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Integration of a perfusion reactor and continuous precipitation in an entirely membrane-based process for antibody capture 在完全基于膜的抗体捕获过程中整合灌注反应器和连续沉淀。
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-09-07 DOI: 10.1002/elsc.202300219
Gabriele Recanati, Magdalena Pappenreiter, Christoph Gstoettner, Patrick Scheidl, Elena Domínguez Vega, Bernhard Sissolak, Alois Jungbauer

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl2 precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab-scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand-alone capture and as an integrated perfusion-capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow-through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host-cell proteins and host-cell DNA by the combination of the precipitation-based capture and the first polishing step.

连续沉淀与连续切向流过滤相结合是从粗细胞培养上清液中捕获重组抗体的一种具有成本效益的替代方案。通过采用管式反应器,在单元操作之间移除缓冲罐,保持了产品的连续收获和质量流,具有窄停留时间分布(RTD)的优点。我们开发了一种实施两种正交沉淀方法的连续工艺,CaCl2沉淀用于去除宿主细胞DNA,聚乙二醇(PEG)用于捕获重组抗体,对糖基化谱没有影响。我们的实验室规模的原型由两个管式反应器和两级切向流微滤组成,以真正连续的方式连续运行长达8天,没有任何产品流中断,无论是作为独立捕获还是作为集成灌注捕获。此外,我们探索了使用带负电荷的膜吸附器进行流通式阴离子交换作为第一抛光步骤。通过基于沉淀的捕获和第一抛光步骤的组合,我们获得了约80%的产品回收率和恒定的产品质量,宿主细胞蛋白质和宿主细胞DNA都具有两个以上的对数还原值(LRV)。
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引用次数: 0
Cover Picture: Engineering in Life Sciences 9'23 封面图片:生命科学工程9’23
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-09-01 DOI: 10.1002/elsc.202370091
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引用次数: 0
Investigation and evaluation of a 3D-printed optical modified cultivation vessel for improved scattered light measurement of biotechnologically relevant organisms 用于改进生物技术相关生物散射光测量的3d打印光学修饰培养容器的研究和评估
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-08-23 DOI: 10.1002/elsc.202300204
Johanna S. Rehfeld, Louis M. Kuhnke, Christian Ude, Gernot T. John, Sascha Beutel

In the field of bioprocess development miniaturization, parallelization and flexibility play a key role reducing costs and time. To precisely meet these requirements, additive manufacturing (3D-printing) is an ideal technology. 3D-printing enables rapid prototyping and cost-effective fabrication of individually designed devices with complex geometries on demand. For successful bioprocess development, monitoring of process-relevant parameters, such as pH, dissolved oxygen (DO), and biomass, is crucial. Online monitoring is preferred as offline sampling is time-consuming and leads to loss of information. In this study, 3D-printed cultivation vessels with optical prisms are evaluated for the use in upstream processes of different industrially relevant microorganisms and cell lines. It was shown, that the 3D-printed optically modified well (OMW) is of benefit for a wide range of biotechnologically relevant microorganisms and even for mammalian suspension cells. Evaluation tests with Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Chinese hamster ovary (CHO) cells were performed, providing highly reproducible results. Growth behavior of OMW cultures was comparable to behavior of shake flask (SF) cultivations and the signal to noise ratio in online biomass measurement was shown to be reduced up to 95.8% by using the OMW. Especially the cultivation phases with low turbidity respective optical densities below 1.0 rel.AU could be monitored accurately for the first time. Furthermore, it was demonstrated that the 3D-printed optics are transferable to different well geometries and sizes, enabling efficient biomass monitoring for individual requirements with tailor-made 3D-printed cultivation vessels in small scale.

在生物过程开发领域,小型化、并行化和灵活性在降低成本和时间方面发挥着关键作用。为了精确满足这些要求,增材制造(3D打印)是一种理想的技术。3D打印能够根据需要快速成型并经济高效地制造具有复杂几何形状的单独设计的设备。为了成功开发生物工艺,监测工艺相关参数,如pH、溶解氧(DO)和生物量,至关重要。由于离线采样耗时且会导致信息丢失,因此首选在线监测。在这项研究中,评估了具有光学棱镜的3D打印培养容器在不同工业相关微生物和细胞系的上游过程中的用途。研究表明,3D打印的光学修饰井(OMW)对各种生物技术相关的微生物,甚至对哺乳动物悬浮细胞都有好处。用大肠杆菌、枯草芽孢杆菌、酿酒酵母和中国仓鼠卵巢(CHO)细胞进行了评估测试,提供了高度可重复的结果。OMW培养物的生长行为与摇瓶(SF)培养物的行为相当,并且通过使用OMW,在线生物量测量中的信噪比降低了95.8%。特别是在浊度较低的培养阶段,即光密度低于1.0 rel.AU的培养阶段可以首次得到准确的监测。此外,研究表明,3D打印的光学器件可以转移到不同的井几何形状和尺寸,通过小规模定制的3D打印培养容器,能够有效监测个人需求的生物量。
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引用次数: 1
Meta-omics assisted microbial gene and strain resources mining in contaminant environment 元组学辅助污染环境中微生物基因和菌株资源的挖掘
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-08-18 DOI: 10.1002/elsc.202300207
Yiqun Huang, Haiyang Hu, Tingting Zhang, Weiwei Wang, Wenzhao Liu, Hongzhi Tang

Human activities have led to the release of various environmental pollutants, triggering ecological challenges. In situ, microbial communities in these contaminated environments are usually assumed to possess the potential capacity of pollutant degradation. However, the majority of genes and microorganisms in these environments remain uncharacterized and uncultured. The advent of meta-omics provided culture-independent solutions for exploring the functional genes and microorganisms within complex microbial communities. In this review, we highlight the applications and methodologies of meta-omics in uncovering of genes and microbes from contaminated environments. These findings may assist in future bioremediation research.

人类活动导致各种环境污染物的释放,引发生态挑战。在这些被污染的环境中,微生物群落通常被认为具有潜在的污染物降解能力。然而,这些环境中的大多数基因和微生物仍未被表征和培养。元组学的出现为探索复杂微生物群落中的功能基因和微生物提供了不依赖培养的解决方案。在本文中,我们重点介绍了元组学在污染环境中发现基因和微生物的应用和方法。这些发现可能有助于未来的生物修复研究。
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引用次数: 0
Cover Picture: Engineering in Life Sciences 8'23 封面图片:生命科学工程8'23
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-07-31 DOI: 10.1002/elsc.202370081
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引用次数: 0
Evaluation of restricted access media for the purification of cell culture-derived Orf viruses 细胞培养源Orf病毒纯化的限制接触培养基的评价
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-07-14 DOI: 10.1002/elsc.202300009
Keven Lothert, Yasmina M. J. Harsy, Patrick Endres, Egbert Müller, Michael W. Wolff

Recently, multimodal chromatography using restricted access media (RAM) for the purification of nanoparticles, such as viruses has regained increasing attention. These chromatography resins combine size exclusion on the particle shell and adsorptive interaction within the core. Accordingly, smaller process-related impurities, for example, DNA and proteins, can be retained, while larger product viruses can pass unhindered. We evaluated a range of currently available RAM, differing in the shells’ pore cut-off and the core chemistry, for the purification of a cell culture-derived clarified model virus, namely the Orf virus (ORFV). We examined impurity depletion and product recovery as relevant criteria for the evaluation of column performance, as well as scale-up robustness and regeneration potential for evaluating a multiple use application. The results indicate that some columns, for example, the Capto Core, enable both a high DNA and protein removal, while others, for example, the Monomix Core 60 (MC60), are more suitable for DNA depletion. Furthermore, column regeneration is facilitated by using columns with larger shell pores (5000 vs. 700 kDa) and weaker binding interactions (anion exchange vs. multimodal). According to these findings, the choice of RAM resins should be selected according to the respective feed sample composition and the planned number of application cycles.

近年来,利用受限存取介质(RAM)进行纳米颗粒(如病毒)纯化的多模态色谱技术越来越受到人们的关注。这些色谱树脂结合了颗粒壳上的尺寸排除和核心内的吸附相互作用。因此,可以保留较小的工艺相关杂质,例如DNA和蛋白质,而较大的产品病毒可以不受阻碍地通过。我们评估了一系列目前可用的RAM,不同于外壳的孔切断和核心化学成分,用于纯化细胞培养衍生的澄清模型病毒,即Orf病毒(ORFV)。我们检查了杂质消耗和产品回收作为评估色谱柱性能的相关标准,以及评估多种用途应用的放大稳健性和再生潜力。结果表明,一些色谱柱(如Capto Core)可以同时去除DNA和蛋白质,而另一些色谱柱(如Monomix Core 60 (MC60))更适合去除DNA。此外,使用具有较大壳孔(5000 kDa vs 700 kDa)和较弱结合相互作用(阴离子交换vs多模态)的柱有助于柱再生。根据这些发现,RAM树脂的选择应根据各自的饲料样品组成和计划的应用周期数量来选择。
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引用次数: 1
Identification and characterization of inulinases by bioinformatics analysis of bacterial glycoside hydrolases family 32 (GH32) 细菌糖苷水解酶家族32 (GH32)生物信息学分析鉴定菊粉酶
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-07-09 DOI: 10.1002/elsc.202300003
Fatemeh Khosravi, Ehsan Mohseni Fard, Marzieh Hosseininezhad, Hadi Shoorideh

The glycoside hydrolase family contains enzymes that break the glycosidic bonds of carbohydrates by hydrolysis. Inulinase is one of the most important industrial enzymes in the family of Glycoside Hydrolases 32 (GH32). In this study, to identify and classify bacterial inulinases initially, 16,002 protein sequences belonging to the GH32 family were obtained using various databases. The inulin-effective enzymes (endoinulinase and exoinulinase) were identified. Eight endoinulinases (EC 3.2.1.7) and 4318 exoinulinases (EC 3.2.1.80) were found. Then, the localization of endoinulinase and exoinulinase enzymes in the cell was predicted. Among them, two extracellular endoinulinases and 1232 extracellular exoinulinases were found. The biochemical properties of 363 enzymes of the genus Arthrobacter, Bacillus, and Streptomyces (most abundant) showed that exoinulinases have an acid isoelectric point up to the neutral range due to their amino acid length. That is, the smaller the protein (336 aa), the more acidic the pI (4.39), and the larger the protein (1207 aa), the pI is in the neutral range (8.84). Also, a negative gravitational index indicates the hydrophilicity of exoinulinases. Finally, considering the biochemical properties affecting protein stability and post-translational changes studies, one enzyme for endoinulinase and 40 enzymes with desirable characteristics were selected to identify their enzyme production sources. To screen and isolate enzyme-containing strains, now with the expansion of databases and the development of bioinformatics tools, it is possible to classify, review and analyze a lot of data related to different enzyme-producing strains. Although, in laboratory studies, a maximum of 20 to 30 strains can be examined. Therefore, when more strains are examined, finally, strains with more stable and efficient enzymes were selected and introduced for laboratory activities. The findings of this study can help researchers to select the appropriate gene source from introduced strains for cloning and expression heterologous inulinase, or to extract native inulinase from introduced strains.

糖苷水解酶家族包含通过水解破坏碳水化合物的糖苷键的酶。菊粉酶是糖苷水解酶32 (GH32)家族中最重要的工业酶之一。本研究为了对细菌菊粉酶进行初步鉴定和分类,利用各种数据库获得了16002个属于GH32家族的蛋白序列。鉴定了菊粉有效酶(内菊粉酶和外菊粉酶)。共检出8个内菊糖酶(EC 3.2.1.7)和4318个外菊糖酶(EC 3.2.1.80)。然后,预测胞内菊粉酶和胞外菊粉酶在细胞中的定位。其中,发现2种胞外菊粉酶和1232种胞外菊粉酶。节肢菌属、芽孢杆菌属和链霉菌属(数量最多)的363种酶的生化特性表明,外菊糖酶由于其氨基酸长度的关系,其酸等电点在中性范围内。即蛋白质越小(336 aa), pI酸性越强(4.39),蛋白质越大(1207 aa), pI处于中性范围(8.84)。此外,负引力指数表明外粉酶的亲水性。最后,考虑到影响蛋白质稳定性和翻译后变化研究的生化特性,选择了1种内酰胺酶和40种具有理想特性的酶来确定它们的产酶来源。为了筛选和分离含酶菌株,随着数据库的扩充和生物信息学工具的发展,可以对不同产酶菌株的大量相关数据进行分类、回顾和分析。尽管在实验室研究中,最多可以检查20至30个菌株。因此,当检测到更多的菌株时,最终选择具有更稳定和高效酶的菌株并引入实验室活动。本研究结果可以帮助研究人员从引进菌株中选择合适的基因源进行异种菊粉酶的克隆和表达,或者从引进菌株中提取原生菊粉酶。
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引用次数: 0
Cover Picture: Engineering in Life Sciences 7'23 封面图片:生命科学工程7’23
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-07-03 DOI: 10.1002/elsc.202370071
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引用次数: 0
Multi-objective Bayesian algorithm automatically discovers low-cost high-growth serum-free media for cellular agriculture application 多目标贝叶斯算法自动发现低成本高生长无血清细胞农业培养基
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-06-28 DOI: 10.1002/elsc.202300005
Zachary Cosenza, David E. Block, Keith Baar, Xingyu Chen

In this work, we applied a multi-information source modeling technique to solve a multi-objective Bayesian optimization problem involving the simultaneous minimization of cost and maximization of growth for serum-free C2C12 cells using a hyper-volume improvement acquisition function. In sequential batches of custom media experiments designed using our Bayesian criteria, collected using multiple assays targeting different cellular growth dynamics, the algorithm learned to identify the trade-off relationship between long-term growth and cost. We were able to identify several media with >100%$>100%$ more growth of C2C12 cells than the control, as well as a medium with 23% more growth at only 62.5% of the cost of the control. These algorithmically generated media also maintained growth far past the study period, indicating the modeling approach approximates the cell growth well from an extremely limited data set.

在这项工作中,我们应用多信息源建模技术来解决一个多目标贝叶斯优化问题,该问题涉及使用超体积改进获取函数同时使无血清C2C12细胞的成本最小化和生长最大化。在使用贝叶斯标准设计的连续批次定制培养基实验中,使用针对不同细胞生长动态的多种分析收集,该算法学会了识别长期增长与成本之间的权衡关系。我们能够识别出与>C2C12细胞的生长速度比对照提高了100%,培养基的生长速度提高了23%,而成本仅为对照的62.5%。这些算法生成的培养基在研究期间也保持了生长,这表明建模方法可以从非常有限的数据集中很好地近似细胞生长。
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引用次数: 0
Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia 评价毛细光生物膜反应器高密度培养混合奖杯人工微生物群落的结垢性
IF 2.7 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2023-06-23 DOI: 10.1002/elsc.202300014
Amelie Kenkel, Rohan Karande, Katja Bühler

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O2, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O2 permeability. High flow rates of 520 μL min−1 prevented biofilm sloughing in 1 m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O2 concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5 m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8 g m−2 and, thus, a successful CBR scale-up by a factor of 25.

Practical application: Cyanobacteria use light energy to upgrade CO2, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O2 accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.

毛细管生物膜反应器(CBRs)对生长光自养细菌具有吸引力,因为它们允许高细胞密度培养。在这里,我们评估了CBR系统生长由聚囊藻属PCC 6803和假单胞菌属VBL120组成的人工群落的适宜性。研究了不同反应器规模下反应器材料、流速、pH、O2和介质组成对生物量发展和生物膜长期稳定性的影响。硅树脂由于其优异的O2渗透性而优于玻璃或PVC等其他材料。520μL min−1的高流速防止了1 m毛细管反应器中的生物膜脱落,与标准操作条件相比,生物量干重增加了54%,反应器内的O2浓度最低。反应器长度进一步增加到5米表明微量元素的限制。将微量元素增加5倍,可以实现完全的表面覆盖,生物质干重为36.8 g m−2,因此,成功地将CBR放大25倍。实际应用:蓝细菌利用光能升级CO2,从而保持碳中和的生产工艺的潜力。持续存在的挑战之一是由于光限制和O2积累导致的低细胞密度,O2积累通常发生在已建立的平板或管状光生物反应器中。与浮游培养物相比,蓝藻生物膜中可以获得更高的细胞密度(因子10-100)。毛细管生物膜反应器(CBR)为蓝藻生物膜提供了良好的生长条件,但其适用性仅在实验室规模上得到证明。在这里,进行了第一次基于规模的放大研究,测试了该系统在大规模应用中的可行性。我们证明,通过优化营养物供应和流动条件,可以通过增加反应器的长度将系统扩大25倍。这种反应器的概念,与蓝藻生物膜和编号相结合,具有作为一种灵活的碳中和的增值化合物生产平台的潜力。
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引用次数: 0
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Engineering in Life Sciences
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