Optimising and adapting perfusion feeds in serum-free medium to intensify CAR-T cell expansion in stirred-tank bioreactors.

IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Frontiers in Bioengineering and Biotechnology Pub Date : 2025-06-02 eCollection Date: 2025-01-01 DOI:10.3389/fbioe.2025.1593895
Pierre Springuel, Tiffany Hood, Fern Slingsby, Timo Schmidberger, Nicola Bevan, Noushin Dianat, Julia Hengst, Qasim A Rafiq
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引用次数: 0

Abstract

The ex vivo expansion of autologous chimeric antigen receptor (CAR) T cells to reach a therapeutic dose significantly prolongs manufacturing time and increases overall costs. The common use of animal- or human-derived serum in T cell expansion culture media further contributes to process variability, costs and introduces additional safety concerns. To address these challenges, this study focused on intensifying CAR-T cell expansion using perfusion processes in xeno-free (XF) and serum-free (SF) culture medium. The impacts of alternative tangential flow (ATF) perfusion rates, perfusion start times and donor variability were evaluated using a Design of Experiments (DOE) approach in the Ambr® 250 High-Throughput Perfusion stirred-tank bioreactor. This allowed the identification of optimal combinations of perfusion parameters on a per-donor basis, enabling 4.5-fold improvements in final cell yields and over 50% reductions in the expansion time required to reach a representative CAR-T dose compared to a fed-batch process. Subsequent process development then established an adaptive perfusion strategy enabling 130 ± 9.7-fold expansions to achieve final cell densities of 33.5 ± 3 × 106 cells/mL while reducing medium requirements by 11% without compromising CAR-T cell quality attributes compared to static well-plate cultures. Harvested cells predominantly expressed naïve and central memory markers, low levels of exhaustion markers, and maintained cytotoxicity and cytokine release in vitro. This study demonstrates the potential of optimising and adapting perfusion strategies in XF/SF-culture medium to enhance CAR-T cell yields, shorten expansion times and reduce medium consumption while addressing patient variability in clinical manufacturing. Key considerations for future implementation and improvement of adaptive perfusion feeds for clinical CAR-T manufacturing are also discussed.

优化和适应无血清培养基中的灌注饲料,以加强搅拌槽生物反应器中CAR-T细胞的扩增。
自体嵌合抗原受体(CAR) T细胞的体外扩增达到治疗剂量显着延长了制造时间并增加了总体成本。在T细胞扩增培养基中普遍使用动物或人源性血清,进一步增加了工艺的可变性、成本,并引入了额外的安全问题。为了解决这些挑战,本研究的重点是通过在无xeno (XF)和无血清(SF)培养基中灌注过程来增强CAR-T细胞的扩增。在Ambr®250高通量灌注搅拌槽生物反应器中,采用实验设计(DOE)方法评估了替代切向流(ATF)灌注率、灌注开始时间和供体可变性的影响。这允许在每个供体的基础上确定灌注参数的最佳组合,使最终细胞产量提高4.5倍,与补料批工艺相比,达到代表性CAR-T剂量所需的扩增时间减少50%以上。随后的工艺开发建立了自适应灌注策略,使130±9.7倍的扩增达到33.5±3 × 106个细胞/mL的最终细胞密度,同时与静态孔板培养相比,在不影响CAR-T细胞质量属性的情况下减少11%的培养基需求。收获的细胞主要表达naïve和中枢记忆标记,低水平的衰竭标记,并维持体外细胞毒性和细胞因子释放。这项研究证明了在XF/ sf培养基中优化和调整灌注策略的潜力,以提高CAR-T细胞产量,缩短扩增时间,减少培养基消耗,同时解决临床制造中的患者差异。本文还讨论了未来实施和改进临床CAR-T制造的自适应灌注饲料的关键考虑因素。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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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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