SUPPLEMENTARY MATERIAL
Purification of biotinylated AAV on immobilized monomeric avidin resin
In this section we provide detailed analysis of several important steps in the purification
procedure. In all cases crude cell lysates containing biotinylated AAV vectors were first
fractionated by a brief 1 h centrifugation on an iodixanol step gradient and were loaded
onto 2-ml bed volume Immunopure immobilized monomeric avidin gel columns (Pierce
Biotechnology, Inc.). After vectors samples were applied to the column it was important
to determine the degree of non-specific cellular protein binding to the column and the
requirements for column washing in order to remove these contaminating cellular
proteins. In a pilot study, crude lystate containing biotinylated AAV was added to an
avidin column, flow-through was collected, the column was washed with 16-ml of PBS,
and vector was eluted with 12-ml of Elution Buffer containing 0.3% sodium
deoxycholate (DOC) and 5 mM D-biotin. Elution Buffers based on either PBS (PBS, pH
8.0 with 150 mM NaCl), or sodium citrate (100 mM sodium citrate, 5 mM MgCl, 10 mM
Tris-HCl, pH 8.3) performed equally well. Protein presented in 1-ml fractions was
estimated by absorbance measurements at 320 nm and AAV particles were quantified by
real-time PCR-based DRP titer analysis (Figure S1). Cellular protein was not retained by
the column and was effectively washed from the column with about 5 column volumes of
PBS. Since the volume of wash buffer needed to remove contaminating cellular proteins
was expected to be dependent on the size of the vector preparation and the amount
cellular protein present, columns were routinely washed with at least 3-times this amount
(>15-ml) of PBS buffer. No loss of bound AAV was observed with increased wash
volumes. Bound AAV was effectively eluted from the column with 10 column volumes
of Elution Buffer (Figure S1). To examine the effect of biotin concentration on elution of
bound AAV, we conducted a second study in which vector was eluted sequentially with
increasing concentrations of biotin (Figure S2). Although 0.5 mM biotin was effective at
eluting the majority of bound biotinylated AAV, increasing the concentration of biotin in
the Elution Buffer to 1.0 mM mediated a slight increase in vector recovery. There was no
increase in vector elution when the concentration of biotin was increased to 5 or 10 mM.
Furthermore, no vector remained bound to the resin, indicating that the majority of
biotinylated AAV is efficiently eluted from monomeric avidin with concentrations of
biotin less than or equal to 5 mM. Interestingly, the concentration of detergent in the
elution buffer had a dramatic effect on our ability to recover biotinylated AAV (Figure
S3). In the example shown, crude lysate containing biotinylated AAV was applied to a
monomeric avidin column by gravity flow. The column was washed with 16-ml of PBS
and eluted sequentially with 10-ml of Elution Buffer containing only 5 mM biotin; 10-ml
of Elution Buffer containing 0.01% DOC, 5 mM biotin; 10-ml of Elution Buffer
containing 0.1% DOC, 5 mM biotin; and 10-ml of Elution Buffer containing 1.0% DOC,
5 mM biotin. Two 5-ml fractions were collected for each elution condition and DRP were
calculated by real-time PCR assay (Figure S3). In the absence of DOC, vector remained
tightly bound to the column and could not be eluted even in the presence of high biotin. A
minimum of 0.1% DOC was needed to elute bound biotinylated AAV. Increasing the
concentration to 1.0% DOC resulted in more rapid elution. Therefore, DOC
concentrations between 0.1% and 1.0% are recommended for effective vector elution.
FIGURE LEGENDS
Figure S1. Purification of biotinylated BAP-modified AAV vectors on immobilized
monomeric avidin affinity resin. Biotinylated AAV1-based eGFP vector was produced in
HEK 293 cells by transfection of pXR1-Cap1.D590_P591insBAP, pEGFP-BirA, and
standard recombinant AAV vector and adenovirus helper plasmids [8]. Packaging cell
lysate was fractionated on an iodixanol step gradient and applied to the avidin affinity
column by gravity flow. Flow-through (FT) was collected, the matrix was washed with
16-ml of PBS, and 1-ml fractions were collected (W1, W2, W3, etc.). Virus was eluted
with 12-ml of Elution Buffer (PBS, pH8.0 containing 150 mM NaCl, 0.3% DOC, and 5
mM biotin), and 1-ml fractions were collected (E1, E2, E3, etc.). The indicated fractions
were analyzed for yield of purified AAV particles by DRP titer, expressed as total DRP
per fraction, and total protein by absorbance at 320 nm.
Figure S2. Elution of biotinylated BAP-modified AAV vectors from immobilized
monomeric avidin affinity resin with biotin. Biotinylated AAV1-based vector was
produced as described above (Figure S1), fractionated on an iodixanol step gradient, and
applied to an avidin column by gravity flow. The column was washed with 16-ml of PBS
and vector was eluted sequentially with two 5-ml fractions of Elution Buffer containing
0.5 mM biotin (E1 and E2), 1 mM biotin (E3 and E4), 5 mM biotin (E5 and E6), and 10
mM biotin (E7 and E8). The indicated fractions and the resin were analyzed for AAV
particles by DRP titer analysis and expressed as total DRP yield per fraction.
Figure S3. Elution of biotinylated BAP-modified AAV vectors from immobilized
monomeric avidin affinity resin is dependent on deoxycholate. Biotinylated AAV1-based
vector was produced as described above (Figure S1), fractionated on an iodixanol step
gradient, and applied to an avidin column by gravity flow. The column was washed with
16-ml of PBS and vector was eluted sequentially with two 5-ml fractions of Elution
Buffer containing 5 mM biotin (E1 and E2); Elution Buffer containing 0.01% DOC and 5
mM biotin (E3 and E4); Elution Buffer containing 0.1% DOC and 5 mM biotin (E5 and
E6); and Elution Buffer containing 1.0% DOC and 5 mM biotin (E7 and E8). The
indicated fractions were analyzed for AAV particles by DRP titer analysis and expressed
as total DRP yield per fraction.