Development of small-scale purification of recombinant adeno-associated virus, AAV

Adeno Associated Virus (AAV) has emerged as the main viral vector for gene therapy given it’s many desirable attributes, such as lack of pathogenicity and efficient infection (1). AAV vector consists of a protein shell protecting the viral genome (VG), approximately 4.7 kb of single stranded DNA. There are various AAVs in nature that vary in tropism, capsid components, and their ability to infect different cells. There are also several synthetic capsid constructs.

Several challenges can arise during purification of AAV. The individual properties of each AAV serotype require different purification approaches and optimization of conditions. During our trials, one challenge we faced was the variability in AAV serotype binding strength to affinity and anion exchange chromatography. We also had to decide how to remove the high amount of empty capsids left in harvest material, as they potentially affect efficacy of the final product.

We focused our studies on chromatography purification of AAV, exemplified with serotype 5, using affinity capture on HiTrap™ Capto™ AVB chromatography resin. The ligand of HiTrap Capto AVB is a single domain antibody fragment that specifically binds rAAV 1, 2, 3, and 5 serotypes. The elution condition of the affinity step was optimized for rAAV5.

HiTrap Capto AVB does not discriminate between full and empty capsid, instead the small difference in pI (5.9 for full capsids and 6.3 for empty capsids on average) can be utilized for separation in anion exchange resins. Capto Q resin is a strong ion exchanger with high capacity, dextran extenders, and shows good mass transfer properties. Conditions for separation of full and empty capsids of AAV5 were optimized on Capto Q chromatography resin packed into Tricorn™ 5/100 GL chromatography column.

The following steps were taken and are described in more detail below:

• Optimization of affinity capture of AAV
• Optimization full/empty capsid separation on anion exchange
• Screening procedure to identify two-step elution conditions

Optimization of affinity capture of AAV

Following concentration and buffer exchange, the rAAV5 sample was applied to HiTrap Capto AVB affinity chromatography column. The initial elution conditions were established for AAV2 (2). The influence of NaCl on the elution was investigated through a run with 50 mM citrate at pH 3.0 with gradient elution from 500 to 0 mM NaCl (Fig 1). In contrast to AAV2, rAAV5 eluted to some degree at high salt but the majority of the rAAV5 was eluted at the end of the gradient when the salt concentration was close to zero, which shows that rAAV5 binds more strongly to HiTrap Capto AVB and the elution conditions were further optimized.

Fig 1. The effect of elution pH and elution strength for the recovery of rAAV5

Column: HiTrap Capto AVB column, 1 mL
Sample: rAAV5 (TFF retentate)
Sample load: ~ 1 × 10¹³ VP/mL resin
Buffer A: 20 mM Tris pH 7.8 + 200 mM NaCl
Buffer B: 100 mM citrate pH 3.0, 500 to 0 mM NaCl gradient
Flow rate: 1 mL/min
System: ÄKTA pure™ 25
Detection: 280 nm

We investigated if there were differences between 100 mM glycine and 100 mM citrate buffer with and without the addition of 500 mM NaCl. A concentration of 100 mM glycine without NaCl led to a more efficient elution (Table 1). This may be because of the higher buffer capacity of glycine at lower pH, but the conductivity is also increased which may reduce the elution of rAAV5 which could be compensated by reducing the glycine concentration to 50 mM. Elution with 500 mM NaCl regardless of the buffer used resulted in the poor recovery of rAAV5. More details of the optimization can be found in reference 3.

Table 1. Recovery (%) of AAV5 particles in screening of buffer and salt conditions for elution

The following final elution conditions: 50 mM glycine and pH 2.7, resulted in good VP recoveries (ranging from 60% to 90%) (Fig 2 A) and high purity (Fig 2 B). The binding capacity was ~ 2–5 ×10¹⁴ VP/mL.

Column: HiTrap Capto AVB column, 1 mL
Sample: rAAV5 (TFF retentate)
Buffer A: 20 mM Tris pH 7.8 + 200 mM NaCl
Buffer B: 50 mM Glycine pH 2.7
Flow rate: 1 mL/min
System: ÄKTA™ pure 25
Detection: 280 nm

Fig 2. (A) Affinity capture of rAAV5 using 50 mM Glycine pH 2.7 for elution on HiTrap Capto AVB 1 mL. (B) Purity analysis of HiTrap Capto AVB eluates of rAAV5 using multiplex fluorescence Western Blot. HCP was detected by Cy5 pre-labeling and rAAV5 proteins were targeted by primary anti-AAV5 antibody and Cy3 labeled secondary antibody.

Optimization full/empty capsid separation on anion exchange

Critical buffer conditions for AAV binding and separation of full and empty capsids

During our studies we found that buffer conditions are critical for binding and efficient separation of full and empty AAV capsids. To ensure binding of AAV to the Capto Q resin it is critical to reduce the conductivity to 1-3 mS/cm either by dilution in buffer A or by buffer exchange.

It has been shown that MgCl₂ is critical for the separation of full and empty AAV capsids on Capto Q ImpRes (3). The mechanism of how MgCl₂ enhances the separation is unclear, but it may be due to the differential binding of Mg²⁺ ion between full and empty capsids, which affects binding to the anion exchange ligand. Increasing the MgCl₂ concentration causes increased elution of empty capsids, but once the concentration is too high full capsids start to elute. In the final protocol presented below, 2 mM proved to be enough for elution complete separation of full and empty capsids on Capto Q resin.

Choice of anion exchange resin

Due to the differences in anion exchanger properties, the choice of anion exchanger resin is important for separation of empty and full capsids. Capto Q is a resin with dextran extenders and showed improved separation of full and empty capsids in linear gradient elution compared to Capto Q ImpRes with no extenders (Fig 3). Capto Q resin was used for further optimization.

Fig 3. Separation of AAV full and empty capsids using Capto Q ImpRes (A) Capto Q resin with dextran surface extenders (B) in the presence of 2 mM MgCl₂.

Step elution versus linear gradient elution

Linear gradient elution is often the first choice in lab scale separations while step elution is used in bioprocessing. Separation of full and empty capsids with a linear NaCl gradient resulted in overlapping of full and empty capsids as seen by the ratio of UV280/A260 (Fig 4A). Steps with a pulse in the conductivity enhanced the separation and resulted in complete separation of full and empty capsids (Fig 4B).

Fig 4. Separation of rAAV5 full and empty capsids using Capto Q resin with linear gradient elution (A) and a step elution (B) with high constant concentration of MgCl₂ (18 mM) and NaCl as elution salt.

Screening procedure to identify two-step elution conditions

Using a pre-screening procedure of small steps with increments of 5% B with 3 CV, the conditions for a 2-step gradient protocol were determined. For AAV5 empty capsids, without any full-capsid leakage, occurred at 35%B (Fig 5A). For the study to be successful it was important to run blank runs to discriminate artefact peaks, we used a 10 mm UV cell for sensitivity and to bypass the mixer to get sharp increments of the steps.

The final conditions and separation of full and empty capsids of AAV5 is shown in Figure 5B. The first step was determined to 35% B for 20 CV by prescreening and second step 100% B for 5 CV. The long first step maximized the removal of empty capsids. The full and empty capsids were completely separated with 100% full capsids in the second peak and the VG recovery was 80% (Table 1).

Pre-screening protocol
Column: Capto Q resin packed in Tricorn 5/100, 2 mL column
Sample load: ~ 1 × 10¹² VP/mL resin
Buffer A: 20 mM BTP, pH 9.0, 2 mM MgCl₂
Buffer B: 20 mM BTP, pH 9.0, 2 mM MgCl₂, 250 mM Na acetate
Equilibration: Buffer A, 5 CV
Wash: Buffer A, 5 CV
Gradient: Step elution, 5% increments, 3 CV each
Flow rate: 2 mL/min
System: ÄKTA pure™ 25

2 step protocol
Column: Capto Q Tricorn 5/100, 2 mL column
Sample load: (1 × 10¹² VP/mL resin)
Buffer A: 20 mM BTP pH 9.0, 2 mM MgCl₂
Buffer B: 20 mM BTP pH 9.0, 2 mM MgCl₂, 250 mM Na acetate
Equilibration: Buffer A, 5 CV
Wash: Buffer A, 5 CV
Gradient: Two-step elution for empty and full
rAAV5 capsids: Step 1 35% buffer B, 20 CV Step 2 100% buffer B, 5 CV

Fig 5. (A) Prescreening using 5% incremental steps with buffer B and Capto Q resin with the goal to select two step elution conditions for AAV5. During prescreening, AAV5 was divided into three peaks. Judged by the UV 260:280 ratios peak 1 contained the empty capsids, peak 2 had some full capsids mixed with empty, and peak 3 contained the full capsids. (B) For the final AAV5 2 step protocol, 20 CV of 35% buffer B was used in step one (corresponding to peak 1 in pre-screening), followed by 5 CV of 100% buffer B in step two.

Table 2. ELISA and qPCR analysis results from Capto Q resin AAV5 elution peak fractions

The procedure to determine step conditions to elute empty capsids was used to optimize elution of other AAV serotypes. During this pre-screening procedure to determine elution steps, we found that the same buffer, 20 mM BTP, pH 9, 2 mM MgCl2 with 250 mM sodium acetate, could be used for serotypes AAV2, AAV8, AAV9, and possibly others.

Conclusions

HiTrap Capto AVB resin proved to have efficient affinity capture of rAAV5. The optimal elution conditions of the affinity step rAAV5 required low pH and no NaCl for good recovery.

Using Capto Q resin with dextran surface extenders, MgCl2 and step elution allowed for complete separation of full and empty capsids and 80% VG recovery of rAAV5.

Pre-screening to identify isocratic two-step conditions (%B) using a single buffer for elution of several serotypes, 20 mM BTP, pH 9, 2 mM MgCl₂ with 250 mM sodium acetate.

Both capture and polishing protocol are suitable for scale-up.

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