用于基于mRNA的疫苗生产中与磁性微粒配合使用的3D打印一次性分离腔的开发。
Development of a 3D-printed single-use separation chamber for use in mRNA-based vaccine production with magnetic microparticles.
作者信息
Wommer Lars, Meiers Patrick, Kockler Isabelle, Ulber Roland, Kampeis Percy
机构信息
Trier University of Applied Sciences Environmental Campus Birkenfeld Institute for biotechnical Process Design Hoppstädten-Weiersbach Germany.
Technical University Kaiserslautern Institute of Bioprocess Engineering Kaiserslautern Germany.
出版信息
Eng Life Sci. 2021 May 14;21(10):573-588. doi: 10.1002/elsc.202000120. eCollection 2021 Oct.
Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)-functionalized magnetic beads after cell lysis. The mRNA-loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high-gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow-through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)-functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c = 1.6-2.1 g·L in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min with the developed SU-HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min, the retained particle mass is even more than 99.99%.
使用磁珠的实验室方案在疫苗mRNA纯化中变得越来越重要。在此,细胞裂解后产生的mRNA与寡聚(dT)功能化磁珠特异性杂交。负载mRNA的磁珠可以用磁铁选择性分离。随后,通过洗涤步骤去除杂质并洗脱mRNA。在每个步骤中都利用磁分离,使用不同的缓冲液,如裂解/结合缓冲液。为了减少临床试验中大量mRNA疫苗纯化所需的时间,高梯度磁分离(HGMS)是合适的。由此,磁珠被选择性地保留在流通式分离室中。为了满足生物制药生产的要求,开发了一种具有认证材料(美国药典VI类)的一次性HGMS分离室,其可以使用3D打印制造。由于特殊设计,过滤基质本身不与产品接触。该分离室用负载有合成mRNA的寡聚(dT)功能化Dynabeads MyOne悬浮液进行了测试。在裂解/结合缓冲液中浓度为c = 1.6 - 2.1 g·L时,使用开发的SU - HGMS分离室,这些1μm的磁性颗粒在高达150 mL·min的体积流量下保留率超过99.39%。当使用体积流量低于50 mL·min的分离室时,保留的颗粒质量甚至超过99.99%。