Virus Processing

What's the recommended filtration setup for concentrating viral vectors?
The recommended filtration setup for concentrating viral vectors is an automated Tangential Flow Filtration (TFF) system, such as the µPulse or aµtoPulse, configured with a low-adsorption, 100–300 kDa molecular weight cut-off (MWCO) membrane. This setup typically uses a modified PES or regenerated cellulose membrane in a single-use chip format to prevent cross-contamination. Operate the system with gentle transmembrane pressure (TMP) and cross-flow velocity to minimize shear stress and preserve viral infectivity. The low hold-up volume (e.g., 0.65 mL in µPulse and 0.25 mL in aµtoPulse) and automated product recovery feature maximize yield. This closed, scalable process efficiently removes impurities and allows for integrated diafiltration into final formulation buffers, supporting consistent, GMP-ready production of AAV, lentiviral, and other viral vectors.

Which membranes are best for viral particle clarification vs concentration?
For viral particle clarification, use a 0.1–0.65 µm pore-size microfiltration (MF) membrane to remove cell debris and large impurities.
For viral concentration, use a 100–300 kDa ultrafiltration (UF) membrane to retain intact viral vectors while removing smaller contaminants.
Clarification membranes are designed for high throughput and minimal fouling during initial harvest. Concentration membranes are selected for viral compatibility, low adsorption, and precise molecular separation. Systems like the µPulse and aµtoPulse support both steps with optimized, single-use flow paths to ensure high recovery and maintain infectivity.

What are typical processing times for AAV or VLP purification using TFF compared to chromatography methods?
Tangential Flow Filtration (TFF) significantly reduces processing times for AAV and VLP purification compared to chromatography. While chromatography often requires sequential column steps, equilibration, and regeneration, TFF consolidates multiple steps into one continuous, automated run on systems like the µPulse or aµtoPulse, which perform simultaneous sample concentration and buffer exchange. In contrast, chromatography involves separate capture, polishing, and buffer exchange cycles, extending both hands-on time and overall duration. This makes TFF the preferred method for rapid, scalable processing, while chromatography remains valuable for high-resolution purification where purity demands justify the additional time and complexity.

What membrane MWCO should I choose for viral vector purification?
For viral vectorprocessing, select a TFF membrane with a 100–300 kDa molecular weight cut-off (MWCO). This range is optimal for retaining viral particles (typically 50–200 nm) while allowing smaller contaminants to pass through. Use low-adsorption regenerated cellulose or modified PES membranes to minimize vector loss. Systems like the µPulse and aµtoPulse offer gentle, automated processing that preserves infectivity and enables high recovery. Always validate the MWCO and process parameters at small scale to confirm purity, yield, and scalability for your specific vector.

How does membrane choice (RC vs. mPES) affect recovery of viral vectors, VLPs, or bacteriophages?
Membrane choice directly impacts viral vector, VLP, and bacteriophage recovery: Regenerated Cellulose (RC) membranes minimize nonspecific binding through their hydrophilic, low-binding surface, preserving the integrity and activity of sensitive enveloped particles, while Modified Polyethersulfone (mPES) membranes offer robust chemical compatibility but may require optimization to minimize adsorption.. mPES membranes provide durability and higher flux but can exhibit greater adsorption; using appropriate buffers or membrane pre-treatment can mitigate losses. For optimal recovery with either membrane, systems like the µPulse and aµtoPulse enable gentle, automated processing with single-use flow paths. Always validate recovery and infectivity at small scale using your specific viral sample to confirm performance before scaling.

What is the recovery efficiency for AAV or lentivirus with TFF?
Yes, TFF typically achieves recovery efficiencies of 80–90% for AAV and lentiviral vectors when optimized, with some processes reaching >90%. This high recovery is accomplished using low-adsorption membranes (100–300 kDa MWCO) and gentle, automated control of pressure and cross-flow in systems like the µPulse or aµtoPulse to preserve viral integrity and infectivity. Key factors influencing recovery include sample quality, membrane selection, hold-up volume, and the effectiveness of the product recovery step. Employing a low hold-up volume system with an optimized recovery protocol is critical to minimize final product loss. Due to the high value of these vectors, process validation at small scale is recommended to maximize yield and ensure scalability to GMP manufacturing.

How does TFF compare to ultracentrifugation for viral vector purification in terms of yield and scalability?
TFF consistently outperforms ultracentrifugation in both yield and scalability for viral vector purification. While ultracentrifugation can achieve high purity, it often results in lower yields due to pelleting losses, aggregation, and shear damage during resuspension. In contrast, TFF systems like the µPulse and aµtoPulse deliver recoveries exceeding 90% by using gentle, controlled cross-flow and integrated product recovery that minimizes stress on viral integrity.
In terms of scalability, ultracentrifugation is notoriously difficult to scale beyond lab use due to equipment limitations, lengthy run times, and significant batch-to-batch variability. TFF, however, is inherently scalable—process parameters optimized at benchtop scale translate directly to manufacturing, supported by automated, closed systems that ensure reproducibility and meet GMP requirements. TFF also reduces processing time from days to hours and enables simultaneous concentration and buffer exchange, making it the preferred choice for high-value viral vector manufacturing in gene therapy and vaccine development.

How does shear affect viral infectivity during TFF?
Excessive shear during TFF can significantly reduce viral infectivity by damaging the viral envelope or capsid, particularly for delicate vectors like AAV and lentivirus. High shear rates from aggressive pumping or turbulence can disrupt structural integrity, leading to decreased functional titer. Optimized TFF systems like the µPulse and aµtoPulse minimize shear impact through precise, automated control of cross-flow velocity and transmembrane pressure. By maintaining a laminar flow regime and avoiding pressure spikes, these systems preserve viral activity while achieving efficient concentration and purification.

What strategies help maintain viral infectivity and particle integrity during TFF processing?
Viral infectivity and particle integrity during TFF processing are maintained through optimized shear control, compatible membrane selection, and gentle system automation. Key strategies include operating at lower cross-flow velocities and controlled transmembrane pressure to minimize mechanical stress on viral structures like envelopes or capsids. Using low-adsorption, hydrophilic membranes with an appropriate molecular weight cut-off reduces non-specific binding and preserves activity. Systems like the µPulse and aµtoPulse ensure consistency through single-use flow paths, precise parameter control, and integrated product recovery, which collectively prevent aggregation, contamination, and sudden pressure changes. Additionally, maintaining cold-chain conditions, validated buffer exchanges, and scalable, reproducible protocols supports high recovery of functional viral vectors, making TFF a robust choice for gene therapy and vaccine manufacturing.

Are µPulse and aµtoPulse systems compatible with processing both enveloped and non-enveloped viruses?
Yes, both the µPulse and aµtoPulse systems are fully compatible with processing both enveloped and non-enveloped viruses. Their design allows for gentle, automated handling that preserves the integrity of sensitive viral structures. For enveloped viruses (e.g., lentivirus, HSV), the systems’ low-shear pumping and precise pressure control prevent disruption of the lipid envelope, maintaining infectivity. For non-enveloped viruses (e.g., AAV, adenovirus), the same gentle processing avoids capsid damage or aggregation.

What are the minimum and maximum sample volumes that can be processed for viral particles using µPulse or aµtoPulse?
The µPulse processes volumes as low as 1 mL, while the aµtoPulse can accommodate volumes from 0.5 mL. Both systems achieve high recovery for precious samples due to their minimal hold-up volumes—0.65 mL for µPulse and 0.25 mL for aµtoPulse—which actively limit product loss during concentration and diafiltration. To ensure optimal results with minimal sample volumes, validate the method using each system’s automated protocols, adjusting transmembrane pressure and cross-flow to preserve the particle integrity.