Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, India.
Nat Protoc. 2013 May;8(5):821-35. doi: 10.1038/nprot.2013.027. Epub 2013 Apr 4.
Low cost and high efficiency make disposable bioreactors feasible for small-scale therapeutic development and initial clinical trials. We have developed a cryogel-based disposable bioreactor matrix, which has been used for production of protein therapeutics such as urokinase and monoclonal antibodies (mAbs). The protocol discusses the application of a cryogel bioreactor for mAb production. Cryogels composed of either polyacrylamide (PAAm) coupled to gelatin or semi-interpenetrating PAAm-chitosan are synthesized by free-radical polymerization at -12 °C. Hybridoma cells are immobilized over the cryogel bioreactor and incubated for 48 h. Medium is circulated thereafter at 0.2 ml min(-1) and bioreactors can be run continuously for 60 d. The cryogel-based packed-bed bioreactor can be formulated as a monolith or as beads; it also has an efficiency four times what can be obtained using a tissue-culture flask, a high surface-to-volume ratio and effective nutrient transport. After incubation, the bioreactor setup will take about 60 min using a pre-prepared sterilized cryogel.
低成本和高效率使一次性生物反应器能够用于小规模治疗开发和初步临床试验。我们已经开发出了一种基于冷冻凝胶的一次性生物反应器基质，该基质已用于生产尿激酶和单克隆抗体（mAbs）等蛋白治疗药物。该方案讨论了使用冷冻凝胶生物反应器进行 mAb 生产的应用。通过在-12°C 下进行自由基聚合，合成了与明胶偶联的聚丙烯酰胺（PAAm）或半互穿 PAAm-壳聚糖组成的冷冻凝胶。将杂交瘤细胞固定在冷冻凝胶生物反应器上，并孵育 48 小时。此后，以 0.2ml min(-1)的速度循环培养基，生物反应器可以连续运行 60 天。基于冷冻凝胶的填充床生物反应器可以制成整体或珠粒形式；与使用组织培养瓶相比，它具有四倍的效率、高的表面积与体积比和有效的营养物质传输。孵育后，使用预先准备好的消毒冷冻凝胶，生物反应器设置大约需要 60 分钟。
