Leibiger Thomas M, Remmler Luke A, Green Erica A, Lee Kelvin H
University of Delaware, Department of Chemical and Biomolecular Engineering, Newark, DE, USA.
Mol Ther Methods Clin Dev. 2024 Jul 25;32(3):101306. doi: 10.1016/j.omtm.2024.101306. eCollection 2024 Sep 12.
Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, biolayer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalizing the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet AAVX biosensors across four rAAV serotypes (rAAV2, -5, -8, and -9) and applied in an upstream and downstream processing context. AAVX-BLI measured the capsid titer across a wide concentration range (1 × 10-1 × 10 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for the total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS CaptureSelect AAVX affinity chromatography, showing resin breakthrough dependence on the operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.
需要更快、更准确的分析方法来支持重组腺相关病毒（rAAV）生产系统的发展。最近，生物层干涉术（BLI）已被开发用于通过将AAVX配体功能化到生物传感器探针上进行高通量AAV衣壳滴度测量（AAVX-BLI）。在这项工作中，使用Octet AAVX生物传感器对四种rAAV血清型（rAAV2、-5、-8和-9）评估了AAVX-BLI方法，并将其应用于上游和下游加工环境中。与酶联免疫吸附测定（ELISA）方法相比，AAVX-BLI在较宽的浓度范围（1×10⁻¹×10⁹衣壳/mL）内测量了不同rAAV血清型和样品背景下的衣壳滴度，测量方差和误差降低。生物传感器可进行再生以重复使用，与纯化样品和上清液样品相比，裂解液样品的再生能力降低。AAVX-BLI方法应用于转染优化研究，其中对培养上清液进行直接衣壳滴度测量生成了总载体基因组（VG）滴度的代表性响应面。对于rAAV纯化，AAVX-BLI用于测量与POROS CaptureSelect AAVX亲和色谱法之间的动态结合能力，显示树脂穿透对操作流速的依赖性。测量准确性、血清型和样品背景灵活性以及高样品通量使AAVX-BLI成为其他衣壳滴度测量技术的有吸引力的替代方法。
