摘要
前言:肿瘤病毒治疗目前被认为是一种很有前途的癌症治疗方法。腺病毒是众所周知的和广泛的特点,作为一种肿瘤剂.T型他增加了使用该病毒的临床试验的数量,产生了对建立良好的纯化方法的需求。TritonX-100通常用于细胞裂解缓冲液Prepar艾兹。在“化学品注册、评价、授权和限制(REACH)条例”高度关注的物质清单中加入这种表面活性剂,促进了研究有效的替代品。 方法:采用经批准的表面活性剂-多聚山梨酸盐20,建立了一种与Ⅰ期临床试验兼容的肿瘤学腺病毒的纯化方法。拟议下游t雨,由澄清、切向流过滤浓缩、阴离子交换层析中间净化、第二浓度和最终抛光组成。对TritonX-100和Polysorbate 20工艺的STEP进行了评价.从产品回收的角度评价了聚山梨酯20和TritonX-100对下游各步细胞裂解的影响。以及去除杂质。感染病毒颗粒的恢复率为61±4%。宿主细胞蛋白和ds-DNA的缺失率分别为99.9%和97.1%. 结果与结论:聚山梨酯20可替代TritonX-100细胞裂解,对产物回收率、效价及纯度无影响。此外,开发的工艺是可扩展的,并能够提供一个高度纯化的产品,用于第一和第二阶段的临床试验。
关键词: 腺病毒,细胞裂解,下游加工,GMP过程开发,肿瘤学病毒,多山梨酸盐20。
Current Gene Therapy
Title:Clinical-Grade Oncolytic Adenovirus Purification Using Polysorbate 20 as an Alternative for Cell Lysis
Volume: 18 Issue: 6
关键词: 腺病毒,细胞裂解,下游加工,GMP过程开发,肿瘤学病毒,多山梨酸盐20。
摘要: Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer preparations. The addition of this surfactant in the list of substances with the very high concern of the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives.
Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorbate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impurities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell proteins and ds-DNA was 99.9% and 97.1%, respectively.
Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the developed process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.
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Clinical-Grade Oncolytic Adenovirus Purification Using Polysorbate 20 as an Alternative for Cell Lysis, Current Gene Therapy 2018; 18 (6) . https://dx.doi.org/10.2174/1566523218666181109141257
DOI https://dx.doi.org/10.2174/1566523218666181109141257 |
Print ISSN 1566-5232 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5631 |
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