Leading virus purification. Enveloped or not.
Virus purification strategies using chromatographic methods have been proven to be one of few choices when high purity and good product recovery is required. CIMmultus chromatographic offer scalable, fast and economically favorable purification processes with selection of chemistries and channel sizes to meet the needs for various virus particles.
Dynamic binding capacities on monoliths range from 10 to 100 times higher than porous particle columns, and 2-10 times higher than membrane adsorbers, plus virus recoveries on monoliths are often twice as high as alternative media. Add to that flow rates 20–50 times higher than porous particle columns and it becomes clear why monoliths are so popular for virus purification.
Process development can be done on 100 µL monoliths to conserve sample, and developed applications can be scaled up with monoliths up to 40 L. Those volumes may seem small but keep in mind that they represent the capacity as packed columns with volumes of 10 mL and 4,000 L respectively. Keep in mind too that column volume determines buffer volume, and buffer volume determines process time. The high capacity of monoliths is a huge asset for your virus purification challenges.
BIA monoliths for virus purification include a full line of activated affinity supports, anion exchangers, cation exchangers, hydrogen bonding and hydrophobic interaction chromatography media.
BIA’s monoliths are well established throughout the fields of gene therapy, vaccines, and bacteriophage purification. In addition to providing monoliths, BIA offers expert fully-integrated process development services, including both purification and analytics. We have developed a high-recovery non-affinity platform for many AAV that provides outstanding separation of empty and full capsids for every serotype evaluated to date, and extensive experience with influenza, including oncolytic vaccines.
The unique architectural and performance properties of monoliths are a perfect match for the unique requirements of purifying lipid-enveloped viruses, including influenza virus.
Fast, effective, scalable template procedures have been developed for industrial purification of both A and B serotypes. Scale-down variations of those templates also support easy, fast, reliable analysis to guide process development and document process control of manufacturing processes .
Shear stress during downstream processing is documented to strip the envelopes from lipid enveloped viruses. This makes absence of turbulent shear from monoliths one of their most compelling features. Flow through monoliths is laminar, with no formation of the eddies that create turbulent shear stress in porous particle columns.
The efficiency of convective flow through monolithic channels is another major attraction. Viruses are big, especially compared to the proteins for which porous particle columns are designed. Big virus particles translate to slow diffusion constants. That is a problem for particle columns but not for monoliths because mass transport in monoliths is convective. Binding efficiency and elution kinetics are both independent of product size.
Binding efficiency and elution kinetics in monoliths are also independent from flow rate. This enables monoliths to be run at flow rates 20–50 times higher than porous particle columns with no loss of performance. This explains why dynamic binding capacity for influenza virus on monoliths is typically 10–100 times higher than for porous particle columns.
The third key feature is that monoliths lack dead ends. All the channels are highly interconnected. This, along with the absence of turbulent shear, explains why monoliths enable 2–3 times higher virus recovery than other chromatography formats [2,3].
The combination of low shear, rapid flow rate, and high recovery also enables monoliths to achieve major process simplifications and improvements in efficiency. For example, instead of requiring a separate process step for harvest concentration by tangential flow filtration (TFF), the sample can be diluted then loaded directly onto a monolith with greater than 99% 1-step reduction of host DNA and proteins in less time than the TFF step alone, and without the product losses.
Monoliths used for purification of influenza virus can be applied in a single-use format or sanitized to process multiple product lots. Recently, we developed highly intensified Influenza virus purification using cation-exchange (SO3) monoliths, for which we filed a patent application. The process key advantage and the basis on which we developed the process, is highly efficient Influenza upstream process using Vero cells in serum-free conditions in combination with low speed centrifugation clarification.
Adenovirus (and Adenovirus Like-Particles)
There has been a long lasting interest in purification of adenoviruses (Ad) in the past years since their wide use as a viral vector for gene therapy, vaccine and cancer gene therapy. Because of adenovirus large size (90-100 nm), monoliths are ideal choice for developing purification process for different adenovirus serotypes. Another important reason is the absence of shear stress when using monoliths that supports high functional recovery, especially for large, shear-sensitive infectious particles.
Anion exchange chromatography using monoliths was shown as an optimal purification media in several adenovirus processes. CIMmultus QA is used for capture and purification and usually further concentration and buffer exchange is done using tangential flow filtration (TFF). GMP compliant process was published for Canine adenovirus type 2 (CAV-2), reporting double yield when using monoliths compared to alternative chromatographic media. Similarly, monoliths of different media compositions were tested for adenovirus like-particles purification and confirmed anion-exchange QA column was the most suitable media choice for adenovirus like-particles purification.
For adenovirus quantification purposes we developed a fully dedicated analytical monolithic column CIMac Adeno-0.1 Analytical Column. It is an anion exchange column with optimized pore size, designed for fast and reproducible analysis of adenoviruses. Several protocols are available for implementing HPLC quantification assay as a fast alternative to your traditional viral assays [1,2,3], especially for monitoring upstream and downstream process of adenoviruses.