Sterile Does Not Necessarily Mean Clean

It is well established and indeed obvious that any medical device that punctures the skin or any medication that enters the bloodstream must be sterile. Sterilization procedures are fairly well established in the pharmaceutical and medical device industries and suitable testing is relatively straight-forward. What may not be as obvious is that these devices and products should also be free of foreign particles (i.e., clean). Control of unwanted particulate matter in products requires knowledge of potential sources of particles and processes that might generate particles to avoid potential problems. Identification of particulate matter is important in determining the root cause and to address safety concerns.

Sterilization is the process used to remove or kill any microbiological activity, such as fungi, bacteria, viruses, and spores, on surfaces or in solution. Steam sterilization and sterile filtration are most commonly used in the pharmaceutical industry. The sterile filtration process involves passing the product through a 0.2 µm filter, and has the added benefit of removing unwanted particulate that may be present. The filtration and filling processes are carried out in cleanroom facilities to further reduce the chance of introducing particles. The containers—vials or syringe cartridges—are cleaned prior to filling. These processes are usually well controlled and are very effective in reducing or eliminating unwanted particles in parenteral products.

However, even in the most well regulated manufacturing industries, particles may be introduced into containers or devices, or develop over time within the product. Sources of particles include environmental debris, such as fibers and fiber fragments. Processing equipment may generate foreign particles. Particles may also originate from the container or packaging itself, or from an interaction of the product with the packaging. Less commonly, sometimes the product itself will react and form particulate matter over time. These types of particles often manifest themselves during accelerated stability studies while undergoing drug development.

Parenteral containers are inspected for the presence of particles, either manually by trained inspectors, and/or using automated methods (the size range for visible particles has not been fully established but is often defined as greater than 100 µm). These samples are inspected in their end use containers, for example, vials or syringes. Appearance testing may also be conducted on aliquots drawn from bulk drug substances at time intervals as part of stability testing. If such sampling takes place in an uncontrolled environment (not in a clean room), or if containers used are not particle-free, particles from the environment may be introduced into the sample. Thus, it is crucial to use sample containers that are particle-free, not simply sterile. A sample with visible particles may contain colorless fibers and particulate from environmental exposure that are not readily visible until the sample is examined with a microscope.

Sub-visible particles are also a potential safety concern and there are defined limits for the amount of particles in products for injection. Parenteral products are tested for the presence of subvisible particles using the USP <788> method. Part I of the method outlines the light obscuration method to test for particles in the ≥ 10 µm and ≥ 25 µm size ranges. The second part of the USP <788> is a microscopic method that utilizes filtration of the product onto a gridded filter membrane and then particle counting using a microscope. The second part of the method is often used when high particle counts are obtained, or the product is not suitable for the light obscuration method.

Products with high failure rates are often submitted for particle identification to identify the root cause and prevent further problems. Forensic analysis or particle in drug (PID) identification requires proper isolation of particulate matter. Optimally, the particles should be isolated in a certified ISO/IEC 14644 Class 5 cleanroom environment with procedures and controls in place to avoid introducing environmental particles. Isolated particles should be characterized and prepared for chemical analysis using a stereomicroscope in the cleanroom.

Silicone lubricants are not particles, but they are used in pharmaceutical containers as a lubricant for vial stoppers and cartridge (syringe) plungers, and are also applied to the tops of the stoppers to aid in processing. Under normal conditions, the silicone used in the packaging does not present a problem. However, if higher amounts of silicone are present, it interferes with the light obscuration method and can result in high particle counts. The samples are then analyzed using the microscopic method; the oil is absorbed into the filter and no particles are observed. In these instances, it is often assumed that silicone is the cause of the high particle counts. The silicone can be confirmed by filtration of the product onto a different type of filter membrane, usually a polycarbonate filter membrane. The oil, if present, can be observed more easily and can be extracted from the membrane and analyzed using FTIR to confirm its identification as silicone. High amounts of silicone may also be detected visually; it appears as a haze in the solution. Silicone tends to react with protein-based drugs and forms amorphous stringy particulate that collapses onto the filter membrane. The amorphous residue is not counted as a particle using the USP <788> method and is not readily observed on the gridded filter membranes. Using proper filters and lighting methods, the protein/silicone residue can be isolated and confirmed using FTIR.

Particles can also be generated from the glass containers. Glass provides a relatively stable material for sterile packaging, but it is not an entirely inert material and can be subject to degradation, especially if the sterilization process is particularly severe. In severe cases, glass delamination flakes, or lamellae, appear in solution. These flakes are very thin; vials are often described as having a “twinkling” appearance when examined using a fiber optic light source. The flakes themselves are difficult to see on the filter membrane. If glass delamination is suspected, it is crucial to use the proper filter membrane and lighting to see the flakes. It is also important to inspect the inside surfaces of the vial to look for evidence of delamination. Glass delamination may be due to irregularities in the glass container, sterilization processing, or to interactions with the product. Processing of the vials and products may also lead to delamination. Some active ingredients may attack the glass.

By the time the glass flakes appear in solution, the problem is hard to fix and can result in product recalls and possible shortages of vital drugs. It is important to check for possible product interactions with glass during the early stages of new drug development. Vial manufacturers offer many different types of glass vials, and they can be tested for compatibility with the drug product as part of the stability study.

Particles can be generated during the use of the product. One fairly common particle type is generated when a drug product is administered as an infusion. The contents of the product vial are removed using a syringe with a hypodermic needle and then added to the infusion bag. The needle may remove a portion of the stopper rubber, which is then added to the bag along with the drug. The stopper coring particle is then observed in infusion bag as a potential contaminant, and the product is not administered to the patient. The coring particle can be isolated and analyzed to confirm that it was related to the packaging and is not an extraneous particle.

As mentioned above, vials should be cleaned prior to sterile filling, and these processes should be carried out in certified cleanroom environments. Remember, sterile is not the same as particle free. On rare occasions, fibers may be introduced during filling and observed during the inspection of the vial. The most common type of fibers observed are cotton and paper fibers, and fiber fragments (linters), but polyester fibers are observed, as well. Glass vials are commonly cleaned prior to use and then heated at high temperature (depyrogenation) to kill endotoxins. If fibers or plastic wrap are not removed from the vials prior to depyrogenation, they are charred and then may appear in the filled vials as charred organic material. The charring may prevent the identification of the original material, but the presence of charred material usually indicates that it was present prior to depyrogenation.

In summary, pharmaceutical products and devices that enter the blood stream must be sterile and particle free. Sterilization does not ensure that products are clean. Monitoring for particles is important and should be included in all phases of drug development and stability studies. Forensic analysis for identification of particles when they appear is necessary to determine the source (root cause), prevent future problems, and evaluate patient safety.