Thanks to scientific and technical advances, revolutionary developments are taking place in medicines derived from biotechnology, for example for the treatment of cancer, autoimmune diseases or rare diseases that only affect a small patient population. However, these highly effective and sometimes highly toxic drugs require special safety precautions and hermetically sealed production processes to protect humans and pharmaceuticals from each other. At the same time, newly developed drugs in particular, which are often produced in smaller batches, require a high degree of flexibility and modularity. Integrated air management and optimized bio-decontamination play a fundamental role in the flexible integration of isolators into existing building and cleanroom concepts, while ensuring safe production processes.
Gassing with hydrogen peroxide (H2O2) has become a standard for automated bio-decontamination of isolators. H2O2 is a fairly stable liquid compound of hydrogen and oxygen – a powerful oxidizing agent which is particularly suitable for decontamination due to its broad-spectrum effect. After the decontamination cycle, the remaining H2O2 is either broken down by catalysts or driven out of the isolator by intense ventilation with fresh air to reach an acceptable residual concentration. The rapid growth of the biotechnology sector has increased the requirements for fillers and isolators, as many products are quite sensitive to residual H.2O2. The goal is to reach a level inside the isolator typically below 0.5 ppm (parts per million) before filling can begin. However, the exact limit largely depends on the sensitivity of the products and can be much lower, even up to approx. 0.03 ppm.
Despite a long aeration phase, part of the H2O2 remains in the atmosphere of the isolator and may even condense on surfaces such as the inner side of the isolator or the filling equipment. Once it enters the pharmaceutical liquid, it may lead to oxidation. While a residual concentration of 0.5 ppm can be achieved in a standard isolator with an aeration time of approximately one hour, several hours may be required to reduce the residual concentration to 0.03 ppm for particularly biopharmaceuticals. sensitive. This leads to filling line downtime which must be kept as short as possible, especially in small batch applications with frequent product changeovers and/or campaign mode manufacturing.
Biological molecules such as hormones or antibodies are easily oxidized. A modification of sensitive amino acid residues like methionine, tryptophan and cysteine affects their physico-chemical properties and possibly also the secondary and tertiary structure of the protein with a potential impact on the efficacy and/or safety of the product . The sensitivity of a drug product depends on many factors, such as the individual properties of the active ingredient, e.g. the type, number and location of oxidizable amino acid residues and their specific impact on the pharmacodynamics and/or or pharmacokinetics. Formulation-related parameters such as the concentration of the active ingredient and the presence of oxidation-sensitive or antioxidant excipients such as polysorbates and L-methionine, respectively, also have an impact. In addition, the diameter of the container and especially the (effective) size of its opening influence the diffusion of H2O2 in the product solution.
Besides these product-related factors, filling equipment, technology and the process itself also play an important role. For example, special attention should be paid to the exposure time of open and partially capped products to residual H2O2 during filling, machine stops or buffering filled units inside the isolator before loading into the freeze dryer. Silicone tubing is known to slowly absorb and release H2O2 and could lead to a relevant migration of H2O2 in the product solution, for example during line stoppages. During filling, nitrogen flushing and layering can help reduce H2O2 residues inside the container.
What to consider when decontaminating with H2O2? At first sight, it seems reasonable to define a universal target according to the most sensitive product. Depending on the specific exposure situation, 0.03 ppm can already affect certain molecules. If there is no experience with these types of products and risks, pharmaceutical manufacturers may want to be cautious with this conservative approach and usually end up well below the required concentration level. By implication, however, this leads to a longer than necessary aeration phase, which costs time and limits the availability of the system.
The best and by far the most effective solution is to familiarize yourself with the most important parameters. How does the product react to H2O2? What residual concentration is acceptable while avoiding a risk of oxidation? Unfortunately, only the “airborne” concentration can be determined during the ongoing process by means of online measuring systems, which continuously monitor the decontamination, aeration/ventilation and production phases. Also, sensors for routine monitoring usually have a limited sensitivity of 0.1 ppm, which is not enough for very sensitive products. Here, the decontamination and aeration cycle must be validated using special and highly sensitive sensors that are not usually present on manufacturing equipment.
On the other hand, the concentration of H2O2 in the product solution can only be determined by off-line experiments and is difficult to track in ongoing production. Nevertheless, studies can establish a relationship between the concentration in the air and in the solution. Using an Airborne Fixed H2O2 concentration and a variable exposure time, it is possible to determine the absorption in the product or a substitute and to simulate the conditions on the machine. This allows pharmaceutical manufacturers and equipment suppliers to refine the process of decontaminating existing production lines. In the case of new lines, an in-depth knowledge of the products makes it possible to adapt the insulator even more precisely to the specific requirements.
When designing or optimizing an isolator, it is important to know all relevant product, process and equipment parameters. Once the H is acceptable2O2 concentration has been determined for a specific product and process, it must not be exceeded in the qualified and validated decontamination process. Again, many factors play a role – from the type of container and its fill volume, the temperature inside the isolator, changes in air volume and H2O2 concentration over time, process time and exposure time of caps and containers. The materials used for filling machines and insulators also offer optimization potential. For example, some materials like tubing or silicone gaskets are known to absorb H2O2. As they only release it very slowly, their use should be kept to a minimum if very sensitive products are to be processed.
The ideal situation, which is not within the reach of all development laboratories, is the simulation of exposure to “airborne” H2O2 in a test isolator. Opened products may be exposed to defined concentrations of H2O2 for different durations. In addition, process parameters such as time between filling and capping, line stops due to interventions and buffering of partially capped containers during loading of the freeze dryer can be taken into account. However, due to handling issues (mainly manual sample preparation), such a study often does not generate enough samples for subsequent stability study. Therefore, dividing the evaluation into an adoption study and a separate doping study is the most feasible alternative.
In this case the absorption study is used to determine the amount of H2O2 absorbed by a surrogate liquid (usually water) under the same exposure conditions and in the same configuration (fill volume, container, partial capping) as the product in the test isolator. Only the concentration of dissolved H2O2 is quantified using a sensitive analytical assay, for example a peroxidase assay with a fluorogenic substrate. The data is then used to enrich the product solutions for the stability study with dilute H2O2 giving the same final concentrations as those measured in the surrogates. From H2O2 can be consumed by the product fairly quickly, a control of water treated in the same way as the product samples makes it possible to check whether the doping procedure was successful.
This split approach also allows pharmaceutical manufacturers to outsource adoption studies to their equipment vendor, provided that the latter has the required process and analytical technology and expertise. In this case, only the doping and stability studies are carried out in-house, an approach that Merck and Syntegon have used successfully for different projects.
Despite the high sensitivity of some biopharmaceuticals, hydrogen peroxide remains the method of choice for decontaminating insulators. Based on experience and appropriate studies, it is possible to determine very precisely how a particular filling line should be designed and operated to decontaminate it safely and effectively. Especially with new lines, decontamination, product oxidation and allowable residual concentration of H2O2 must be taken into account during the design and engineering phase in order to minimize its effects on cycle times.
The more biopharmaceuticals come to market, the more experience there will be in dealing with these molecules and their challenges in the fill-finish process. Therefore, working with a reliable partner with many years of experience in process and measurement technologies, insulator design and product testing helps to successfully meet these challenges.
Dr. Thomas Kosian is Senior Barrier Systems Expert at Syntegon Technology. He can be contacted by email at [email protected]
Dr. Felix Heise is Head of Global MSAT DP Biologics for Merck KGaA – Healthcare (EMD Serono). He can be contacted by email at [email protected]