Figure 3.
AAV Purification and Characterization
(A) TEM image of affinity-purified AAV5 showing a mixed population of full particles (F) and empty capsids (E) with a characteristic particle diameter of 20–23 nm. (B) VP stoichiometry analysis via a Flamingo-stained SDS-PAGE gel of an affinity-purified sample of three production runs of AAV in batch mode (shake flask [SF]) and fedbatch mode (shake flask: SF and 3 L bioreactor). The total VG loading per well was in the range of 5 × 108–7 × 108. The VP band intensities in all samples are similar and comparable to that of the 1 L bioreactor sample. (C) AUC histogram representing a 260 nm absorbance profile of representative samples of an affinity-purified AAV5 from production in (1) batch mode in shake flask, (2) fedbatch mode in shake flask, (3) fedbatch mode in 3 L bioreactor, and (4) fedbatch mode in 1 L bioreactor. Different components in an affinity-purified AAV5 sample as a function of sedimentation coefficient are shown. A very sharp peak around 62–67 s represents the presence of an empty capsid, and a major peak around 93–98 s and the intermediate population between these two major peaks represent genomic capsids. The peaks corresponding to the intermediate population may represent the form of a particle with encapsidation of partial AAV genome or collaterally packaged contaminating DNA. (See also Figures S4–S6, and Tables S1 and S2.) SF, shake flask.