Non-Affinity Platforms 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
The most critical subset of product related impurities include:
- Empty Capsids
- Capsid Aggregates
- Capsid DNA Complexes
Pete Gagnon, CSO BIA Separations: “The most serious troublemakers are characterized by the presence of residual chromatin debris. During cell culture, dying cells degrade to the point of becoming undetectable within 24 hours, but some of their contents survive for months after harvest. Those survivors include chromatin; chromosomal remnants in the form of degraded nucleosomal arrays and fragments.They act as nucleation centers for accretion of other contaminants, leading to formation of chemically “sticky” aggregates. Some refer to them as complexes.They bind to all chromatography and filtration media, modify their surfaces, and interfere with their function. They sometimes bind to drug products as well. On top of that, extracellular chromatin is antigenic.”
- Chromatin interferes with all AAV purification methods including affinity chromatography, at any step
- It depresses binding capacity.
- It inflates host protein contamination.
- It inflates host DNA contamination.
- It decreases final product (full AAV capsid) recovery.
- Most important - if not properly managed - it resides at all steps of purification
BIA Separations has developed fully scalable monolith-based AAV DSP platforms to eliminate process specific and product specific impurities.
Common to all AAV process solutions is the combination of two IEX chromatographic steps in order to reduce Chromatin related impurities.
As an example, BIA Separations first generation 2-step AAV downstream process (see below) represents a basic building block and a good starting point for optimization. BIA Separations is currently focussing its capacities on developing novel, innovative chromatin management tactics in order to further optimize overall process performance.
Mandatory for the successful process developent of AAV based ATMPs are powerful process analytics.
BIA Separations has developed a broad portfolio of HPLC based analytical methods for fast, accurate, and meaningful characterization of
- Mammalian or Insect cell culture harvests
- Intermediate / in-process samples at any unit-operational step
- Purified AAV vector lots
Applications of novel CIMac™ based HPLC column protocols employing innovative, orthogonal Detector assemblies allow for holistic interpretation of samples, with respect to
- Impurity profile
- Chromatin burden
- Total AAV content
- Full / Empy Capsid ratios
- Free - and Capsid-associated DNA
Please follow the links of some recent publications and posters below to learn more about applications of CIMac based AAV analytics:
- Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development Dr. Xiaotong Fu, Dr. Wei-Chiang Chen, C. Argento, R. Dickerson, P. Clarner, V. Bhatt, G. Bou-Assaf, Dr. M. Bakhshayeshi, Dr. Xiaohui Lu, Dr. S. Bergelson, Dr. J. Pieracci Human Gene Therapy (2019)
- Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2 Wang Chunlei, Mulagapati Sri Hari Raju, Chen Zhongying, Du Jing, Zhao Xiaohui, Xi Guoling, Chen Liyan, Linke Thomas, Gao Cuihua, Schmelzer Albert, Liu Dengfeng Molecular Therapy Methods & Clinical Development, Volume 15, September 26 2019, Pages 257-263
- Orthogonal characterization of AAV samples by simultaneous monitoring of multiple analytical parameters during chromatography B. Goricar, S. Peljhan, P. Gagnon, A. Strancar BIA Separations d.o.o., Mirce 21, 5270 Ajdovscina, Slovenia
- Estimation of empty and full AAV10 particle ratio using the multi angle light scattering detector B. Goricar, S. Peljhan, P. Gagnon BIA Separations d.o.o., Mirce 21, 5270 Ajdovščina, Slovenia
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.
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.