Biosafety Considerations For Single-Use Bioreactors

Originally published by BioProcess International

Single-use bioreactors are widely accepted in the pharmaceutical industry and are increasingly being used to perform mammalian cell cultures in commercial manufacturing applications. They address some key challenges the industry faces by decreasing time-to-market, reducing validation efforts, increasing flexibility, reducing investment costs, and optimizing cost of goods.

Vaccine manufactures require a high level of flexibility from a bioreactor platform because of the large variety of cell lines they use and the number of different vaccines they produce within one facility. The flexibility offered by single-use bioreactors means that they are becoming the preferred tool for cell culture in the vaccine industry.

Vaccine manufacturers must consider various design aspects when selecting the proper single-use bioreactor technology. Single-use bioreactors have a much lower degree of automation compared with stainless steel solutions. Operators make a large number of manual connections instead of working with transfer lines and automated valves. The transport of the single-use bag into the cleanroom and installation in the bag holder also require a high degree of manual interaction. If not performed properly, those activities may result in damage to the bioreactor or incorrect connections that lead to a break in the sterile barrier. Ensuring bag integrity is of critical importance during cell cultivation. A production batch must be discarded if the sterile barrier is broken, resulting in financial loss, a negative influence on production planning, and even product shortages in the market.

Critically, when infectious viruses are present within the bioreactor, a break in the sterile barrier poses a biosafety risk to an operator and the environment. Such processes are typically classified at biosafety risk level 2 and 3. From a biosafety perspective, the cell culture vessel is the first barrier protecting the environment against pathogenic organisms, whereas the room in which the system is installed is described as the secondary containment. An elevated risk is associated with failures of the first barrier when using single-use bioreactors because the materials of construction are inherently more fragile than stainless steel.

Manufacturers should challenge single-use bioreactor suppliers on how they mitigate potential risks of bag rupture during transportation, storage, installation, and use. Furthermore, they should understand how ruptures can be detected and how proper containment can be ensured in the event of a bag failure.

Biosafety Risk Assessment of Biostat STR and flexsafe STR Technology

Sartorius Stedim Biotech (SSB) focused heavily on robustness and risk mitigation measures during the development of the single-use, stirred bioreactor BIOSTAT STR and Flexsafe STR bags. The sources of bag ruptures can have a number of root causes. Appropriate safety measures were put in place to mitigate the risk of a rupture depending on the specific source of risk. During product development, four critical questions were considered when evaluating the risk of bag ruptures:

1. What could cause damage to the single-use bioreactor?
2. How could the risk of damage be mitigated?
3. How could potential damage be detected?
4. How can the hazardous liquid be contained in the event a rupture occurs?

FMEA Methodology

A Failure Mode and Effect Analysis (FMEA) methodology allowed a thorough evaluation of risks and areas on which to focus. The criticality of a risk was assessed by multiplying four factors that had to be quantified:

• I: Impact (1–3)
• O: Occurrence (1–3)
• D: Detectability (1–3)
• A: Action response time (1–3)

Products can be classified according to the quantified risk as follows:

• Low risk: 1–3
• Medium risk: 4–6
• High risk: 7 and above

Risk Analysis BIOSTAT STR with Single-Use Flexsafe STR Bags

Figure 1 describes sources of potential bag damage. Proper protection of the plastic material is required against high temperatures, cutting objects, friction and overpressure.

Biosafety Risk Mitigation Approach with Biostat STR and Flexsafe STR Technology

Risk Mitigation Preventing Bag Ruptures

Temperature Protection
Two heat sources must be considered when a single-use bioreactor is operated: the filter heater used for filter protection against filter blockage and the temperature control system used to heat up the bioreactor through the double wall bag holder. The filter heater is in direct contact with the exhaust filter whereas the heated bag holder is in direct contact with the Flexsafe film. Safety measures are required based on the temperature resistance of the filter and Flexsafe film.

Exhaust Filter
The filter heater is installed around the exhaust filter to protect against filter blockage due to excessive humidity. Standard Sartofluor filters are used with the Flexsafe STR that withstand sterilization cycles of up to 134 °C for 30 minutes. However, resistance to long-term heat exposure at temperatures above 65 °C has yet to be demonstrated. Therefore, to protect against overheating, a safety shut down mechanism should be installed that is activated at the appropriate temperature. In the BIOSTAT STR technology, a three-staged safety mechanism is in place, ensuring that the filter heater control loop is limited to a defined maximum temperature. Table 1 shows the temperature shut-off levels. An automatic and reversible filter heater shut-off is activated at the first safety shut-off temperature limit. An independent and nonreversible shut-off at the second safety temperature limit occurs if the first limit is breached. The final mechanical shut off ensures safe use of the filter heater even in the event of a malfunction of the first two safety levels.

Flexsafe Film
During development of the Flexsafe STR bags, various tests were performed to evaluate both short-term and long-term stability against temperatures that can occur during cell culture processes. The long-term stability was evaluated to ensure safe usage of the Flexsafe STR bags under normal cell culture conditions. Testing confirmed that the Flexsafe STR bags can be used safely for a minimum of 21 days at a temperature range between 16–40 °C.

The short-term stability was determined to ensure that the single-use bag could withstand higher temperatures for a limited time during the initial heating phase of the medium in the bioreactor. Tests have been performed to help define the temperature safety shutdown limits in case of malfunction of the temperature control system. These tests confirmed the resistance of the film material to temperatures of 2–43 °C for 24 hours. Further tests confirmed the film is resistant to short-term temperature peaks of up to 60 °C for two hours.

During the heating phase, a temperature overshoot beyond the set point has been limited to ±1 °C inside of the bag. The jacket, however, is allowed to reach higher temperatures, below the determined limits that the film can withstand, for a defined period of time.

Temperature safety measures have been established to ensure immediate correction and stabilization of permissive temperatures in case of unexpected overheating. The critical temperature based on these tests is 60 °C across the film material. However, in the event of overheating of the double wall, there is a time lapse to be considered because it takes some time for the temperature to transfer from the temperature circulation system through the stainless steel of the bag holder to the film material of the bag. Safety measures have been designed in such a way that they automatically switch off the heating elements of the electrical heater. The temperature linked to the activation of these safety interlocks is measured directly in the jacket loop by a standard pT100 sensor. When a temperature of 60 °C is reached, a first shut-off is initiated. In case of malfunctioning of the first shut-off, an alarm signal is generated by a mechanical switch inside the heating element at a temperature of 70 °C (± 5 °C). A second, independent safety temperature limiter measures the temperature inside the electrical heater and turns the heater off if 90 °C (± 8 °C) is reached. This mechanical limiter can be reset only manually.

These multistage cascaded safety measures offer full protection against overheating, not only in case a temperature control failure occurs, but also in case the first safety measure fails. This back-up safety measure has been established to offer maximum reliability and risk mitigation.

Overpressure
The three main reasons a pressure increase may occur are linked to gas flow, specific feeding strategies, or filter blockage.

Both short-term and long-term stability tests were performed to evaluate the robustness of the Flexsafe film against high pressures. The long-term film stability was evaluated to ensure safe usage of the Flexsafe STR bag during normal cell culture conditions. The short-term resistance defines the worst-case scenario in the event of an abnormal overpressure event.

Long-term Pressure Resistance
Worst-case robustness trials were performed during the qualification of the Flexsafe STR bags. Bags of all sizes were tested in a standard STR bag holder under the following test conditions:

• Maximum working volume
• Maximum stirrer speed
• Temperature: 40 °C
• 30 mbar pressure
• Duration: 21 days

All Flexsafe STR bags passed these tests successfully. At the end of the robustness test, the pressure was increased in 5 mbar steps to 50 mbar. The pressure was maintained for one hour at each 5 mbar increment. The results are documented in the Flexsafe STR Validation Guide and are available on request.

These robustness trials confirm that Flexsafe STR bags can be used safely at operating pressures below 30 mbar.

Short-term Pressure Resistance
Safety measures are required to avoid abnormal pressure increases beyond critical values during cell culture processes. Exhaust filter blockages often cause such pressure increases. In such an event, the pressure will increase rapidly and exponentially as shown in Figure 2. Therefore, short-term pressure resistance tests are sufficient to define critical pressure values.

To define the short-term pressure resistance of the film, further tests were performed on selected bags from the successfully completed, worst-case robustness tests described above. After the 21-day robustness test, the pressure was increased gradually to 110 mbar and maintained at 110 mbar for 1 hour. Tests performed on the Flexsafe STR 2,000-L bag can be considered worst-case conditions because pressure resistance increases as bag sizes decrease. Figure 3 describes the test for a Flexsafe STR 2,000-L bag. The test is successful because the bag does not leak or burst.

Similar tests have been performed on Flexsafe STR bags, and the results are shown in Table 2. To ensure operator safety, no burst test was performed on a Flexsafe STR 2,000-L bag. The tests show the pressure resistance of the bags up to 110 mbar, which is above that at which the last safety measure is activated (70 mbar).

The Flexsafe film shows superior performances in comparison to other PE films (Figure 4).

Bag bursting pressure are far above the pressure at which safety systems are activated. Also, S80 film robustness has been compared with other PE films and demonstrate superior mechanical characteristics. Results are shown in Figure 4.

Based on this data it was decided to implement a three-stage cascaded pressure safety approach. An electrical safety switch will shut down gassing when a pressure of 50 mbar is reached inside the bag. Gassing is automatically reactivated in case the pressure returns below 30 mbar. This reactivation will avoid cells becoming short of oxygen in the event of shortterm pressure peaks.

The pressure could also increase due to the action of pumps that are adding fresh media or other feeds to the single-use bag. Therefore, in the event that the pressure continues to rise after the first shut-off, a second electrical safety switch
is activated at 60 mbar that will deactivate all connected pumps. The pumps will automatically be reactivated once the pressure recovers to below 60 mbar. As a final safety measure, a pressure relief valve is installed in the system, it will open at 70 mbar.

Filter Blockage
Various solutions to mitigate the risk of filter blockage on Flexsafe STR bags are available. The most commonly used solution is the use of a heater around the exhaust filter. The filter heater heats up the condensate in the exhaust gas entering the filter and hence reduces the relative humidity. This allows the gas to pass through the filter without causing blockage. The use of such a filter heater has proven successful for the vast majority of cell culture processes. However, a traditional filter heater might not be sufficient for highly demanding processes for which high f low rates are used for a long period of time (e.g., continuous processing). To illustrate this, Figure 2 shows the speed of filter blockage when continuously applying a gas flow of 0.1 vvm in a STR 200-L bag filled with deionized water and heated to 37 °C.

To offer a second option of risk mitigation for more demanding processes, SSB has developed a single-use exhaust cooler (Photo 1), which is installed upstream of the exhaust filter.

The single-use exhaust cooler is a plate heat exchanger in which cooling water circulates in the opposite direction to the exhaust gas f low and cools the gas leading to vapor condensation (Figure 5). The condensate is collected in a condensate trap from which it can either be pumped back to the bag chamber or alternatively to a waste bag.

Figure 2 describes the enhanced efficiency of the single-use exhaust cooler when protecting the filter against humidity in the exhaust gas at normal to medium f low rates. Gas flow rates of 0.4 vvm were used to fully test the capacity of the device. This is twice as high as the maximum possible flow rate in a BIOSTAT STR unit.

Figure 6 shows that even under such extreme conditions, the single-use exhaust cooler offers a unique solution against filter blockage caused by high humidity in the exhaust gas. The protection offered by a traditional filter heater under these conditions is not efficient.

Finally, excessive foaming can cause filter blockages. To mitigate the risk of foam reaching the exhaust filter, the Flexsafe STR bags were designed with a large headspace representing 23–29% of the total bag volume (Table 3). This results in a single-use bag design with one of the largest headspaces of all single-use bioreactors on the market. This provides maximum protection to the filter from foam.

In the event that filter blockage does occur, every Flexsafe STR exhaust line is designed so that a new, second exhaust filter line can be manually connected to the single-use bag. This avoids a premature end to the cell culture. SSB also offers an option where a valve automatically opens a preinstalled, second exhaust line without the need for human intervention. This is triggered at a specific pressure limit in the exhaust line. It further mitigates the risk of bag rupture in the event that no operators are present in the production facility when filter blockage occurs.

We conclude that the sum of these safety measures, in combination with the pressure resistance of the Flexsafe STR film, ensures proper protection against overpressure that could occur, especially during high−cell-density cell culture processes.

Friction and cutting objects
Contact of bags with cutting objects should be avoided at all times during manufacturing, transportation, and handling. The use of sharp objects is not permitted in the manufacturing area of SSB to avoid accidental damage to the bags.

Furthermore, SSB has developed a transportation box for the STR bags with a specially designed support frame that not only ensures overall protection during transport and storage, but also ensures that no sharp objects can accidentally damage the bag (Photo 2). During transport, single-use bags are exposed to various vibrations and shocks. This could result in friction where tubing and connectors are in direct contact with the bag chamber, leading to damage over time. The specifically designed packaging frame includes special compartments to hold the different feeding and harvesting lines on the top and bottom. In this way, contact between parts is prevented, and no friction can occur during transport.

During unpacking, special markers indicate the exact location where the inner plastic protection can be opened without risk of bag damage (Figure 7).

No specific measures are required to protect the bag chamber against friction during use because no moving parts contact the bag chamber, and hence, no bag rupture due to friction can occur.

Finally, to ensure full protection against cutting objects, we recommend users to avoid having sharp objects in close proximity of the bioreactor. This helps prevent accidental damage during bioreactor operation once the bag is installed.

Risk Mitigation Concept in Case of Bag Rupture

Bag Damage Before the Start of the Cell Culture Process
The quality assurance programs of bag manufacturers are designed to ensure leak-free single-use bags upon delivery. After delivery, however, various risk factors still exist that can affect the integrity of a single-use bag. In contrast to a stainless steel bioreactor, a high degree of manual operation is required to transport a single-use bioreactor bag from storage to the cleanroom and prepare the single-use bag for installation and use. Various manual connections must be made to connect feeds to the bioreactor bag. This implies the need for stringent operator training and the implementation of suitable standard operating procedures (SOPs). Additional safety measures may be useful to consider along with clear instructions and regular training.

SSB has developed a unique, automated tool that allows the testing of Flexsafe STR bioreactor bags for leaks after installation but before use. This helps detect a damaged bag or bad connection. The pressure-decay test is performed using the Sartocheck 4 Plus bag tester after installation of the Flexsafe STR bag in the corresponding bag holder and once all feed lines have been connected (Photo 3). This device can measure a pressure drop over time and enables detection of potential leaks that might have been introduced during storage, unpacking, and installation.

This device detects damage until the point at which tubes are clamped. That allows for detection of improper manual connections that could result in a loss of integrity. It helps to detect potential damages accidentally introduced through human failure and offers a unique solution to increase the risk mitigation level when working in biosafety level 2 environments.

A patented fleece is available from SSB that can be installed inside the bag holder during the test. The fleece is made of a porous material that allows gas to pass through even when the fleece is pushed against the stainless steel surface. Hence, masking caused by the bag being pushed against the stainless steel bag holder wall during the pressure decay test is prevented, and damage to the bag can be detected regardless of its location. The minimum detectable leak sizes are shown in Table 4. Further details on this have been published (1).

Bag Damage Occurring During the Cell Culture Process
To ensure full risk mitigation and offer a maximum level of operator safety, a worst-case scenario where bag rupture occurs during the cell culture process must also be considered.

In such cases, it is important that the contamination be contained as efficiently as possible with limited or no exposure to operators and the environment. The BIOSTAT STR has been designed so that in the event of a bag rupture, the liquid can leave the surrounding bag holder either only through the bottom opening or through the sensor windows at the side of the bag holder. In such cases, a downward flow of liquid is most likely to occur with liquid leaving the bag holder through the bottom port.

Finally, it is critical that the spilled cell culture liquids do not spread across the cleanroom area but instead are collected and inactivated appropriately. SSB has developed a containment tray that can be placed directly under the bag holder (Photo 4). This containment tray can collect up to 25% of the maximum working volume of the bioreactor. This is usually enough to cover the volume of cell culture broth that could leak out of a single-use bioreactor before detection. Furthermore, the containment tray has been designed such that the spilled liquid is brought into a groove in the tray from where the contaminated culture can be evacuated through a tri-clamp connection to a kill tank. This ensures that no spillage can occur in the clean room area, and collected liquid can be safely removed for further processing.

Ensuring Operator safety
Figure 8 summarizes the most important safety measures established in the Flexsafe STR and BIOSTAT STR technology for safe use of singleuse bioreactors in a Biosafety Level 2 or 3 environment.

Considering the biosafety risks that are inherent to a vaccine manufacturing process, the proper selection of a single-use bioreactor vendor is critical. Operator safety should be a significant concern is selection of a single-use bioreactor technology for use the vaccine manufacturing. It is extremely important for users to challenge singleuse bioreactor vendors on their approaches to biosafety risk mitigation to allow a company to make well-founded decisions regarding its future bioreactor platform.

The different safety measures offer a holistic approach covering the different identified risks when working in a biohazardous environment. Irrespective of whether problems are linked to human interactions, system failures, or process parameters, the BIOSTAT STR and Flexsafe STR technology is designed to mitigate these risks. Moreover, the fact that safety measures are incorporated through a staged and complementary approach provides full protection in a worst-case situation when biohazardous material leaks from the primary containment of the bioreactor. This holistic approach makes the BIOSTAT STR and Flexsafe STR bag technology one of the most secure solutions for the vaccine industry and the preferred solution for many vaccine manufacturers around the world.

Reference
1. Stering M, et al. Pressure Decay Method for Post-Installation Single-Use Bioreactor Bag Testing. BioProcess Int. 12 (Supplement 5): 58‒61. c Davy De Wilde is Director of Commercial Operations EMEA, Joerg Weyand is Director of Marketing Fermentation Technologies, Ute Husemann is Manager STR Characterization, Bernward Husemann is R&D Manager Bioprocess Instruments, and Gerhard Greller is R&D Director Upstream Technology at Sartorius Stedim Biotech.