Virus Purification Strategies
Improved virus purification strategies with advanced chromatographic techniques and optimal resin selection.
Introduction
Viral particles are an increasingly important class of biologics, with applications in vaccine development, disease prevention, and as delivery mechanisms for gene therapy and the treatment of diseases such as cancer. Ensuring that virus production and purification are of the highest quality for effective recovery of particles is vital for both research and therapy. This application eBook explores methodologies for optimized virus process purification to achieve increased productivity and improved economics with chromatography systems and innovative resins.
Challenges in Virus Production
Viruses are large and complex, presenting barriers to production and purification. Traditional methods like ultracentrifugation, precipitation, and filtration are inefficient, yielding variable quality and recovery. Column chromatography has become a key technique to improve recovery and impurity removal while maintaining virus infectivity.
Contents
- Single-step purification for influenza and dengue virus using a mixed-mode chromatography resin
- High-capacity IEX resin for virus purification
- Adenovirus purification strategies
- High recovery and purity for mammalian virus purification
- Purification of challenging or sensitive molecules
- Featured Products
- Virus purification selection guide
Choosing the Right Chromatography Media
High-capacity or high-affinity chromatographic resins bind viruses through various mechanisms and can be fine-tuned for binding capacity and selectivity. Bio-Rad offers several resins:
- High-performance strong anion exchange resins (e.g., Nuvia HP-Q): Suitable for downstream purification of large biomolecules at fast flow rates.
- CHT Ceramic Hydroxyapatite Type II Media: Combines calcium affinity and cation exchange interactions for high purity, recovery, and viral activity.
Method development is crucial for improved chromatography performance and productivity. Resin selection, lifespan, stability, scalability, and single-step purification functionalities are important considerations for process economics.
Single-Step Influenza and Dengue Virus Purification with Mixed-Mode Media
This study demonstrates single-step purifications of influenza and dengue viruses using CHT XT Media. CHT Media are mixed-mode calcium affinity/cation exchange supports that bind viruses via electrostatic interactions.
Materials and Methods
The study involved culturing viruses (Influenza strain A/California/07/2009 and Dengue virus Type 2 strain ThNH7/93) and preparing them for chromatography. Protocols for purification and analysis (HA assay, TCID50, dsDNA, protein analysis) were followed.
Influenza Virus Purification
Chromatography using CHT XT Media showed viral activity eluting at approximately 250 mM phosphate, well-separated from DNA and UV-absorbing material. Hemagglutination activity (HA) confirmed viral recovery. Analytical assays on pooled samples indicated significant clearance (>99%) of total and host cell proteins and >60% removal of dsDNA.
Figure 1 (Replicate elution of influenza virus from CHT XT) shows chromatograms with elution volume on the x-axis and conductivity, absorbance (A280, A260), and HA titer on the y-axes. Viral activity peaks are observed between 26-31 ml elution volume.
Figure 2 (Infectivity of the influenza virus purified on CHT XT) shows similar trends for infectivity and HA titer.
Figure 3 (SDS-PAGE and immunoblotting analysis) confirmed purification of HA proteins in the active pooled fractions.
| Log Reduction in dsDNA | Log Reduction in Total Protein | Log Reduction in HCP Content |
|---|---|---|
| 0.42 | 2.82 | 2.54 |
Dengue Virus Purification
Chromatographic results for dengue virus purification showed elution at approximately 400 mM phosphate. HA activity was observed between 28-38 ml elution volume, with approximately 87% viral activity recovery. Analytical assays showed significant clearance of protein (>93%) and dsDNA (>91%).
Figure 4 (Replicate elution of dengue virus from CHT XT) displays chromatograms with elution volume on the x-axis and conductivity, absorbance (A280, A260), and HA titer on the y-axes. Viral activity peaks are observed between 28-38 ml elution volume.
Figure 5 (SDS-PAGE and immunoblotting analysis) confirmed the presence of dengue E and C proteins in the purified fractions.
| Log Reduction in dsDNA | Log Reduction in Total Protein |
|---|---|
| 1.07 | 1.15 |
High Dynamic Binding Capacity for Efficient Downstream Purification of Large Biomolecules (Nuvia HP-Q)
Nuvia HP-Q is a high-performance strong anion exchange resin designed for downstream purification of large molecules like viruses. Its UNOsphere epoxide base bead offers fast mass transfer kinetics and low nonspecific binding. The resin's particle size is optimized for high dynamic binding capacity (DBC) at fast flow rates.
| Resin | Matrix Material | Particle Size, µm | Pressure, bar | Recommended Flow Rate, cm/hr | DBC (IgM) |
|---|---|---|---|---|---|
| Nuvia HP-Q | UNOsphere epoxide | 50 | <3 | 300 | +++ |
| Resin 1 | Dextran beads | 50 | <3 | 30 | +++ |
| Resin 2 | Agarose | 50 | <3 | 300 | + |
| Resin 3 | PS/DVB | 75 | <3 | 300 | + |
| Resin 4 | PMMA | 50 | <3 | 300 | + |
Nuvia HP-Q offers excellent pressure flow properties, allowing high flow rates without significant backpressure, leading to increased productivity. It also demonstrates robust performance, stability, and reusability over many cycles.
Figure 1 (Dynamic binding capacity vs. flow velocity of Nuvia HP-Q) shows DBC increasing with flow velocity.
Figure 2 (Dynamic binding capacity vs. residence time) compares DBC of Nuvia HP-Q with other resins.
Figure 3 (Pressure/flow performance of Nuvia HP-Q Resin) illustrates low backpressure at high flow rates.
Figure 4 (Stability, reusability, and recovery with Nuvia HP-Q Resin) shows consistent performance over multiple cycles and exposure to NaOH.
Practical Guide: Selecting the Optimal Resins for Adenovirus Process Purification
Adenovirus is a complex virus, making traditional purification methods inefficient. Column chromatography has become a preferred method for adenoviral purification. Bio-Rad offers a two-column capture and polish strategy for recombinant adenovirus, yielding an active, concentrated product with high purity.
Design of Experiment (DoE) for the Process Purification of Adenoviruses
Initial screening of five Bio-Rad resins (four IEX and one MM) was performed. Nuvia cPrime (MM), Nuvia Q (AEX), and UNOsphere Q (AEX) showed potential for virus capture. Nuvia cPrime was selected for mass capture due to its ability to flow through albumin and negatively charged impurities, reducing column fouling and improving productivity.
Selection of the Polish Resin
Nuvia Q was chosen as the polish resin, suitable for adsorbing virus at high NaCl concentrations present in the Nuvia cPrime capture eluate.
Figure 1 (Representative chromatogram from Nuvia cPrime mass capture) shows OD 260, OD 280, and conductivity over elution volume.
Figure 2 (Representative chromatogram from Nuvia Q polish purification) shows similar data for the polish step.
Figure 3 (SDS-PAGE analysis of intermediates and final product) shows significant reduction in product volume and improvement in purity after the two-step process.
| Sample | Total virus (x10¹¹ particles) | Impurity levels (ng/10¹⁰ particles) | |
|---|---|---|---|
| DNA | HCP | ||
| Bulk harvest | 30.6 | 3,144 | n/d |
| Nuclease-treated harvest | 31.8 | 30 | 3,022 |
| Nuvia cPrime eluate | 18.4 | n/d | 58 |
| Nuvia Q eluate | 16.4 | <0.02 | 2 |
Mammalian Virus Purification Using Ceramic Hydroxyapatite
This paper reports the use of ceramic hydroxyapatite (CHT) media for purifying a variety of mammalian viruses, offering a simple, scalable method for obtaining highly active virus preparations. CHT media provides high purity, recovery, and viral activity for seven mammalian viruses.
Materials and Methods
The study lists various viruses used and their properties. Chromatography was performed using Bio-Rad's BioLogic DuoFlow System with CHT Ceramic Hydroxyapatite Type II Media.
| Virus | Family | Genus | Genome | Envelope | Size, nm |
|---|---|---|---|---|---|
| Dengue | Flaviviridae | Flavivirus | ssRNA+ | + | 50 |
| Japanese encephalitis | Flaviviridae | Flavivirus | ssRNA+ | + | 50 |
| Influenza | Orthomyxoviridae | Influenzavirus | ssRNA+ | + | 80-120 |
| Mouse hepatitis | Coronaviridae | Coronavirus | ssRNA+ | + | 100-150 |
| Adenovirus | Adenoviridae | Mastadenovirus | dsDNA | - | 90 |
| Poliovirus | Picornaviridae | Enterovirus | ssRNA | - | 30 |
| Feline calicivirus | Caliciviridae | Vesivirus | ssRNA | - | 30-38 |
Results
Dengue Virus: CHT Type II Media recovered dengue virus type 2 with HA activity separated from contaminants. Decreasing flow rate improved elution peak sharpness and separation. Recovery of HA activity was over 95%. Figure 1 (Chromatograms of dengue virus type 2 by CHT Type II Media) shows elution volume on the x-axis and conductivity, absorbance (A260, A280), and viral activity (HA test) on the y-axes. Type 2 dengue virus elutes around 250 mM sodium phosphate.
Influenza Virus: Chromatography of influenza virus strains showed viral activity separated from BSA and unbound material, with 98% recovery for one strain. Higher salt concentrations were needed for another strain. Figure 2 (Chromatography of influenza virus) shows elution volume on the x-axis and conductivity, absorbance (A260, A280), and HA titer on the y-axes.
Mouse Hepatitis Virus: MHV strains bound to CHT Type II Media and eluted at 26-28 minutes. Figure 3 (Chromatography of two strains of MHV) shows elution volume on the x-axis and conductivity, absorbance (A260, A280), viral infectivity (TCID50), and dsDNA on the y-axes.
Nonenveloped Viral Particles: Nonenveloped viruses like Adenovirus, Feline Calicivirus, and Poliovirus can also be purified using CHT Type II Media, showing different elution times. Figure 4 (Chromatograms of cell lysate and culture fluid containing nonenveloped viral particles) illustrates these purifications.
Figure 5 (JEV chromatography) shows Japanese encephalitis virus purification.
Effect of Hydroxyapatite Type on Separation: Testing different apatites is crucial for optimal separation. CHT Type II Media provided the best separation for dengue virus compared to other apatite media.
Figure 6 (Chromatograms of dengue virus type 2 on different apatites) compares CHT Type I, CHT Type II, CFT Ceramic Fluorapatite Type II, and MPC Media.
Figure 7 (Chromatograms of culture fluid of poliovirus) compares CHT Type II and CFT Type II Media for poliovirus purification.
New Selectivity and Large Design Space for Downstream Purification Processes (Nuvia cPrime)
Nuvia cPrime is a mixed-mode hydrophobic cation exchange media offering unique selectivity, high recovery, and versatility for large-scale purification of therapeutic proteins. It is effective for initial capture and polish applications, especially for challenging molecules.
Mixed-Mode Ligand
The mixed-mode ligand provides a balance of hydrophobic and charged characteristics, enabling straightforward method development.
Properties of Nuvia cPrime Media
| Property | Description |
|---|---|
| Functional group | Hydrophobic weak cation exchange |
| Base matrix composition | Macroporous highly crosslinked polymer |
| Particle size | 70 µm ± 10 µm |
| Dynamic binding capacity* (hlgG) | >40 mg/ml |
| Dynamic binding capacity (lactoferrin) | >60 mg/ml |
| Ligand density | 123± 20 µeq/ml |
| Recommended linear flow rate | 50-600 cm/hr |
| pH stability | Short-term: 3-14, Long-term: 4-13 |
| Shelf life* | 5 years |
Chromatographic Performance for Novel Therapeutics
Nuvia cPrime is effective for purifying proteins that lack an affinity handle, are salt- and pH-sensitive, or have a high propensity for aggregation.
Figure 2 (Chromatographic performance and unique selectivity) illustrates effective separation.
Large Design Space for Binding and Elution
Nuvia cPrime offers a large design space for binding and elution, allowing for direct loading without dilution and integration into multicolumn processes.
Figure 3 (Large design space afforded by Nuvia cPrime media) shows binding capacity and recovery across different pH and salt concentrations.
Simple Method Development
The mixed-mode nature of the Nuvia cPrime ligand facilitates a directed and intuitive approach to method development.
Figure 4 (Recommended approach to method development) outlines steps for elution by pH, salt, or modifiers.
Built to Meet the Demands of Commercial Operations
Nuvia cPrime is built on a porous polymeric base matrix providing low backpressure at high flow rates, ensuring efficient chromatography for commercial-scale applications.
Figure 5 (Nuvia cPrime displays low backpressure at high flow rates) shows pressure vs. linear velocity and DBC vs. linear velocity.
Featured Products
Chromatography Resins and Screening Tools
Bio-Rad offers a wide range of resins for each phase of virus process purification, from nanograms to kilograms. A Virus purification resin selection card is available to guide resin selection.
NGC Quest 10 Chromatography System is highlighted as a versatile piece of equipment adaptable for various detectors.
Virus Purification Resin Selection Guide
This guide helps select the optimal resin for virus process purification.
| Process Purification Resin Type | Adenovirus | Other Small to Midsized Mammalian Viruses | ||||
|---|---|---|---|---|---|---|
| Nuvia cPrime | Nuvia Q | UNOsphere Q | CHT Ceramic Hydroxyapatite | CFT Ceramic Fluoroapatite | MPC Ceramic Hydroxyfluoroapatite | |
| Resin type | Mixed-mode (HIC and cation exchange) | Anion exchange | Anion exchange | Mixed-mode (cation exchange and metal affinity) | Mixed-mode (cation exchange and metal affinity) | Mixed-mode (cation exchange and metal affinity) |
| Particle size | 70 ± 10 µm | 85 ± 15 µm | 120 µm | 20 ± 2, 40 ± 4, 80 ± 8 µm | 40 ± 4 µm | 40 ± 4 µm |
| Ionic capacity | 110-150 µeq/ml | 100-170 µeq/ml | 120 µeq/ml | |||
| Dynamic binding capacity (DBC) | >40 mg hlgG/ml at 300 cm/hr* | ≥170 mg/ml at 300 cm/hr* | ≥180 mg BSA/ml at 600 cm/hr* | 25-60 mg IgG/ml at 300 cm/hr* | 25-50 mg IgG/ml* | 25-50 mg IgG/ml* |
| Recommended linear flow rate | 50-600 cm/hr | 50-600 cm/hr | 50-300 cm/hr | 50-300 cm/hr | 50-300 cm/hr | 50-300 cm/hr |
| pH stability | Short-term: 3-14, Long-term: 4-13 | Short-term: 2-14, Long-term: 4-12 | 1-14 | 6.5-14 | 6.5-14 | 6.5-14 |
Note: BSA, bovine serum albumin; HIC, hydrophobic interaction chromatography.
Ordering Information
Details on catalog numbers and sizes for various resins and plates are provided by Bio-Rad.
Conclusion
Ceramic hydroxyapatite media provides high purity, recovery, and viral activity for mammalian viruses. Optimizing parameters like flow rate and gradient slope is important. Nuvia cPrime offers new selectivity and a large design space for downstream purification processes, meeting commercial demands with its robust performance and scalability.
Acknowledgements
The document acknowledges contributions from various researchers and institutions for supplying viral particles and conducting experiments.
References
A list of cited references is provided, including publications on virus purification methods and safety.
