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 × 10 ¹¹ 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.

Recombinant adeno-associated virus (rAAV) has emerged as a prominent vector for in vivo gene therapy, owing to its distinct advantages. Accurate determination of the rAAV genome titer is crucial for ensuring the safe and effective administration of clinical doses. The evolution of the rAAV genome titer assay from quantitative PCR (qPCR) to digital PCR (dPCR) has enhanced accuracy and precision, yet practical challenges persist. This study systematically investigated the impact of various operational factors on genome titration in a single-factor manner, aiming to address potential sources of variability in the quantitative determination process. Our findings revealed that a pretreatment procedure without genome extraction exhibits superior precision compared with titration with genome extraction. Additionally, notable variations in titration results across different brands of dPCR instruments were documented, with relative standard deviation (RSD) reaching 23.47% for AAV5 and 11.57% for AAV8. Notably, optimal operations about DNase I digestion were identified; we thought treatment time exceeding 30 min was necessary, and there was no need for thermal inactivation after digestion. And we highlighted that thermal capsid disruption before serial dilution substantially affected AAV genome titers, causing a greater than ten-fold decrease. Conversely, this study found that additive components of dilution buffer are not significant contributors to titration variations. Furthermore, we found that repeated freeze–thaw cycles significantly compromised AAV genome titers. In conclusion, a comprehensive dPCR titration protocol, incorporating insights from these impact factors, was proposed and successfully tested across multiple serotypes of AAV. The results demonstrate acceptable variations, with the RSD consistently below 5.00% for all tested AAV samples. This study provides valuable insights to reduce variability and improve the reproducibility of AAV genome titration using dPCR.

Adeno-associated virus (AAV) is a promising gene therapy vector due to its high transduction efficiency, low pathogenicity, low immunogenicity, and the ability to mediate the long-term stable expression of exogenous genes. The viral particle titer is an essential quality attribute of recombinant adeno-associated virus (rAAV) gene therapy products. Multiangle light scattering (MALS) is an important means of directly measuring the absolute molecular weight and distribution of macromolecular drugs. This study established and validated a method based on SEC-UV-MALS-RI tandem technology for accurately determining rAAV particle titers. The verification results indicated that the method exhibited good specificity, linearity, precision, accuracy, and durability. Several collaborative laboratories used this method to calibrate the standard substances needed for rAAV particle titer determination. The results suggested that combining the SEC-MALS method with standard substances enables the rapid and accurate measurement of the viral particle titers in rAAV gene therapy products.

Currently, the most commonly used methods for detecting Mycoplasma are the culture method and the indicator cell culture method. However, both approaches exhibit low sensitivity and are incapable of detecting low-concentration contamination. In addition, the detection period may extend up to 28 days, which is unsuitable for rapid screening and may delay timely contamination control measures. To address these limitations, a Mycoplasma detection method based on nucleic acid amplification technology (NAT) was developed following a comparative analysis of gene sequences from various Mycoplasma species. The method was validated with respect to its detection performance and its applicability to biological product samples. DNA was extracted from Mycoplasma-contaminated samples using a magnetic bead-based nucleic acid extraction method. Universal primers were designed based on the highly conserved 16S rRNA gene sequence of Mycoplasma, and amplification was performed using multiplex quantitative PCR (qPCR) with fluorescent probes. The limit of detection (LOD) was established based on statistics of 24 replicates. Method specificity and robustness were evaluated according to the guidelines set by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH Q2), while sample applicability was assessed in accordance with the European Pharmacopoeia (EP) . The NAT-based Mycoplasma detection method enabled rapid, qualitative identification of Mycoplasma contamination. The validated LOD was 10 CFU/mL, and the method met predefined requirements for sensitivity, specificity, and robustness. To assess applicability, real biological product samples, including monoclonal antibodies, antibody fusion proteins, bispecific antibodies, and trispecific antibodies, were spiked with 10 CFU/mL of standard Mycoplasma strains. All spiked samples tested positive. These findings confirm that the NAT-based Mycoplasma detection method is suitable for process control and product release testing in the production of biological products.