Viruses

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.

virus purification

Adeno associated virus

A Non-affinity Platform for Purification of All AAV Serotypes

Therapeutic application of AAV-based gene therapy vectors requires that host-cell contaminants and product related impurities be removed. A critical subset of product related impurities includes empty capsids; capsids lacking their therapeutic DNA payload. This is a particular challenge because of the chemical similarity of the capsid exteriors.

We have developed a fully scalable monolith-based platform to fulfil these requirements. It commences with a capture step immediately after upstream production, using a monolith with hydroxyl (-OH) coating for initial capture and concentration. It continues with a cation exchange monolith (SO3) remove the bulk of remaining host contaminants. It finishes with anion exchange chromatography (QA) for separation of empty and full capsids.

Product recovery is very high at each of the individual steps. This enables total process recovery values of 50–75%, depending on the serotype, production method, and harvest method. The process has been demonstrated to be effective with several serotypes. The examples shown below were produced with AAV 9 and AAV 10 serotypes.

aavkrom1

aavkrom2Capture is performed using a CIMmultus™ OH monolith in the presence of precipitating salts. The virus binds. Most of the small molecule contaminants and proteins are eliminated in the flow-through. AAV co-elutes with a highly reduced population of contaminating proteins. A subset of stable DNA-protein complexes is very strongly retained and requires  1 M NaOH for removal.

Intermediate purification of AAV is performed using a CIMmultus™ SO3 cation exchange monolith. The AAV fraction from the capture step is titrated to a lower pH and diluted to

binding conditions. Sugars and surfactants are added to suppress non-specific interactions with tubing and containers, and the product is eluted in a salt gradient. Another subset of stable DNA-protein A complexes remains strongly bound and requires 1 M NaOH for removal.

Empty/Full separation is conducted on a CIMmultus™ QA anion exchange monolith which separates empty capsids from full capsids. The efficiency of this step benefits from the removal pf DNA-protein complexes in the previous steps. If not removed previously, they interfere with Empty/Full Separation and inflate contamination by host cell proteins and DNA.

Process analytics

We have customized two rapid HPCL methods to monitor total virus (titer) and empty versus full capsid content. Both are conducted with 100 µL monoliths that require very small amounts of sample. Both provide fast results that can be used to aid process development and optimization, validation, and near-real time in-process monitoring. The total AAV assay employs a specially optimized cation exchanger and gives best results with a fluorescence monitor to amplify sensitivity. Our empty full assay employs a specially optimized anion exchanger. Comparison of UV absorbance ratios at 260 and 280 nm allows clear identification of empty and full capsids. Simultaneous monitoring with flurorescence and other methods can permit robust estimates of empty/full ratios.

Total AAV analysis with CIMac™ SO3 0.1 mL (P/N 110.6157-1.3). This assay is valuable for in-process control after the CIMmultus™ OH capture step and CIMmultus™ SO3 intermediate purification steps.

virus purification

Empty/Full analysis with CIMac™ AAV empty/full (P/N 110.8503-1.3). This assay is valuable during process development to guide optimization towards conditions that support more effective removal of empty capsids, and to evaluate empty/full ratios in the final product. Divalent metal cations provide useful modification of selectivity.

aavkrom4

Influenza

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 [1].

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.

virus purification

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.

virus purification

Refer to references [4,5,6,7,8] for more information concerning purification of influenza virus.

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