Aseptic Processing: A Primer

In this fifth part of a series of primers with training experts from the National Institute for Bioprocessing Research and Training (NIBRT), Operations Consultant Ray O'Connor discusses the basics of biopharmaceutical facility design and operation. NIBRT provides training, educational, and research solutions for the international bioprocessing industry in state-of-the-art facilities. Located in South Dublin, it is based on an innovative collaboration between University College Dublin, Trinity College Dublin, Dublin City University, and the Institute of Technology Sligo.

BioPharm: Why is aseptic processing crucial for injectables, such as vaccines and other biologic-based products?

O'Connor: Certain pharmaceutical products must be sterile as they're introduced to the patient by injection because this mode of drug delivery bypasses the body's natural defenses. Therefore, with biologics, there is an increased risk of infection being introduced to the patient. Sterile drug products can be manufactured using two techniques: terminal sterilization or true aseptic processing. Terminal sterilization usually involves heat or irradiation; however, quite a large proportion of vaccines and biological-based drugs can be destroyed by exposure to heat or irradiation, hence, the requirement to manufacture in an aseptic manner.

According to the FDA guidance for industry on sterilized drug products produced by aseptic processing issued in September 2004, in an aseptic process, the drug product and container/closure are subjected to sterilization methods separately, as appropriate, and then brought together. Because there is no process to sterilize the product in its final container, it is crucial the containers be filled and sealed in an extremely high-quality environment. Therefore, aseptic processing involves more variables than just terminal sterilization. Before aseptic assembly into a final product, the individual parts of the final product must be subjected to various sterilization processes.

Similarly, in the European Union, GMP guidelines state that the manufacture of sterile products is subject to special requirements in order to minimize the risk of microbial contamination and of particulate and pyrogen contaminant. So, as a result, much of the aseptic process depends on the scale of training and attitudes of the people involved.

It's particularly important that this type of manufacture strictly follow established and validated methods of preparation and procedure. Sole reliance for sterility or other quality aspects would not be placed on any terminal process or finished product testing. To summarize, aseptic processing is minimizing the risk of introducing any microbial contaminant into your product as you move it through the manufacturing process.

BioPharm: Aseptic processing and the term "fill/finish" are often used interchangeably throughout the industry. Are these two terms truly the same or are there unique steps for each?

O'Connor: Fill/finish is a discrete part of the manufacturing process. If you have an upstream or downstream process, that will be where you manufacture your active ingredient. Fill/finish is the filling and packaging part of this process. It comes after the product has been manufactured and is ready to be put into its final package container that the patient will see.

Aseptic processing refers to the various techniques that go into ensuring that the product is free of contaminants, thereby reducing the risk of infection to the patient. Aseptic processing, is the processing of drug components, drug product containers, and excipients in a manner that precludes microbial contamination of the final sealed product.

PRODUCT CONTAMINATION

BioPharm: What types of product contamination can occur during bioprocessing?

O'Connor: Contamination in the biopharmaceutical industry can have serious health effects on the patient, so it is crucial to monitor and avoid. The different types of contamination one might find include bacteria, which could cause an infection in someone who is already ill, thus making the actual condition worse; chemicals, which could cause poisoning or other effects on the patient; and physical contamination, which could be particulates that can cause serious problems, such as cuts, blockage, or even death in the patient. If work is being done in a multiproduct facility, cross-contamination from one product to another can occur as well.

With regard to bacteria, viable particles are of particular concern in the biopharmaceutical industry. If they enter a product, they will multiply rapidly (e.g., they can double in under 20 minutes in the right conditions). If bacteria get into the system, they can actually overpower the product being made, and you may end up losing the product.

Most bacterial contamination comes from human beings. Hence, it's vitally important that, when staff walk into a cleanroom or work in a cleanroom, they be appropriately garbed to ensure minimal exposure of skin to the environment.

Overall, there are five main routes of entry into the product of any type of contaminant. First is raw materials. All the raw materials used in the manufacturing of the product are potential sources of contamination. Quality systems associated with the supply and release of raw materials into the manufacturing processes are critical. A second source is the plant. Poorly sanitized equipment can lead to contamination. A third source is the environment. The cleanroom design, as described below, must be executed properly. Fourth is movement of personnel. It's important that people move in a controlled and deliberate fashion in a cleanroom. Erratic behavior can generate particles. A fifth source is gowning. People represent 80 to 90% of common contamination sources. Proper gowning behavior and training in aseptic technique and aseptic processing is vital.

EQUIPMENT AND CLEANROOMS

BioPharm: What type of equipment is typically used for aseptic processing?

O'Connor: The major piece of equipment, so to speak, is a cleanroom. Cleanrooms contain laminar airflow hoods, isolators or restricted access barrier systems (RABS), filling lines, sterilizing filters, autoclaves for sterilizing the equipment, depyrogenation tunnels for removing any endotoxins, as well as other equipment. Cleanrooms also have automated cleaning systems for reducing any low levels of contaminants prior to sterilizing, and isolators that have filling lines installed in them. These units are totally enclosed and have a localized area with what's called "Grade-A air" inside of them; this is where the filling line fills the product into final containers such as vials, syringes or bags.

A cleanroom could also have various environmental monitoring devices; filters, which are important for removing contaminants; and finally, single-use disposable systems, which are becoming more prevalent these days.

BioPharm: Can you provide more details about cleanrooms and the garments that are required?

O'Connor: There are a number of different standards around cleanrooms, but the International Organization for Standardization (ISO) 14644-1 defines a cleanroom as a room in which the concentration of airborne particles is controlled and which is constructed and used in a manner to minimize the introduction, generation, and retention of particles inside the room and which other relevant parameters (e.g., temperature, humidity, and pressure) are controlled as necessary.

Within the cleanroom, the critical components include high-efficiency particulate air filters on the ceilings that filter all the air coming into the room, as well as exhaust vents at the floor level. These vents ensure that laminar airflow and effective removal of air so that there are many air changes per hour, typically from 5 to 100. There should not be any drains inside an aseptic processing area in a cleanroom. Airlocks are important to facilitate the movement of people and equipment from one cleanroom into another.

It's important that cleanrooms are designed appropriately to ensure seamless and rounded floor-to-wall junctions to prevent buildup of any contamination or water. Floors and walls and ceilings must be constructed of smooth, hard surfaces that can be easily cleaned. One should limit the amount of fixtures and fittings on the walls to facilitate the ease of cleaning of walls, and layout of equipment must be optimized for the comfort, and movement of people.

In addition to ISO 14644, in Europe, cleanroom classifications are divided into Class A, B, C, and D, where A is the most stringent and D is the least exacting. In the US, room classifications are referred to as Class 100, Class 1000, Class 10,000, and Class 100,000. The ISO standards then would be 5, 6, 7, and 8. The standards in general apply to particle sizes in the range of 0.5 microns to 5 microns.

The standards are used interchangeably, depending on where your site is located and where you're being regulated. So, if your site is regulated by FDA, it would use the Class 100, 10,000 100,000 standards. Similarly, if your site follows the European standard, that means using Class A, B, C, and D. A Class-A air area would be used to perform a high-risk operation such as filling or making aseptic connections. A Grade-B area is typically used for aseptic preparation and as a backround environment for traditional filling operation. Grade C is used for preparation of solutions and equipment.

Microbial monitoring is also required to demonstrate the cleanliness of the cleanroom during production. The recommended limits are specific for the each room classification. Cleanrooms should have settle plates where air is allowed to settle to support the growth of bacteria. Staff can then see how much bacteria might be in the environment. Contact plates can be put up against walls and surfaces, and gloveprints can be used in Grade A and B areas. Gloveprints are where people actually place their gloved fingers to see whether there is any contamination present.

With each area, there are standardized limits. In a Grade-B room, for example, one can have no more than 10 colony forming units. It's important to trend these results, otherwise one cannot determine whether the room is under control. If there is no control, the product is at risk.

ENVIRONMENTAL MONITORING

BioPharm: What sort of environmental monitoring program needs to be used in aseptic processing?

O'Connor: The goal of an environmental monitoring program is to provide meaningful information on the quality of the aseptic processing. Typical controls are for airborne particles and nonviable contaminant monitoring (i.e., to analyze the amount of microbes in the room). Viable contaminant monitoring of surfaces involves touching surfaces with agar plates. Viable monitoring of personnel and temperature/humidity monitoring are also typical controls to have in place.

Environmental monitoring must be done across all processing shifts (i.e., day and night). All floors, walls, and equipment surfaces need to be tested. The location of the surfaces to be sampled, and the timing and frequency of the sampling, should be specified in writing, so that it is not just a random process, but rather, a risk-based process. It's also important for staff to ensure reproducible results.

The heating and ventilation and air conditioning (HVAC) unit should be under control of the building management system. This system controls the amount of air coming in to the cleanrooms and the differential pressure across the HEPA filters. If any changes are observed, actions and alerts should be raised to start an investigation and corrective/preventive action plan.

ANALYTICS AND TESTING

BioPharm: Various analytics and testing are involved in aseptic processing, including sampling, validation, and identification. Can you talk about some of the major considerations manufacturers need to keep in mind for this part of aseptic processing?

O'Connor: Sampling is an important part that involves testing to determine whether your process is in control throughout manufacturing. Before approving a process, it must be validated, which requires an extensive battery of tests on the product, including characterization, identification, and contamination minimization. One key factor for taking samples of biologics is to ensure that the product is not contaminated while samples are taken, which could lead to false positives.

Samples are also taken after the product has been sterile-filtered for sterility testing. If the product does not pass sterility test, it will be a failed batch and will be rejected.