Adeno-associated virus (AAV), valued for its low immunogenicity, broad tropism, and long-lasting in vivo gene expression, has emerged as the leading vector for gene therapy delivery.

For AAV vectors intended for clinical use, the FDA requires stringent quality control to mitigate potential risks. This involves evaluating the ratio of empty to full capsids and monitoring impurity levels. Transmission electron microscopy (TEM) is one of the most effective QC methods, offering direct visualization. Using specific staining, TEM clearly determines the full-to-empty capsid ratio, establishing itself as a standard in quality control processes.

While gradient ultracentrifugation generally produces highly purified AAV preparations, some unknown contaminants can still persist in the final product—a common issue in many commercial AAV preps. These contaminants often manifest as small complexes or particles that are difficult to eliminate through standard purification techniques (Figure 1). Their presence can be particularly problematic for downstream research, as they have the potential to interfere with experimental outcomes, reduce the efficacy of the AAV vectors, and introduce variability in results.

Figure 1
Figure 1. Image showing small particles (white arrow) and empty AAV capsids (dashed white box) under transmission electron microscopy (Ref.1)

What were these particles? And where did they originate?

Researchers and manufacturers must remain vigilant in identifying and addressing these impurities to ensure the integrity and reliability of AAV-based research and applications. Ongoing improvements in purification processes and more stringent quality control measures are essential to mitigate the impact of these contaminants and enhance the overall success of AAV-based therapies and experiments.

We, at PackGene, a reputable provider of AAV services and solutions, consistently uphold the highest industry standards for AAV production and quality testing. Not only were we curious about these findings, but we also wanted to understand the substance and eliminate any impurities from a quality assurance perspective. As a result, this observation prompted a thorough investigation, and we decided to conduct further testing on the samples.
Silver staining of SDS-PAGE gels from these samples revealed distinct contaminated protein bands, which were clearly clustered around the 20 kDa range, as shown in Figure 2. This finding prompted further investigation, and subsequent analysis indicated that these bands corresponded to the same contaminant particles observed in the TEM images.

Figure 2
Figure 2. SDS-PAGE staining results of AAV samples. M: Protein molecular weight marker; S1: AAV sample 1; S2: AAV sample 2; VP1-3: AAV capsid proteins.
Additionally, mass spectrometry analysis provided further confirmation that the protein bands identified in the silver-stained SDS-PAGE gels were indeed human ferritin (Figure 3). This protein is a key component of cellular lysate, originating from the host cells used in the AAV production process. Human ferritin, being an abundant protein found in the cytoplasm and lysosomes of mammalian cells, was inadvertently introduced into the final product during the manufacturing process.
Figure 3
Figure 3. Mass spectrometry results for particles.
Its identification highlights the importance of thorough analytical methods in ensuring the purity of the AAV preparations. We later discovered that most standard host cell protein (HCP) ELISA assays are often insufficient in detecting ferritin, as they lack the necessary antigens to identify this particular impurity. This limitation makes it easy for ferritin to go undetected, leading to an underestimation of host cell protein (HCP) levels in the final product. To further investigate, we enriched the ferritin fraction from the electrophoretic gels and performed TEM analysis. The TEM images confirmed that ferritin was indeed the source of the small particles (Figure 4).
Figure 4
Figure 4. Ferritin negative staining TEM results.
Ferritin contamination in AAV production is rarely discussed in the literature, but a 2018 study by Sean M. Crosson brought attention to the potential impact of ferritin on AAV titer, a crucial factor for researchers, particularly in early-stage studies. Recognizing the importance of this issue, our QC team launched an investigation into the nature of ferritin contamination, while our process development team simultaneously worked on strategies to minimize its presence. Through a series of innovative optimizations to our purification process, we were able to achieve a multi-log reduction in ferritin levels, successfully eliminating the contaminated particles. This was confirmed by the TEM images, which showed a marked improvement in the quality of the final AAV preparations (Figure 5).
Figure 5
Figure 5. AAV negative staining TEM image after further purification.

Conclusion:

Since identifying ferritin contamination, we have implemented an optimized purification process across all our standard AAV production. This enhancement further ensures the superior quality and purity of PackGene’s AAV products, reinforcing our commitment to delivering reliable solutions for advanced research and therapeutic applications.

Reference:
1.Sean M. Crosson, Peter Dib, J. Kennon Smith, Sergei Zolotukhin,
Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation,Molecular Therapy – Methods & Clinical Development,Volume 10,2018,Pages 1-7
About PackGene

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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