Effective removal of host cell-derived nucleic acids bound to hepatitis B core antigen virus-like particles by heparin chromatography.

IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Frontiers in Bioengineering and Biotechnology Pub Date : 2024-10-03 eCollection Date: 2024-01-01 DOI:10.3389/fbioe.2024.1475918
Angela Valentic, Jürgen Hubbuch
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Abstract

Virus-like particles (VLPs) show considerable potential for a wide array of therapeutic applications, spanning from vaccines targeting infectious diseases to applications in cancer immunotherapy and drug delivery. In the context of hepatitis B core antigen (HBcAg) VLPs, a promising candidate for gene delivery approaches, the naturally occurring nucleic acid (NA) binding region is commonly utilized for effective binding of various types of therapeutic nucleic acids (NAther). During formation of the HBcAg VLPs, host cell-derived nucleic acids (NAhc) might be associated to the NA binding region, and are thus encapsulated into the VLPs. Following a VLP harvest, the NAhc need to be removed effectively before loading the VLP with NAther. Various techniques reported in literature for this NAhc removal, including enzymatic treatments, alkaline treatment, and lithium chloride precipitation, lack quantitative evidence of sufficient NAhc removal accompanied by a subsequent high VLP protein recovery. In this study, we present a novel heparin chromatography-based process for effective NAhc removal from HBcAg VLPs. Six HBcAg VLP constructs with varying lengths of the NA binding region and diverse NAhc loadings were subjected to evaluation. Process performance was thoroughly examined through NAhc removal and VLP protein recovery analyses. Hereby, reversed phase chromatography combined with UV/Vis spectroscopy, as well as silica spin column-based chromatography coupled with dye-based fluorescence assay were employed. Additionally, alternative process variants, comprising sulfate chromatography and additional nuclease treatments, were investigated. Comparative analyses were conducted with LiCl precipitation and alkaline treatment procedures to ascertain the efficacy of the newly developed chromatography-based methods. Results revealed the superior performance of the heparin chromatography procedure in achieving high NAhc removal and concurrent VLP protein recovery. Furthermore, nuanced relationships between NA binding region length and NAhc removal efficiency were elucidated. Hereby, the construct Cp157 surpassed the other constructs in the heparin process by demonstrating high NAhc removal and VLP protein recovery. Among the other process variants minimal performance variations were observed for the selected constructs Cp157 and Cp183. However, the heparin chromatography-based process consistently outperformed other methods, underscoring its superiority in NAhc removal and VLP protein recovery.

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用肝素层析法有效去除与乙型肝炎核心抗原病毒样颗粒结合的宿主细胞衍生核酸。
病毒样颗粒(VLPs)在广泛的治疗应用中显示出相当大的潜力,从针对传染病的疫苗到癌症免疫疗法和给药应用,无所不包。乙型肝炎核心抗原(HBcAg)VLP 是一种很有希望的基因递送方法,在乙型肝炎核心抗原(HBcAg)VLP 中,通常利用天然存在的核酸(NA)结合区来有效结合各种类型的治疗核酸(NAther)。在 HBcAg VLPs 的形成过程中,宿主细胞衍生的核酸(NAhc)可能与 NA 结合区相关联,从而被包裹到 VLPs 中。收获 VLP 后,需要先有效去除 NAhc,然后再将 NAther 装入 VLP。文献中报道的各种去除NAhc的技术,包括酶处理、碱处理和氯化锂沉淀等,都缺乏足够去除NAhc的定量证据,同时也缺乏随后高VLP蛋白回收率的定量证据。在本研究中,我们提出了一种基于肝素层析的新型工艺,可有效去除 HBcAg VLP 中的 NAhc。六种 HBcAg VLP 构建物的 NA 结合区长度各不相同,NAhc 负载也各不相同,我们对它们进行了评估。通过NAhc去除率和VLP蛋白回收率分析,对工艺性能进行了全面检测。为此,采用了反相色谱法结合紫外/可见光谱法,以及硅胶旋柱色谱法结合染料荧光测定法。此外,还研究了硫酸层析和额外核酸酶处理的替代工艺变体。与氯化锂沉淀和碱性处理程序进行了比较分析,以确定新开发的色谱法的功效。结果显示,肝素色谱法在实现高 NAhc 去除率和同时 VLP 蛋白回收率方面表现出色。此外,还阐明了 NA 结合区长度与 NAhc 去除效率之间的微妙关系。因此,在肝素工艺中,构建体 Cp157 的 NAhc 去除率和 VLP 蛋白回收率均高于其他构建体。在其他工艺变体中,所选构建体 Cp157 和 Cp183 的性能差异极小。然而,基于肝素色谱法的工艺始终优于其他方法,凸显了其在去除 NAhc 和回收 VLP 蛋白方面的优势。
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来源期刊
Frontiers in Bioengineering and Biotechnology
Frontiers in Bioengineering and Biotechnology Chemical Engineering-Bioengineering
CiteScore
8.30
自引率
5.30%
发文量
2270
审稿时长
12 weeks
期刊介绍: The translation of new discoveries in medicine to clinical routine has never been easy. During the second half of the last century, thanks to the progress in chemistry, biochemistry and pharmacology, we have seen the development and the application of a large number of drugs and devices aimed at the treatment of symptoms, blocking unwanted pathways and, in the case of infectious diseases, fighting the micro-organisms responsible. However, we are facing, today, a dramatic change in the therapeutic approach to pathologies and diseases. Indeed, the challenge of the present and the next decade is to fully restore the physiological status of the diseased organism and to completely regenerate tissue and organs when they are so seriously affected that treatments cannot be limited to the repression of symptoms or to the repair of damage. This is being made possible thanks to the major developments made in basic cell and molecular biology, including stem cell science, growth factor delivery, gene isolation and transfection, the advances in bioengineering and nanotechnology, including development of new biomaterials, biofabrication technologies and use of bioreactors, and the big improvements in diagnostic tools and imaging of cells, tissues and organs. In today`s world, an enhancement of communication between multidisciplinary experts, together with the promotion of joint projects and close collaborations among scientists, engineers, industry people, regulatory agencies and physicians are absolute requirements for the success of any attempt to develop and clinically apply a new biological therapy or an innovative device involving the collective use of biomaterials, cells and/or bioactive molecules. “Frontiers in Bioengineering and Biotechnology” aspires to be a forum for all people involved in the process by bridging the gap too often existing between a discovery in the basic sciences and its clinical application.
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