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.

反應器培養CHO細胞生產單抗過程中過低的二氧化碳分壓導致抗體半乳糖基化水平升高
目的：研究過低的二氧化碳分壓（pCO2）對中國倉鼠卵巢（CHO）細胞培養產生的單克隆抗體N-糖基化的影響。
結果：在反應器流加培養過程中，通過增加表層通氣來降低培養基中的 pCO2 (<25 mmHg)這一方式會降低CHO細胞的細胞活力及單抗產量。此外，在過低的pCO2條件下，單抗糖基的半乳糖化為27.45±2.13%，顯著高于正常pCO2條件下收獲的單抗糖基的半乳糖化水平(21.36±1.66%)。然而，所有相關的胞內核苷糖濃度在第7天均急劇下降到正常pCO2培養條件下的約50%。實時PCR顯示，在過低pCO2條件下，與半乳糖基化相關的糖基轉移酶基因及底物轉運子基因的上調對改善半乳糖基化起著關鍵作用。
結論：在反應器培養過程中，過低的pCO2會提高單抗N糖鏈的半乳糖化水平。

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.

一種基于高斯核非參數回歸算法的識別CHO細胞培養基關鍵組分及流加優化的新方法
動物細胞培養基的組成日益復雜，關鍵變量的識別對于簡化和指導隨后的培養基優化非常重要。然而，傳統的實驗設計方法在探索由大量變量所形成的特性空間時能力有限。因此，本研究開發了NRGK（基于高斯核的非參數回歸）方法，旨在培養基開發過程中確定對抗體產量有影響的關鍵組分。通過這一非參數模型，我們發現了被經典最小二乘算法所忽略的關鍵組分。本方法的優越性通過方差分析也得到了進一步的印證。此外，NRGK由于同時對培養基組分和抗體產量之間的非線性和線性關系進行建模，因此具有更高的選擇準確性。本方法為精確識別關鍵組分并進而在更短時間內完成培養基優化提供了新的方向。

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.

培養基中添加?；撬峤档蛦慰寺】贵w的堿性電荷變體水平
目的：探討?；撬釋HO細胞培養中單克隆抗體堿性電荷變異體的影響。
結果：在流加培養過程中，于流加培養基中添加?；撬峥梢月晕⒃黾覥HO細胞的最高活細胞密度和抗體濃度。而更為重要的是，?；撬犸@著降低了賴氨酸變體和氧化變體的含量，使堿性變體含量從32%進一步下降到27%。添加?；撬岬臈l件下賴氨酸變體含量比對照條件下低約4%，這是堿性變體含量下降的主要原因。實時 PCR 和無細胞分析法顯示，?；撬嵩趬A性羧肽酶表達量的上調以及刺激胞外堿性羧肽酶活性方面都發揮了關鍵作用。
結論：?；撬釋Φ蛪A性電荷變體表現出明顯的作用，這主要是由于賴氨酸變體和氧化蛋白變體的減少。

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懸浮細胞高效生產H9N2流感疫苗
H9N2亞型禽流感疫苗的使用是控制禽流感病毒在禽類間傳播的有效途徑，而細胞培養生產平臺能夠克服傳統雞蛋平臺的局限性并對其替代，從而實現疫苗生產工藝的升級。無血清懸浮細胞培養技術的發展，特別是高產懸浮細胞株的開發和培養策略的優化，能夠有助于提高病毒的產量。本研究將一株貼壁型MDCK細胞系進行了無血清懸浮馴化，批培養條件下最高細胞密度可達12×106 cells/mL。隨后對H9N2流感病毒在MDCK懸浮細胞系中的擴增情況進行了評估，并從MOI和感染時的細胞密度等方面對感染條件進行了優化。此外，為感染期提高病毒滴度，本研究考察了不同的補料策略，并發現采用1:2培養基稀釋策略時能獲得最大的HA滴度13 log2 (HAU/50μL)。進一步對比經操作條件優化的生物反應器和搖瓶培養體系，MDCK細胞的性能和病毒產量相當，但在反應器中單位細胞的病毒產量更高，達到13000 virions/cell以上。最后，生物反應器中收獲并經純化后的H9N2病毒，也即MDCK細胞生產的疫苗能夠在雞體內誘導高滴度的中和抗體。綜上，這些結果顯示了MDCK細胞高效生產平臺在禽流感疫苗生產應用中的廣闊前景。

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懸浮細胞高產甲型流感病毒的灌注培養工藝
與最近的COVID-19大流行相似，甲型流感病毒對全球社會構成了持續的威脅。對于流感疾病的治療，抗病毒藥物和疫苗都是可用的，但疫苗是最有效和最安全的方法。以動物細胞培養為基礎的生產工藝，克服了雞胚生產工藝的局限性，而要進一步提高全球疫苗的生產能力，則可以從細胞的懸浮培養技術和高密度灌注培養策略等方面進行工藝的強化。本研究展示了利用MDCK懸浮細胞生產甲型H1N1流感病毒的灌注培養工藝的開發過程，從搖瓶規模到實驗室規模的生物反應器。半灌注模式下，搖瓶培養的MDCK細胞最高密度可達41×10⁶ cells/mL，并獲得4.25 log₁₀(HAU/100 μL)的高產量病毒。規模放大至采用交變切向流（ATF）灌注系統的生物反應器需要優化pH的控制并在感染實施溫度轉變。使用電容探頭進行在線的灌注控制，能夠最大限度減少培養基消耗，并同時保持病毒最大滴度為4.37 log₁₀(HAU/100 μL)和感染病毒滴度1.83×10¹⁰ virions/mL，有利于工藝控制和經濟性能的提高。綜上，本研究展示了基于懸浮細胞的灌注培養工藝在下一代高產流感疫苗制造方面的優勢，以隨時應對大流行的發生。

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懸浮細胞培養的人用甲型流感疫苗的生產工藝
季節性流感每年都會在北半球和南半球流行。盡管不同流感病毒表面抗原和不同季節性流感病毒亞型的毒力存在差異，但疫苗制造商目前已較好地適應了這種周期性的疫苗需求?；跀凳甑牧鞲胁《狙芯?，新型流感疫苗的開發也變得相對簡單。與持續的新型冠狀病毒大流行相似，疫苗生產是快速供應全球數十億劑疫苗的主要瓶頸。彼時，基于雞胚法的疫苗生產將難以在短時間內生產數十億劑疫苗，而且基于雞胚法生產的其他疫苗也將出現短缺。細胞培養生產系統能夠在短時間內生產大量疫苗，并可以顯著提高人們應對流行病和新出現的病毒性疾病的能力。在這項研究中，我們開發了一種在化學成分確定培養基中培養的Madin-Darby Canine Kidney (MDCK) 懸浮細胞株生產滅活甲型流感病毒疫苗的工藝。研究表明，在 1 L 攪拌式生物反應器中，MDCK 細胞在化學成分確定培養基中可生長至 9.5×106 cells/ml，在A/PR/8/34 H1N1 流感病毒感染后病毒滴度可高達3.6 log10(HAU/100 µl)。膜空間排阻層析和基于硫酸化纖維素膜吸附親和層析的組合使病毒純化后的回收率高達 80%。純化的病毒顆粒顯示出均勻良好的尺寸分布，平均直徑為 80 nm。根據流感疫苗中單價疫苗需含有15 µg 血凝素（單放射免疫擴散試驗測定）的要求計算，該工藝制備的單價疫苗中總蛋白和宿主細胞 DNA 水平分別為 58 µg 和 10 ng。此外，所有工藝步驟都可以完全擴大到工業規模，以便用于季節性或大流行性流感病毒疫苗的產業化生產。該工藝可實現每升培養液在 4 到 5 天內快速生產300 劑疫苗，使該工藝不僅可以與其他基于細胞培養的流感疫苗生產工藝競爭，而且可以與雞胚工藝相競爭。

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.

通過毛細管電泳同時檢測煙酰胺腺嘌呤核苷酸和腺苷酸池來量化生產單抗的CHO細胞的氧化還原和能量狀態
中國倉鼠卵巢(CHO)細胞是生產用于治療各種疾病的治療性蛋白的主要宿主細胞。對CHO細胞代謝進行簡單快速的表征和研究有利于促進過程開發和細胞系構建。因此，我們建立了一種毛細管電泳同時檢測CHO細胞中7種氧化還原和能量代謝相關化合物的方法。采用50 μm× 44 cm的無涂層熔融二氧化硅毛細管對代謝物進行在線檢測改善了峰形并提高了分辨率。采用實驗設計軟件(DoE)對關鍵參數及其相互作用進行了研究，通過期望函數確定BGE的最優條件為24℃，95 mM, pH 9.4。并對該方法進行驗證，以確保其靈敏度、線性和重復性。7種代謝物的檢出限(lod)范圍為0.050 ~ 0.688 mg/L，線性相關系數均大于0.996。除NADPH外，其它六種代謝物的遷移時間和峰面積的相對標準偏差(RSD)分別小于0.872%和5.5%，加樣回收率在97.5 ~ 101.2%之間。該方法成功應用于兩種不同培養條件下CHO細胞提取物的分析。

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.

轉錄組學表征揭示MDCK細胞高產流感病毒的屬性
Madin-Darby犬腎(MDCK)細胞系是流感病毒疫苗生產最常用的細胞系之一。隨著基于細胞培養的流感病毒生產過程逐漸取代傳統的基于雞胚的生產過程，如何增加細胞的病毒生產能力至關重要。為闡明影響細胞的病毒生產能力的因素，我們通過細胞克隆化篩選分離得到一個子代克隆H1，其生產A型流感病毒（IAV）的能力是母本細胞(P)的兩倍，并且在物理和分子水平上分別詳細表征了H1和P的差異。轉錄組學分析結果顯示，在IAV感染后數小時內，病毒mRNA可達總mRNA水平的20%以上，且其中幾個病毒基因在H1中的表達顯著高于P。轉錄組動力學功能分析顯示，H1和P對IAV感染產生的應激反應相似，這些應激反應主要來源于宿主關閉效應和炎癥效應的影響。重要的是，H1在病毒感染后其蛋白翻譯和RNA加工能力較好，且其炎癥和抗病毒反應更弱。綜上所述，我們推測IAV的高生產力取決于抑制宿主部分功能來合理利用細胞資源以及維持胞內足夠的病毒復制活力之間的平衡。對病毒生產力機制的探究有利于過程優化和細胞工程改造以推進流感疫苗工業化生產進程。