AAV Packaging - NHP Grade

Premium AAV Packaging specifically designed for Non-Human Primates studies


Non-human primate (NHP) studies often involve larger animals that require higher doses of adeno-associated virus (AAV) vectors. Low-quality AAV vectors can trigger immune responses or increase toxicity in animals. To mitigate these risks and improve the quality and reproducibility of NHP studies, PackGene offers high-quality NHP-grade AAV vectors with accurate measurement of virus quantity, genome integrity, empty capsid rate, and strict contamination control. By using PackGene's high-quality NHP-grade AAV vectors, researchers can lower the safety risks, enhance efficacy, and generate more reliable and reproducible results in their NHP studies.

Preclinical non-human primate (NHP) studies are an essential step in the development of adeno-associated virus (AAV) based gene therapies before they can be tested in human clinical trials. However, there are several challenges associated with conducting preclinical NHP studies for AAV-based gene therapy. NHP studies can be expensive to conduct due to the high cost of animal care, housing, and maintenance. Additionally, the cost of AAV vector production and characterization can also be significant. In addition, the use of NHPs in research is strictly regulated and requires a significant amount of paperwork and approvals, which can delay study timelines. The availability of NHPs can be limited, particularly for certain species or strains, which can make it difficult to conduct large-scale studies. NHPs can develop an immune response against the contaminants in AAV vector, which can affect the safety and efficacy of the therapy. This immune response can be difficult to predict, particularly in cases where the AAV vector is delivered systemically.

Conducting preclinical NHP studies for AAV-based gene therapy requires careful consideration of these challenges and a rigorous approach to study design, execution, and data analysis to ensure that the therapy is safe and effective for human use.

Providing high quality AAV is important for NHP studies because it directly impacts the safety, efficacy, and reproducibility of the study.

Firstly, AAV vectors can be immunogenic and provoke an immune response in the host, particularly in NHPs. This can complicate the interpretation of results, and can even impact the safety of the study, particularly in the case of systemic administration. High-quality AAV vectors with low levels of impurities and empty capsids can reduce the immune response to the therapy and improve the reproducibility of the results.

Secondly, AAV vectors with poor quality or purity can lead to variability between NHPs, making it challenging to draw accurate conclusions from small sample sizes. High-quality AAV vectors can improve the consistency of the study and reduce the variability between individual animals.

Thirdly, the accuracy and reproducibility of preclinical studies depend on the quality of the AAV vectors used. Any variability or uncertainty in the quality of the AAV vector can impact the results, and it can be challenging to attribute any observed effects to the therapy itself. By providing high-quality AAV vectors, researchers can ensure that any observed effects are due to the therapy itself and not confounded by issues related to vector quality.

High-quality AAV vectors is essential for preclinical NHP studies of AAV-based gene therapy. It ensures that the study is conducted with the highest level of rigor, reduces the risk of confounding factors impacting the results, and improves the accuracy, reproducibility, and translatability of the study.


Endotoxin and Contamination control


Precisely quantified Titer, Genome copy and integrity


Lower safety risks and enhance efficacy


Reproducible results

What We Offer
AAV type Scale Turnaround time
Standard capsids (guaranteed yield) 5E+13 GC~8E+15 GC Start from 17 business days
Custom capsids(by volume) Up to 100L with 30mL-200mL small scale test Start from 25 business days

The yield of AAV is dependent on various factors, such as serotypes, the gene of interest, and other variables. We offer a guaranteed package for commonly used AAV serotypes that are known to have high yields, such as 1, 3b, 5, 7, 8, 9, and Rh10. For other serotypes with low or unknown yields, we produce them based on volume. To determine the necessary production volume to achieve the target yield, we can conduct a small-scale test. If you’re unsure whether your serotypes qualify for our guaranteed package, please reach out to our technical support team for more information.

NHP process
Process of how we make NHP grade AAV

The AAV NHP (non-human primate) grade production process involves several steps to ensure the production of a high-quality and safe product. The process begins with clone screening and ITR (inverted terminal repeat) verification to ensure the correct vector sequence. Plasmid preparation and ITR and GOI (gene of interest) verification are then carried out to confirm the correct expression of the gene.

Next, co-transfection is performed in HEK 293T cells, which involves introducing the AAV cis-plasmid, Helper plasmid and Rep-Cap plasmid into the cells to produce the AAV vectors. Small scale culture may be conducted to determine the optimal conditions for growth and vector production. Following this, the cells are cultured or fermented to produce the AAV vectors.

The crude purification step separates the AAV vectors from cellular debris and other contaminants. Concentration and ultracentrifugation are then carried out to concentrate and further purify the AAV vectors. The sterile filtration step removes any remaining microorganisms.

The final step is a series of quality control (QC) tests, including AAV genome integrity, empty capsid rate, endotoxin tests, and contamination tests, etc. The results of these tests are reviewed by the quality assurance (QA) team, who then release the AAV vectors for use.

The benefits of this production process are that it minimizes safety risks, enhances efficacy, and ensures reproducible results, making it suitable for use in non-human primate studies.

QC standards
AAV Packaging – NHP Grade specifications
Test Method QC Standard
Standard QC ddPCR Measure titer, normalized to 1e13vg/ml Standard capsids: Concentration and total quantity meet needs. Custom capsids: quantity based on production scale.
AAV Capsid size* SDS-PAGE silver stain Match capsid protein size
Guarantee endotoxin LAL <1EU/ml
Mycoplasma Detection qPCR Negative
Bioburden Direct inoculation No growth
AAV Genome integrity CE(titer>1e+12vg/ml, volume >50ul) Report
Empty Capsid Rate TEM <20%
Additional QC AAV Genome sequencing Nanopore Report
Empty Capsid Rate AUC Report
Endotoxin removal LAL <0.2EU/ml
Residual Triton Analysis(bundle with endo removal) HPLC 5ppm
Sterility test Direct inoculation No growth

NHP grade AAV undergoes stringent QC tests before being released to ensure the quality of the AAV product and minimize the adverse effects in animal studies.



This test measures the titer of the AAV product and normalizes it to 1e13vg/ml. The concentration and total quantity of the product will meet the guaranteed yield package. For non-guaranteed yield, the quantity is based on the production scale.


Empty Capsid Rate

This test uses TEM (transmission electron microscopy) to measure the percentage of empty capsids in the AAV product. The empty capsid rate must be below 20%.


Guarantee Endotoxin

This test uses LAL (limulus amebocyte lysate) to detect endotoxin levels, which must be below 1EU/ml.


Mycoplasma Detection

This test uses qPCR to ensure that there is no mycoplasma contamination in the AAV product.



This test uses direct inoculation to detect the presence of any microbial growth in the AAV product.


AAV Genome Integrity by CE

This test uses CE (capillary electrophoresis) to detect the integrity of the AAV genome. The results of this test are reported.


AAV Capsid Size

This test uses SDS-PAGE silver stain to verify the capsid protein size of the AAV product.

In addition to the above tests, there are several optional QC tests that can be carried out, including AAV genome sequencing by Nanopore, empty capsid rate by AUC (analytical ultracentrifugation), endotoxin removal and verify by LAL, residual Triton analysis via HPLC, and sterility tests to detect both fast growing and slow growing microorganisms.
Plasmids used for AAV NHP Grade exceeds industrial standards.

To ensure the fidelity of the gene of interest and efficient AAV packaging, the plasmids utilized for NHP grade AAV packaging undergo strict QC. The basic standards consist of requirements for appearance, A260:280 ratio, homogeneity (supercoil >90±10%), restriction analysis, residual RNA and E. coli DNA, endotoxin (≤0.01 EU/µg), and ITR and GOI Sanger sequencing, which are crucial for ensuring the efficient delivery of therapeutic genes via AAV.

In addition to the basic standards, there are also advanced requirements that are not included in the default package and are available upon request with additional cost. These advanced requirements include residual host protein, advanced endotoxin removal, bioburden, mycoplasma contamination, ITR Nanopore sequencing, animal-free production, material archiving, pH (7.5~8.0), residual Kan, USP sterility, dedicated purification resin or filtration membrane, etc.

Test Method QC Standard
Basic standards Appearance Visual inspection Clear, colorless, no visible particulates
A260:280 Nanodrop  1.8 - 2.0
Homogeneity Agarose gel, supercoil >90±10% Supercoil >90±10%
Restriction Analysis SmaI/AhdI Conforming to reference pattern
Residual RNA Agarose gel Not visible with 200ng load
Residual E. coli DNA Agarose gel <15% of total band with 200ng load
Endotoxin LAL ≤0.01 EU/µg
ITR and GOI Sequencing Sanger 100% match ITR and GOI correct and clean peak
Additional QC Residual Host Protein Nano orange <0.1% by HCP ELISA
Advanced Endotoxin Removal <0.005 EU/µg Quantitative endotoxin test <0.005 EU/µg
Bioburden Testing LB plate No growth
Mycoplasma Contamination Quantitative PCR Negative
Competent cell JM108, Sure, Sure2, Stbl3, NEBStbl Customer define
ITR integrity Nanopore sequencing Report
Animal Free Production* TSE/BSE
Material Archiving Yes (plasmid+bacteria)
pH 7.5~8.0
Kan ELISA no detectable
USP sterility Yes
Dedicated purification resin Upon request
Buffer ddH2O, TE or customer defined
Dispensing Upon request
Dedicated filtration membrane Upon request
Case Study
The test methods described are necessary to ensure the quality and purity of AAV (adeno-associated virus) produced by PackGene, especially for its use in non-human primates (NHP). The different test methods evaluate various aspects of AAV quality, including its genome integrity, capsid size, and the presence of contaminants.
  • ddPCR (droplet digital PCR) is a highly sensitive method for quantifying the amount of AAV in a sample. It allows accurate and precise measurement of AAV genome copies per milliliter.
  • qPCR (quantitative PCR) is a method for detecting and quantifying Mycoplasma contamination in a sample. Mycoplasma is a common contaminant in cell cultures and can affect AAV quality, so confirming its absence is important.
  • Capillary Electrophoresis (CE) is a technique used to analyze the integrity of the AAV genome. CE can detect the presence of truncated or rearranged AAV genome fragments, which can affect AAV potency and safety.
  • SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and silver stain are methods for analyzing the size and purity of the AAV capsid protein. These methods can detect the presence of impurities and ensure that the capsid size is consistent with the expected size for the AAV serotype.
  • TEM (transmission electron microscopy) and AUC (analytical ultracentrifugation) are methods for determining the proportion of empty capsids in the AAV sample. Empty capsids are undesirable as they do not contain the therapeutic payload and can reduce the efficacy of the AAV vector. Monitoring the empty capsid rate is important to ensure the potency and quality of the AAV product.
The combination of these tests allows for a comprehensive assessment of the AAV quality, purity, and safety, ensuring that the AAV produced by PackGene is suitable for use in NHP studies.
NHP studies
Upon receiving the AAV cis plasmid template from our customers, we conduct a double-check of the ITR integrity in addition to verifying the GOI size during clone screening before scaling up the plasmid. After the plasmid preparations are finished, another round of ITR and GOI sequencing is performed to ensure maximum fidelity. You can rest assured that the plasmid we used for NHP grade AAV are of the highest quality and integrity.

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