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The authors give special consideration factors affecting blow–fill–seal technology.
For therapeutics administered by certain routes such as pulmonary delivery, plastic ampuls can offer many advantages over glass ampuls, vials, or syringes. Plastic ampuls are manufactured using blow–fill–seal (BFS) technology. The BFS process involves plastic extrusion, molding, aseptic filling, and hermetic sealing in one sequential operation. Unlike small molecules, biological drug products, such as proteins or monoclonal antibodies, are more prone to degradation during processing, which may result in loss of activity or safety concerns. The operating conditions for a BFS process and the nature of plastic ampuls pose many challenges to the stability and integrity of biological drug products. In this article, the authors discuss considerations in the development and manufacturing of biological products using the BFS process, including potential product exposure to elevated temperature, requirements for leak detection, and packaging operations. They also highlight challenges and strategies for BFS process characterization and validation in the context of biopharmaceutical manufacturing.
Biological drug products, such as proteins or monoclonal antibodies, are predominately packaged into vials or prefilled syringes for intravenous or subcutaneous administration. However, some biological drug products must be administered by alternative routes, such as pulmonary delivery in the form of a mist using a nebulizer. In such a case, using plastic ampuls as the primary drug container offers many advantages over vials or syringes. Plastic ampuls are convenient, simple to use, are unbreakable, and child-friendly. One example of biological product supplied in plastic ampuls is Pulmozyme (dornase alfa, Genentech), prescribed for the treatment of cystic fibrosis. Pulmozyme is a sterile, clear, colorless, highly purified solution of recombinant human deoxyribonuclease I (rhDNase), an enzyme which selectively cleaves DNA, and is available as single-use 2.5-mL ampuls. The patient self-administers the drug by simply emptying the entire content of an ampul (one dose) into the nebulizer bowl for delivery. The development of the Pulmozyme formulation and manufacturing process has been previously reported (1).
Plastic ampuls are manufactured using blow–fill–seal (BFS) technology (2). BFS has gained wide acceptance for pharmaceutical solutions (e.g., eye, nose, and ear drops, contact-lens solutions, inhalations, oral, or topical solutions) and household chemicals (e.g., insecticides, detergents, and disinfectants) because it offers many manufacturing advantages, such as high output and reduced human intervention. The use of BFS in biopharmaceutical manufacturing, however, has been scarce. Unlike small molecules, biological drug products are more prone to degradation, which may result in loss of activity. The unique operating conditions and requirements of BFS technology also pose many challenges to the development and manufacturing of biological drug products.
Conventional fill–finish unit operations such as mixing, filtration, and filling, and their potential impact on the product have been previously reviewed (3). This article focuses on specific challenges and considerations associated with the development and manufacturing of plastic ampuls using the BFS process, including elevated temperature, leak detection, packaging, as well as process characterization and validation.
The BFS process involves plastic extrusion, molding, aseptic filling, and hermetic sealing in one sequential operation, as illustrated in Figure 1. The resin material for primary containers is typically received as plastic granules (e.g., low-density polyethylene [LDPE] or polypropylene [PP]). In the extrusion step, plastic granules are fed through an extruder, where they are melted at temperatures above 160 °C. The melted plastic is pressed and extruded through an orifice, resulting in a continuous hollow tube of molten plastic, referred to as a parison (see Figure 1, Step 1). The metal mold then moves to enclose the parison and forms the plastic container of desired shape, aided by either application of vacuum to the mold cavity through small orifices or by blowing sterile air into the container (see Figure 1, Step 2). Subsequently, a parison dye cut will cut the parison at the top, and the mold carrying the unclosed plastic container (at this stage referred to as an ampul) will move to the filling cabinet. In the filling step (see Figure 1, Step 3), the filling needle is inserted into the top opening and solution is discharged into the ampul. The filling needle retracts after the fill completion. The top portion of the mold then comes together to press the plastic and to form hermetically sealed ampuls (see Figure 1, Step 4). The mold then opens and the ampuls are released from the BFS machine and conveyed to the inspection station (see Figure 1, Step 5).
Figure 1: Overview of the blowâfillâseal process.
BFS steps in biological manufacturing
Fill–finish operations of biological drug products, particularly proteins or monoclonal antibodies, usually begin with drug substance (or concentrated bulk) supplied either refrigerated at 2–8 °C for temporary storage, or frozen at <–20 °C for extended storage. The drug substance is thawed, if necessary, at a defined temperature and process. If the drug substance has a different concentration from the final drug product, a formulation buffer can be added and mixed together with the drug substance in a mixing tank, to reach the target concentration as final drug product. The solution is then transferred into a holding tank through a filter to reduce bioburden level. The holding tank containing formulated product can be used to store the product for an extended period of time to accommodate manufacturing scheduling. These operation steps, including thawing, dilution, mixing, and filtration are typically conducted in a Grade C area. Figure 2 illustrates the process flow.
Figure 2: The blowâfillâseal process in the context of biological manufacturing.
Most parts of the BFS machine are located in a Grade C room; only the filling cabinet is controlled under a Grade A condition. Time-pressure filling, where a pinch valve is used to control the fill volume, is often used in the filling operation. The flow rate is determined by the pressure applied to the liquid reservoir (or surge tank), and the pinch valve opening time determines the final fill volume. During the filling process, solution sterilization is achieved by sterilizing-grade filters.
The connection of the BFS machine with filling needles, surge tank, and sterilizing filter usually takes place in a Grade C environment. The BFS machine is first assembled and cleaned through clean-in-place (CIP) procedures. It then gets connected to the sterilizing filter, and the entire connection is sterilized under a validated steam-in-place (SIP) cycle, from the filling needle, all the way to the point of connection with the product tank.
Aseptic processing requires contamination control and sterilization of three main aspects: drug-product solution, primary container components, and operation environment. The sterilization method for drug product solution in a BFS process is similar to a conventional fill–finish process, where typical sterilizing grade filters are used. Unlike the component processing steps in the conventional fill–finish process (i.e., washing, depyrogenation, and siliconization of vials, syringes, and stoppers), the primary container is formed in the BFS process, and the molding of plastic resin at elevated temperature provides a container "free" of viable microorganisms and with acceptable endotoxin levels. The ability for a BFS extrusion process to yield product with acceptable quality has been demonstrated by controlled challenges studies, where LDPE granulates were contaminated with characterized levels of bacteria spores and endotoxins, then pressessed through a BFS machine using Tropton Soy Broth and Water for Injection as the filling media (4). Higher fractions of contaminated units were observed with increased challenge level (or amount of spores and endotoxin in the plastic granulates). The above study demonstrated it is critical to establish appropriate acceptance limits for bioburden and endotoxin levels on plastic granulates to assure final product quality.
The overall extrusion and molding process of the BFS operation is conducted in a Grade C area. However, the air shower for the filling shroud of the BFS machine should provide a Grade A condition. European Union guidelines state that, "The condition should comply with the viable and nonviable limits at rest and viable limit only when in operation" (5). There is potential risk for airborne contamination before the mold moves to the filling shroud area, where the air shower maintains a Grade A environment. Recent studies have shown that most of the airborne contamination occurs between the time when the top is cut from molded containers and when they pass under the air shower (6). The microbial and particulate controls are highly equipment and site specific. Therefore, a media fill has to be conducted for each specific machine, process, or container type. A recent survey of the BFS industry provides an excellent overview of the current practices in aseptic BFS technology, showing that the BFS process has a much lower frequency (about one-tenth) of contaminated media fills compared with conventional processes (7).
Product temperature in a BFS process
One of the major challenges for the BFS operation compared with a conventional filling process is the high temperature involved in the plastic extrusion step. As illustrated in Figure 1, the plastic granules are melted and extruded at temperature in excess of 160 °C. The hot parison will come into contact with a metal mold, be transported to the filling shroud, and be filled with drug product. Cool water circulating inside the mold helps to lower the ampul temperature. The cooling process, however, is limited by the contact time between the ampul and the mold (a total of about 10 s). In addition, the ampul cooling is restricted by the minimum temperature needed to form a hermetic seal at the end of the filling step. The ampul temperature near the neck area has to remain high enough to ensure an appropriate seal at the end of the filling process.
In the development phase of a biological drug product, when using a plastic ampul as the primary container, it is important to understand the temperature to which the product could be exposed. The folded conformations of proteins are only marginally stable, and a rise in temperature can denature the molecule leading to loss of activity. Elucidating the temperature profile during the BFS process will enable formulation scientists to develop more robust formulations.
In a BFS manufacturing process, product temperature during the filling process is not an easily controlled parameter, and it cannot be directly measured. Instead, the controllable parameters are the starting-product solution temperature in the bulk tank, plastic extrusion temperature, mold-cooling water temperature, and the cycle time for each BFS step (e.g., molding, filling, and sealing).
The authors have used a computational fluid dynamic (CFD) model (ANSYS Fluent 12) to simulate the BFS process using the Pulmozyme ampul as a model. Initial simulation results (not shown) showed that ampul-wall temperature reaches mold temperature quickly (within 1 s) during Step 2 of Figure 1. If the actual mold temperature is at cooling-water temperature (usually near room temperature of ~ 25 °C), the finished ampuls at the end of BFS process should not be significantly hotter than the mold cavity. It is the authors' experience, however, that product ampuls usually feel warm right after the BFS process. This observation suggests that the actual mold temperature is much higher than the cooling-water temperature (the actual mold temperature during BFS production is not usually measured).
For the purpose of the CFD simulation, the authors adjusted the mold temperature to different settings. Figure 3 illustrates the product-temperature profile when the mold temperature was set at 60 °C. The simulation assumes that it takes approximately 11 s for the entire BFS process including ~3 s for the molding step, ~3 s for filling, and ~5 s for forming a hermetic seal. The upper portion of Figure 3 shows the liquid volume in the ampul, as it is being filled (from time = 3 s). The temperature profiles of the entire ampul (including wall, solution, and air) throughout the filling and sealing step are shown in the bottom sequence of Figure 3. It should be noted that in the actual manufacturing process, the filling nozzle is retracted from the ampoule after filling is complete to allow for sealing. Since this is a nonproduct contact part the current CFD simulation simply turns off the solution flow from the inlet nozzle, instead of modeling the actual nozzle retraction.
Figure 3: Simulation of product temperature in a blowâfillâseal process.
The simulation revealed that the ampul is cooled from the initial extrusion temperature of ~160 °C to 60 °C (assumed actual mold temperature) in less than 2 s. Forced convection dominates heat transfer during filling (from 3 s to 6 s), thereafter natural convection becomes more prominent. By the time the filled ampul was ejected from the mold (~ 11 s), only the fluid layer near the ampul walls had been heated to within 10 °C of the mold temperature. The majority of the liquid remains considerably cooler.
In the current simulation the authors assumed a worst-case scenario of liquid being filled at ~25 °C. In the actual production process, the drug substance is typically maintained at 2–8 °C and would be introduced in the ampoule close to these temperatures. Therefore, in a production BFS process, it is expected that the drug substance would experience temperatures that are lower than these shown in the CFD simulation results.
Based on the observation of higher actual mold temperature compared with that of the cooling water from the simulation results, it is highly recommended to monitor the actual mold temperature during the development or characterization phase of the BFS process. Monitoring could potentially be achieved by attaching thermocouples to the mold or by infrared temperature measurement of the mold surface.
Leak detection in plastic ampuls
The formation of the primary container is an integral part of the BFS operation and container-closure defects can be a major problem. US regulatory authorities require that "as a final measure, the inspection of each unit of a batch should include a reliable, sensitive, final product examination that is capable of identifying defective units (e.g. "leakers")"(8). It is also stated in the EU guidelines that "containers closed by fusions, e.g., glass or plastic ampoules should be subject to 100% integrity testing"(5).
There are multiple leak-detection technologies available, such as the vacuum-decay method, high-voltage leak detector, or near infrared leak detection. Among these methods, the vacuum-decay method is often selected for biological manufacturing becauese it has no product quality impact (even though there is no documented product quality impact from other methods to the authors' knowledge). During vacuum decay leak detection, the test article is placed inside a chamber, which is then evacuated to a known pressure. If the product container exhibits a leak, the chamber pressure will rise at a rate greater than a predetermined baseline value.
The qualification of a leak-detection machine can be achieved by challenging the system with calibrated needle valves, or by means of "leaky" samples, created by either laser microdrilling or capillary tube insertion into ampuls. In the authors' experience, creation of "leaky" samples of ampuls is not reproducible, and needle valve is a preferred method to qualify the leak detection machine. Leaky samples created by laser microdrilling or capillary tube insertion in plastic materials have many challenges. Most plastic resins, such as LDPE, do not have the required material characteristics (too soft and poor adhesion) to support reliable mounting of a capillary or to maintain a consistent hole size. The drilling process with LDPE yields a conical-shaped orifice that results in flow rates that are difficult to predict. Furthermore, the verification process is destructive in that the flow rate is measured under vacuum, which can result in obstruction of the orifice by the product. Therefore, the actual test sample used cannot be verified prior to testing. The variables of shipping, handling, and pressure as a result of air transportation could also result in inconsistent results.
Alternatively, the needle valves can be calibrated with a flow meter to a specific flow rate corresponding to a specific orifice size. Calibrated needle valves represent a suitable qualification approach to the system that will demonstrate the system's ability to detect an actual leaking sample with a known hole size and flow rate.
Another main challenge with in-line leak detection operation is the high false rejection rate. Due to the high temperature required for plastic extrusion, the ampuls, immediately after BFS process, are notably warmer than room temperature. When the products are immediately placed in a vacuum-detection chamber, the heat from the ampul can cause a slight increase in pressure which the instrument may interpret as a leaky sample, which will trigger a false rejection signal. Therefore, it is highly advisable to have the product equilibrated to room temperature prior to subjecting the product to vacuum leak detection.
Packaging requirements, extractables and leachables
The primary containers of ampuls manufactured using BFS process are plastic materials, such as LDPE or PP, which are gas-permeable to some degree. During long-term storage, water vapor may diffuse out of the ampul resulting in alterations in drug concentration. Conversely, oxygen permeation could cause protein oxidation. Gas permeation can be minimized by sealing the plastic ampuls inside laminated foil pouches (as a secondary packaging layer). For example, in the development of the Pulmozyme drug product, studies showed that protein concentration increased by more than 30% due to water loss when the naked product was stored at 37 °C for ~700 days (1). Such water loss was prevented by adding an aluminum foil pouch as a secondary packaging layer. In addition, the aluminum foil pouch protects the drug product from light-induced degradation. Furthermore, oxygen-sensitive products can be sealed into pouches under a nitrogen blanket (overlay) to provide additional protection against oxidation.
Secondary packaging materials are typically comprised of multiple layers of polymers (i.e., polyester, PP, or polyethylene), inks, adhesives, as well as possibly unreacted monomers and oligomers derived from adhesive materials. When in direct contact with primary containers, these materials have the potential to penetrate through plastic ampuls. In addition, the direct contact between the liquid product and the primary container (even though the primary container material is typically made of much cleaner polymers) could lead to extraction of chemicals. These chemical extracts and leachates from plastic packaging materials could act as adjuvants in stimulating an immune response in the patient (9). Leachates would especially be of concern if the product is to be injected subcutaneously, as the localized concentration of product and adjuvant is more likely to stimulate the production of neutralizing antibodies than intravenous injection or inhalation (9). Therefore, it is imperative that the extractable and leachable program for plastic ampul products be conducted with both pouched and unpouched samples to understand the respective extracts and leachates from both the primary and the secondary packaging materials (10–12).
A biological manufacturing validation program encompasses equipment qualification, cleaning validation, and process validation. The BFS equipment suppliers and manufacturers usually have well-established practices for performing equipment qualification and cleaning validation. Process validation, however, could be a challenge for biological manufacturing using BFS technology.
The goal of process validation is to provide a high degree of assurance that the specific manufacturing process will consistently produce product meeting predetermined acceptance criteria. Modern-day process validation programs consist of process characterization studies and full-scale qualification lots produced under GMP conditions. Process characterization can be performed on each unit operation to evaluate and characterize an acceptable range of operating conditions that yields acceptable product quality and process performance. A quality-by-design (QbD) approach, which systematically uses risk assessments and multivariate characterization studies, is the current state of the art. Process characterization may be carried out at different scales, and it may also include engineering runs to mimic the overall process flow. Based on the outcome of process characterization, full scale GMP batches are manufactured under normal settings. Both physical attributes (such as ampul appearance, integrity, fill volume, and wall thickness) and chemical attributes of the product are checked to ensure that the process performs as expected.
It is important to recognize the uniqueness of the BFS process when applying the above process characterization and process-validation approach. The BFS machine is a custom-built machine. The equipment setup and operation often involves both automation control and manual adjustments. The operator experience or "know how" generally is very important to the proper operation of the BFS equipment. Such operator-dependent performance, to a certain extent, is in contrast to the QbD paradigm of pharmaceutical manufacturing, where critical process parameters (CPPs) are identified, understood, controlled, and monitored to achieve a robust manufacturing process. The large number of controllable and uncontrollable parameters in the BFS process makes process characterization and process validation much more challenging than conventional biological filling processes. For instance, the filling speed for a peristaltic or rotary piston pump is the typical process parameter that can be evaluated during stand-alone process-characterization studies. The filling speed, however, for a BFS process is mostly fixed, due to constraints from synchronized movement of the extruder, mold, filling, and seal operations. Only minor adjustments can be made to the duration of each of the BFS steps (extruding, molding, and filling), and such adjustment may be necessary to achieve optimum product appearance.
Another example of manually adjusted parameters is the plastic-resin weight. The resin weight determines how much plastic is extruded to form the container and directly correlates with the container wall thickness. Too much resin may result in containers with "bulky" appearance or problems with deflash while too little resin may cause issues for container integrity or ampoule leakage. The appropriate amount (or range) of resin is typically determined from equipment qualification. It can only be manually adjusted through a few mechanical parts at the beginning of each product run. Therefore, the process and product development for BFS process needs to balance an empirical approach and a systematic approach. The biological manufacturer needs to collaborate closely with the BFS equipment supplier or contract manufacturer to design a robust program for process characterization and validation.
Figure 4 provides an example of the potential roadmap for process characterization and process validation. Risk assessments are highly recommended for each unit operation as well as for each of the steps from the BFS operation in order to identify potential failure modes from equipment and mechanics, as well as product-impact aspects. The output from the risk assessments would be a list of process parameters that could affect the overall process or product quality. Further development and characterization studies should be designed for these parameters either as stand-alone studies or overall engineering runs. CPPs are selected based on the outcome of process characterization. Finally, full-scale qualification lots should be produced (typically at target settings of the process parameters) to demonstrate the validated state of the end-to-end process.
Figure 4: Process flow for blowâfillâseal (BFS) process characterization and validation (CPP is critical process parameter; PC is process characterization).
BFS technology has great potential in the field of biopharmaceutics because of reduced human intervention during the production process, convenience, and ease of use offered by its final product in plastic ampul form. The operating conditions of the BFS process and the nature of plastic ampuls pose many challenges to the stability of biopharmaceutical drug products. Biopharmaceuticals may experience elevated temperature during the BFS process. CFD could be a useful tool for better understanding the temperature dynamics during the BFS operation. The unique aspects of BFS operation call for a balanced empirical and systematic approach during process development and process validation.
Finally, the BFS process may not be suitable for many proteins, especially large, complex proteins with multiple sites for activity and proteins that are highly temperature-sensitive. In addition, chemical extracts or leachates from the direct contact between the product and the primary container, as well as volatile chemicals from secondary packaging layers could act as adjuvants in stimulating an immune response in the patient, which would be of particular concern if the product were to be injected subcutaneously. These factors should be evaluated and addressed in the early-development phase if plastic ampuls are selected as the product containers.
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