AAV Genome Integrity by CE

The %RSD of general biochemical methods is usually 15%-25%, and the accepted standard of ddPCR is 10%. In most cases the %RSD for PackGene’s QC ddPCR verification data is lower than 5%, and the %RSD of TCID50 is lower than 10%.

Viral empty shell rate can be determined using several techniques including anion chromatography HPLC, Analytical Ultracentrifugation (AUC), Transmission Electron Microscopy (TEM), CyroTEM, or VG Titer/Capsid titer. AUC, TEM and CyroTEM are typically not suitable for quantitative quality control determinations and thus PackGene’s standard method for empty shell rate determination is anion chromatography HPLC. PackGene can provide additional CyroTEM and AUC analytical services to serve as a secondary verification of the results derived from anion chromatography HPLC.

For research-grade plasmids, we perform the QC tests listed below.

Appearance: A visual inspection of the plasmid solution to assess its clarity and the absence of particles or discoloration.
A260/280: Measures the purity of plasmid DNA by comparing absorbance at 260 nm (nucleic acids) and 280 nm (proteins). A ratio of ~1.8 indicates pure DNA.
Homogeneity by Agarose Gel: Assesses the uniformity of plasmid DNA by running it on an agarose gel to ensure consistent molecular weight and the ratio of supercoiled plasmids.
Restriction Analysis: Verifies the identity and integrity of plasmid DNA by cutting it with specific restriction enzymes and analyzing the resulting fragments via gel electrophoresis.
Endotoxin by LAL: Detects bacterial endotoxins in plasmid preparations using the Limulus Amebocyte Lysate (LAL) assay, ensuring plasmids are safe for sensitive applications.

Additionally, upon request, we offer extra QC tests, which are also included in our preclinical plasmid quality control.

Homogeneity by HPLC: Uses High-Performance Liquid Chromatography (HPLC) to evaluate the uniformity and purity of plasmid DNA, and measure the ratio of supercoiled plasmid DNA.
Residual RNA by SYBRGold: Quantifies any remaining RNA in the plasmid preparation by staining with SYBRGold and analyzing fluorescence intensity.
Residual E. coli DNA by qPCR: Detects and quantifies residual E. coli genomic DNA in plasmid preparations using quantitative PCR (qPCR).
Bioburden Testing by Direct Inoculation: Assesses the microbial contamination level of the plasmid preparation by inoculating samples in growth media and monitoring for microbial growth.
Sequencing by Sanger: Confirms the accuracy of the plasmid sequence by using Sanger sequencing to verify the inserted DNA or the entire plasmid.
Residual Host Protein by ELISA: Detects any leftover host proteins in plasmid preparations using an ELISA assay specific to E. coli proteins.
Mycoplasma Contamination by qPCR: Screens for mycoplasma contamination in the plasmid sample using sensitive qPCR techniques.
pH by Potentiometry: Measures the pH of the plasmid solution to ensure it is within the acceptable range for stability and application.
Residual Kanamycin by ELISA: Detects any remaining kanamycin antibiotic from the plasmid selection process using an ELISA assay.
Sterility: Confirms the absence of viable microorganisms in the plasmid preparation, ensuring it is sterile and suitable for sensitive applications.
Osmolality: Measures the osmolality (concentration of solutes) in the plasmid solution to ensure it is within acceptable limits for biological compatibility.

Yes, we provide a capping efficiency assay as part of our mRNA QC services, using enzyme digestion and LC-MS methods. For more information, please contact our technical support team.

Typically, methods such as AUC, TEM, CyroTEM, or VG TITER/CAPSID TITER are employed. However, AUC, TEM, and CyroTEM are not suitable for QC release; they are better suited for quality research and process development. Currently, anion exchange chromatography HPLC is utilized for method development. We at PackGene can provide analysis method development services and validate them against CyroTEM and AUC test results.

Empty capsid rates vary for each serotype obtained through column chromatography.For research-grade and NHP-grade, we guarantee <30% or <20% empaty capsid rate by TEM for most common serotypes. For GMP production, we will test the empty capsid rate during procss development. For example, PackGene’s AAV9 production can be optimized through process improvements to maintain levels below 10%.

PackGene produces high-purity AAV, purified using iodixanol density gradient centrifugation and multiple steps of filtration and sterilization.

PackGene’s offers QC tests for our AAV product depend on the AAV-grade you choose, the detailed QC test comparison are listed here

Here are some QC tests we offer:

① Purity: Assessed by running SDS-PAGE followed by Coomassie blue stainig (research grade) or silver staining (NHP-grade) to observe purity.
② Titer: For research-grade or HT-grade AAV, PackGene calibrates using ATCC standard, then employs SYBR Green QPCR to detect titer, achieving a titer of 1E+13GC/ml or higher.For NHP-grade, we determing the titer via ddPCR with primers specifically complement to 5′ and 3′ ITR region.
③ Endotoxin detection: Endotoxin content does not exceed 10 EU/ml for research-grade and 1EU/ml for NHP-grade.You may request more stringent endotoxin threshold with additional fees.
④ TEM electron microscopy (optional): Observation of empty/full ratio through electron microscopy, with a rate below 30% for most common serotypes in research-grade AAV, or below 20% for most common serotypes in NHP-grade AAV.
⑤ HPLC based purity assessment (optional): We offer AEC-HPLC (Anion Exchange Chromatography – High Performance Liquid Chromatography) and SEC-HPLC(Size Exclusion Chromatography – High Performance Liquid Chromatography). Both methods are used for measuring the purity of Adeno-Associated Virus (AAV) preparations.
AEC-HPLC is particularly useful for separating and measuring AAV from impurities such as host cell proteins and nucleic acids, which may have a different net charge compared to the AAV particles.
SEC-HPLC separates molecules based on their size. SEC-HPLC is valuable for assessing the size distribution of AAV particles and separating AAV from aggregates, fragmented particles, or other macromolecules present in the sample.
⑥ Mass spectrometry analysis (optional): Determination of AAV serotype. We offer AAV Capsid Peptide Mapping test by HPLC-MS/MS, and Capsid Protein Molecular Weight and Ratio test by RP-HPLC-MS. AAV Capsid Peptide Mapping by HPLC-MS/MS involves analyzing the peptides within the capsid proteins of the Adeno-Associated Virus (AAV) using High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (HPLC-MS/MS). Capsid Protein Molecular Weight and Ratio by RP-HPLC-MS” involves determining the molecular weight and ratio of the capsid proteins of the AAV using Reversed-Phase High-Performance Liquid Chromatography coupled with Mass Spectrometry (RP-HPLC-MS).
⑦ ddPCR titer detection (optional): compared to qPCR titer measurement, ddPCR offers high sensitivity, accuracy, and reproducibility.

For more additional QC test we provide, please check our AAV analytical serivce page.

Our AAV products are subjected to standard release testing procedures, including endotoxin assessment using Limulus Amebocyte Lysate (LAL) assay, purity analysis via Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), and titer determination using quantitative Polymerase Chain Reaction (qPCR) or droplet digital PCR(ddPCR). Moreover, we conduct restriction enzyme digestion for the Gene of Interest (GOI) plasmids utilized in packaging. Different grade AAV may include different QC tests as listed here.

In addition, we offer 40+ analytical tests to measure titer, AAV genome integrity, characterization, purity, aggregation, contamination and safty, including TEM, AUC, TCID50, Nanopore deep sequencing and may others. Please refer to our anlytical tests webpage.