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.

Human granulocyte colony-stimulating factor (hG-CSF) is primarily used to treat neutropenia induced by cancer chemotherapy and bone marrow transplantation. The current identification test for hG-CSF relies on Western blot (WB), a labor-intensive and technically demanding method. This study aimed to screen and prepare an anti-hG-CSF nanobody to identify and quantify hG-CSF, with the ultimate goal of developing colloidal gold-labeled nanobody test strips for rapid identification. An alpaca was immunized with hG-CSF, and the VHH gene sequence encoding the anti-hG-CSF nanobody was obtained through sequencing following phage display library construction and multiple rounds of biopanning. The nanobody C68, obtained from screening, was expressed by E. coli, and its physicochemical properties such as molecular weight, isoelectric point, and affinity were characterized after purification. WB analysis demonstrated excellent performance of the nanobody in identification tests in terms of specificity, limit of detection (LOD), applicability with products from various manufacturers, and thermal stability. Additionally, we established an ELISA method for hG-CSF quantification utilizing the nanobody C68 and conducted methodological validation. Finally, colloidal gold-based test strips were constructed using the nanobody C68, with a LOD of 30 μg/mL, achieving rapid identification for hG-CSF. This study represents a novel application of nanobodies in pharmaceutical testing and offers valuable insights for developing identification tests for other recombinant protein drugs.

Recombinant adeno-associated viruses (rAAVs) are pivotal gene therapy vectors due to their safety and stable transduction, yet comprehensive characterization of capsid post-translational modifications (PTMs)-critical for potency, immunogenicity, and manufacturing consistency-remains limited across production platforms. This study employs microflow LC-MS/MS coupled with electron-activated dissociation (EAD) to analyze PTMs in clinically relevant rAAV5 and rAAV9 serotypes produced via mammalian (HEK293) and insect (Sf9) cells, with parallel cellular-level evaluation of vector potency and infectivity, conducted under matched purity and capsid thermal stability conditions to isolate PTM-specific effects. Intact mass analysis revealed conserved N-terminal acetylation in VP1/VP3 across both platforms, while PTM profiling identified six distinct modification types, including deamidation, oxidation, and phosphorylation, with Sf9-derived vectors exhibiting 14 % more PTMs than HEK293-produced counterparts. Despite comparable purity and thermostability, HEK293-derived vectors demonstrated superior in vitro potency (1.9-fold higher eGFP expression) and lower physical-to-infectious particle ratios (P:I, 1.8-3.2-fold reduction), linking PTM patterns to enhanced infectivity. EAD fragmentation mapped isoaspartate (IsoAsp) formation to specific asparagine residues, implicating deamidation-driven instability, while analysis of four Sf9-produced rAAV9 batches revealed ≤ 5 % lot-to-lot variability in PTM site counts. Preliminary data identified low-variance PTM sites (e.g., N57, N452; coefficient of variation, CV ≤ 15 %) and IsoAsp levels (CV ≤ 10 %) as potential stability markers for batch consistency monitoring, though their definitive utility as critical quality attributes requires further validation. These findings establish serotype- and platform-specific PTM landscapes under controlled biophysical parameters, providing actionable insights for optimizing production systems and establishing PTM-driven quality control in gene therapy.