Abstract
Adventitious viral contamination in mammalian cell culture manufacturing facilities can lead to loss of product due to regulatory concerns regarding potential health risks. These events can also result in manufacturing shutdowns for extended periods of time. Numerous measures are currently taken to minimize these risks. Nonetheless, raw materials remain a high-risk entry point for viral contamination of mammalian cell cultures. Two virucidal technologies, ultraviolet radiation in the C band and high-temperature short-time pasteurization, were tested for the treatment of mammalian cell culture media. The results demonstrated no impact to the cell culture process or the quality of the products produced at the chosen dosage while providing robust viral protection.
Introduction
Measures are in place to protect against adventitious viral contaminations in all cell culture manufacturing facilities. These include cell line characterization, material sourcing, in-process intermediate testing, and control of material and personnel flow.
These measures are effective but do not address raw materials as a potential source of viral entry. Media suppliers do not adhere to the same rigors as a current good manufacturing practice (cGMP) manufacturing facility and have little control over the materials once they leave their facility. Low-level viral contamination of a raw material or the raw material container could result in a contaminated cell culture process. Mammalian cell culture media are routinely protected from microbial contamination through sterilization-grade filtration; however, a similar prophylactic use of viral retentive filters is not economically feasible. A robust inactivation technology that can be conducted after all raw materials have been added and the media is ready for use is a viable option to mitigate the viral contamination risk posed by raw materials. There are two proven technologies for controlling virus contamination in large volume applications: high-temperature short-time (HTST) pasteurization and treatment with ultraviolet radiation in the C range (UVC). These technologies have been used in the biotechnology, food, plasma, and water treatment industries with success.
Experiments were conducted to assess the impact of these technologies on process and product for three mammalian cell culture processes. Viral inactivation data were also generated with two model viruses over a range of conditions for both UVC and HTST. The viruses tested included murine minute virus (MMV), which is a small non-enveloped virus, and Cache Valley virus (CVV), which is a medium-sized enveloped virus.
Materials and Methods
Each cell culture process was run using a qualified scale-down model for both cell culture and purification. The HTST media treatment targeted 102 °C for 10 s with robustness testing at 115 °C for 30 s. Limited data was available for UVC treatment of mammalian cell culture media, so a broad range (15 mJ/cm2 to 3000 mJ/cm2) of doses was evaluated. The highest treatment dose that had no impact to the various cell culture processes and that was practical from a process duration perspective was chosen for the final testing.
UVC treatment was completed using a bench-scale UVC device employing a helical flow path. The device had an output maximum at wavelength 254 nm. The dose applied to media was determined using a quenchable fluorescent microsphere assay that could be used to correlate the dose delivered based on standard curves generated with a collimated beam-type device.
HTST treatments were performed using a bench-scale processing system. This instrument employed a heat exchanger system for treating liquids at a high temperature for a short amount of time for virus inactivation and disinfection. The unit is a scalable tubular heat exchanger with sections configured for preheating, heating, and cooling of the solution to be treated. A pressurized hot water system is used for heating and chilled water is used for cooling. The flow of the heat exchange fluid is counter-current to product flow for more efficient heat transfer.
Impact to the molecules tested was assessed using several analytical assays. These included size exclusion chromotography, oligosaccharide mapping, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), peptide mapping, cation exchange chromotograpy, capillary zone electrophoresis for isoform distribution, and biological activity assay.
Viral inactivation studies used common cell culture-based tissue culture infective dose 50% infectivity (TCID50) viral assays, as well as quantitative polymerase chain reaction (qPCR) technology.
Results
The impact to the cell culture process was minimal in the three processes tested for both HTST and UVC. Extreme time-temperature HTST conditions of 115 °C for 30 s resulted in a minor impact to cell growth but were acceptable. This demonstrated that the processes were not on a performance edge under set point conditions of 102 °C for 10 s and supported that excursions beyond the set point would not likely result in process failure.
The UVC testing at the extreme of 3000 mJ/cm2 also demonstrated a minor impact to cell growth, but provided acceptable results and indicated that the process tolerance for UVC is high. The final UVC dosage chosen was 125 mJ/cm2. This fluency dose provided significant viral inactivation while allowing for the media to be treated in a time frame that would be acceptable for large-scale production. Figures 1⇓–3 depict growth, viability, and product titer data, respectively, from one of the processes tested. These data are representative of the two other cell culture processes evaluated.
Growth data from HTST- and UVC-treated cell culture media.
Cell viability data from HTST- and UVC-treated cell culture media.
Protein production data from HTST- and UVC-treated cell culture media.
Representative analytical data to assess product impact are presented in Figures 4, 5, and 6. These data are from HTST experiments, but similar results were observed in those experiments that employed UVC media treatment. All quality testing including high temperature and time robustness for HTST and high UVC treatment conditions demonstrated no impact to product. It is worth noting that product quality was comparable to control even under the extreme conditions evaluated where minor cell culture impact was observed.
Sialyated glycan profile from representative HTST study.
SE-HPLC profile from representative HTST study.
Lys-C peptide map from representative HTST study.
Viral inactivation studies with MMV and CVV revealed robust viral inactivation under set point condition for each technology. Viral inactivation studies carried out with media treated under lower than set point stringencies still produced effective inactivation. This illustrates the robustness of the two technologies. Figures 7 and 8 illustrate representative viral inactivation results. A summary of the UVC inactivation results achieved with MMV is presented in Figure 7. These data demonstrate that ≥6 logs of MMV reduction is achievable with a UVC dose as low as 15 mJ/cm2. Figure 8 demonstrates significant viral inactivation using HTST treatment conditions below the set point of 102 °C for 10 s.
Viral inactivation summary from UVC viral studies.
HTST robustness viral inactivation study.
Discussion
Media treated using HTST or UVC technology were observed to be relatively benign in the cell culture processes tested with respect to process and product impact. Both of these technologies can be controlled to a very tight tolerance around set point conditions. As such, the low/high conditions that were tested are likely to be well outside that range experienced in a manufacturing setting. Interestingly, even when the process experienced slight impact related to cell growth, the quality of the therapeutic protein being produced was not compromised. Testing of three different cell culture processes with similar results provides confidence that both technologies are potentially viable options. A fourth mammalian cell culture process that contains fetal bovine serum (FBS) in the media was also tested. The FBS-containing media was not compatible with HTST treatment. The same process was minimally impacted (titer decrease) by UVC treatment. As such, a lower UVC dose relative to the other processes evaluated may be necessary to make this a viable option.
The viral inactivation studies demonstrated the ability of these treatment technologies to provide robust virus protection. The primary goal of such treatment is to significantly reduce risk of viral entry into cell culture processes. The introduction of viral particles to cell culture media would more than likely be at a very low initial level; however, cell culture propagation of the virus would exacerbate these low-level contamination events. High viral loads are not expected in any one component, and subsequent dilution via other components would further dilute the titer. The viral clearance data in conjunction with the process and product impact data provide a compelling reason to expect that these technologies are viable risk mitigation strategies.
Conclusion
The repercussions of a viral contamination event in a manufacturing facility are considerable. Efforts to reduce this risk are likely to provide benefits from a financial as well as a regulatory perspective. The multitude of components that typically go into mammalian cell culture media provide multiple entry points for virus contamination from either the components or the containers that they are shipped in. Viral agents can be introduced on the supplier end before blending the media, during shipment to the manufacturing site, or during media preparation at a manufacturing facility. Once a viral particle enters the media, removal via sterilization-grade filtration is not an effective option. A media treatment method that could be applied after all media components have been compounded is likely to provide the most robust barrier to viral entry into cell culture processes.
HTST and UVC media treatments were tested on several mammalian cell culture processes to assess the impact to process and product quality. The effectiveness of these technologies for viral inactivation was also assessed. The results indicated that both of these technologies provide a safe and robust method of media treatment to reduce the risk of a viral contamination in a cell culture manufacturing facility.
- © PDA, Inc. 2010
