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Rational design of short-chain dehydrogenase/reductase for enantio-complementary synthesis of chiral 1,2-diols by successive hydroxymethylation and reduction of aldehydes 合理设计短链脱氢酶/还原酶,通过醛的连续羟甲基化和还原反应对手性 1,2 二醇进行对映互补合成
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-10 DOI: 10.1002/bit.28841
Xiu-Xin Ren, Bing-Mei Su, Xin-Qi Xu, Lian Xu, Juan Lin
Enantiopure 1,2-diols are widely used in the production of pharmaceuticals, cosmetics, and functional materials as essential building blocks or bioactive compounds. Nevertheless, developing a mild, efficient and environmentally friendly biocatalytic route for manufacturing enantiopure 1,2-diols from simple substrate remains a challenge. Here, we designed and realized a step-wise biocatalytic cascade to access chiral 1,2-diols starting from aromatic aldehyde and formaldehyde enabled by a newly mined benzaldehyde lyase from Sphingobium sp. combined with a pair of tailored-made short-chain dehydrogenase/reductase from Pseudomonas monteilii (PmSDR-MuR and PmSDR-MuS) capable of producing (R)- and (S)-1-phenylethane-1,2-diol with 99% ee. The planned biocatalytic cascade could synthesize a series of enantiopure 1,2-diols with a broad scope (16 samples), excellent conversions (94%–99%), and outstanding enantioselectivity (up to 99% ee), making it an effective technique for producing chiral 1,2-diols in a more environmentally friendly and sustainable manner.
对映体纯 1,2-二醇作为重要的基本成分或生物活性化合物被广泛应用于药品、化妆品和功能材料的生产中。然而,开发一条温和、高效、环保的生物催化路线,从简单的底物中生产出不对映的 1,2 二醇,仍然是一项挑战。在这里,我们设计并实现了一种分步生物催化级联法,以芳香醛和甲醛为起始原料,通过一种新近从 Sphingobium sp.结合一对定制的假单胞菌短链脱氢酶/还原酶(PmSDR-MuR 和 PmSDR-MuS),能够生成(R)-和(S)-1-苯基乙烷-1,2-二醇,ee值高达 99%。计划中的生物催化级联法可以合成一系列对映体纯的 1,2-二醇,范围广泛(16 个样品),转化率高(94%-99%),对映体选择性好(ee高达 99%),是一种以更环保、更可持续的方式生产手性 1,2- 二醇的有效技术。
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引用次数: 0
Application of microbubble air flotation to harvest Microcystis sp. from agriculture wastewater: The regulation and mechanisms 应用微气泡气浮收获农业废水中的微囊藻:调节和机制
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-09 DOI: 10.1002/bit.28836
Jianfeng Ye, Zhihao Zhu, Zhaofeng Song, Huiting Xu, Tianchen Xu, Hui Liu
The harvesting of microalgae is the main bottleneck of its large-scale biomass production, and seeking an efficient, green, and low-cost microalgae harvesting technology is one of the urgent problems to be solved. Microbubble air flotation has been proven to be an effective measure, but the mechanisms of microbubbles-algal cell attachment are still unclear. In this study, microbubble air flotation was used as a harvesting method for Microcystis cultured in agricultural wastewater. The process mechanism of microbubble air flotation harvesting microalgae in wastewater was fully revealed from three aspects (the design of bubble formation, the adhesion law, and the recovery rate of microalgae under different working conditions). The results show that the length of the release pipe is the main factor affecting the proportion of microbubbles with a particle size of less than 50 μm. In the process of adhesion, when the particle size of microbubbles is 0.6–1.7 times the size of Microcystis, the adhesion efficiency of microbubbles to Microcystis is the highest. Under the conditions of pressure 0.45 MPa, gas–liquid ratio 5%, and release pipe length 100 cm, the harvesting performance of Microcystis was the best. Microbubble air flotation has better harvesting performance (63.5%, collection rate) of Microcystis with higher density. By understanding the mechanism of microbubble flotation, the technical parameters of microbubble flotation for harvesting energy microalgae are optimized to provide support for the development of efficient and low-cost devices and equipment for collecting microalgae.
微藻收获是大规模生物质生产的主要瓶颈,寻求一种高效、绿色、低成本的微藻收获技术是亟待解决的问题之一。微气泡气浮已被证明是一种有效的措施,但微气泡-藻细胞附着的机理仍不清楚。本研究采用微气泡气浮作为农业废水中培养的微囊藻的收获方法。从气泡形成设计、附着规律和不同工况下微囊藻回收率三个方面全面揭示了微气泡气浮收获废水中微囊藻的过程机理。结果表明,释放管的长度是影响粒径小于 50 μm 的微气泡比例的主要因素。在粘附过程中,当微泡粒径为微囊藻粒径的 0.6-1.7 倍时,微泡对微囊藻的粘附效率最高。在压力为 0.45 兆帕、气液比为 5%、释放管道长度为 100 厘米的条件下,微囊藻的收获效果最好。微气泡气浮对密度较大的微囊藻有较好的收获效果(收集率为 63.5%)。通过对微泡气浮机理的认识,优化了微泡气浮收获能源微藻的技术参数,为开发高效、低成本的微藻收集装置和设备提供了支持。
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引用次数: 0
Auto-transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy 慢病毒载体生物处理中的自动转导:定量评估和新型抑制策略
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-08 DOI: 10.1002/bit.28834
Thomas Williams-Fegredo, Lee Davies, Carol Knevelman, James Miskin, Kyriacos Mitrophanous, Qasim A. Rafiq
Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7–6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.
慢病毒载体是一种高效的基因递送载体,广泛应用于快速发展的细胞和基因治疗领域。随着越来越多的疗法进入后期临床试验阶段并实现商业化,对高效、大规模慢病毒载体生物处理的需求也在不断增长。然而,尽管取得了重大进展,但仍存在一些工艺效率低下的问题。在生产过程中,新合成的慢病毒载体颗粒会对病毒载体生产细胞产生意外的自动转导,这就是效率低下的原因之一,而这一问题在很大程度上仍未得到解决。在这项研究中,我们发现在上游生产过程中产生的功能性慢病毒载体颗粒有 60% 以上因自动转导而丢失,这凸显了行业内可能普遍存在的主要工艺低效问题。在载体生产过程中,通过降低细胞外 pH 值,抑制了以广泛使用的水泡性口炎病毒 G 蛋白为假型的颗粒对细胞的自动转导,从而损害了载体与其目标受体相互作用的能力。将转染后的 pH 值调至 6.7-6.8 后,病毒载体生产细胞群中由慢病毒载体介导的转导引起的载体基因组整合事件减少了七倍,并最终改善了慢病毒载体的生产动力学。所提出的策略具有可扩展性和成本效益,为提高慢病毒载体的生产效率提供了一种与工业相关的方法。
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引用次数: 0
Higher efficiency of vanadate iron in heterogeneous Fenton-like systems to pretreat sugarcane bagasse and its enzymatic saccharification. 在类似芬顿的异质系统中使用钒酸盐铁对甘蔗渣进行预处理和酶法糖化的效率更高。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-09-01 Epub Date: 2024-05-06 DOI: 10.1002/bit.28733
Ju Liang, Huiying Zeng, Yuting Zhang, Wenbing Zhou, Naidong Xiao

Pretreatment is crucial for effective enzymatic saccharification of lignocellulose such as sugarcane bagasse (SCB). In the present study, SCB was pretreated with five kinds of heterogeneous Fenton-like systems (HFSs), respectively, in which α-FeOOH, α-Fe2O3, Fe3O4, and FeS2 worked as four traditional heterogeneous Fenton-like catalysts (HFCs), while FeVO4 worked as a novel HFC. The enzymatic reducing sugar conversion rate was then compared among SCB after different heterogeneous Fenton-like pretreatments (HFPs), and the optimal HFS and pretreatment conditions were determined. The mechanism underlying the difference in saccharification efficiency was elucidated by analyzing the composition and morphology of SCB. Moreover, the ion dissolution characteristics, variation of pH and Eh values, H2O2 and hydroxyl radical (·OH) concentration of FeVO4 and α-Fe2O3 HFSs were compared. The results revealed that the sugar conversion rate of SCB pretreated with FeVO4 HFS reached up to 58.25%, which was obviously higher than that under other HFPs. In addition, the surface morphology and composition of the pretreated SCB with FeVO4 HFS were more conducive to enzymatic saccharification. Compared with α-Fe2O3, FeVO4 could utilize H2O2 more efficiently, since the dissolved Fe3+ and V5+ can both react with H2O2 to produce more ·OH, resulting in a higher hemicellulose and lignin removal rate and a higher enzymatic sugar conversion rate. It can be concluded that FeVO4 HFP is a promising approach for lignocellulose pretreatment.

预处理对于木质纤维素(如甘蔗渣)的有效酶法糖化至关重要。本研究分别使用五种异相芬顿类催化剂体系(HFS)对甘蔗渣进行预处理,其中α-FeOOH、α-Fe2O3、Fe3O4 和 FeS2 是四种传统的异相芬顿类催化剂(HFC),而 FeVO4 则是一种新型的 HFC。然后比较了不同异相芬顿类预处理(HFP)后 SCB 的酶促还原糖转化率,并确定了最佳 HFS 和预处理条件。通过分析 SCB 的组成和形态,阐明了糖化效率差异的机理。此外,还比较了 FeVO4 和 α-Fe2O3 HFS 的离子溶解特性、pH 值和 Eh 值的变化、H2O2 和羟基自由基(-OH)的浓度。结果表明,用 FeVO4 HFS 预处理的 SCB 糖转化率高达 58.25%,明显高于其他 HFP。此外,用 FeVO4 HFS 预处理的 SCB 表面形态和成分更有利于酶糖化。与 α-Fe2O3 相比,FeVO4 能更有效地利用 H2O2,因为溶解的 Fe3+ 和 V5+ 都能与 H2O2 反应产生更多的 -OH,从而导致更高的半纤维素和木质素去除率以及更高的酶糖转化率。因此,FeVO4 HFP 是一种很有前景的木质纤维素预处理方法。
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引用次数: 0
Molecular mechanisms of natural antifreeze phenomena and their application in cryopreservation 天然防冻现象的分子机制及其在低温保存中的应用
IF 3.8 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-30 DOI: 10.1002/bit.28832
Lingyu Shi, Chuanbao Zang, Zhicheng Liu, Gang Zhao
Cryopreservation presents a critical challenge due to cryo‐damage, such as crystallization and osmotic imbalances that compromise the integrity of biological tissues and cells. In contrast, various organisms in nature exhibit remarkable freezing tolerance, leveraging complex molecular mechanisms to survive extreme cold. This review explores the adaptive strategies of freeze‐tolerant species, including the regulation of specific genes, proteins, and metabolic pathways, to enhance survival in low‐temperature environments. We then discuss recent advancements in cryopreservation technologies that aim to mimic these natural phenomena to preserve cellular and tissue integrity. Special focus is given to the roles of glucose metabolism, microRNA expression, and cryoprotective protein modulation in improving cryopreservation outcomes. The insights gained from studying natural antifreeze mechanisms offer promising directions for advancing cryopreservation techniques, with potential applications in medical, agricultural, and conservation fields. Future research should aim to further elucidate these molecular mechanisms to develop more effective and reliable cryopreservation methods.
由于结晶和渗透失衡等低温损伤会损害生物组织和细胞的完整性,因此低温保存是一项严峻的挑战。与此相反,自然界中的各种生物却表现出卓越的耐冻能力,它们利用复杂的分子机制在极寒环境中生存下来。本综述探讨了耐冻物种的适应策略,包括对特定基因、蛋白质和代谢途径的调控,以提高它们在低温环境中的生存能力。然后,我们讨论了旨在模拟这些自然现象以保存细胞和组织完整性的低温保存技术的最新进展。我们将特别关注葡萄糖代谢、microRNA 表达和低温保护蛋白调节在改善低温保存结果方面的作用。从研究天然防冻机制中获得的启示为冷冻保存技术的发展提供了前景广阔的方向,并有可能应用于医学、农业和自然保护领域。未来的研究应旨在进一步阐明这些分子机制,以开发出更有效、更可靠的低温保存方法。
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引用次数: 0
Insights into yeast response to chemotherapeutic agent through time series genome-scale metabolic models 通过时间序列基因组尺度代谢模型深入了解酵母对化疗药物的反应
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-28 DOI: 10.1002/bit.28833
Muhammed E. Karabekmez

Organism-specific genome-scale metabolic models (GSMMs) can unveil molecular mechanisms within cells and are commonly used in diverse applications, from synthetic biology, biotechnology, and systems biology to metabolic engineering. There are limited studies incorporating time-series transcriptomics in GSMM simulations. Yeast is an easy-to-manipulate model organism for tumor research. Here, a novel approach (TS-GSMM) was proposed to integrate time-series transcriptomics with GSMMs to narrow down the feasible solution space of all possible flux distributions and attain time-series flux samples. The flux samples were clustered using machine learning techniques, and the clusters' functional analysis was performed using reaction set enrichment analysis. A time series transcriptomics response of Yeast cells to a chemotherapeutic reagent—doxorubicin—was mapped onto a Yeast GSMM. Eleven flux clusters were obtained with our approach, and pathway dynamics were displayed. Induction of fluxes related to bicarbonate formation and transport, ergosterol and spermidine transport, and ATP production were captured. Integrating time-series transcriptomics data with GSMMs is a promising approach to reveal pathway dynamics without any kinetic modeling and detects pathways that cannot be identified through transcriptomics-only analysis. The codes are available at https://github.com/karabekmez/TS-GSMM.

生物特异性基因组尺度代谢模型(GSMMs)可以揭示细胞内的分子机制,通常用于合成生物学、生物技术、系统生物学和代谢工程等多种应用领域。将时间序列转录组学纳入 GSMM 模拟的研究还很有限。酵母是一种易于操作的肿瘤研究模式生物。本文提出了一种新方法(TS-GSMM),将时间序列转录组学与 GSMMs 结合起来,缩小所有可能通量分布的可行解空间,并获得时间序列通量样本。利用机器学习技术对通量样本进行聚类,并利用反应集富集分析对聚类进行功能分析。酵母细胞对化疗试剂--多柔比星--的时间序列转录组学反应被映射到酵母GSMM上。我们的方法获得了 11 个通量簇,并显示了通路动态。我们捕捉到了与碳酸氢盐形成和运输、麦角甾醇和亚精胺运输以及 ATP 生产相关的通量诱导。将时间序列转录组学数据与 GSMMs 相结合是一种很有前途的方法,它能揭示通路动态,而无需任何动力学建模,并能检测到仅通过转录组学分析无法确定的通路。代码可在 https://github.com/karabekmez/TS-GSMM 网站上获取。
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引用次数: 0
Biotechnology and Bioengineering: Volume 121, Number 9, September 2024 生物技术与生物工程第 121 卷第 9 号,2024 年 9 月
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-13 DOI: 10.1002/bit.28825
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引用次数: 0
A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics 用于生产生物治疗药物的高滴度可扩展中国仓鼠卵巢瞬时表达平台。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-05 DOI: 10.1002/bit.28817
Juan C. Gonzalez-Rivera, Alberto Galvan, Todd Ryder, Monica Milman, Kitty Agarwal, Lakshmi Kandari, Anurag Khetan

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application—cell growth, buffer exchange, and cell mass concentration—and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

中国仓鼠卵巢(CHO)细胞中的瞬时基因表达(TGE)提供了一条加速生物制剂开发的途径,与传统的细胞系开发相比,它能提前数周至数月提供材料。然而,生产率低、产品质量不稳定以及可扩展性方面的挑战阻碍了它的广泛应用。在这项研究中,我们开发了一种可扩展的基于 CHO 的 TGE 系统,可在未修饰的宿主体内实现 1.9 克/升的单克隆抗体产量。我们整合了连续流-电穿孔和交替切向流(ATF)灌注,实现了从 N-1 灌注到 50 升生物反应器生产的端到端封闭系统。通过优化三合一应用(细胞生长、缓冲液交换和细胞质量浓缩)的 ATF 操作和流电穿孔工艺,我们开发出了一个利用瞬时转染细胞生产生物治疗药物的平台。我们证明了这一平台的可扩展性,它的生物反应器容量可达 50 升,滴度保持在 1 克/升以上。我们还展示了瞬时和稳定生产材料之间的可比质量,以及不同批次之间的一致性。结果证实,纯度、电荷变体和 N-聚糖特征都是相似的。我们的研究证明了基于 CHO 的 TGE 平台在加快生物制剂工艺开发进度方面的潜力,并提供了支持其生产早期临床材料可行性的证据,旨在加强对 TGE 更广泛应用的认可。
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引用次数: 0
Chemically defined production of engineered cardiac tissue microspheres from hydrogel-encapsulated pluripotent stem cells 利用水凝胶包裹的多能干细胞,以化学方法生产工程化心脏组织微球。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-05 DOI: 10.1002/bit.28818
Ferdous B. Finklea, Mohammadjafar Hashemi, Yuan Tian, Hanna Hammons, Caroline Halloin, Wiebke Triebert, Robert Zweigerdt, Elizabeth A. Lipke

Chemically defined, suspension culture conditions are a key requirement in realizing clinical translation of engineered cardiac tissues (ECTs). Building on our previous work producing functional ECT microspheres through differentiation of biomaterial encapsulated human induced pluripotent stem cells (hiPSCs), here we establish the ability to use chemically defined culture conditions, including stem cell media (E8) and cardiac differentiation media (chemically defined differentiation media with three components, CDM3). A custom microfluidic cell encapsulation system was used to encapsulate hiPSCs at a range of initial cell concentrations and diameters in the hybrid biomaterial, poly(ethylene glycol)-fibrinogen (PF), for the formation of highly spherical and uniform ECT microspheres for subsequent cardiac differentiation. Initial microsphere diameter could be tightly controlled, and microspheres could be produced with an initial diameter between 400 and 800 µm. Three days after encapsulation, cardiac differentiation was initiated through small molecule modulation of Wnt signaling in CDM3. Cardiac differentiation occurred resulting in in situ ECT formation; results showed that this differentiation protocol could be used to achieve cardiomyocyte (CM) contents greater than 90%, although there was relatively high variability in CM content and yield between differentiation batches. Spontaneous contraction of ECT microspheres initiated between Days 7 and 10 of differentiation and ECT microspheres responded to electrical pacing up to 1.5 Hz. Resulting CMs had well-defined sarcomeres and the gap junction protein, connexin 43, and had appropriate temporal changes in gene expression. In summary, this study demonstrated the proof-of-concept to produce functional ECT microspheres with chemically defined media in suspension culture in combination with biomaterial support of microsphere encapsulated hiPSCs.

化学定义的悬浮培养条件是实现工程心脏组织(ECT)临床转化的关键要求。在我们之前通过分化生物材料封装的人类诱导多能干细胞(hiPSCs)生产功能性ECT微球的工作基础上,我们在这里建立了使用化学定义培养条件的能力,包括干细胞培养基(E8)和心脏分化培养基(含三种成分的化学定义分化培养基,CDM3)。我们使用定制的微流体细胞包被系统,将不同初始细胞浓度和直径的hiPSCs包被在聚乙二醇-纤维蛋白原(PF)混合生物材料中,以形成高度球形和均匀的ECT微球,用于随后的心脏分化。微球的初始直径可严格控制,可制成初始直径在400至800微米之间的微球。封装三天后,通过小分子调节 CDM3 中的 Wnt 信号,启动了心脏分化。结果表明,这种分化方案可使心肌细胞(CM)含量超过 90%,但不同分化批次的 CM 含量和产量差异相对较大。ECT微球在分化第7至10天开始自发收缩,ECT微球对高达1.5赫兹的电起搏有反应。分化出的CM具有清晰的肌节和缝隙连接蛋白(connexin 43),基因表达也有适当的时间变化。总之,本研究证明了在悬浮培养中使用化学定义的培养基结合生物材料支持微球封装的 hiPSCs 生产功能性 ECT 微球的概念验证。
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引用次数: 0
Peptide ligands for the universal purification of exosomes by affinity chromatography 通过亲和层析法普遍纯化外泌体的肽配体。
IF 3.5 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2024-08-04 DOI: 10.1002/bit.28821
Ryan E. Kilgore, Brandyn D. Moore, Sobhana A. Sripada, Wenning Chu, Shriarjun Shastry, Eduardo Barbieri, Shiqi Hu, Weihua Tian, Heidi Petersen, Mohammad Mohammadifar, Aryssa Simpson, Ashley Brown, Joseph Lavoie, Driss Elhanafi, Steffen Goletz, Ke Cheng, Michael A. Daniele, Stefano Menegatti

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.

作为药物输送、疫苗接种和再生医学的载体,外泌体的作用日益突出。由于外泌体的表面生物化学反映了母细胞膜,因此这些纳米级生物制剂具有免疫原性低、组织滋养性可调以及能够携带各种有效载荷穿越生物屏障等特点。然而,由于外泌体的大小和组成具有异质性,因此对其进行纯化具有挑战性。超速离心和过滤等传统技术的产物产量和纯度都很低,而且会危及颗粒的完整性。亲和色谱法是外泌体纯化的绝佳途径。然而,目前的亲和介质依赖于抗体配体,这种配体的选择性可获得高纯度的产品,但却要求针对不同表面生化性质的外泌体定制吸附剂,而其结合强度所带来的洗脱条件可能会损害产品的活性。为了解决这些问题,本研究首次推出了用于从重组原料中通用纯化外泌体的多肽亲和配体。这些肽的设计目的是:(1)对外泌体标记物具有杂乱的生物识别能力,不会结合与过程相关的污染物;(2)在保障产品稳定性的条件下洗脱产品。所选配体 SNGFKKHI 和 TAHFKKKH 证明能够捕获 14 种细胞来源分泌的外泌体,并纯化了来自 HEK293、PC3、MM1、U87 和 COLO1 细胞的外泌体。最后,SNGFKKHI-Toyopearl 树脂被用于两步纯化过程,从 HEK293 细胞液中分离出外泌体,产率达 68%,HCPs 的滴度降至 68 ng/mL。分析色谱、Western 印迹分析、透射电子显微镜和纳米粒子追踪分析证实了分离出的外泌体的生物分子和形态特征。
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