 6: Overview of ammonia leak testing of half-suits and waste containers. |
The benchtop air inlet valve was opened, and when the test cloth inside the waste container began to turn green, the sterilization outlet valve was closed. When the waste container reached the desired inflation, the benchtop air inlet valve was closed. But use caution. The pressure can contain some residual pressure and will continue to inflate the waste container for a few seconds. It may be advantageous to close the air line before complete inflation, then adjust as needed later. The inflated waste container is shown in Figure 5. A yellow test cloth was used to inspect the waste container for leaks, and holes were patched as necessary. Now, the ammonia vapor had to be cleared out of the waste container.
First, the sterilization exhaust valve was opened to release the pressure from the waste container. The benchtop air inlet valve was confirmed closed, and any residual pressure was relieved from the pressure can by using the pressure relief valve. The air line was disconnected from the benchtop inlet line, and the air line from the outlet of the pressure can was removed and attached to the benchtop air inlet. To contain any residual vapor, the free end of the air hose was attached to the outlet of the pressure can to make a closed loop.
Now the benchtop air valve was opened, and the sterilization outlet valve was confirmed to still be open. The air flow was adjusted at the benchtop to keep the waste container approximately halfway inflated, and air was allowed to purge the container until the test cloth in the sleeve changed from dark green to light yellow. When this occurred (approximately 30 minutes), the benchtop air inlet was closed and the waste container was allowed to deflate. The waste container lid was removed, the small piece of test cloth was removed, and the lid replaced. The sterilization outlet valve was closed, the inlet and outlet connections were removed from the waste container, and the waste container inlet and outlet caps replaced.
Summary
Half-suits and waste containers are critical components of isolators, and thorough leak testing has not always been easily performed. This method of leak testing half-suits and waste containers is a relatively quick, efficient way to find holes. See Figure 6 for the overview diagram.
Thomas P. Burns is the lead scientist of the Sterility Laboratory for Eli Lilly and Co. in Indianapolis. In more than 15 years at Lilly, he has been involved with various areas of parenteral product testing, including the chemical and biological laboratories, technical services and sterilization validation. Mr. Burns is a graduate of the University of Pittsburgh at Johnstown with a B.S. in Chemistry. He can be contacted at (317) 276-9207 or [email protected].
Reprinted with permission from ISPE's Pharmaceutical Engineering, January/February 2001, Volume 21, Number 1.