Author: Chen Wang, Jiaqi Wang, Min Chen, Li Fan, Liang Zhao*, Wen-Song Tan.
Biotechnology Letters, 2018, 40(8): 1201-1208

Abstract
Objective To explore the influence of ultra-low carbon dioxide partial pressure (pCO2) on the monoclonal antibody (mAb) N-glycosylation profile in Chinese hamster ovary (CHO) cell culture. Results In fed-batch bioreactor cultures, lowering the pCO2 in the medium (<25 mmHg) via increasing headspace aeration decreased the cell viability and mAb production in CHO cells. Additionally, mAb galactosylation under low pCO2 was approximately 27.45 ± 2.13%, noticeably higher than that observed under normal pCO2 (21.36 ± 1.66%) at harvest. However, all of the relevant intracellular nucleotide sugar concentrations were dramatically decreased to approximately 50% of the levels found under normal pCO2 on day 7. Real-time PCR revealed that the upregulation of galactosylationrelated glycosyltransferase genes and substrate transporter genes played a critical role in the improved galactosylation under the ultra-low pCO2 condition. Conclusions In the bioreactor culture processes, ultra-low pCO2 demonstrated a positive effect on mAb galactosylation.

Author: Mao Zou*, Zi-Wei Zhou, Li Fan, Wei-Jian Zhang, Liang Zhao, Xu-Ping Liu, Hai-Bin Wang, Wen-Song Tan.
Journal of Industrial Microbiology & Biotechnology, 2020, 47(1): 63-72

Abstract
As the composition of animal cell culture medium becomes more complex, the identification of key variables is important for simplifying and guiding the subsequent medium optimization. However, the traditional experimental design methods are impractical and limited in their ability to explore such large feature spaces. Therefore, in this work, we developed a NRGK (nonparametric regression with Gaussian kernel) method, which aimed to identify the critical components that affect product titres during the development of cell culture media. With this nonparametric model, we successfully identified the important components that were neglected by the conventional PLS (partial least squares regression) method. The superiority of the NRGK method was further verified by ANOVA (analysis of variance). Additionally, it was proven that the selection accuracy was increased with the NRGK method because of its ability to model both the nonlinear and linear relationships between the medium components and titres. The application of this NRGK method provides new perspectives for the more precise identification of the critical components that further enable the optimization of media in a shorter timeframe.

Author: Mengjuan Liu, Jiaqi Wang, Hongping Tang, Li Fan, Liang Zhao, Hai-Bin Wang, Yan Zhou*, Wen-Song Tan.
Biotechnology Letters, 2018, 40(11): 1487-1493

Abstract
Objective To explore the impact of taurine on monoclonal antibody (mAb) basic charge variants in Chinese hamster ovary (CHO) cell culture. Results In fed-batch culture, adding taurine in the feed medium slightly increased the maximum viable cell density and mAb titers in CHO cells. What’s more, taurine significantly decreased the lysine variant and oxidized variant levels, which further decreased basic variant contents from 32 to 27%. The lysine variant content in the taurine culture was approximately 4% lower than that in control condition, which was the main reason for the decrease in basic variants. Real-time PCR and cell-free assay revealed that taurine played a critical role in the upregulation of relative basic carboxypeptidase and stimulating extracellular basic carboxypeptidase activities. Conclusion Taurine exhibits noticeable impact on lower basic charge variants, which are mainly due to the decrease of lysine variant and oxidized protein variants.

Author: Yixiao Wu, Hanjing Jia, Hanzhang Lai, Xuping Liu*, Wen-Song Tan.
Bioresources and Bioprocessing, 2020, 7: 63

Abstract
The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.

Author: Yixiao Wu, Thomas Bissinger, Yvonne Genzel, Xuping Liu*, Udo Reichl, Wen-Song Tan.
Applied Microbiology and Biotechnology, 2021, 105(4): 1-14

Abstract
Similar to the recent COVID-19 pandemic, influenza A virus poses a constant threat to the global community. For the treatment of flu disease, both antivirals and vaccines are available with vaccines the most effective and safest approach. In order to overcome limitations in egg-based vaccine manufacturing, cell culture–based processes have been established. While this production method avoids egg-associated risks in face of pandemics, process intensification using animal suspension cells in high cell density perfusion cultures should allow to further increase manufacturing capacities worldwide. In this work, we demonstrate the development of a perfusion process using Madin-Darby canine kidney (MDCK) suspension cells for influenza A (H1N1) virus production from scale-down shake flask cultivations to laboratory scale stirred tank bioreactors. Shake flask cultivations using semi-perfusion mode enabled high-yield virus harvests (4.25 log₁₀(HAU/100 μL)) from MDCK cells grown up to 41 × 10⁶ cells/mL. Scale-up to bioreactors with an alternating tangential flow (ATF) perfusion system required optimization of pH control and implementation of a temperature shift during the infection phase. Use of a capacitance probe for on-line perfusion control allowed to minimize medium consumption. This contributed to a better process control and a more economical performance while maintaining a maximum virus titer of 4.37 log₁₀(HAU/100 μL) and an infectious virus titer of 1.83 × 10¹⁰ virions/mL. Overall, this study clearly demonstrates recent advances in cell culture–based perfusion processes for nextgeneration high-yield influenza vaccine manufacturing for pandemic preparedness.

Author: Thomas Bissinger, Yixiao Wu, Pavel Marichal-Gallardo, Dietmar Riedel, Xuping Liu*, Yvonne Genzel*, Wen-Song Tan.
Biotechnology and Bioengineering, 2021, 118(10): 3996-4013

Abstract
Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well-adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg-based vaccine production would be difficult to schedule and shortages of other egg-based vaccines with high demands also have to be anticipated. Cell culture-based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin-Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10(HAU/100 μl) were achieved using fast-growing MDCK cells at concentrations up to 9.5×106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane-based stericexclusion chromatography followed by pseudo-affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 μg hemagglutinin (single-radial immunodiffusion assay), the level of total protein and host cell DNA was 58 μg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4-5 days makes this process competitive not only to other cell-based processes but to egg-based processes as well.

Author: Jiaqi Wang, Chen Wang, Li Fan, Liang Zhao*, Wen-Song Tan.
Analytical and Bioanalytical Chemistry, 2019, 411(13): 2971-2979

Abstract
Chinese hamster ovary (CHO) cells are predominant in the production of therapeutic proteins to treat various diseases. Characterization and investigation of CHO cell metabolism in a quick and simple way could boost process and cell line development. Therefore, a method to simultaneously detect seven redox- and energy-related metabolites in CHO cells by capillary electrophoresis has been developed. An on-line focusing technique was applied to improve the peak shape and resolution by using a 50 μm× 44 cm uncoated fused silica capillary. Key parameters and their interactions were investigated by design of experiments (DoE) and optimized conditions were determined by desirability function as follows: 24 °C, 95 mM, and pH 9.4 of BGE. The method was validated to ensure sensitivity, linearity, and reproducibility. The limits of detection (LODs) ranged from 0.050 to 0.688 mg/L for seven metabolites, and correlation coefficients of linearity were all greater than 0.996. The relative standard deviations (RSD) of migration time and peak area were smaller than 0.872% and 5.5%, respectively, except for NADPH, and the recoveries were between 97.5 and 101.2%. The method was successfully applied to analyze the extracts from CHO cells under two different culture conditions.

Author: Qian Ye, Thu Phan, Wei-Shou Hu*, Xuping Liu, Li Fan, Wen-Song Tan, Liang Zhao*.
Viruses, 2021, 13(11): 2200

Abstract
The Madin–Darby Canine Kidney (MDCK) cell line is among the most commonly used cell lines for the production of influenza virus vaccines. As cell culture-based manufacturing is poised to replace egg-based processes, increasing virus production is of paramount importance. To shed light on factors affecting virus productivity, we isolated a subline, H1, which had twice the influenza virus A (IAV) productivity of the parent (P) through cell cloning, and characterized H1 and P in detail on both physical and molecular levels. Transcriptome analysis revealed that within a few hours after IAV infection, viral mRNAs constituted over one fifth of total mRNA, with several viral genes more highly expressed in H1 than P. Functional analysis of the transcriptome dynamics showed that H1 and P responded similarly to IAV infection, and were both subjected to host shutoff and inflammatory responses. Importantly, H1 was more active in translation and RNA processing intrinsically and after infection. Furthermore, H1 had more subdued inflammatory and antiviral responses. Taken together, we postulate that the high productivity of IAV hinges on the balance between suppression of host functions to divert cellular resources and the sustaining of sufficient activities for virus replication. Mechanistic insights into virus productivity can facilitate the process optimization and cell line engineering for advancing influenza vaccine manufacturing.