Innovations in vaccine development, gene therapy, and oncolytic treatments often rely on advanced purification methods to ensure the safety and efficacy of viral platforms. Recombinant measles viruses (MVs), derived from the widely used and highly effective measles vaccine strain, have emerged as a powerful tool in these fields. However, the path to scalable production of these delicate, enveloped viruses is riddled with challenges—particularly when it comes to removing impurities like host-cell DNA while maintaining high recovery rates.
In this blog post, we summarize and explore Highly pure measles virus generated by combination of salt-active nuclease treatment and heparin affinity chromatography, Mayer et al. in the Journal of Chromatography A, a groundbreaking study that demonstrates how integrating ArcticZymes' M-SAN HQ nuclease with heparin affinity chromatography transforms MV purification. This streamlined approach not only simplifies workflows but also achieves unparalleled levels of purity and yield.
Dive into the details to see how this innovative solution is setting a new benchmark for viral purification.
Summary of Mayer et al.
Recombinant measles viruses (MV) are an invaluable platform for developing vaccines, gene therapies, and oncolytic treatments. Based on the safe, widely used measles vaccine strain, these viruses can be engineered to carry additional genes, enabling them to target various diseases, including chikungunya, SARS-CoV-2, and dengue. However, the path to scalable production of MV is not straightforward. Purification processes are complicated by MV's fragility, requiring careful handling to maintain viral integrity and effectiveness.
Purification Challenges and the Need for Innovation
Purifying MV's is inherently challenging due to their delicate envelope and the size range of their particles, which are vulnerable to damage from temperature shifts, shear forces, and UV light. Conventional purification strategies often involve multiple unit operations, which risk viral loss and reduce yield—often to as low as 20-50%. Additionally, current methods frequently struggle to eliminate host cell proteins and DNA without multiple processing steps.
To overcome these issues, researchers seek a streamlined purification method that not only ensures high yield but also meets regulatory standards for purity, particularly for contaminants like host cell DNA.
Addressing DNA Contamination with Affinity Chromatography and Nuclease Treatment
An attractive approach to purifying fragile MV's would be to use only one affinity column that binds only the virus, while all contaminants end up in the flow through. This would require an affinity column not only specific for the viral vector, but also gentle enough to avoid share forces to cause unacceptable loss of viable viruses.
Two affinity columns were tested for these properties.
1. Capto™ DeVirS provided to strong MV binding resulting in low recoveries of infective particles, limiting its usability.
2. Capto™ Heparin demonstrated gentler binding, which allowed better recovery rates but led to more chromatin contamination in the eluate.
A critical contaminant in measles virus (MV) production is chromatin from host cells, which closely resembles MVs in size and charge, complicating the purification process. To tackle this, nuclease treatments with Benzonase and M-SAN HQ were tested to assess their effectiveness in degrading host cell DNA and chromatin.
A significant breakthrough in MV purification was achieved by combining Capto™ Heparin affinity chromatography with ArcticZymes' M-SAN HQ.
Why M-SAN HQ Outperforms Other Nucleases
M-SAN HQ demonstrated superior performance in DNA removal compared to Benzonase:
1. Chromatin Removal: M-SAN HQ effectively breaks down chromatin into smaller fragments, removing chromatin particles that can otherwise interfere with MV purification.
2. DNA Reduction Below Regulatory Limits: As shown in Figure 1, M-SAN HQ reduces DNA content to levels well below the FDA’s maximum allowable limit of 10 ng per dose.
3. High Recovery Rates: Both Benzonase and M-SAN HQ preserved MV yield, but M-SAN HQ achieved superior DNA clearance without sacrificing recovery rates.
4. Simplified Workflow: The enhanced efficacy of M-SAN HQ may eliminate the need for additional DNA-removal steps, enabling a more efficient, streamlined process.
In contrast, Benzonase, while helpful in reducing DNA content, did not achieve the same level of DNA removal. Residual chromatin fragments from Benzonase treatment were similar in size to MVs, complicating further purification efforts with size-exclusion methods.
Refining Purity with Polishing Steps
Though M-SAN HQ dramatically reduces chromatin and DNA levels, a small amount of histone contamination may remain. This can be addressed by incorporating a polishing step, such as batch incubation with Capto Core 700, a resin that can selectively remove proteins and small contaminants without binding MVs. This step helps ensure a high-purity product, aligning with the stringent standards of gene therapy and vaccine production.
Conclusion
Integrating Capto™ Heparin affinity chromatography and M-SAN HQ nuclease treatment represents a highly efficient, single-step process for MV purification. Host cell DNA contamination, a common challenge in viral vector production, must be reduced to levels below the FDA threshold of 10 ng/dose to ensure compliance and therapeutic efficacy. Traditional nucleases, included in the study, struggle to consistently meet these standards without adding complexity and cost, e.g. additional filtration, to the purification process.
M-SAN HQ nuclease addresses this challenge head-on, achieving DNA clearance far below the 10 ng/dose while also maintaining exceptionally high recovery of virus particles. This unique combination of performance and efficiency enables manufacturers to streamline their workflows, reduce the need for additional purification steps, and improve overall production yields.
For patients, this means faster access to safe, effective therapies; for manufacturers, it translates into lower costs, greater reliability, and confidence in meeting regulatory expectations. M-SAN HQ is paving the way for more robust, scalable, and accessible solutions in biopharmaceutical production.
Q&A about the publication
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Related:
Read this whitepaper to see how current salt-active nucleases compare in removing chromatin, with insights drawn from recent studies.
