AAV

CORNERSTONE AAV Process Solution: A Non-Affinity Platform for Purification of All AAV Serotypes

Therapeutic applications of AAV-based gene therapy vectors require, that process - and product related impurities be removed, as they represent serious safety threats as well as burden the economics of manufacturing.

The most critical subsets of process related impurities include:

  • Host Cell Nucleic Acids
  • Host Cell Proteins
  • Chromatin

The most critical subset of product related impurities include:

  • Empty Capsids
  • Capsid Aggregates
  • Capsid DNA Complexes

BIA Separations has developed a fully scalable monolith-based platform to fulfill these requirements. It commences with a propriatery extraction technology followed by either a monolith with strong cation exchange (SO3) or hydroxyl (OH) coating for initial capture, complex removal  and concentration. It finishes with anion exchange chromatography (QA) for separation of empty and full capsids and residual chromatin depletion.

Product recovery is very high at each of the individual steps. This enables total process recovery values of 60–80%, 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 2/8 and AAV 10 serotypes.

Contact us to initiate your AAV process development project with BIA Separations.

Get access to the slide-deck of Pete Gagnon’s latest presentation on AAV at BPI 2019.

    Backbone of the CORNERSTONE AAV Process Solution

    is a novel tool, CIMasphere™, which is a particulate solid phase, that binds DNA so strongly, it becomes dissociated from its pre-existing associations.

    Chromatin reduction in advance of chromatography

    The process

    Advance chromatin extraction creates multiple non-affinity options for high performance AAV purification.

    Capture with CEX and alternatively capture with preferrential exclusion chromatography.

    Capture with cation exchange provides the more streamlined option because only dilution is required before polishing. The two can be combined for highly demanding separations.

    High performance platforms for all AAV serotypes

    CIMasphere advanced extraction technology

    Conventional nuclease processing of SF9 lysate, AAV 2/8.

    Analytical SEC with monitoring of intrinsic tryptophan fluorescence.

    Nuclease lyses accessible DNA but works poorly with chromatin because DNA’s strong histone associations protect it.
    Nuclease lyses accessible DNA but works poorly with chromatin because DNA’s strong histone associations protect it.
     
    Advance chromatin reduction from filtered harvest

    CIMasphere processing of nuclease-treated SF9 lysate, AAV 2/8.

    Analytical SEC with monitoring of intrinsic tryptophan fluorescence.

    CIMasphere takes advantage of the strong associations between Host Cell proteins, Nucleic Acids and histones, and uses them to selectively remove the contaminants. After incubation the particles are removed by any convenient method; centrifugation, membrane or depth filtration.
    CIMasphere takes advantage of the strong associations between Host Cell proteins, Nucleic Acids and histones, and uses them to selectively remove the contaminants. After incubation the particles are removed by any convenient method; centrifugation, membrane or depth filtration.
     

    Capture option by cation exchange chromatography

    100 mL SF9 lysate, AAV2/8, post-extraction with CIMasphere.

    CIMmultusTM SO3, 1 mL, 2 μm channels, 10 CV/min.

    Chromatin left after solid phase extraction binds strongly to SO3 because of its histone component. Its removal at this step is essential for enabling good separation of empty and full capsids by anion exchange.
    Chromatin left after solid phase extraction binds strongly to SO3 because of its histone component. Its removal at this step is essential for enabling good separation of empty and full capsids by anion exchange.
     

    Capture option by preferential exclusion chromatography

    100 mL HEK293 lysate, AAV10, post-extraction with CIMasphere.

    CIMmultusTM OH, 1 mL, 2 μm channels, 10 CV/min.

    Preferential exclusion chromatography uses high concentrations of precipitating salts to promote retention on a non-hydrophobic solid phase. Proteins are mostly unretained except low levels of HMW nucleoproteins. They are more prominent on the chromatogram because of their DNA component. PAGE reveals only protein.
    Preferential exclusion chromatography uses high concentrations of precipitating salts to promote retention on a non-hydrophobic solid phase. Proteins are mostly unretained except low levels of HMW nucleoproteins. They are more prominent on the chromatogram because of their DNA component. PAGE reveals only protein.
    ​​​

    Analytical characterization of empty/full content

    Analytical determination of % full capsids after anion exchange.

    AAV 2/8 SF9 lysate after CIMasphere extraction and cation exchange.

    Fluorescence supports reliable estimates of empty/full proportions because it measures only capsid proteins. UV overestimates full capsids because absorbance by DNA inflates protein measurement.
    Fluorescence supports reliable estimates of empty/full proportions because it measures only capsid proteins. UV overestimates full capsids because absorbance by DNA inflates protein measurement.

     

    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 Analytical Column. 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-0.1 Analytical Column. 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

    Most often used