Burnham Brenda, Nass Shelley, Kong Elton, Mattingly MaryEllen, Woodcock Denise, Song Antonius, Wadsworth Samuel, Cheng Seng H, Scaria Abraham, O'Riordan Catherine R
Gene Therapy, Genzyme, a Sanofi Company , Framingham, Massachusetts.
Hum Gene Ther Methods. 2015 Dec;26(6):228-42. doi: 10.1089/hgtb.2015.048. Epub 2015 Oct 15.
Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.
重组腺相关病毒（rAAV）载体代表了一类新型生物制药。用于基因治疗的临床级rAAV载体的生产将受益于能够在均匀性、纯度和生产一致性方面监测药品质量的分析方法。在此，我们展示了分析超速离心法（AUC）在表征rAAV载体制剂均匀性方面的新应用。我们表明，rAAV载体的单次沉降速度运行检测并定量了许多不同的病毒种类，例如携带完整基因组的载体、缺乏载体基因组的（空颗粒）以及包含片段化或不完整载体基因组的。该信息是通过使用计算机程序SEDFIT对由折射或紫外检测系统生成的AUC数据进行直接边界建模获得的。使用AUC，我们表明多个参数对载体质量有影响，包括AAV基因组形式（即自我互补与单链）、载体基因组大小以及生产和纯化方法。因此，AUC是确定最佳生产和纯化工艺以及监测rAAV载体物理属性以确保其质量的关键工具。
