After decades of research, gene therapy is finally on track to deliver solutions to myriad diseases once considered untreatable. More than 400 gene therapy trials are currently underway in the United States, investigating methods ranging from CRISPR gene editing to the use of viral vectors.1 Adeno-associated viruses (AAVs) are an extremely versatile vehicle for transporting healthy gene copies into a patient’s cells. However, producing a safe and effective AAV-based treatment is not easy: The steps taken to extract and purify AAV vectors are not perfect. And ultimately, an imperfect product can put patients at risk.
Many of the quality issues associated with AAV development stem from the fact that AAVs are produced in live cells in cell culture. To extract the viruses, the host production cells must be lysed, which introduces several challenges. First, this process often fails to yield sufficient viral titers, especially for AAVs. Second, the extract might contain residual host cell components, such as proteins, nucleic acids, bacteria, and other viruses, as well as proteins from the cell culture media that were derived from serum and animals.2–4 Finally, some of the AAV capsids might not contain the intended sequence—they might be empty, or they might contain mispackaged host cell DNA. All of these challenges pose risks to patients.
The common denominator in all of these challenges is the need for rigorous quality control. Manufacturers must test all AAV batches to ensure that they contain the proper viral titer and are free from hazardous contaminants, even as production is scaled.
Below is a deeper dive into the top five bioprocessing challenges that AAV developers face. It details how established technologies, such as droplet digital PCR (ddPCR), can help guarantee the quality of each batch.
Top five challenges to AAV vector purification
- A dearth of vectors
A gene therapy is effective only if the vector is present at a sufficient concentration. If the dose is too low, the treatment will not work. Unfortunately, the standard upstream bioprocessing approach used to grow these vectors yields a concentration of up to 2 × 1011 vectors/mL—less than the 1 × 1014 vectors/mL needed to treat patients. To reach an effective dose in a reasonable volume, the batch needs to be concentrated between 100 and 10,000 times.5
However, most commercial concentration devices are not built to concentrate AAV vectors into such small volumes. Additionally, by concentrating the vectors, the manufacturer is also concentrating any host cell contaminants that remain in the batch. Consequently, AAV developers must test their batches for vector concentration as well as for the presence of impurities to ensure that the batches are safe and effective.
- The threat of empty capsids
One such impurity that could impact the effectiveness of an AAV-based gene therapy is empty capsids. Up to 90% of the capsids in a batch could be empty, reducing the overall vector concentration.5,6 To compensate, one would have to increase the volume of the dose, but that can make it challenging to deliver therapies to smaller spaces in the body, such as the brain or spinal cord.7 To generate an effective dose in a practical volume, AAV developers must monitor and minimize empty capsids.
- The persistence of oncogenic host cell DNA
The virus is not the only barrier to generating a quality gene therapy; the cells in which the vectors are grown also contain impurities that could make their way to a final batch and into patients. One such molecule is oncogenic DNA, which is common in manufacturing cell lines.
To reduce the risk of treating a patient with a gene therapy that contains oncogenic material, AAV developers must reduce residual DNA as much as possible. It is nearly impossible to remove these genetic sequences if they have been mispackaged in the AAV vector, as the capsid shields the DNA from nucleases.3,5 Therefore, AAV developers need to test for the presence of oncogenes in their therapeutic batches and then also perform specific tests to determine if their vectors are likely to be oncogenic.
- Immune system–provoking proteins
Other impurities that might pose a risk to the quality of an AAV vector are immunogenic proteins. Scientists believe that the material contained within AAV capsids is unlikely to trigger an immune reaction because it does not contain engineered lipids or other chemical compounds.
However, the capsid protein itself can elicit a response that could ultimately prevent the vector from infecting the patient cells and reduce the effectiveness of the treatment.5,8 Beyond this innate immune response, many patients’ immune systems have been exposed to AAV before, potentially priming them to mount a stronger, adaptive immune response to the treatment.8,9
Currently, researchers do not know whether empty capsids themselves elicit an immune response. In fact, empty capsids might serve as decoys, pulling anti-AAV antibodies away from active virions.10 Scientists need to investigate this further so developers can adopt proven methods for minimizing the presence of protein contaminants that can elicit an unwanted immune reaction.
- Contamination by mycoplasmas
Approximately 30% of cell lines across the world—including cell lines used to culture batches of recombinant AAV—are contaminated by mycoplasmas.11 AAV developers must minimize mycoplasma contamination to avoid causing a respiratory illness in patients.
Mycoplasmas pose a challenge because they are very small; hence, they are hard to detect via standard light microscopy. They are gram-negative and resistant to the β-lactam antibiotics used for cell line maintenance. Manufacturers, therefore, need to use an alternative method such as traditional cell culture methods to detect mycoplasma-contaminated batches so they can be removed from production.
Quality control with Droplet Digital PCR
The multitude of issues associated with AAV development means that developers need to incorporate reliable quality control methods into the production process.
Several of these challenges, including measuring viral titer, identifying oncogenic DNA, and detecting mycoplasma contamination, can be addressed using accurate and precise nucleic acid quantification methods. These purposes are commonly achieved with the quantitative PCR (qPCR) technique. However, this technique comes with several important drawbacks that limit its value for gene therapy quality control.
Specifically, qPCR results are relative: this technique works by amplifying the target genetic sequence and counting the number of cycles it takes for fluorescence to reach a certain threshold. To convert cycles to concentration, a user must generate a standard curve, a process that is often fraught with human error that can cause results to vary by as much as a factor of two.12 This variability limits the technique’s accuracy and sensitivity.
A more sensitive alternative is Droplet Digital PCR (ddPCR). This technology does not depend on a standard curve; instead, it counts nucleic acids directly.13 As a result, it has the potential to measure more precisely the concentration of a target nucleic acid sequence in a batch, as well as the concentration of host cell DNA remnants and mycoplasma. Additionally, since ddPCR does not rely on measuring whether amplification reaches a particular threshold, it is less affected by secondary DNA structures and host cell contaminants that might interfere with amplification.14
In one direct comparison of qPCR and ddPCR technology, ddPCR was up to four times more sensitive than qPCR in the absolute quantification of single-stranded AAV vector genomes.15 In another study, researchers examined the ability of ddPCR technology to detect several mycoplasma species. The limits of detection were 4.19, 6.29, and 5.63 genome copies/well among the three species they tested. They also confirmed that ddPCR did not produce false positives.16 In a third study where qPCR yielded a negative result, ddPCR was able to detect A. laidlawii standards in suspension at 1 colony forming unit/mL.4 Altogether, these data demonstrate how useful an accurate measure of DNA concentration can be.
Why quality control is important
For decades, pharmaceutical companies could rely on chemical formulas and mathematics to design, produce, and predict the effects of a drug. Gene therapies change the equation. An AAV developer must rely on a natural, biological process to develop therapeutics, and with this comes greater uncertainty in the composition of the final product. To ensure these treatments are safe and effective, developers must employ quality control technologies such as ddPCR. Although the development of these therapies poses additional challenges, their potential to treat the untreatable makes the process worth it.
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Mark White, PhD is Associate Director, Biopharma Product Marketing, Digital Biology Group at Bio-Rad Laboratories.