1
Technical Note A fit for purpose AAV purification method: Introduction AAVs or adeno-associated viruses, are small, single strand DNA viruses from the family Parvoviridae that have become a popular viral vector for gene therapy. AAVs were initially discovered as a contaminant of adenovirus preparations. Because of their low risk of insertional mutagenesis and their proven clinical success, they have become the leading platform for gene delivery for the treatment of a variety of diseases. Recent advances in developing AAV capsids and optimizing genomic payload have contributed substantially to the growth of the gene therapy vector, bolstered by preclinical and clinical successes. AAV2 is the most widely used AAV serotype for gene delivery. However, there are many other AAV serotypes. Many of which, including AAV5, AAV8 and AAV9 have been used in viral vector gene therapy trials. As gene therapy technologies become more advanced, and more companies enter the market, there is now an increasing demand for recombinant or rAAV to be used as the foundation for platform technologies.
Technical note: A fit for purpose AAV purification method
1
How are AAVs purified? AAV purification typically requires the use of bioprocessing resins, and affinity-based methods are an established chromatography step in many workflows. These resins are based upon camelid ligand technologies and are used to interact with different binding sites on the surface of the capsid. These affinity ligands can have broad applicability across a range of serotypes, but are not without their drawbacks, including incompatibility with caustic solutions, tendency to coelute with ad5 and high price. Additionally, AAV mutants to overcome pre-existing immunity also have poor affinity for protein-based affinity columns. After the AAVs have been captured from the feedstock, the empty and full capsids must be separated. In some instances, ultracentrifugation with a cesium gradient is used, however this is not scalable to large manufacturing needs.
An Astrea Bioseparations Solution To meet the growing needs of viral vector gene therapy companies, Astrea Bioseparations has developed a complete purification solution applying our cutting-edge ligand technology and the proprietary PuraBead® base matrix. By utilizing our proven resin technologies, a workflow can be developed that is fully scalable, serotype agnostic and reusable. The first step in the Astrea Bioseparations purification workflow is primary capture. To accomplish this, our SP PuraBead® P6HF was selected and was used to purify AAV8 and AAV9 serotypes. Columns were initially loaded with 9.4 e12 of AAV8 and 1.14 e13 AAV9, which had been acidified to pH 4 and clarified. After passing through the column at a 3.3-minute residence time, the retained AAVs were eluted off the column using 0.4 M NaCl (AAV8) or 0.5 M NaCl (AAV9). The samples were then analyzed by ELISA to determine the amount of AAV particles in the different fractions. After elution, we were able to obtain 7080% recovery of our AAV capsids, which were functional as measured by infectivity assay and able to be exchanged into a neutral storage solution for further processing.
Technical note: A fit for purpose AAV purification method
2
900 800 700 600 500 400 300 200 100 0
0
10
20
30
40
50
60
200 180 160 140 120 100 80 60 40 20 0
mS/cm
mAU
AAV 8 on P6HF SP
CV UV280
UV260
mS/cm
Figure 1: Chromatogram of AAV8 feedstock loading on to SP PuraBead® P6HF
60 50
mAU
40 30 20 10 0
22
27
32
37
42
200 180 160 140 120 100 80 60 40 20 0
mS/cm
AAV 8 on P6 HF SP zoom in on elution peak
CV UV280
UV260
mS/cm
Figure 1a: Zoom in on elution of AAV 8 from SP PuraBead® P6HF
Technical note: A fit for purpose AAV purification method
3
Table 1: AAV8 SP PuraBead® P6HF column performance data from ELISA
ELISA Results for AAV8 Total Capsids Loaded
9.40E+12
Total Capsids Bound
9.23E+12
% Total Capsids Bound
98.2
% Recovery from Bound
70.1
% Recovery from Load
68.8
% Strip Recovery from Load
1.5
% Mass Balance
72.1
200 180 160 140 120 100 80 60 40 20 0
1000 900 800 700 600 500 400 300 200 100 0 0
5
10
15
20
25
30
35
40
mS/cm
mAU
AAV 9 on P6 HF SP
45
CV UV280
UV260
mS/cm
Figure 2: Chromatogram of AAV9 feedstock loading on to SP PuraBead® P6HF
Technical note: A fit for purpose AAV purification method
4
200 180 160 140 120 100 80 60 40 20 0
60 50
mAU
40 30 20 10 0 21
23
25
27
29
31
33
35
37
mS/cm
AAV 9 on P6HF SP zoom in on elution peak
39
CV UV280
UV260
mS/cm
Figure 2a: Zoom in on elution of AAV 9 from SP PuraBead® P6HF
Table 2: AAV9 SP PuraBead® P6HF column performance data from ELISA
ELISA Results for AAV9 Total Capsids Loaded
1.14E+13
Total Capsids Bound
9.28E+12
% Total Capsids Bound
81.2
% Recovery from Bound
123.6
% Recovery from Load
100.3
% Strip Recovery from Load
2.9
% Mass Balance
122.1
Like all primary capture steps, capturing our AAV capsids with SP PuraBead® P6HF removes a significant number of contaminants including HCPs and HCDNA. However, it still needs further purification before the empty and full capsids can be separated. Removing impurities in flowthrough mode before the resolution step can improve the efficiency of that final empty full separation, and formulation or dilution must be carried out before the resolution step. To remove these impurities, HCPure™ host cell protein clearance resin was selected for its mixed mode chemistry and multiple binding interactions to be able to remove the most contaminants possible.
Technical note: A fit for purpose AAV purification method
5
Since AAV2 is challenging to purify, due to product losses that can occur on formulation and diafiltration, it was chosen as the serotype for this phase of the workflow. For this step, the AAV2 serotypes were purified with a commercially available affinity resin, however a significant amount of low molecular weight protein remained. HCPure™ was used in flowthrough mode, which does not require moving away from the neutral storage buffer after primary capture. An initial column run was performed to determine the amount of the AAV2 intermediate which could be loaded on to an 8 mL column. SDS-PAGE analysis of the flow through fractions (Figure 3) shows that low molecular weight contaminant proteins start to break through after the first 2.5 mL fraction.
Samples 1. 2. 3. 4. 5. 6. 7.
MW marker AAV2 Intermediate Load FT1 FT2 FT3 FT4 Eluate
Figure 3: SDS-PAGE showing break though of contaminants from the HCPure™ in flowthrough mode HCPure™ flow-through fractions which contained contaminant proteins were pooled and re-processed through the HCPure™ column loading 2.5 mL. The flow-through and PLW were collected in 2 mL fractions. The 5 flow-through fractions from the column run were run on SDS-PAGE and can be seen in Figure 4. The first 2 Flowthrough fractions show excellent purity and could be pooled to remove the troublesome contaminant proteins with only a small loss of AAV2 which could be further optimized to minimize any losses.
Technical note: A fit for purpose AAV purification method
6
Figure 4: SDS-PAGE showing removal of contaminants following HCPure™ in flowthrough mode ®®®The last step before having functional AAV that can be used as gene therapies is to separate the empty and full capsids. Without this important step, the potency and effectiveness of the therapy can be compromised. To separate the empty and full capsids, our Q PuraBead® P6HF adsorbent was used. Samples containing 5.9 e 13 AAV 8/ mL of adsorbent were loaded at low conductivity in BTP pH 9 onto a column containing Q PuraBead® and eluted using a linear gradient to 250 mM NaOAc. Using this method, 70% of the enriched AAV8 full capsids could be recovered.
20
100
18
90
16
80
14
70
12
60
10
50
8
40
6
30
4
20
2
10
% empty particles
conductivity mS/cm
% of Empty AAV 8 capsids Vs conductivity in elution pool from Q PuraBead
Conductivity % Empty particles
0
0 0
5
10
15
20
CV Figure 5: Empty AAV 8 particles (by HPLC determination) vs conductivity in elution gradient
Technical note: A fit for purpose AAV purification method
7
Conclusions As demonstrated, standard adsorbents from Astrea Bioseparations can be used to form a fit for purpose purification platform for multiple different AAV serotypes. By utilizing adsorbents, this platform can be scaled from initial discovery through to manufacturing and sized to fit the needs of the customer. Unlike other commercially available AAV affinity adsorbents, Astrea Bioseparations’ resins are caustic stable, allowing for multiple cleaning cycles without a decrease in performance. When looking for a cost effective, scalable platform for AAV purification, Astrea Bioseparations provides platform products to fit every need.
Materials and Methods Acidified to pH 4 and conductivity 15-20 mS/c by the addition of 100 Mm sodium acetate for 25% of the total volume if required added acetic acid. Clarified by centrifugation and 0.45 uM filtration. SP PuraBead® P6HF column Equilibration 25 mM NaOAc pH 4 100 mM NaCl 0.001% F68 pluronic and 1% Sucrose Elution 25 mM NaOAc pH 4 1M NaCl 0.001% F68 Pluronic and 1% Sucrose CIP 0.5M NaOH Elution pools buffer exchanged in PBS, 200 mM sodium chloride, 0.001% Pluronic F-68, pH 7.5 AAV 2 Flowthrough purification AAV 2 expressed in HEK 293 cells, clarified by centrifugation and 0.22 uM filtration capture on AAV X buffer exchanged in PBS, 200 mM sodium chloride, 0.001% Pluronic F-68, pH 7.5 HCPure™ column P6HF Equilibration PBS, 200 mM sodium chloride, 0.001% Pluronic F-68, pH 7.5 Strip 50mM Citrate pH 3 CIP 0.5M NaOH AAV 8 resolution AAV 8 expressed in HEK 293 cells, clarified by centrifugation and 0.22 uM filtration capture on AAV X buffer exchanged in PBS, 200 mM sodium chloride, 0.001% Pluronic F-68, pH 7.5 then diluted to ~1.2 mS/cm. Q PuraBead® P6HF column Equilibration buffer 20 mM BTP, 5 mM MgSO4, 0.001% Pluronic F-68 pH 9.5 Elution buffer 20 mM BTP, 5 mM MgSO4, 0.001% Pluronic F-68 250 mM NaOAc pH 9.5 Strip buffer 2M NaCl CIP 1M NaOH
Technical note: A fit for purpose AAV purification method
8
This product is covered by or for use under one or more patents: www.astreabioseparations.com/patents All trademarks, trade names, trade dress, product names and logos appearing on this document are the property of Astrea UK Services Ltd. Copyright © 2023 Astrea Bioseparations Ltd. All rights reserved.