Dave Abram is manager of validation and technical services at BioConvergence LLC, 4320 West Zenith Drive, Bloomington, IN 47404, tel. 812.961.1700, fax 812.961.1733.

Dave Abram

The formulation process for parenteral, or injectable, solutions often follows the overly simplified method of combining water for injection (WFI), active pharmaceutical ingredient (API) and excipients in a formulation vessel (FV) located within a formulation room and mixing until dissolved. The formulated solution is filtered through one or two sterilizing-grade (0.22 μm) membrane filters into a sterilized receiving vessel located in a traditional cleanroom with an International Organization for Standardization (ISO) Class 5 unidirectional airflow, isolator, or restricted access barrier system (RABS) (1). Occasionally, however, the formulated product includes a component that cannot be sterilized through filtration because the act of filtration would render the final product ineffective. These nonfilterable components may be insoluble particles suspended in a solution or they could be molecules too large to pass through a filter membrane. With the exception of terminal sterilization of the formulated or filled product, devising a means to formulate the product aseptically may be the only solution to ensure product sterility. Performing aseptic formulation requires consideration of several aspects for the entire process.  

In making these evaluations, the analysis in this article assumes that the facility and equipment have been qualified and approved for parenteral production under good manufacturing practices (GMPs). The analysis takes the perspective of using a traditional cleanroom for manufacturing with the understanding that these approaches can be applied to advanced systems such as isolators or RABS and tailored as necessary. 

The first step in determining the approach for an aseptic-formulation project is to learn the characteristics and limitations of the product. This determination will help guide the formulation process. The key elements are: the total volume to be formulated because this will influence the formulation vessel size and design; and the various components of the product capable and incapable of being sterile filtered. 

The type, brand, and model of sterilizing filter selected may influence the sterilization process for the filters (autoclave or sterilization-in-place [SIP]) and may also influence the formulation process. Of those components that can be sterile filtered, it must be determined whether they should be filtered in series through the same set of filters or whether separate filters are needed for some or all components. This selection may influence the sterilization process for the filters and may also influence the formulation process. 

For those components that cannot be sterile filtered, it is important to understand how those will be sterilized and where such sterilization will occur (presterilized or sterilized in-house). Sterilizing the material in house could add another layer of complexity and cost to the project, depending on the characteristics of the material. Also important to know are any specific temperature conditions required for the formulation process because these conditions will influence the FV design and potential heating and cooling equipment. 

The existing facilities and available utilities may be adequate, may need to be modified, or may need to be designed and built to support aseptic formulation project needs. A source of clean, ISO 5-compliant, unidirectional airflow is critical for making aseptic connections and performing general aseptic operations. Additionally, some aseptic connection points may require the use of horizontal laminar airflow. Making the decision requires a good understanding of the aseptic formulation process as well as the intended sterilization process. 

From a utilities perspective, if the FV will be sterilized through SIP, qualified pure-steam ports and inert-gas ports will be needed in the area intended for SIP. The pure steam-system pressure should be high enough (e.g., ≥ 40 psig) to ensure that sterilization within the FV can occur at the same time as supplying other steam-consuming equipment (e.g., autoclaves, sterilizers, and lyophilizers). The inert-gas distribution system pressure will likely exceed 100 psig. Therefore, regulators will be needed to reduce the pressure for use in drying the FV following SIP. 

The FV needs to be designed for its collective intended uses, which include sterilization (autoclave or SIP), preservation of sterility following the sterilization, the aseptic-formulation process, and a robust means of aseptically transferring the sterile contents of the FV to the filling machine. The product contact surfaces of the FV should be composed of electropolished 316-L stainless steel, or they should be glass-coated if the product is not compatible with stainless steel. The FV must be sized to thoroughly mix and contain the final volume of formulated product but also able to fit through door openings within the facility. The design of the FV should include load cells for weighing materials required by the formulation. Consideration should be given to aseptic connections, in-process samples, and transferring formulated product from the FV to the filling machine (

 a dip tube or bottom-outlet port). The FV may need to be jacketed and rated for pressure and vacuum depending on the product and process needs (e.g., cooling during formulation and SIP). To facilitate the performance of the process steps, various fittings need to be connected to the FV (see Table I).  

In the event that SIP is the chosen sterilization method for the FV, the items identified in Table II should facilitate the process.  

Table II: Components for sterilization in place. 

The existing facility, utilities, equipment, and regulations establish boundaries for the chosen aseptic formulation process. In-house expertise in manufacturing, process development, and validation all factor into the success of the project. The primary goal of the aseptic formulation process is to produce a sterile product. The following guidelines improve the chances of success:  

Minimizing the number of aseptic connections.

Designing the FV in such a way so when sterilized, that all fittings needed in the aseptic formulation process are also sterilized (e.g., filters, sampling devices and valves).

Exposing the aseptic connection points to a constant supply of ISO 5 unidirectional airflow. Avoid having connection points near the floor or in other areas where there is turbulent airflow (i.e., bottom-outlet valves may present aseptic connection difficulties).

Whenever the aseptic formulation process has been developed and validated, training is critical. Train multiple operators on the "how's" as well as the "why's" of the process. If the operators fail to understand the "whys," recognizing deviations and their impact during manufacturing will be difficult. It may be helpful to include these operators in the validation activities.

Leveraging, whenever possible, existing processes and validations.

Although not directly related to sterility assurance, one also must put into place a filter integrity-testing strategy. Filter and immediately remove the filters to be postuse integrity-tested before proceeding; filter and proceed with the remainder of the formulation before integrity-test results are known or redundantly filtered using a series of two filters. Table III outlines advantages and disadvantages of the options.

Table III: Options in filter integrity testing. 

The sterilization method for the FV must be decided upon and the process developed. Preservation of sterility following the cycle is equally critical as the act of sterilization.  Common sterilization methods that could be employed are autoclave sterilization and SIP. If the FV can fit inside the autoclave, this is the simplest option. If the FV is too large for autoclave sterilization, SIP using steam from the plant's pure-steam system will fulfill the need. SIP is more difficult than autoclave sterilization, and it presents some hazards for operators. SIP will generate substantial humidity in the area where it is being performed. This moisture presents slip–and–fall hazards, and the heat of the external surfaces of the FV can scald. 

Regardless of the method chosen, the sterilization cycle needs to be developed for the FV. If autoclave sterilization will be used, the same cycle used to sterilize durable goods may be the most reasonable starting point for FV sterilization. It is suggested to place hydrophobic vent filters on the ports to be used for aseptic connections to facilitate air removal and steam penetration as well as preserving sterility following the autoclave cycle. If vent filters are not used, these connection points will be dead legs, which make air removal and steam penetration more difficult and sterilization less efficient. 

If SIP will be used, cycle development will be more involved. The FV needs to be on-hand with all fittings and any other necessary equipment. Having a good idea of the general aseptic formulation process and the connections to be made will help to set the fittings on the FV as well as determine inlet and outlet points. The use of temperature measurement tools (i.e., thermocouples or data loggers) is necessary for the development of the SIP cycle. It is assumed that the SIP will be performed in a lower classified area and the sterile FV ultimately moved into an ISO 5-compliant cleanroom for the aseptic formulation. 

The flow of steam through the system needs to be set up to ensure steam penetration and contact with all interior surfaces and product pathways throughout the FV and fittings. Dead legs should be avoided because air can become trapped, thereby reducing sterilization efficiency. When the SIP cycle starts, the steam will condense on the cold interior surfaces of the FV and exit through the outlet ports as water. Once the FV surfaces heat up, the condensate will gradually turn to steam. The inlet and outlet valves need to be adjusted to build the desired internal pressure to achieve sterilization. The valves to the jacket should be opened to prevent the buildup of pressure caused by the temperature increase within the vessel. After the steaming portion is complete, the FV will need to be cooled and dried with a sterile-filtered inert gas. The transition from steam to inert gas should be made gradually while maintaining positive pressure within the FV. 

Following the cooling and drying cycle, preservation of sterility is critical. The manner in which sterility is preserved depends on the method of sterilization. If the FV is autoclave sterilized, it will be dried but still quite warm following the cycle. Removing the FV from the autoclave should happen under the protection of unidirectional airflow. Check all clamps for tightness while the FV is still warm. Once the FV cools to room temperature, it can be removed from the unidirectional airflow and stored in lower classification environment until use. The exterior should be sanitized before aseptic formulation begins.   

If the FV is SIP sterilized, the following two options exist for preserving sterility:   

Pressurize the FV with sterile-filtered inert gas following the cool-and-dry cycle. All outlet valves will be closed, first the outermost outlet valves and last the inlet valve. All clamps should be tightened. The exterior of the pressurized FV will be sanitized (sanitization method should be qualified). The sanitized FV will be moved into an ISO 5 environment until use. The FV could remain in lower classified environments provided that the internal pressure is maintained until use. If the internal pressure is lost before being placed in an ISO 5 environment, contamination concerns will need to be addressed.

Pressurize the FV with sterile-filtered inert gas following the cool–and–dry cycle and perform an integrity test of the FV with all fittings. This integrity test will involve closing the valves and measuring the pressure decay as a result of cooling. If the FV has leaks, the pressure loss will be greater than the pressure loss due to cooling alone. Once passing results are obtained, the FV could be stored in a lower classified environment until needed for use. The exterior would still need to be sanitized.

Performance qualification of FV sterilization

Regardless of the method selected for sterilizing the FV, performance qualification (PQ) must be performed. If autoclave sterilization was the chosen method, the PQ of the cycle will follow the same process that has been used for other durable loads. If SIP was the chosen method, there are two basic parts of the SIP cycle to consider: sterilization and drying. To qualify the sterilization portion, the FV will need to be assembled as in manufacturing and probed with thermocouples, data loggers, and biological indicators (BI). The placement of the thermocouples and BIs should be in locations thought to represent the worst-case areas regarding steam penetration. The SIP cycle should be run at worst-case parameters when compared to those used for routine manufacturing. For example, if the parameters used for manufacturing are anticipated to be 30 min at a steam pressure of ≥ 18 psig, the parameters used for the PQ should use a shorter timeframe and lower pressure to provide additional sterility assurance. One method for controlling the pressure during the cycle at a specific pressure is to replace the outer diaphragm valve with a needle valve, allowing for more precise control of steam flow.   

Following the steam portion of the SIP cycle, the FV should be dried using sterile filtered inert gas. Following the drying portion, the FV will need to be closed in a manner to retain positive pressure and preserve sterility. If the strategy for manufacturing is to simply pressurize the FV and transport into the ISO 5 cleanroom, this aspect is low risk and does not need to be qualified. If the strategy is to perform a pressure hold test on the FV and relieve the internal pressure after passing results have been achieved, those success criteria need to be derived from qualification. Perform the SIP as in manufacturing (higher pressure and longer timeframe). The additional temperature and time will raise the temperature of the FV beyond that found using the sterilization-qualification parameters, which will impact the pressure loss behavior as it cools. Once the FV has been dried and cooled, close all valves to retain a positive internal pressure. Allow the FV to remain undisturbed for a short timeframe and then record the internal chamber pressure.  Allow the FV to continue cooling for several hours until it reaches room temperature. Record the internal pressure and ensure that the FV indeed has retained pressure. The pressure decay as a result of cooling will be the parameters used for integrity testing during routine batch manufacture. 

Validation of the aseptic formulation process

The process developed for aseptically formulating the product requires validation. The method for this process validation is the process simulation (i.e., media challenge). Running a process simulation for the recently developed aseptic formulation process uses the same basic concept as used for  the aseptic-filling process simulations. 

The process simulation should be performed per written batch record using the same manufacturing personnel, equipment, and facilities that will be involved in the manufacture of the actual product. Where the manufacturing process uses inert gases, these will be substituted with oil-free compressed air to promote microbial growth in the event contamination occurs. A total of three batches will be performed in which each step of the proposed manufacturing process will be included with media substituting for the product. Upon completion of the process simulation, the entire FV can be closed and incubated or a portion can be transferred as it will be performed in routine batch manufacture and that portion incubated. The media vessels should be sealed to prevent contamination from occurring during incubation. 

Assessing worst cases in process simulations is a balancing act of weighing the benefits of success against the risks and consequences of failure. Building flexibility into the process, covering worst-case and some nonroutine interventions to allow the manufacturing to operate within a larger design space is desirable. This objective can only be achieved by including these worst-case scenarios and interventions in successful media challenges. In the event the media challenge fails, time and effort will be lost on the media challenge in addition to the time and effort required of an investigation. If multiple interventions were included in a failed process simulation batch, identifying the root cause of the contamination will be challenging. 

Drug products that require aseptic formulation can present challenges. These challenges can be overcome with experienced personnel and a good understanding of the project objectives, regulatory requirements, facilities and equipment. Once the processes have been developed and validated and personnel training completed, manufacturing the aseptically formulated product will be able to proceed with relative ease. 

is manager of validation and technical services at BioConvergence LLC, 4320 West Zenith Drive, Bloomington, IN 47404, tel. 812.961.1700, fax 812.961.1733, 

Cleanrooms and Associated Controlled Environments—Part 1: Classification of Air Cleanliness,

 First Edition, Sec. 2.4, p. 3, May 1, 1999. 

Articles

The author details the factors in formulation design, requirements in facilites and equipment, and validation criteria for aseptic formualtions.