Increased productivity using IsoTag AAV at various harvest scales
Increased productivity of AAV9 affinity capture using IsoTag-AAV at various harvest scales
Correspondence: mdzuricky@isolerebio.com
Abstract: Adeno-associated viruses (AAV) have high potential for use as a gene therapy vehicle because of their superior safety profile, but low expression titers, shockingly inefficient purification, and increasingly scrutinized regulatory requirements are slowing development and casting doubt on its commercial feasibility. While traditional unit operations enable target biologic purification by either size or affinity interaction, the IsoTag™ technology consolidates both mechanisms into one simple, elegant protein reagent. The IsoTagTM-AAV technology is unique because it is based on harvest volume, not titer and therefore can be scaled up linearly by simply increasing the filter surface area
Materials:
· Size 16 Masterflex® L/S Platinum-Cured Silicone Tubing, Cole Parmer
· Size 13 Masterflex® L/S Platinum-Cured Silicone Tubing, Cole Parmer
· 0.2µm Bottle Top filter
· 2 Stir plates with magnetic stir bars
· Pendotech retentate vessels 140ml and 600 ml
· Spectrum Hollow Fiber Filters (various surface areas)
Buffers:
· Wash Buffer - 20mM Tris pH7, 0.6M NaCl, 0.001% Pluronic Acid, 0.01% Tween 20
· Elution Buffer - 100mM Glycine pH 3, 0.6M MgCl2, 0.001% Pluronic Acid, 0.01% Tween 20
· 2X Elution Buffer - 200mM Glycine pH 3, 0.001% Pluronic Acid, 0.01%Tween 20 (Keep on Ice to add cold)
· 5M NaCl
· 5M MgCl2
Results:
The IsoTag™-AAV TFF process was implemented on harvest volumes of 0.2L to 1L and 8L by linearly and proportionately increasing the filter area to keep volumetric loading constant. The permeate controlled flux was also kept constant at all volumes. The resulting runs had nearly identical processing times of ~3.5 hours despite the 16X increase in volumetric capacity with very little increase in transmembrane pressure, suggesting high transmission and easy filtering (Figure 1). This was confirmed by residual host cell protein and host cell DNA quantification that demonstrates consistent 3.8-4.0 log removal of both species for each of the three runs. The capture of the IsoTag™-AAV process was consistent at each scale with average capture of 96.6% and 98.2% by qPCR analysis for vector genomes and ELISA analysis for total capsids (cp) respectively. The total elution yield for the process across each scale was 75.3% and 74.7% by qPCR and ELISA respectively (Figure 2). These results were achieved without any elution buffer or diavolume optimization, and as the vast majority of the “unrecovered” AAV9 is present in the retentate, there is still room to push yields higher for individual molecules.
Figures:
Figure 1 – Flux and TMP profiles for 200mL, 1L and 7L TFF runs. The Flux profile is controlled and kept constant 40-50LMH to keep total run time consistent across scales. The TMP is measured throughout the run as an indication of filter performance.
Figure 2 – A) AAV9 capture and elution percentages with IsoTag™-AAV TFF across 200mL, 1L and 8L scales by qPCR (vg) and total capsid ELISA (cp). Percentages are based on total AAV9 present in cell culture harvest prior to TFF. B) Log reduction values for host cell protein and DNA contaminants by IsoTag™-AAV TFF process across 200mL, 1L and 8L scales.
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