整体
色谱法
下游加工
背景(考古学)
化学
九氟化硫
重组DNA
整体式高效液相色谱柱
高效液相色谱法
生物化学
生物
古生物学
夜蛾
基因
催化作用
作者
Kaja Bažec,Marjan Krašna,Andrej Mihevc,Maja Leskovec,Aleš Štrancar,Mojca Tajnik Sbaizero
标识
DOI:10.1002/elps.202300104
摘要
Adeno-associated virus (AAV) vectors are crucial tools for gene therapy applications. As AAVs are administered in vivo, stringent purity requirements must be met, necessitating the development of various downstream processing strategies in accordance with regulatory guidelines. In this context, we focus on the non-affinity serotype-independent recombinant AAV (rAAV) capture step, which involves the use of Convective Interaction Media (CIM) cation-exchange SO3 monoliths. We analyzed differentially pretreated viral samples obtained from the Sf9 cell line and applied these samples to the capture SO3 chromatography step. We conducted screening experiments using CIM SO3 0.05 mL monolithic 96-well plates with buffers of varying pH, sodium chloride concentrations, and the inclusion of poloxamer 188, aiming to select the optimal binding mobile phase. Dynamic binding capacity was defined for different pretreatments and the optimal conditions were subsequently retested using the industrial purification CIMmultus line. The results demonstrated a high overall vector recovery (51%) and a significant reduction in impurities (99.98% for protein reduction and 99.25% for DNA reduction) using the selected capture step parameters, thereby confirming the successful optimization of the rAAV capture step in the downstream process using monoliths.
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