RESUMEN
Recombinant proteins are gaining increasing popularity for treating human diseases. The clinical effectiveness of recombinant proteins is directly related to their biological activity, which is an important indicator in drug development and quality control. However, certain recombinant proteins have unclear or complex signal pathways, making detecting their activity in vitro difficult. For instance, recombinant human endostatin (endostatin), a new antitumor drug developed in China, lacks a sensitive and stable assay for its biological activity since being market approval. To address this issue, we performed a genome-wide screening of immortalized human umbilical vein endothelial cells (HUVECs) using a CRISPR/Cas9 knockout library containing 20,000 targeted genes. We identified two potential endostatin-resistant genes, NEPSPP and UTS2, and successfully constructed a highly sensitive cell line, HUVEC-UTS2-3#, by knocking down the UTS2 gene. Based on the optimized parameters of HUVEC-UTS2-3# cells, we established a new method for detecting the biological activity of endostatin. The method was validated, and it produced results consistent with primary HUVEC cells but with higher sensitivity and more stable data. The use of gene-editing technology provides a novel solution for detecting the biological activity of recombinant proteins that other methods cannot detect.
RESUMEN
Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.