基于GMP级可溶性多孔微载体的大规模电池生产

摘要

在这里，我们提出了一种方案，通过基于GMP级可溶解微载体的全封闭系统实现贴壁细胞的大规模制造。人间充质干细胞、HEK293T细胞和Vero细胞的培养经过验证，符合细胞和基因治疗行业的数量要求和质量标准。

摘要

长期以来，细胞和基因治疗（CGT）行业的研究人员在细胞的高效和大规模扩增方面面临着巨大的挑战。针对二维平面培养系统的主要缺点，我们创新性地开发了一种基于GMP级可溶解多孔微载体的自动化封闭式工业规模细胞生产（ACISCP）平台，用于贴壁细胞的3D培养，包括人间充质干细胞/基质细胞（hMSCs）、HEK293T细胞和Vero细胞。为了实现大规模扩增，使用5 L和15 L搅拌罐生物反应器进行了两级扩增，在9天内产生了1.1 x^{10 10} hMSCs，整体扩增了128倍。通过完全溶解微载体收获细胞，浓缩，洗涤和配制基于连续流离心机的细胞处理系统，然后与细胞填充系统分装。与2D平面培养相比，从3D培养中收获的hMSCs的质量没有显著差异。我们还将这些可溶解的多孔微载体应用于CGT领域的其他流行细胞类型;具体而言，HEK293T 细胞和 Vero 细胞的峰值细胞密度分别为 1.68 x 10 7 个细胞/mL 和 1.08 x 10⁷ 个细胞/mL。本研究提供了一种使用生物工艺工程平台的方案，利用GMP级可溶解微载体和先进的封闭设备的特性来实现贴壁细胞的工业规模生产。

引言

在过去的二十年里，CGT行业呈指数级增长。下一代药物的发展有望治疗和治愈许多难治性疾病¹.自 2017 年美国食品药品监督管理局 （FDA） 首次批准 CGT 产品 Kymriah 以来，全球 CGT 相关研发持续快速增长，FDA 看到 CGT 的积极研究性新药申请在 2018 年增加到 500 项²。据预测，到 2030 年，美国 CGT 产品的批准数量可能达到 54-74 个²。

虽然CGT研究和创新的快速增长令人兴奋，但实验室研究和工业规模制造之间仍然存在巨大的技术差距，这些技术可以以可承受的成本提供这些有前途的药物，以达到尽可能多的患者。这些临床试验目前采用的流程是在实验室中建立的，用于小规模实验，需要付出巨大的努力来改进和创新CGT制造³。CGT产品有很多种，其中大多数基于活细胞，可以是同种异体的、自体的、工程的或天然的。这些活体药物比小分子实体或生物制剂复杂得多，因此使大规模生产成为一项重大挑战^4,5,6。在这项工作中，我们展示了一种广泛应用于CGTs的三种锚定依赖性细胞的大规模细胞生产方案....

研究方案

人体脐带购自北京清华长庚医院。所有关于人脐带间充质干细胞 （UCMSCs） 的获取、分离和培养的程序和方案均在知情同意下进行，并经北京清华长庚医院伦理委员会批准（备案号 22035-4-02），程序和方案符合 1964 年赫尔辛基宣言及其后来的修正案或类似的伦理标准。

1. hMSCs、HEK293T细胞和Vero细胞的单层培养

1. 根据报告的方法²¹从沃顿氏果冻中分离原代人UCMSC。使用无血清 （SF） hMSC 培养基分离和生长原代 UCMSC，在第 2 代收获细胞，并作为主细胞库 （MCB） 冷冻保存。
2. 在一个 T75 烧瓶中接种 6 x 10⁵ 人 UCMSC（优选传代 2 或传代 3 个细胞）（即，2D 平面血管上的接种密度为 8,000 个细胞/cm²），用于单层培养，用 15 mL 完全 SF hMSC 培养基。通过烧瓶的八字形运动确保均匀播种。在37°C下在5%CO₂ 细胞培养箱中培养至80%-90%汇合度。
3. 收获时，倒出细胞培养基，并用 3-5 mL 磷酸盐缓冲盐水 （PBS） 冲洗细胞两次。然后，加入2mL 0.25%胰蛋白酶-EDTA，并在37°C下孵育1-2分钟，直到大多数细胞变圆并从烧瓶中分离，如在明场倒置显微镜下观察到的那样4倍物镜。加入 3 m....

讨论

免疫疗法和干细胞疗法都使用活细胞作为药物;然而，它们的最终产品不应像小分子或病毒那样进行纯化或灭菌。因此，应始终牢记质量源于设计 （QbD） 的原则，并将其实际应用于电池生产过程中的化学制造和控制 （CMC） 过程²³。优先考虑完全封闭的细胞培养系统以及处理系统和灌装系统，以满足要求。在这项研究中，我们提出了一种利用基于 GMP 级、可溶解和多孔 3D 微载体的.......

材料

Name	Company	Catalog Number	Comments
0.25% trypsin EDTA	BasalMedia	S310JV	Used for 2D cell harvest.
3D FloTrix Digest	CytoNiche Biotech	R001-500	This is a reagent that specifically dissolves 3D TableTrix microcarriers.
3D FloTrix MSC Serum Free Medium	CytoNiche Biotech	RMZ112	This is a serum-free,animal-free medium for mesenchymal stem cell expansion and maintenance in 2D planar culture as well as 3D culture on 3D TableTrix microcarriers.
3D FloTrix Single-Use Filtration Module	CytoNiche Biotech	R020-00-10	This module contains 0.22 μm capsule filters used for filtration of culture medium and digest solution.
3D FloTrix Single-Use Storage Bag (10 L)	CytoNiche Biotech	R020-00-03	Used as feed bag for 5 L bioreactor.
3D FloTrix Single-Use Storage Bag (3 L)	CytoNiche Biotech	R020-00-01	Used as cell seeding or transfer bags.
3D FloTrix Single-Use Storage Bag (50 L)	CytoNiche Biotech	R020-00-04	Used as feed bag for 15 L bioreactor.
3D FloTrix vivaPACK Disposable Fill&Finish Consumable Kit	CytoNiche Biotech	PACK-01-01	This is a standard kit adapted to 3D vivaPACK fill and finish system.
3D FloTrix vivaPACK fill and finish system for cells	CytoNiche Biotech	vivaPACK	This system is a closed liquid handling device, with automated mixing and gas exhausting functions. Cells resuspended in cryopreservation buffer can be rapidly and evenly aliquoted into 20 bags per batch.
3D FloTrix vivaPREP PLUS cell processing system	CytoNiche Biotech	vivaPREP PLUS	This system is a continuous flow centrifuge-based device.Cells can be concentrated, washed, and resuspended under completely closed procedures.
3D FloTrix vivaPREP PLUS Disposable Cell Processing Kit	CytoNiche Biotech	PREP-PLUS-00	This is a standard kit adapted to 3D vivaPREP PLUS cell processing.
3D FloTrix vivaSPIN  bioreactor 15 L	CytoNiche Biotech	FTVS15	This bioreactor product employs a controller, a 15 L glass stirred-tank vessel, and assessories. A special perfusion tube is available.
3D FloTrix vivaSPIN  bioreactor 5 L	CytoNiche Biotech	FTVS05	This bioreactor product employs a controller, a 5 L glass stirred-tank vessel, and assessories.A special perfusion tube is available.
3D FloTrix vivaSPIN Closed System Consumable Pack (10/15 L)	CytoNiche Biotech	R020-10-10	This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 15 L, containing sampling bags.
3D FloTrix vivaSPIN Closed System Consumable Pack (2/5 L)	CytoNiche Biotech	R020-05-10	This is a standard tubing kit adapted to 3D vivaSPIN bioreactor 5 L, containing sampling bags.
3D TableTrix microcarriers G02	CytoNiche Biotech	G02-10-10g	These porous and degradable microcarriers are suitable for HEK293T cell culture. They come pre-sterilized in 10g/bottle with C-Flex tubings for welding to tubes on bioreactors.
3D TableTrix microcarriers V01	CytoNiche Biotech	V01-100-10g	These porous and degradable microcarriers are suitable for adherent cell culture, they come as non-sterilized microcarriers that need to be autoclaved in PBS before use. They are especially suitable for vaccine production.
3D TableTrix microcarriers W01	CytoNiche Biotech	W01-10-10g (single-use packaging); W01-200 (tablets)	These porous and degradable microcarriers are suitable for adherent cell culture, especially for cells that need to be harvested as end products. They come pre-sterilized in 10g/bottle with C-Flex tubings for welding to tubes on bioreactors.The product has obtained 2 qualifications for pharmaceutical excipients from CDE, with the registration numbers of [F20200000496; F20210000003]. It has also received DMF qualification for pharmaceutical excipients from FDA, with the registration number of [DMF:35481]
APC anti-human CD45 Antibody	BioLegend	368512	Used in flow cytometry for MSC identity assessment
Calcein-AM/PI Double Staining Kit	Dojindo	C542	Calcein-AM/PI Double Staining Kit is utilized for simultaneous fluorescence staining of viable and dead cells. This kit contains Calcein-AM and Propidium Iodide (PI) solutions, which stain viable and dead cells, respectively.
Cap for EZ Top Container Closures for NALGENE-containers (500mL)	Saint-Gobain	CAP-38	Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.
C-Flex Tubing, Formulation 374 (0.25 in x 0.44 in)	Saint-Gobain	374-250-3	Used for tube welding and disconnection.
CryoMACS Freezing Bag 50	Miltenyi Biotec	200-074-400	Used for expanding the 3D FloTrix vivaPACK Disposable Fill&Finish Consumable Kit.
Dimethyl Sulfate (DMSO)	Sigma	D2650-100mL	Used for preparation of cryopreservation solution.
Dulbecco's Modified Eagle Medium (DMEM)	BasalMedia	L120KJ	Used for cultivation of HEK293T and Vero cells.
DURAN Original GL 45 Laboratory bottle (2 L)	DWK life sciences	218016357	Used for waste collection from the 5 L bioreactor.
DURAN Original GL 45 Laboratory bottle (5 L)	DWK life sciences	218017353	Used for waste collection from the 15 L bioreactor.
DURAN Original GL 45 Laboratory bottle (500 mL)	DWK life sciences	218014459	Used for supplementary bottle of 0.1 M NaOH.
EZ Top Container Closures for NALGENE-containers (500mL)	Saint-Gobain	EZ500 ML-38-2	Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.
Fetal bovine serum (FBS) superior quality	Wisent	086-150	Used for cultivation of HEK293T cells.
FITC anti-human CD14 Antibody	BioLegend	301804	Used in flow cytometry for MSC identity assessment.
FITC anti-human CD34 Antibody	BioLegend	343504	Used in flow cytometry for MSC identity assessment.
FITC anti-human CD90 (Thy1) Antibody	BioLegend	328108	Used in flow cytometry for MSC identity assessment.
Flow cytometry	Beckman Coulter	CytoFLEX	Used for cell identity assessment.
Fluorescence Cell Analyzer	Alit life science	Countstar Rigel S2	Used for cell counting. Cell viability can be calculated by staining with AO/PI dyes.
GL 45 Multiport Connector Screw Cap with 2 ports	DWK life sciences	292632806	Brands and catalogue numbers are only for example, similar products are available from various suppliers and as long as they have the same functionality, items could be substituted with other brands.
Glucose Meter	Sinocare	6243578	Used for detecting glucose concentration in cell culture medium and supernatant.
Hank's Balanced Salt Solution (HBSS), with calcium and magnesium	Gibco	14025092	Used for preparation of digest solution.
Human Albumin 20% Behring (HSA)	CSL Behring	N/A	Used for preparation of wash buffer.
Inverted fluorescent microscope	OLYMBUS	CKX53SF	Used for brifgt field and fluorescent observation and imaging.
Nalgene Measuring Cylinder (500 mL)	Thermo Scientific	3662-0500PK	Used for calibrating the liquid handling volume speed of peristaltic pumps.
Newborn calf serum (NBS) superfine	MINHAI BIO	SC101.02	Used for cultivation of Vero cells.
OriCell human mesenchymal stem cell adipogenic differentiation and characterization kit	Cyagen	HUXUC-90031	Used for tri-lineage differentiation of hUCMSCs.
OriCell human mesenchymal stem cell chondrogenic differentiation and characterization kit	Cyagen	HUXUC-90041	Used for tri-lineage differentiation of hUCMSCs.
OriCell human mesenchymal stem cell osteogenic differentiation and characterization kit	Cyagen	HUXUC-90021	Used for tri-lineage differentiation of hUCMSCs.
PE anti-human CD105 Antibody	BioLegend	800504	Used in flow cytometry for MSC identity assessment.
PE anti-human CD19 Antibody	BioLegend	302208	Used in flow cytometry for MSC identity assessment.
PE anti-human CD73 (Ecto-5'-nucleotidase) Antibody	BioLegend	344004	Used in flow cytometry for MSC identity assessment.
PE anti-human HLA-DR Antibody	BioLegend	307605	Used in flow cytometry for MSC identity assessment.
Phosphate Buffered Saline (PBS)	Wisent	311-010-CL	Used in autoclaving of glass vessel and V01 microcarriers, and replacement of culture medium.
Sani-Tech Platinum Cured Sanitary Silicone Tubing (0.13 in x 0.25 in)	Saint-Gobain	ULTRA-C-125-2F	Used for solution transfering driven by peristaltic pumps.
Sterile Saline	Hopebiol	HBPP008-500	Used for preparation of wash buffer.
Trypzyme Recombinant Trypsin	BasalMedia	S342JV	This reagent is used for bead-to-bead transfer of HEK293T and Vero cells.
Tube Sealer	Yingqi Biotech	Tube Sealer I	This sealer is compatible with both C-Flex tubing and PVC tubing.
Tube Welder for PVC tubing	Chu Biotech	Tube Welder Micro I	Used for welding of PVC tubing.
Tube Welder for TPE tubing	Yingqi Biotech	Tube Welder I-V2	Used for welding of TPE tubing.
ViaStain AO / PI Viability Stains	Nexcelom	CS2-0106-25mL	Dual-Fluorescence Viability, using acridine orange (AO) and propidium iodide (PI), is the recommended method for accurate viability analysis of primary cells, such as PBMCs, and stem cells in samples containing debris.