Lyophilizer Sterilization And Design Of Lyophilizers

Lyophilizer Sterilization And Design Of Lyophilizers

       The sterilization of the lyophilizer is one of the more frequently encountered problems noted during inspections. Some of the older lyophilizers cannot tolerate steam under pressure, and sterilization is marginal at best. These lyophilizers can only have their inside surfaces wiped with a chemical agent that may be a sterilant but usually has been found to be a sanitizing agent. Unfortunately, piping such as that for the administration of inert gas (usually nitrogen) and sterile air for backfill or vacuum break is often inaccessible to such surface "sterilization" or treatment. It would seem very difficult for a manufacturer to be able to demonstrate satisfactory validation of sterilization of a lyophilizer by chemical "treatment".

       Another method of sterilization that has been practiced is the use of gaseous ethylene oxide. As with any ethylene oxide treatment, humidification is necessary. Providing a method for introducing the sterile moisture with uniformity has been found to be difficult. 

       A manufacturer has been observed employing Water For Injection as a final wash or rinse of the lyophilizer. While the chamber was wet, it was then ethylene oxide gas sterilized. As discussed above, this may be satisfactory for the chamber but inadequate for associated plumbing. 

       Another problem associated with ethylene oxide is the residue. One manufacturer had a common ethylene oxide/nitrogen supply line to a number of lyophilizers connected in parallel to the system. Thus, there could be some ethylene oxide in the nitrogen supply line during the backfilling step. Obviously, this type of system is objectionable.

       A generally recognized acceptable method of sterilizing the lyophilizer is through the use of moist steam under pressure. Sterilization procedures should parallel that of an autoclave, and a typical system should include two independent temperature sensing systems. One would be used to control and record temperatures of the cycle as with sterilizers, and the other would be in the cold spot of the chamber. As with autoclaves, lyophilizers should have drains with atmospheric breaks to prevent back siphonage. 

       As discussed, there should also be provisions for sterilizing the inert gas or air and the supply lines. Some manufacturers have chosen to locate the sterilizing filters in a port of the chamber. The port is steam sterilized when the chamber is sterilized, and then the sterilizing filter, previously sterilized, is aseptically connected to the chamber. Some manufacturers have chosen to sterilize the filter and downstream piping to the chamber in place. Typical sterilization-in-place of filters may require steaming of both to obtain sufficient temperatures. In this type of system, there should be provisions for removing and/or draining condensate. The failure to sterilize nitrogen and air filters and the piping downstream leading into the chamber has been identified as a problem on a number of inspections. 

       Since these filters are used to sterilize inert gas and/or air, there should be some assurance of their integrity. Some inspections have disclosed a lack of integrity testing of the inert gas and/or air filter. The question is frequently asked how often should the vent filter be tested for integrity? As with many decisions made by manufacturers, there is a level of risk associated with the operation, process or system, which only the manufacturer can decide. If the sterilizing filter is found to pass the integrity test after several uses or batches, then one could claim its integrity for the previous batches. However, if it is only tested after several batches have been processed and if found to fail the integrity test, then one could question the sterility of all of the previous batches processed. In an effort to minimize this risk, some manufacturers have resorted to redundant filtration.

       For most cycles, stoppering occurs within the lyophilizer. Typically, the lyophilizer has some type of rod or rods (ram) which enter the immediate chamber at the time of stoppering. Once the rod enters the chamber, there is the potential for contamination of the chamber. However, since the vials are stoppered, there is no avenue for contamination of the vials in the chamber which are now stoppered. Generally, lyophilizers should be sterilized after each cycle because of the potential for contamination of the shelf support rods. Additionally, the physical act of removing vials and cleaning the chamber can increase levels of contamination.

       In some of the larger units, the shelves are collapsed after sterilization to facilitate loading. Obviously, the portions of the ram entering the chamber to collapse the shelves enters from a non-sterile area. Attempts to minimize contamination have included wiping the ram with a sanitizing agent prior to loading. Control aspects have included testing the ram for microbiological contamination, testing it for residues of hydraulic fluid, and testing the fluid for its bacteriostatic effectiveness. One lyophilizer fabricator has proposed developing a flexible "skirt" to cover the ram. 

       In addition to microbiological concerns with hydraulic fluid, there is also the concern with product contamination. 

       During steam sterilization of the chamber, there should be space between shelves that permit passage of free flowing steam. Some manufacturers have placed "spacers" between shelves to prevent their total collapse. Others have resorted to a two phase sterilization of the chamber. The initial phase provides for sterilization of the shelves when they are separated. The second phase provides for sterilization of the chamber and piston with the shelves collapsed.

batches 

processed. 

In 

an 

effort 

to 

minimize 

this 

risk, 

some 

manufacturers 

have 

resorted to redundant filtration.

       Typically, biological indicators are used in lyophilizers to validate the steam sterilization cycle. One manufacturer of a Biopharmaceutical product was found to have a positive biological indicator after sterilization at 121oC for 45 minutes. During the chamber sterilization, trays used to transport vials from the filling line to the chamber were also sterilized. The trays were sterilized in an inverted position on shelves in the chamber. It is believed that the positive biological indicator is the result of poor steam penetration under these trays. 

       The sterilization of condensers is also a major issue that warrants discussion. Most of the newer units provide for the capability of sterilization of the condenser along with the chamber, even if the condenser is external to the chamber. This provides a greater assurance of sterility, particularly in those situations in which there is some equipment malfunction and the vacuum in the chamber is deeper than in the condenser.

      Malfunctions that can occur, which would indicate that sterilization of the condenser is warranted, include vacuum pump breakdown, refrigeration system failures and the potential for contamination by the large valve between the condenser and chamber. This is particularly true for those units that have separate vacuum pumps for both the condenser and chamber. When there are problems with the systems in the lyophilizer, contamination could migrate from the condenser back to the chamber. It is recognized that the condenser is not able to be sterilized in many of the older units, and this represents a major problem, particularly in those cycles in which there is some equipment and/or operator failure.

      As referenced above, leakage during a lyophilization cycle can occur, and the door seal or gasket presents an avenue of entry for contaminants. For example, in an inspection, it was noted that during steam sterilization of a lyophilizer, steam was leaking from the unit. If steam could leak from a unit during sterilization, air could possibly enter the chamber during lyophilization.

      Some of the newer lyophilizers have double doors - one for loading and the other for unloading. The typical single door lyophilizer opens in the clean area only, and contamination between loads would be minimal. This clean area, previously discussed, represents a critical processing area for a product made by aseptic processing. In most units, only the piston raising/lowering shelves is the source of contamination. For a double door system unloading the lyophilizer in a non-sterile environment, other problems may occur. The non-sterile environment presents a direct avenue of contamination of the chamber when unloading, and door controls similar to double door sterilizers should be in place. 

       Obviously, the lyophilizer chamber is to be sterilized between batches because of the direct means of contamination. A problem which may be significant is that of leakage through the door seal. For the single door unit, leakage prior to stoppering around the door seal is not a major problem from a sterility concern, because single door units only open into sterile areas. However, leakage from a door gasket or seal from a non-sterile area would present a significant microbiological problem. In order to minimize the potential for contamination, it is recommended that the lyophilizers be unloaded in a clean room area to minimize contamination. For example, in an inspection of a new manufacturing facility, it was noted that the unloading area for double door units was a clean room, with the condenser located below the chamber on a lower level. 

       After steam sterilization, there is often some condensate remaining on the floor of the chamber. Some manufacturers remove this condensate through the drain line while the chamber is still pressurized after sterilization. Unfortunately, some manufacturers have allowed the chamber to come to and remain at atmospheric pressure with the drain line open. Thus, non-sterile air could contaminate the chamber through the drain line. Some manufacturers have attempted to dry the chamber by blowing sterile nitrogen gas through the chamber at a pressure above atmospheric pressure. 

      In an inspection of a biopharmaceutical drug product, a Pseudomonas problem probably attributed to condensate after sterilization was noted. On a routine surface sample taken from a chamber shelf after sterilization and processing, a high count of Pseudomonas sp. was obtained. After sterilization and cooling when the chamber door was opened, condensate routinely spilled onto the floor from the door. A surface sample taken from the floor below the door also revealed Pseudomonas sp. contamination. Since the company believed the condensate remained in the chamber after sterilization, they repiped the chamber drain and added a line to a water seal vacuum pump.