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Development of an Anion Exchange Chromatography Method to Assess Percent Full Capsids for Chimeric Capsid AAV-LK03
W Lee, N DiGioia, N Walsh, H
Reuter, L M Drouin, M Hebben
October 19-22, 2021
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Introduction to LK03 Chimeric Capsid
William Lee
- AAV-LK03vectors demonstrate improved transgene expression in primary hepatocytes in culture compared to other AAV serotypes [1]
- Currently a part of the LB-001 drug product in the SUNRISE clinical trial for children with methylmalonic acidemia.
- During development, this product has been subject to extensive process development and stability/forced degradation experiments to optimize unit operations and final formulation.
Parental Origin of LK03[1] | Relative Transgene Expression[1] |
[1] Lisowski et. al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature. 2014
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Empty Capsids and %Full
Empty Partial Full
William Lee
- Empty capsids containing no genome are generated during AAV production and are considered a process impurity.
- Reduced transduction efficiency [1]
- Increased immune response as a result of more capsids delivered per dose [1]
- The percentage of empty and filled capsids needs to be monitored from batch to batch.
- High throughput methods to measure %Full are necessary to support extensive process development experiments.
- Gao et. al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Molecular Therapy - Methods & Clinical Development, 2014
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EF-HPLC Method Parameters
- Strong anion exchange chromatography with a quaternary ammonium salt gradient can be used to separate empty from full AAV capsid particles.
- Tetramethylammonium chloride salt has been shown to improve separation of AAV capsids compared to sodium chloride. [1]
- Intrinsic tryptophan fluorescence detection can be used for direct quantitation of capsids by determining the area under the curve.
- Only 0.2% of the fluorescence signal is from encapsidated DNA, so full capsids are assumed to produce the same signal as empty capsids [2]
- Note: this method cannot distinguish partial and full capsids
William Lee
Affinity Eluate Chromatogram (29% Full)
Empty Peak
A260/A280= 0.83Full Peak
A260/A280= 1.78
- Wang et. al. Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2, Molecular Therapy - Methods & Clinical Development, 2019.
- Gagnon et. al. Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification. Pharmaceutics. 2021
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Assessing EF-HPLC Analytical Procedure Repeatability | William Lee |
- The repeatability of the method was tested with replicate injections of AAV-LK03 capsid.
- The sample was transferred to an HPLC vial and injected six times in series.
Waterfall Plot of Replicate Injections
Data Table of Replicate Injections
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LogicBio Therapeutics Inc. published this content on 21 October 2021 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 21 October 2021 21:33:19 UTC.