Analysis of Fortem bioprocessing film

Why end users want better bioprocessing films

While single-use technology is becoming more mainstream in the biopharmaceutical industry, several challenges remain, and system requirements continue to evolve. Over the past several years, additional focus has been placed on the materials used to construct bioprocess films. It is now known that extractable compounds throughout the film development process have the potential to negatively impact the growth of sensitive cell lines (1, 2).

As a result, the industry is moving towards developing single-use films better suited to the needs of biomanufacturing. Specifically, end users have highlighted the need for improved film performance, broader knowledge of material properties, and a more reliable supply chain.

When developing a bioprocess film, it is critical to map all the required material performance attributes across the entire workflow. Then, the film must be designed with the correct balance of these attributes to achieve the desired performance and reliability.

Optimizing film design for biomanufacturing

This article shows how we optimized resin selection and film architecture to design a bioprocess film built from the ground up for bioprocessing. Here details are provided on how the new Fortem™ film maintains critical performance attributes, such as container integrity and gas barrier properties, under the significant forces associated with rocking bioreactors and liquid transport.

Fortem technology is forged from a multi-year development effort with Sealed Air Corporation that enables a robust security of supply.

Material selection and film structure

The film is composed of ten discrete layers, which have been thoughtfully designed and placed to optimize the film architecture. In addition, the antioxidant package was designed to contain additives in specific locations minimizing their impact on mammalian cell culture performance.

The most internal layer, the fluid contact layer, is a blend of linear low-density polyethylene (LLDPE) and cyclic olefin copolymer (COC). This blend is compliant with EP 3.1.3, EP 3.1.5, JP 7.02, and USP Class VI regulations. The COC in the fluid contact layer also acts as a macromolecular slip agent, which eliminates the need for traditional small molecule additives. The combination of LLDPE and COC in the fluid contact layer allows for a clean, inert surface with advantageous welding properties.

The external layer is made of a nylon that provides strong abrasion and puncture resistance. This nylon layer is effective even in humid conditions and can withstand flex fatigue in rocking applications. Two gas barrier layers are in the middle of the film architecture. This allows for optimal gas exchange in both wet and dry conditions due to varying ratios of ethylene vinyl alcohol (EVOH).

Two larger layers composed of a polyethylene blend sandwich the gas barrier and tie layer complex. These larger layers are designed for robustness and flexibility over a wide temperature range. By placing the gas barrier layers in the neutral plane of the film width, they are shielded from any flexural forces that may be placed on the distal layers.

Overall, the film is 300 µm thick and each layer can be identified in Figure 1. The film is co-extruded as a mirror image double-ply film in a Class 8 cleanroom at Sealed Air Corporation’s US-based facility. Multiple film prototypes were developed and tested until the final composition was optimized for manufacturability and application performance.

Fig 1. Ten layers in Fortem film.

In addition, the material construction of the Fortem technology makes it well-suited for multiple applications that require a balance of robustness and flexibility. Figure 2 demonstrates how the unique mechanical forces of rocking bioreactors (WAVE Bioreactor) and fluid transportation applications can minimize impact on the film’s internal layers.

Fig 2. Placement of the less flexible gas barrier layers reduces the impact of external forces.

Housing the less flexible gas barrier layers in the center of the film’s cross-sectional thickness minimizes the impact of external forces. This is what differentiates Fortem technology from others available in the market.

Chemical development and manufacturing testing

A key attribute of a bioprocessing film is that it must not leach harmful compounds into the bags made from the film. One potential leachable, bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP), is a degradant of Irgafos™ 168, a polymer additive in polyethylene formulations. Literature shows that bDtBPP can adversely affect CHO cell line growth (1, 2). Therefore, bDtBPP presence in the film must be minimized and monitored. Throughout the film development and launch process, testing was performed to quantitate the amount of Irgafos (TBPP), oxidized Irgafos, and the degradant bDtBPP.

bis(2,4-di-tert-butylphenyl) phosphate (bDtBPP) analysis

In a preliminary study, Fortem film was gamma irradiated at 40–55 kGy. Samples of the film were added to glass bottles and extracted in water at 50°C for 3 and 7 days. A glass bottle was used as a negative control. The surface area to volume (SAV) ratio for the extraction was 0.37 cm²/mL. Analysis was performed using liquid chromatography-mass spectrometry (LC-MS) with a limit of detection (LOD) of 2 ppb.

Table 1. Concentration of bDtBPP found in extract

LOD = 2 ppb.

The results in Table 1 show that the concentration of bDtBPP found in the extract of Fortem film was below the LOD (2 ppb) at both the 3-day and 7-day interval.

Irgafos 168, oxidized Irgafos 168, and bDtBPP analysis

When determining the total amount of bDtBPP present, it is important to test for its precursors: Irgafos 168 and oxidized Irgafos 168 forms. Each lot of Fortem film is tested according to internal SOPs. Extractions are performed on two Fortem samples across the full span of the film lot using 50 mL of dichloromethane (DCM), for a final weight to solvent ratio of 0.5 g/10 mL.

LC/MS/MS in ACPI positive mode is used to quantitate Irgafos 168 and its oxidized forms. An LC/Q Exactive™ method is used in electrospray ionization mode to quantitate bDtBPP. These extractions and analyses are performed following the validated methods of Nelson Labs in Belgium.

Each lot of Fortem film is subjected to extraction and quantitation of Irgafos 168, oxidized Irgafos 168, and bDtBPP as part of the incoming inspection lot release criteria. Samples are taken throughout the manufacturing process. This analysis is done to ensure the total amount of these three compounds is ≤ 11 ppm per sample.

Results from two lots of Fortem film are shown in Table 2.

Table 2. Quantitation of Irgafos 168, oxidized Irgafos 168, and bDtBPP in Fortem film

Method detection limit (MDL) = 1 ppm; Method quantitation limit (MQL) = 0.4 ppm.

The results show that all test samples taken throughout the manufacturing process of two film lots contain acceptably low levels of Irgafos 168, oxidized Irgafos 168, and bDtBPP.

Cell growth analysis

To confirm that the levels of Irgafos and its degradants were not harmful to cell cultures, a confirmation study was performed. This study used the CHO DG44 cell line, which is sensitive to bDtBPP down to 0.1 mg/L (0.1 ppm).

Two 2 L Cellbag containers made with Fortem film were tested at our facility in Logan, Utah, USA. Each bag was filled with 200 mL (8 cm² of film/mL) of cell culture medium and incubated on a WAVE rocker at a 6° angle, 20 rpm speed, at 37°C for three days. This was followed by four days rocking incubation at room temperature.

The glass bottle control was filled with medium to achieve the same SAV ratio as the bag. The bottle was then incubated in a laboratory refrigerator for seven days at 4°C. All test articles and controls were protected from light during the study. After incubation, the medium from each bottle was harvested and used to run a three-day cell culture with mAb-producing CHO DG44 cells in shaker flasks. The initial viable cell density was 0.3 × 10⁶ cells/mL.

Population doubling was calculated using the following equations:

Cell viability was calculated using the following equation:

The average population doubling of the cultures exposed to the medium incubated with Fortem film was 98 ± 3%. The average cell viability was 94 ± 1%.

When combining the Irgafos extraction values with the lot release cell culture testing, we can verify that the film does not contribute levels of Irgafos degradant that impact cell cultures.

Film property testing

Multiple verification tests involving carbon dioxide transmission rate, oxygen transmission rate, and product shelf life were performed to ensure robust product development.

Carbon dioxide transmission rate

Many cell culture media contain carbonate buffers to help stabilize cell culture pH, because shifts in pH can impact cell growth and viability. Carbonate buffers are sensitive to the level of carbon dioxide. Therefore single-use systems, especially those used to store media, should provide a sufficient barrier to CO₂ exchange.

One of the film’s application requirements is that it must maintain a carbon dioxide transmission rate (CO₂TR) of < 1.35 cc/m²/d. To generate this data, samples from three lots of film were gamma irradiated to 42.0–51.3 kGy and tested according to ASTM F2476 Standard Test Method for the Determination of Carbon Dioxide Transmission Rate (CO₂TR) Through Barrier Materials Using an Infrared Detector. The test temperature was set at 23˚C, and the internal and external relative humidity (RH) were set at 0%, as stated in the Fortem film validation guide^*.

^*Access is limited.

Table 3. Carbon dioxide transmission rate of three Fortem film lots

LOD = 1 cc/m²/d.

The results in Table 3 show that all three lots of Fortem have a CO₂TR less than the instrument's LOD of 1 cc/m²/d.

Oxygen transmission rate

Fortem film must maintain an oxygen transmission rate (O₂TR) of < 1 cc/m²/d. For this study, three lots of film were gamma irradiated to 42.0–51.3 kGy. Testing was executed according to ASTM D3985 Standard Test Method for Oxygen Gas Transmission Rate (O₂TR) Through Plastic Film and Sheeting Using a Coulometric Sensor.

The temperature was set at 22.8°C and internal and external relative humidity (RH) was set at 0%. Then, an additional test was performed after changing the RH to 100% inside and 50% outside, as stated in the Fortem film validation guide.

Table 4 outlines the results of this testing. All three lots were below the limit of detection of < 0.2 cc/m²/d for the instrument used in this study.

Table 4. Oxygen transmission rate of three Fortem film lots

LOD = 0.2 mL/m²/d.

Certain proteins and excipients commonly used in biopharmaceutical formulations are at risk of oxidative degradation. In order to mitigate this risk, Fortem film must provide a robust enough barrier to oxygen gas. The results in Table 4 show that Fortem consistently has an O₂TR of < 0.2 mL/m²/d regardless of humidity levels. This low rate helps prevent oxidative degradation of the bag contents.

Shelf life involving flex fatigue and cell growth analysis for rocking bioreactors

Understanding product shelf life is key to ensuring a product will meet customer needs at the point of use. The WAVE rocking bioreactor product line (Cellbag containers) is available with Fortem technology and includes a well-substantiated 2-year post-manufacture shelf life claim.

For this testing, multiples of 50 L and 2 L WAVE bag assemblies were made from two different Fortem film lots. All samples were gamma irradiated at 40–55 kGy and subjected to accelerated aging at a Toxikon facility. Samples were kept at 50˚C for 21 weeks to model 2.25 years of real-time aging. A total of five 50 L WAVE bags made from two Fortem film lots were tested for flex fatigue endurance according to internal verification procedures.

This testing involves mounting the bags on 50 L WAVE Bioreactor rockers, filling each with 25 L of distilled water. Bags were subjected to rocking for 35 d at 25 rpm, a 9˚ angle, 37˚C, and 0.25 L/min compressed air flow. Each bag was inspected twice daily for film whitening, delamination, seal quality, and visual defects.

After the 35-day testing timeline, each 50 L Cellbag container was inspected comprehensively for defects. All bags were visibly intact. One spot of slight whitening was observed on each of two bags in areas that see the most stress from the rocking motion. The appearance of slight whitening did not compromise the integrity of the test articles.

Additionally, four of the 2 L Cellbag containers subjected to accelerated aging were sent to our Logan facility for cell growth testing. Each 2 L bag was filled with 200 mL of HyClone CDM4CHO medium to achieve an SAV ratio of 8 cm² of film/mL. The test articles were mounted on WAVE Bioreactor trays and rocked at an 8˚ angle, 20 rpm speed, and 37°C for three days. After this incubation period, the test articles were protected from light at room temperature for four days.

Two controls were performed. Control 1 was a bottle containing CDM4CHO medium stored in the dark in a refrigerator (2°C–8°C). Control 2 was a bottle containing CDM4CHO medium and incubated on the WAVE bioreactor alongside the test samples. Media from both controls and each test article was harvested in triplicate. CHO-S cells were seeded with a concentration of 3 × 10⁵ cells/mL into each flask. The cultures were grown for 72 h at 37°C, 5% CO₂ in an incubator with an orbital shaker set at 120 rpm.

Results of the cell growth are reported as averages in Table 5.

The following equations were used for calculations, where PD refers to population doubling:

Table 5. Growth performance and cell viability of medium incubated with Fortem film

As shown in Table 5, growth of CHO-S cells in medium incubated with Fortem film is comparable to growth of controls. Also, cell viability remains high at > 98%.

A film designed for biologic manufacturing

To keep pace with the needs of the biomanufacturing industry, the films used in single-use bioprocess technology should be purposefully designed for bioprocess applications. With Fortem technology, the combination of resin selection and film architecture has been selected and optimized to meet end user needs. It achieves critical to quality attributes across applications as evidenced by data included in this article.

Learn more about Fortem platform film for bioprocessing.

References

1. Steiger, N. and Eibl, R.  Interlaboratory test for detection of cytotoxic leachables arising from single-use bags. Chemie Ingenieur Technik 85, 26–28 (2013).
2. Hammond M. et al.  Identification of a leachable compound detrimental to cell growth in single-use bioprocess containers. PDA Journal of Pharma. Sci. and Technol. 67, 123–134 (2013).