Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation

Molecular Therapy(2005)

引用 123|浏览14
暂无评分
摘要
Aggregation of recombinant AAV2 results in reduced yield during purification and may have deleterious effects on vector transduction efficiency, biodistribution and immunogenicity following in vivo administration. Studies to elucidate the mechanism of vector aggregation and methods to prevent its occurrence are reported. In excipient screening studies, the sugars sorbitol, sucrose, mannitol, trehalose, or glycerol at concentrations of up to 5% (w/v), or surfactants Tween 80 or Pluronic F68, did not prevent aggregation. Aggregation was prevented by the use of various salts at concentrations corresponding to solution ionic strengths of >200 mM. AAV2 vectors purified by double cesium chloride gradient centrifugation, cation-exchange chromatography, or combined chromatography and gradient centrifugation each demonstrated a similar requirement for ionic strength to prevent aggregation. AAV2 vectors concentrated to 6.7 × 1013 vector genome (vg)/mL in neutral-buffered isotonic saline resulted in 59 ± 6.0% recovery of nonaggregated material compared to 96 ± 4.4% recovery in an isotonic formulation with elevated ionic strength. The latter showed no aggregation following storage or after 10 freeze–thaw cycles at −20°C. AAV2 vectors stored for an extended period in an elevated ionic strength formulation retained a high infectivity titer (13 vg/infectious unit) and transduction efficiency. Nuclease digestion of purified AAV2 vectors reduced aggregation, implicating trace amounts of vector surface nucleic acids in interparticle binding.
更多
查看译文
关键词
adeno-associated virus,vector aggregation,ionic strength
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要