Improved AAV Downstream Processing - Cytiva (2023)

Scalable, cost-effective and robust filtration and chromatography-based processes are required for the purification of adeno-associated viruses (AAV) in bioprocess engineering. In this article, we show how we developed an efficient purification process for recombinant AAV (rAAV). The chromatographic purification described includes affinity capture to maximize recovery with efficient removal of impurities. The optimal elution conditions of the affinity step were different for rAAV2 and rAAV5 serotypes. Recovery of rAAV5 was significantly improved by removing NaCl from the low pH elution buffer. We used anion exchange polishing to reduce empty capsids and maximize enrichment of complete rAAV capsids. In the polishing step, we developed an anion exchange protocol for rAAV5 in which empty capsids with high MgCl content were eluted first₂Concentration and complete capsids eluted when a NaCl gradient was applied. Total capsid enrichment was 40% to 65% with viral genome (VG) recovery of 60% to 80%, depending on the clustering strategy used.

introduction

Key factors for an effective AAV purification process are high overall yields and maximum reduction of impurities from the empty capsid product. After harvesting by detergent cell lysis and DNA degradation by nuclease treatment, the feed is clarified, followed by a concentration and buffer exchange step that reduces the volume to be prepared for the capture step. Affinity capture is an efficient way to specifically bind and concentrate the target while achieving high removal of contaminants.

In this study, we adapted an affinity capture step for rAAV2 and rAAV5, followed by a polishing step with rAAV5 to improve the yield of complete rAAV capsids (Fig. 1). Check out these articles to see how the upstream cell culture steps were performedupstream process developmentand rAAV production in HEK293 suspension cells using disposable bioreactors.

Capto™ AVB is a resin for affinity chromatographydeveloped for the purification of rAAV.O-binders make Capto™ AVB resinis a single-domain antibody fragment that specifically binds rAAV serotypes 1, 2, 3 and 5, and in addition genetically engineered recombinant variants dependent on the presence of the AVB epitope-binding region. The affinity ligand does not discriminate between complete rAAV capsid (containing the VG and gene of interest) and empty rAAV capsids (product-related contamination lacking viral genomes).

The proportion of complete capsid product in the harvested crop is generally well below 50%. High levels of empty capsids reduce potency and contaminate the product and must therefore be reduced. In addition to empty capsids, partially filled capsids with truncated genes or process-related impurities can also be packaged into the capsid during upstream production. Traditionally, CsCl gradient ultracentrifugation, which separates based on density differences, has been used to purify whole rAAV capsids on a small scale. However, for scalable production, methods based on chromatography are preferred.

Complete capsids have a lower isoelectric point (pI) than empty capsids with approximately pI 5.9 versus 6.3, and the charge difference can be used to separate them by ion exchange chromatography using salt or pH elution. In addition, depending on the serotype, other properties of the rAAV capsid can influence binding behavior. In cation exchange, using a lower pH buffer below the average pI of the capsid, the net charge on the capsid is positive. Full capsids therefore elute first in a salt gradient since they are less positively charged compared to empty capsids. If anion exchange is used instead, the net charge of the capsid is negative at a buffer pH greater than the average pI of the capsid. In this case, empty capsids elute first in a salt gradient because they are less negatively charged compared to full capsids (Fig. 2). Additives such as detergents, carbohydrates and metal ions can improve separation.

Achieving the accuracy of full and empty rAAV capsid analysis is challenging. However, it is essential to optimize the polishing step. The determination of percent complete capsids with qPCR:ELISA relies on two independent assays, both of which have significant inherent variability, leading to uncertainties. We recommend using at least one additional orthogonal analysis method for percent full capsids to confirm the results and increase accuracy.

In this study, we used the UV 260:280 ratio in the chromatograms to indicate the percentage of full capsids (approximately 0.6 to 0.7 for empty capsids and 1.1 to 1.3 for full capsids). To detect VG, we also determined the proportion of full and empty capsids in the fractions by quantitative polymerase chain reaction (qPCR). We used ELISA for viral particle (VP) detection to determine total capsid percentage and VG recovery.

Illustration 1.Overview of the purification steps of rAAV2 and rAAV5.

Figure 2.Principles of separation of full and empty capsids by ion exchange.

results and discussion

Starting materials for all experiments were obtained from rAAV production by triple plasmid transfection into HEK-293T cells, followed by harvest using detergent lysis (Tween™ 20), clarification and concentration or buffer exchange by tangential flow filtration using hollow fibers (HF). for details,verMaterials and Methods and Figure 1.

Optimization of the affinity capture step

Concentrated and buffer exchanged rAAV2, rAAV5 and rAAV8 samples were applied to Capto™ AVB affinity chromatography and eluted in 50mM citrate at pH 3.0 with gradient elution from 500 to 0mM NaCl (Fig. 3). rAAV2 elutes completely in 500 mM NaCl. rAAV5 eluted with some degree of high salt content, but most of the rAAV5 eluted at the end of the gradient when the salt concentration was near zero, indicating that rAAV5 binds more tightly to Capto™ AVB. It was clear that the recovery of rAAV5 (but not rAAV2) was improved as the NaCl concentration in the buffer was reduced during elution at pH 3.0. The results in Figure 3 show that rAAV8 binds to Capto™ AVB at very low levels.

Split:Capture™ AVB HiTrap™ Column, 1mL
Probe:rAAV2, 5 and 8 (TFF preserved)
Sample fee:~ 1 × 10¹³VP/ml resin
Initial Buffer:Puffer 20 mM Tris, 200 mM NaCl, pH 7,8
Elutionspuffer:Citrate 100mM pH 3.0, gradient NaCl 500 and 0mM
flow quotient:1 ml/min (150 cm/h)
System:ÄKTA™ pure 25
Recognition:280 nautical miles

Figure 3.The effect of elution pH and elution strength on recovery of rAAV2, 5 and 8.

Most AAV5 did not elute using the AAV2 protocol (elution in 50mM citrate pH 3.5, 500mM NaCl, 500mM arginine) as elution in the presence of NaCl reduced the yield as seen in Figure 3 . Therefore, we further optimized the conditions for rAAV5, since this serotype was the focus of the process to be scaled (verAlsoscalable AAV production process).

We examined whether there were differences between 100 mM glycine and 100 mM citrate buffer with and without the addition of 500 mM NaCl (Fig. 4). A concentration of 100 mM glycine without NaCl resulted in the most efficient elution at pH 3.0. This may be due to glycine's greater buffering capacity at lower pH, but conductivity is also increased, which may reduce rAAV5 elution (Fig. 3). To compensate for this, we chose these final elution conditions: 50 mM glycine and pH 2.7, which resulted in good PV recoveries (ranging from 60% to 90%). Elution with 500 mM NaCl resulted in poor recovery of rAAV5 regardless of the buffer used, even when the pH was reduced to 2.1 (Figure 4). Removal of arginine did not adversely affect the yields (analyzed by ELISA and transduction assay) of eluted AAV5 (data not shown).

Split:Capture™ AVB HiTrap™ Column, 1mL
Probe:rAAV5 (retentado TFF)
Sample fee:~ 1 × 10¹³VP/ml resin
Initial Buffer:Puffer 20 mM Tris, 200 mM NaCl, pH 7,8 bis 8,0
Elutionspuffer:100 mM glycine or 100 mM citrate buffer with and without 500 mM NaCl
flow quotient:1 ml/min (150 cm/h)
System:GENUINE™ 25 reins
Recognition:280 nautical miles

Figure 4.Comparison between glycine and citrate buffers and elution pH for Capto™ AVB with or without NaCl. Percent recovery of total rAAV5 particles is indicated.

Total dynamic binding capacity (DBC) experiments were not performed for rAAV2, but based on four high-load experiments (ranging from 1.0 × 10¹⁴for 1.8×10¹⁴VP/ml resin) with an average VP recovery of 83%. The highest load resulted in 100% PV recovery, so the DBC for rAAV2 was estimated to be over 2 × 10¹⁴VP/ml resin.

Next, we needed to determine DBC for rAAV5 using a classic Breakthrough Experiment (BT) approach, where a 1mL Capto™ AVB HiTrap™ prepacked column was loaded until rAAV5 BT appeared (Fig. 5). The applied material contained 2.1 × 10¹²VP/mL when analyzed by ELISA and the 10% BT point equals 3.8 × 10¹⁴VP/ml Capto™ AVB resin. Maximum capacity was used at 80% load to achieve 10% BT level. This corresponded to a DBC of 3.1 × 10¹⁴rAAV5 particles/ml resin.

Split:Capture™ AVB HiTrap™ Column, 1mL
Probe:rAAV5
Sample fee:1,0 × 10¹⁴for 1.8×10¹⁴VP/ml resin
Initial Buffer:Puffer 20 mM Tris, 200 mM NaCl, pH 7,8
Balance Cap:Puffer 20 mM Tris, 200 mM NaCl, pH 7,8
flow quotient:1 ml (150 cm/h)
System:GENUINE™ 25 reins
Recognition:280 nautical miles

Figure 5.Breakthrough experiment using a 1mL Capto™ AVB HiTrap™ chromatography column for the determination of DBC for rAAV5.

For rAAV2, a protocol (Figure 6) eluting in 50mM citrate pH 3.5, 500mM NaCl, 500mM arginine was considered optimal, resulting in a high purity removal and an average recovery of 90%. No further analysis was performed for rAAV2.

A typical Capto™ AVB (rAAV2) chromatogram and final detailed protocols are shown in Figure 6. The high purity of the eluates for rAAV2 and rAAV5 can be seen in Figure 7. Results are from a multiplex fluorescent Western blot for the detection of total protein or host cell protein (HCP) with prelabeled Cy™5 (sensitivity similar to silver stain) and viral proteins (VP1, VP2 and VP3) detected by a primary antibody and a secondary antibody labeled with Cy™3 on the same blot. Cy5™ HCP signals could not be detected in the eluates, indicating good purity after Capto™ AVB chromatography.

No virus bands were detected in the flow-through fractions, while clearly visible virus bands were detected in the eluate, confirming the ELISA results of approximately 100-200 times the rAAV concentration in the affinity capture step. Transmission electron microscopy (TEM) imaging of the rAAV2 eluate also confirms high purity and unique intact rAAV2 particles (Fig. 7). The absence of salt in the elution buffer did not cause aggregation of rAAV5 as judged by analytical size exclusion chromatography with Superose™ 6 Boost (<1% data not shown).

Split:Capto™ AVB, HiTrap™ Column, 1mL
Probe:rAAV2 is 5
Sample fee:~170 to 215 hp (retained TFF, 1 to 3×10¹⁴VP/ml Harza)
Initial Buffer:Tris 20 mM pH 7.8 + NaCl 200 mM, 10 CV
Elutionspuffer:rAAV2: Citrate 50 mM, pH 3.5, NaCl 500 mM, Arginine 500 mM, 4 CV
rAAV5: Glicina 50 mM pH 2,7, 5 CV
flow quotient:1ml/min or 150cm/hr
System:GENUINE™ 25 reins
Recognition:260 bis 280 Nanometer

Figure 6.Chromatogram of rAAV2 purification in Capto™ AVB.

Figure 7.Analysis of the purity of Capto™AVB in (A) rAAV2 and (B) in rAAV5 eluates by multiplex fluorescence western blot and (C) TEM image (MiniTEM™ by ViroNova) with negative staining. HCP was detected by pre-labeling with Cy™5 and rAAV proteins were targeted by anti-AAV2 or AAV5 primary antibodies and Cy™3-labeled secondary antibodies.

Optimization of the polishing step

The goal of the polishing step is to reduce as much as possible the contamination associated with the empty capsid product. Affinity-purified rAAV5 eluates were neutralized and diluted approximately 10-fold in the equilibration buffer used in the experiment to increase the pH and decrease the conductivity to between 1 and 3 mS/cm. We evaluated different resins for the separation performance of rAAV5 full and empty capsid (Table 1) and found that Capto™ Q ImpRes and Capto™ Q anion exchange resins are the most promising candidates.

We selected Capto™ Q ImpRes to further optimize the operating conditions and the evaluation results are presented in Table 2.

Table 1.Screening of polishing resins to separate solids and voids

*Using screening conditions

Table 2.Conditions and parameters tested with Capto™ Q ImpRes for full and blank deposition

The affinity purified rAAV5 probe was loaded onto Capto™ Q ImpRes equilibrated in 20mM Bis-Tris-Propane (BTP), pH 9.0. After a wash step, the same buffer with 0, 5, 17 or 20 mM MgCl₂was applied followed by a shallow gradient from NaCl (20 CV) to 1 M NaCl containing the same constant concentration of MgCl₂. All buffers contained additives (1% sucrose + 0.1% poloxamer 188).

A critical step was the use of an isocratic MgCl₂Washing step to elute empty capsids before a NaCl gradient to elute full capsids. increase in MgCl₂the concentration causes an increasing proportion of the empty capsids to elute before the NaCl gradient (Fig. 8 A to D). When the shallow NaCl gradient was applied, the complete capsids eluted along with some empty capsids (Figure 8). A concentration of 20 mM MgCl₂is too high since some full capsids also elute along with empty capsids, resulting in decreased VG recovery (Figure 8D). the MgCl₂The concentration must be adjusted to maximize the removal of empty capsids while leaving full capsids for elution on the NaCl gradient. In this experiment, 17 mM MgCl₂(Fig. 8C) Improved separation compared to 20 mM MgCl₂(Fig. 8D). A factor affecting the separation can be the binder density of the anion exchange resin.

From the qPCR and ELISA results in Figure 8 we see that high MgCl₂Concentration can improve the separation of full and empty particles and elute complete capsids at the beginning of the NaCl gradient.

The mechanism of how MgCl₂increases the separation is unclear but may be due to different Mg binding²⁺ion between the full and empty capsids, which in turn affects binding to the anion exchange ligand.

Split:Capto™ Q ImpRes HiScreen™-Säule, 4.7 ml
Probe:rAAV5 (Capto™ AVB-Eluate, 10-fold dilution in Buffer A)
Sample fee:20 CV
Puffer A:BTP or 20mM Tris pH 9.0 +/- additives (1% sucrose + 0.1% poloxamer 188)
Puffer B:20 mM BTP or Tris pH 9.0 + 1 M NaCl + (0 to 20 mM) MgCl₂+/- additives (1% sucrose + 0.1% poloxamer 188) Flow rate: 150 cm/h
Gradient:1ml/Min
System:GENUINE™ 25 reins
Recognition:260 bis 280 Nanometer

Figure 8.The effect of MgCl₂Concentration in separating full and empty capsids. Recovery of VG (using qPCR) and VP (using ELISA) is shown in yellow and dark blue histograms, respectively.

The separation of full and empty particles results in overlapping peaks, and there is a tradeoff between percent full capsids and VG recovery depending on how you group the fractions. We used a procedure to help us define the clustering strategy (Figure 9). We aimed for more than 50% complete capsids and more than 40% VG yield (Fig. 9).

Figure 9.Procedures to support the pooling strategy. The steps involved were: (1) classification of fractions based on the proportion of full and empty capsids; (2) calculation of the proportion of full and empty capsids and VG recovery in theoretical pools, including an increasing number of fractions in the pool; (3) Plot the calculated values ​​for each pool and compare them to the defined criteria (Design Space, see theSection definitions in this articleto learn more about the design space).

The elution fractions were analyzed by qPCR and ELISA and classified based on the percentage of full and empty capsids. Then the full/empty ratio and VG recovery were calculated for the increasing number of fractions included in the pool. Our design space criteria were product and process criteria with more than 50% complete capsids and 40% VG recovery defined by the final clustering strategy (Fig. 9). The final protocol to separate rAAV5 full and empty capsids using Capto™ Q ImpRes resulted in a percentage of full capsid between 40% and 65%. Depending on the clustering strategy, the VG recovery was between 60% and 80% (Fig. 10). We determine the capacity from approximately 1 to 3 × 10¹³VP/ml resin (data not shown).

Split:Capto™ Q ImpRes HiScreen™-Säule, 4.7 ml
Probe:rAAV5, Capto™ AVB HiTrap™, 1 ml Elute 10-fold in Buffer A
Sample fee:5 CV (2 × 10¹²PV/ml)
Puffer A:20mM Tris pH 9.0 + 18mM MgCl2 + additives (1% sucrose and 0.1% poloxamer 188)
(Wash 1: Buffer A, 4 CV)
Puffer B:Puffer A + 1 M NaCl
flow rate:150 cm/h or residence time 2.4 min
Gradient:Linear slope (0% to 20% B), 15 CV. elution maintained at 0% Buffer B, 5 CV; Elution maintained at 20% Buffer B, 5 CV
System:GENUINE™ 25 reins
Recognition:260 bis 280 Nanometer

Figure 10.The definitive streamlined protocol for separating full and empty rAAV5 capsids using Capto™ Q ImpRes resin

Gradient elution allows for small variations in conditions with the flexibility to pool the right fractions. However, an isocratic step-elution protocol can simplify the process in large-scale GMP production. We evaluated options for stepwise elution of rAAV5 using Capto™ Q ImpRes (Fig. 11) and Capto™ Q (Fig. 12) resins. Small isocratic elutions can be optimized by varying pH, MgCl₂and/or NaCl to achieve complete and empty capsid separation. The results show a good separation between full and empty capsids. Capto™ Q resin contains dextran extenders that may improve separation for other serotypes as well (work in progress).

Split:Capto™ Q ImpRes HiScreen™-Säule, 4.7 ml
Probe:rAAV5
Sample fee:5 CV
Puffer A:20mM Tris pH 9.0 + additives (1% sucrose and 0.1% poloxamer 188)
Puffer B:20 mM Tris pH 9,0 + 10 mM MgCl₂and NaCl 1 M, pH 9.0
flow rate:150 cm/h, residence time 2.4 min
Gradient:11 % bis 100 % Puffer B
System:AKTA Pure™ 25
Recognition:280 bis 260 Nanometer

Figure 11.Separation of full and empty rAAV5 capsids using Capto™Q ImpRes resin and isocratic step elution (qPCR VG recovery is shown in dark blue histograms).

Split:Coluna Capture™ Q HiScreen™, 4,7 ml
Probe:rAAV5
Sample fee:5 CV
Puffer A:20 mM BTP pH 9,5 + 2 mM MgCl₂+ additives (1% sucrose + 0.1% poloxamer 188
Puffer B:20 mM BTP pH 9,5 + 2 mM MgCl₂+ additives (1% sucrose + 0.1% poloxamer188) + 1 M NaCl
flow rate:150 cm/h, residence time 2.4 min
Gradient:8 % bis 100 % Puffer B
System:GENUINE™ 25 reins
Recognition:280 nautical miles

Figure 12.Separation of full and empty rAAV5 capsids with Capto™ Q resin and isocratic step elution.

Conclusions

• Capto™ AVB resin enabled efficient capture by affinity chromatography.
• The optimal Capto™ AVB elution conditions depended on the rAAV serotype used.
• Anion exchange polishing steps with Capto™ Q ImpRes or Capto™ Q resins can be used to enrich full AAV capsids. MgCl₂Concentration is crucial in this step.
• The clustering strategy is critical and a trade-off between the proportion of full and empty capsids and the yield of full capsids must be considered.
• The protocol can be converted to an isocratic step elution and is suitable for large-scale clinical-grade production.

recognition

We thank iBET (Instituto de Biologia Experimental e Tecnológica), Oeiras, Portugal for affinity capture discussions and good collaborations.

CY24378-11Abr22-AN
TR29791259, 29792294 and 29646699