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Reducing Risks with Disposable Technology

Mon, 09/15/2008 - 6:12am

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Filtration and mixing characteristics of hydrolysate containing cell culture media

Cell culture media is a complex mixture of synthetic and biological components that, in an aqueous solution, provides the proper chemical environment and necessary

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Figure 1
nutrients for healthy cell propagation and high protein expression. Mammalian cell culture growth media remains an evolving technology in which change is driven by the expanding variety of production cell lines and by concerns for product safety and purity. Mycoplasma contamination in particular, is a great concern for biopharmaceutical manufacturers, as these small bacteria are difficult to detect in cell culture. This case study presents our recommended methods for thoroughly mixing hydrolysate-containing media, and considerations for choosing and evaluating a sterilizing-grade filter to meet your needs for improved process efficiency and reduced risk of microbial contamination.

Media Types

Today’s cell culture media span many media types and formulations to meet the needs of different cell lines. In response to increasing pressure from regulatory agencies to eliminate potential risk from adventitious agents, such as prions and viruses, the industry is moving towards media formulations that do not contain animal-derived components. Plant-derived hydrolysates, such as LucraToneTM Soy P hydrolysate in DMEM basal media, are a commonly used substitute for animal-derived cell culture media additives. Plant hydrolysates are created from plant material that is enzymatically digested to produce a mixture of peptides, amino acids, and other essential elements that support cell survival. Hydrolysates offer an inexpensive method for increasing cell growth and protein production. However, like animal-derived media additives, plant derived hydrolysates are not free from risk of mycoplasma contamination and filterability varies lot to lot, presenting filterability challenges.

Consistent Filtration Performance with Cell Culture Media

Extracts and peptones, commonly used serum substitutes, vary in filterability since they are derived from natural sources and their chemical and physical properties are dependent on environmental, process, and storage conditions. As a result, the robustness and consistency of the sterile filtration process for these variable feed properties is critical for proper production control. A cell culture developer must ensure that the filtration train can handle variation in feed and batch media. To address this need, it is recommended to characterize filter variability and to size the filtration system appropriately.

Towards this objective, Millipore initiated a case study to mimic the way a cell culture developer would assess filter lot variability with cell culture media. Throughput testing (to at least 80% of initial flow) was performed using three samples each (n=3) of Millipore Express SHR 0.1µm membrane from three different non-contiguous lots. Throughput testing was run at 10 psi constant pressure using one batch of 5 g/L LucraTone Soy P hydrolysate mixed with 10g/L DMEM. Results, shown in Figure 1, demonstrate that Millipore Express SHR 0.1µm membranes have minimal lot-to-lot variation in filterability.

Disposable Media Mix Consistency

Careful preparation of cell culture media is important to ensure sterility of the cell culture batch and subsequent purification processing. Media is typically a batch operation. The media is mixed in bulk and transferred under pneumatic pressure from a mix tank to the bioreactor through sterilizing-grade 0.1µm filters. A majority of the media used at production scale is in powdered form and is hydrated on site. Thus, the consistency and efficiency of the media mixing process is a critical component of cell culture fermentation.

The ideal mixing technology would reduce equipment floor space and raise plant productivity, efficiency and output. The Mobius MIX200 system uses a bottom-mounted, magnetically driven impeller

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Figure 2
inside a 3-D conical single-use process container made of Pureflex™ film. This high-purity, medical-grade film provides strength and flexibility and acts as a gas barrier. Mimicking the design of traditional stainless steel vessels, the Mobius MIX200 system minimizes foaming potential, particle generation and vessel contamination. As a disposable single use system, an advantage of the MIX200 is lower capital costs, reduced production floor space requirements, higher facility efficiency/output, elimination of cleaning steps and reduced validation requirements.

To meet these needs, Millipore initiated a case study to mimic the way customers mix and filter hydrolysate-containing media. The results of mixing using the MIX200 Disposable MIX technology and a single lot of Millipore Express SHR sterilizing-grade, mycoplasma removing 0.1 µm filters are presented in Figure 2.

Figure 2 shows the consistency of Mobius MIX200 system when mixing LucraTone hydrolysate. Samples taken from the top and bottom ports of the MIX200 system and filtered through a single lot of Millipore Express SHR membrane, show similar results for 5g/L LucraTone Soy P hydrolysate, demonstrating efficient mixing.

Mycoplasma Removal

Mycoplasma contamination is a critical concern in biopharmaceutical production processes. Mycoplasma are small, cell wall-deficient bacteria that are widely recognized as the smallest bacterium thriving in cell culture. They can be brought into a cell culture process from plant- or animal-derived raw materials as well from humans. Acholeplasma laidlawii, Mycoplasma arginini and M. hyorhinis are isolated from hydrolysates, cell lines and sera. M. fermentans, M. orale, and M. salivarium are isolated from humans, animals and cell lines.

Mycoplasma can be difficult to detect in cell culture because many species do not produce turbidity or cytopathic effects. Due to their diminutive size and deformability, mycoplasma can readily pass through 0.2 µm rated devices. As a result, media preparations increasingly call for the use of 0.1µm sterilizing-grade filters to ensure sufficient log removal of mycoplasma.

For bioreactor feed applications, membrane filters and devices are designed for various performance characteristics, such as capacity, permeability, and mycoplasma removal. Selection of a filtration device for mycoplasma reduction is based on knowledge of the process requirements and the bioburden. Capacity and bioburden reduction must be in balance to ensure a safe as well as efficient process.

Figure 3 illustrates mycoplasma reduction and permeability performance among three different manufacturers of 0.1 µm rated hydrophilic filters using an animal-free medium. In this study, membrane samples were tested simultaneously in three separate experiments using three separate cultures of A. laidlawii ATCC 23206. The average total challenge per membrane was 4.9 x 108 cfu/filter (± 5.4 x 107) and the average challenge per membrane area was 3.5 x 107 cfu/cm2 (± 3.9 x 106).


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Figure 3
Mycoplasma removal and permeability varied under the conditions of this test. The log reduction value among the membranes was different (ANOVA, a 0.05, P=0.000). Millipore Express SHR 0.1µm (n=10) membranes and Manufacturer A’s PES (n=9) membrane performed equivalently (Tukey post-test). However, Millipore Express SHR 0.1µm membranes exhibited less variability than those of Manufacturer A. The PVDF membrane from Manufacturer C (n=11) exhibited lower removal of A. laidlawii.

The flow time was measured using A. laidlawii challenge suspension transformed to permeability (LMH/psid). Millipore Express SHR 0.1µm membrane challenge permeability (n=6) was significantly higher than those of Manufacturer A (n=6) and Manufacturer B (n=8) (ANOVA, a 0.05, P=0.004).

Conclusion

This case study demonstrates the robustness and capability of Millipore’s products, LucraTone animal free cell culture supplements, single-use Mobius MIX200 disposable mixing technology, sterilizing-grade, mycoplasma clearance filters with Millipore Express 0.1µm membranes, used in combination to meet a cell culture developer’s needs of improved process efficiency and reduced risk of microbial contamination.
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