Patrick Casey, Petroff Matthew, Plegaria Jefferson, Razinkov Vladimir, Padmakumar Smrithi, Mallela Krishna M G
Center for Pharmaceutical Biotechnology, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 East Montview Blvd, MS C238-V20, Aurora, CO 80045, United States.
Spark Therapeutics, Inc., a subsidiary of Hoffman-La Roche, 3025 Market Street, Philadelphia, PA 19104, United States.
J Pharm Sci. 2025 Jul;114(7):103825. doi: 10.1016/j.xphs.2025.103825. Epub 2025 May 9.
Adeno-associated viruses (AAVs) are the leading viral vector for in-vivo gene therapy, and their use is expected to grow rapidly in the next decade. While AAVs are promising tools for treating many genetic diseases, vectors are vulnerable to adsorptive losses to surfaces due to the low concentrations (10-100 μg/mL) during manufacturing, storage, and administration. Here, we examined the filter-induced loss of two AAV serotypes of Clade E and Clade C as a function of the filter type (materials of composition such as polyvinylidene (PVDF), polyethersulfone (PES), and nylon), pore size (0.22 vs 0.45 μm), pH and ionic strength of the formulation, and hydrophobicity and ionic charge of the AAVs. Our results show that both the AAV serotype and the filter used result in varied amounts of adsorptive losses. Increased hydrophobicity of the AAV or the filter resulted in increased levels of adsorption. Altering formulation pH from 5 - 8 or ionic strength from 50 mM - 180 mM NaCl did not have a significant impact on adsorption loss. Additionally, no change in adsorptive loss was observed with an increase in the filter pore size. Three surfactants (polysorbate 20 (PS20), polysorbate 80 (PS80), and poloxamer 188 (P188)) are observed to rescue the filter-dependent loss of AAVs, but to different minimum concentrations. P188 was found to be the most efficient in the case of nylon filters, whereas PS20 is the most efficient with PES filters. A comparison of empty vs. full capsids shows that empty capsids can be used to optimize formulation parameters for full capsids. This study highlights the importance of surfactants in mitigating filter-dependent surface-induced losses observed during AAV manufacturing processes.
腺相关病毒（AAV）是体内基因治疗的主要病毒载体，预计在未来十年其使用量将迅速增长。虽然AAV是治疗许多遗传疾病的有前景的工具，但由于在制造、储存和给药过程中浓度较低（10 - 100μg/mL），载体易因吸附到表面而损失。在此，我们研究了滤器导致的E分支和C分支两种AAV血清型的损失，作为滤器类型（如聚偏二氟乙烯（PVDF）、聚醚砜（PES）和尼龙等组成材料）、孔径（0.22对0.45μm）、制剂的pH值和离子强度以及AAV的疏水性和离子电荷的函数。我们的结果表明，AAV血清型和使用的滤器都会导致不同程度的吸附损失。AAV或滤器疏水性的增加会导致吸附水平升高。将制剂pH值从5改变到8或离子强度从50mM改变到180mM NaCl对吸附损失没有显著影响。此外，随着滤器孔径的增加，未观察到吸附损失的变化。观察到三种表面活性剂（聚山梨酯20（PS20）、聚山梨酯80（PS80）和泊洛沙姆188（P188））可挽救AAV的滤器依赖性损失，但所需的最低浓度不同。发现P188在尼龙滤器的情况下最有效，而PS20在PES滤器的情况下最有效。空衣壳与完整衣壳的比较表明，空衣壳可用于优化完整衣壳的制剂参数。这项研究强调了表面活性剂在减轻AAV制造过程中观察到的滤器依赖性表面诱导损失方面的重要性。
