LogicBio Therapeutics : ESGCT 2021 - Analytical development

Delivering the hope of genetic medicine to people impacted by devastating diseases

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.

1. 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

1. 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.
2. 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