Journal: Molecular Therapy. Methods & Clinical Development
Article Title: Highly Efficient Ultracentrifugation-free Chromatographic Purification of Recombinant AAV Serotype 9
Figure Lengend Snippet: Laboratory-Scale Purification of AAV9-dsEGFP by Quaternary Ammonium Anion-Exchange Column and Size-Exclusion Chromatography (A) The AAV9-dsEGFP preparations were analyzed by 5%–20% (v/v) gradient gel SDS-PAGE and stained with Oriole fluorescent gel stain before and after chromatography purification using a HiPrep Q XL 16/10 column. The white arrowhead indicates a 200-kDa impurity. Lane 1, pre-TFF; lane 2, post-TFF; lane 3, after heat treatment; lane 4, 1/3→1/2 AS; lane 5, diluted 1/3→1/2 AS; lane 6, pass-through fraction; lane 7, wash-out fraction; lane 8, column-bound and eluted fraction. (B) The pass-through fraction was subsequently subjected to size-exclusion chromatography using a HiLoad 16/60 Superdex 200 preparation-grade column using an ÄKTA Explorer 100 HPLC system equipped with a 10-mL sample loop, with MHN buffer (pH 6.5) containing 300 mM NaCl and 0.01% (w/v) Pluronic F-68 as the mobile phase. y axis, 280 nm absorbance; x axis, fraction number. The black arrowhead indicates the peak fractions of the rAAV9 (corresponding to lanes 2–14 in C). (C) The elution fraction was analyzed by two 5%–20% (v/v) gradient SDS-PAGE gels with Oriole fluorescent staining; the left gel is from lanes 1–12, and the right gel is from lanes 13–23. Lanes 1–18, fractions 14–31; lane 19, fraction 33; lane 20, fraction 35; lane 21, fraction 37; lane 22, fraction 39; lane 23, fraction 41. Peak fractions (fractions 15–27) were collected to obtain the final product. (D–G) The final AAV9-dsEGFP product was analyzed by 5%–20% (v/v) gradient gel SDS-PAGE with Oriole fluorescent staining (D), western blotting (E, anti-AAV capsid monoclonal antibody B1), EM (F, negative staining), and analytical ultracentrifugation (AUC, G). Shown are the peak fractions of (i) empty particles (68.6 S, 25.7%), (ii) intermediate particles (88.8 S, 32.4%), and (iii) fully packaged virions (102.6 S, 38.8%). The goodness of fit (RMSD) was 0.004635. y axis, continuous-size C(S) distribution; x axis, sedimentation coefficient. Lane 1 and lane 2, the final rAAV9 product. The three bands represent the AAV9 capsid proteins VP1 (82 kDa), VP2 (67 kDa), and VP3 (60 kDa).
Article Snippet: The pass-through fraction was purified by size-exclusion (gel filtration) chromatography using a HiLoad 16/60 Superdex 200 preparation-grade column (GE Healthcare) and an ÄKTA Explorer 100 HPLC system (GE Healthcare) equipped with a 10-mL sample loop running the MHN buffer (pH 6.5) containing 300 mM NaCl and 0.01% (w/v) Pluronic F-68.
Techniques: Purification, Size-exclusion Chromatography, SDS Page, Staining, Chromatography, High Performance Liquid Chromatography, Western Blot, Negative Staining, Sedimentation