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.

反应器培养CHO细胞生产单抗过程中过低的二氧化碳分压导致抗体半乳糖基化水平升高
目的：研究过低的二氧化碳分压（pCO2）对中国仓鼠卵巢（CHO）细胞培养产生的单克隆抗体N-糖基化的影响。
结果：在反应器流加培养过程中，通过增加表层通气来降低培养基中的 pCO2 (<25 mmHg)这一方式会降低CHO细胞的细胞活力及单抗产量。此外，在过低的pCO2条件下，单抗糖基的半乳糖化为27.45±2.13%，显著高于正常pCO2条件下收获的单抗糖基的半乳糖化水平(21.36±1.66%)。然而，所有相关的胞内核苷糖浓度在第7天均急剧下降到正常pCO2培养条件下的约50%。实时PCR显示，在过低pCO2条件下，与半乳糖基化相关的糖基转移酶基因及底物转运子基因的上调对改善半乳糖基化起着关键作用。
结论：在反应器培养过程中，过低的pCO2会提高单抗N糖链的半乳糖化水平。

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.

一种基于高斯核非参数回归算法的识别CHO细胞培养基关键组分及流加优化的新方法
动物细胞培养基的组成日益复杂，关键变量的识别对于简化和指导随后的培养基优化非常重要。然而，传统的实验设计方法在探索由大量变量所形成的特性空间时能力有限。因此，本研究开发了NRGK（基于高斯核的非参数回归）方法，旨在培养基开发过程中确定对抗体产量有影响的关键组分。通过这一非参数模型，我们发现了被经典最小二乘算法所忽略的关键组分。本方法的优越性通过方差分析也得到了进一步的印证。此外，NRGK由于同时对培养基组分和抗体产量之间的非线性和线性关系进行建模，因此具有更高的选择准确性。本方法为精确识别关键组分并进而在更短时间内完成培养基优化提供了新的方向。

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.

培养基中添加牛磺酸降低单克隆抗体的碱性电荷变体水平
目的：探讨牛磺酸对CHO细胞培养中单克隆抗体碱性电荷变异体的影响。
结果：在流加培养过程中，于流加培养基中添加牛磺酸可以略微增加CHO细胞的最高活细胞密度和抗体浓度。而更为重要的是，牛磺酸显著降低了赖氨酸变体和氧化变体的含量，使碱性变体含量从32%进一步下降到27%。添加牛磺酸的条件下赖氨酸变体含量比对照条件下低约4%，这是碱性变体含量下降的主要原因。实时 PCR 和无细胞分析法显示，牛磺酸在碱性羧肽酶表达量的上调以及刺激胞外碱性羧肽酶活性方面都发挥了关键作用。
结论：牛磺酸对低碱性电荷变体表现出明显的作用，这主要是由于赖氨酸变体和氧化蛋白变体的减少。

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.

利用MDCK悬浮细胞高效生产H9N2流感疫苗
H9N2亚型禽流感疫苗的使用是控制禽流感病毒在禽类间传播的有效途径，而细胞培养生产平台能够克服传统鸡蛋平台的局限性并对其替代，从而实现疫苗生产工艺的升级。无血清悬浮细胞培养技术的发展，特别是高产悬浮细胞株的开发和培养策略的优化，能够有助于提高病毒的产量。本研究将一株贴壁型MDCK细胞系进行了无血清悬浮驯化，批培养条件下最高细胞密度可达12×106 cells/mL。随后对H9N2流感病毒在MDCK悬浮细胞系中的扩增情况进行了评估，并从MOI和感染时的细胞密度等方面对感染条件进行了优化。此外，为感染期提高病毒滴度，本研究考察了不同的补料策略，并发现采用1:2培养基稀释策略时能获得最大的HA滴度13 log2 (HAU/50μL)。进一步对比经操作条件优化的生物反应器和摇瓶培养体系，MDCK细胞的性能和病毒产量相当，但在反应器中单位细胞的病毒产量更高，达到13000 virions/cell以上。最后，生物反应器中收获并经纯化后的H9N2病毒，也即MDCK细胞生产的疫苗能够在鸡体内诱导高滴度的中和抗体。综上，这些结果显示了MDCK细胞高效生产平台在禽流感疫苗生产应用中的广阔前景。

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.

基于MDCK悬浮细胞高产甲型流感病毒的灌注培养工艺
与最近的COVID-19大流行相似，甲型流感病毒对全球社会构成了持续的威胁。对于流感疾病的治疗，抗病毒药物和疫苗都是可用的，但疫苗是最有效和最安全的方法。以动物细胞培养为基础的生产工艺，克服了鸡胚生产工艺的局限性，而要进一步提高全球疫苗的生产能力，则可以从细胞的悬浮培养技术和高密度灌注培养策略等方面进行工艺的强化。本研究展示了利用MDCK悬浮细胞生产甲型H1N1流感病毒的灌注培养工艺的开发过程，从摇瓶规模到实验室规模的生物反应器。半灌注模式下，摇瓶培养的MDCK细胞最高密度可达41×10⁶ cells/mL，并获得4.25 log₁₀(HAU/100 μL)的高产量病毒。规模放大至采用交变切向流（ATF）灌注系统的生物反应器需要优化pH的控制并在感染实施温度转变。使用电容探头进行在线的灌注控制，能够最大限度减少培养基消耗，并同时保持病毒最大滴度为4.37 log₁₀(HAU/100 μL)和感染病毒滴度1.83×10¹⁰ virions/mL，有利于工艺控制和经济性能的提高。综上，本研究展示了基于悬浮细胞的灌注培养工艺在下一代高产流感疫苗制造方面的优势，以随时应对大流行的发生。

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.

基于MDCK悬浮细胞培养的人用甲型流感疫苗的生产工艺
季节性流感每年都会在北半球和南半球流行。尽管不同流感病毒表面抗原和不同季节性流感病毒亚型的毒力存在差异，但疫苗制造商目前已较好地适应了这种周期性的疫苗需求。基于数十年的流感病毒研究，新型流感疫苗的开发也变得相对简单。与持续的新型冠状病毒大流行相似，疫苗生产是快速供应全球数十亿剂疫苗的主要瓶颈。彼时，基于鸡胚法的疫苗生产将难以在短时间内生产数十亿剂疫苗，而且基于鸡胚法生产的其他疫苗也将出现短缺。细胞培养生产系统能够在短时间内生产大量疫苗，并可以显著提高人们应对流行病和新出现的病毒性疾病的能力。在这项研究中，我们开发了一种在化学成分确定培养基中培养的Madin-Darby Canine Kidney (MDCK) 悬浮细胞株生产灭活甲型流感病毒疫苗的工艺。研究表明，在 1 L 搅拌式生物反应器中，MDCK 细胞在化学成分确定培养基中可生长至 9.5×106 cells/ml，在A/PR/8/34 H1N1 流感病毒感染后病毒滴度可高达3.6 log10(HAU/100 µl)。膜空间排阻层析和基于硫酸化纤维素膜吸附亲和层析的组合使病毒纯化后的回收率高达 80%。纯化的病毒颗粒显示出均匀良好的尺寸分布，平均直径为 80 nm。根据流感疫苗中单价疫苗需含有15 µg 血凝素（单放射免疫扩散试验测定）的要求计算，该工艺制备的单价疫苗中总蛋白和宿主细胞 DNA 水平分别为 58 µg 和 10 ng。此外，所有工艺步骤都可以完全扩大到工业规模，以便用于季节性或大流行性流感病毒疫苗的产业化生产。该工艺可实现每升培养液在 4 到 5 天内快速生产300 剂疫苗，使该工艺不仅可以与其他基于细胞培养的流感疫苗生产工艺竞争，而且可以与鸡胚工艺相竞争。

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.

通过毛细管电泳同时检测烟酰胺腺嘌呤核苷酸和腺苷酸池来量化生产单抗的CHO细胞的氧化还原和能量状态
中国仓鼠卵巢(CHO)细胞是生产用于治疗各种疾病的治疗性蛋白的主要宿主细胞。对CHO细胞代谢进行简单快速的表征和研究有利于促进过程开发和细胞系构建。因此，我们建立了一种毛细管电泳同时检测CHO细胞中7种氧化还原和能量代谢相关化合物的方法。采用50 μm× 44 cm的无涂层熔融二氧化硅毛细管对代谢物进行在线检测改善了峰形并提高了分辨率。采用实验设计软件(DoE)对关键参数及其相互作用进行了研究，通过期望函数确定BGE的最优条件为24℃，95 mM, pH 9.4。并对该方法进行验证，以确保其灵敏度、线性和重复性。7种代谢物的检出限(lod)范围为0.050 ~ 0.688 mg/L，线性相关系数均大于0.996。除NADPH外，其它六种代谢物的迁移时间和峰面积的相对标准偏差(RSD)分别小于0.872%和5.5%，加样回收率在97.5 ~ 101.2%之间。该方法成功应用于两种不同培养条件下CHO细胞提取物的分析。

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.

转录组学表征揭示MDCK细胞高产流感病毒的属性
Madin-Darby犬肾(MDCK)细胞系是流感病毒疫苗生产最常用的细胞系之一。随着基于细胞培养的流感病毒生产过程逐渐取代传统的基于鸡胚的生产过程，如何增加细胞的病毒生产能力至关重要。为阐明影响细胞的病毒生产能力的因素，我们通过细胞克隆化筛选分离得到一个子代克隆H1，其生产A型流感病毒（IAV）的能力是母本细胞(P)的两倍，并且在物理和分子水平上分别详细表征了H1和P的差异。转录组学分析结果显示，在IAV感染后数小时内，病毒mRNA可达总mRNA水平的20%以上，且其中几个病毒基因在H1中的表达显著高于P。转录组动力学功能分析显示，H1和P对IAV感染产生的应激反应相似，这些应激反应主要来源于宿主关闭效应和炎症效应的影响。重要的是，H1在病毒感染后其蛋白翻译和RNA加工能力较好，且其炎症和抗病毒反应更弱。综上所述，我们推测IAV的高生产力取决于抑制宿主部分功能来合理利用细胞资源以及维持胞内足够的病毒复制活力之间的平衡。对病毒生产力机制的探究有利于过程优化和细胞工程改造以推进流感疫苗工业化生产进程。