Full adeno associated virus (AAV) capsid enrichment using Mustang™ Q membrane

Abstract

This document describes a method for the enrichment of full AAV capsids that contain a DNA payload. Removal of empty capsids that do not contain DNA is achieved through elution from Mustang™ Q membrane. Instead of traditional linear gradients, we show a novel method employing small (~ 1 mS/cm) conductivity steps¹. The small steps result in a series of discrete elution peaks, simplifying assessment of the purification and thereby accelerating process development. Employing Mustang Q membrane along with this step elution strategy is shown to provide ~ 5-fold enrichment in full capsids for AAV serotypes 5, 8, and 9 which makes it suitable as a platform process step.

¹ Patent pending

Introduction

AAV is well suited as a gene therapy vector as it is non-pathogenic and has an acceptable safety profile. Production of AAV particles is dependent upon enzymatic insertion of the ssDNA encoding the gene of interest (GOI) into a stochastically assembled capsid shell. However, the efficiency for producing functional AAV is highly variable resulting in low quantities of full AAV (capsid containing gene of interest) relative to empty AAV (capsid containing no DNA). Given only the full AAV particles can deliver the therapeutic agent, the empty capsids can be viewed as a process impurity and removing them presents a challenge to the manufacturing process (1).

Here we describe a novel method for enrichment of full capsids using Mustang Q membrane chromatography. Mustang membrane chromatography units are ready-to-use chromatography membrane adsorbers with bed volumes ranging from approximately 1 mL to 5 L. They are designed for capturing targeted molecules or for the removal of impurities in bioprocesses, from laboratory to manufacturing scale. The separation takes place at the elution step and is achieved with small conductivity steps of around 1 mS/cm. This elution strategy results in a series of discrete elution peaks. The discrete peaks can accelerate process development of full capsid enrichment as they facilitate rapid assessment of the purification. Initial assessment of the purification can be performed via ultraviolet (UV) relative absorbance at 260 nm, primarily from DNA, and at 280 nm, primarily from protein. When the 280 nm signal is much larger than the 260 nm signal the capsids are predominately empty and when 260 nm signal is higher than 280 nm the capsids are predominately full. Further assessment of the purification can be performed via droplet digital polymerase chain reaction (ddPCR) which can quantitate the virus genome copies (GC) and capsid enzyme-linked immunosorbent assay (ELISA) which can quantitate the number of intact capsids. The ratio of these two measurements—(ddPCR/ELISA) × 100—can give a crude assessment of the full percentage of capsids.

The details below outline the general methods and buffer compositions needed to achieve full AAV capsid enrichment of serotypes 5, 8, and 9. For all three serotypes tested, we determined greater than 50% full capsid post Mustang Q membrane. We also demonstrated that this process is scalable to 5 mL Mustang Q capsules.

Results

The 1 mS/cm elution step method was used to develop purification procedures for AAV 5, 8, and 9. The chromatograms are shown below: AAV8 (Fig 1A), AAV9 (Fig 1B), AAV5 on 0.86 mL Mustang Q XT membrane in Acrodisc™ format (Fig 1C), AAV5 on 5 mL Mustang Q XT capsule (Fig 1D).

Fig 1. Separation of empty and full AAV capsids by Mustang Q anion exchange membrane chromatography utilizing ~ 1 mS/cm conductivity elution steps. The purification can be assessed by UV absorbance at 260 nm and 280 nm along with an ELISA to measure total capsids and a ddPCR assay to measure encapsulate genome content (full capsids).

With all the serotypes, the chromatograms have distinctive shared features. The 1 mS/cm conductivity steps are highly reproducible and for each serotype we have a series of elution peaks. In every case, the first one or two elution peaks look predominantly empty, the ratio of UV absorbance 260 nm:280 nm is relatively low. Following on from the empty peaks we see additional eluates. Peaks 2 and 3 for AAV8 (Fig 1A) and peaks 3 and 4 for serotypes 5 (Fig 1C) and 9 (Fig 1B) appear predominately full with the ratio of UV absorbance 260 nm:280 nm relatively high. Additional peaks elute at higher conductivities, though these typically do not contain much genome content. We speculate that these peaks might be aggregated or damaged AAV capsids. The 260 nm:280 nm absorbance data is corroborated by ddPCR:Capsid ELISA ratio which can give an estimate of the percentage of full capsids. For AAV8 and AAV5 the ddPCR:Capsid ELISA ratio shows that we can get a 4- to 5-fold enrichment of full capsids with Mustang Q membrane. With serotypes 8 and 9 where we have relatively high levels of full capsids initially (20% and 50%), Mustang Q membrane combined with the 1 mS/cm elution steps can result in eluates that are ~ 100% full. For AAV5 we show the purification is scalable from the 0.86 mL Mustang Q Acrodisc capsule(Fig 1C) to the 5 mL Mustang Q capsule (Fig 1D). A similar chromatographic profile can be seen with the two devices upon scale up.

Methods

Experiments were performed on an ÄKTA™ avant 25 chromatography system or ÄKTA avant 150 chromatography system. The mixer and inline filter were removed to minimize hold-up volume, increasing the sharpness in transition of the conductivity steps. The conditions for the chromatography processes are shown in Table 1. The same overall procedure was performed with all three serotypes. Through process development it was discovered that elution salt and excipients could impact the separation of full capsids. For AAV5 NaCl was determined to be the best elution salt, but for AAV8 and AAV9 sodium acetate gave the best resolution of empty and full capsids. For AAV8, addition of 5% glycerol and 0.01% Pluronic F-68 improved the yield and separation of full capsids.

The salt steps were generated using appropriate pump flow ratios of A and B pump of the ÄKTA avant. To simplify this method, B buffer was produced at 20 mS/cm and A buffer was produced without additional salt to minimize conductivity. This way each additional 5% increment in the speed of the B pump, with concomitant 5% slowing of the A pump, resulted in an approximately 1 mS/cm conductivity increase. As an alternative, a series of buffers can be formulated directly at the correct conductivity, but this was found to be more time consuming and challenging to create a series of reproducible steps.

Table 1. Operating conditions for Mustang Q anion exchange membrane chromatography. The method generates a step conductivity gradient, with each step being of ~ 1 mS/cm. Note that AAV8 purification was improved with the inclusion of 5% glycerol and 0.01% Pluronic F-68. Elution salt also impacts purification for all three serotypes. For AAV5, Buffer B is set to 20 mS/cm using ~ 200 mM NaCl, while for serotypes 8 and 9 ~ 250 mM sodium acetate is the preferred elution salt.

¹ Flow rate for all steps = 10 membrane volumes (MV)/min

Conclusion

Mustang Q membrane has the capability to enrich for full AAV5, 8, and 9 capsids. As such, it is suitable as part of an AAV platform purification process. By using the 1 mS/cm step elution strategy alongside common UV absorbance measurements (260 nm/280 nm), the separation of empty and full capsids is observable in real time. Confirmation using analytics such as ddPCR for genomic content and ELISA for capsid count shows Mustang Q membrane capsules can enrich capsids ~ 5-fold and can result in eluates that are ~ 100% full.

Mustang Q membrane can be operated at 10 membrane volumes per minute (MV/min). This rapid operation is beneficial for two reasons. Loading onto anion exchange after affinity chromatography often requires a large dilution to increase pH and lower conductivity. Mustang Q membrane is ideal for this as it has low backpressure at high flow rates, so large dilutions of 20-fold can still typically be loaded in under 10 min. Consequently, a purification process can be performed in less than 30 min. This rapid chromatography performance along with the ability to assess the purification visually allows for intensification of process development and the ability to perform optimization for the different serotypes. For AAV8, we found that improved separation of empty and full capsids could be achieved via a change in elution salt to sodium acetate and the inclusion of 5% glycerol and 0.01% Pluronic F-68 to the load, wash, and elution buffers. For AAV9, simply switching to sodium acetate for the elution salt proved effective.

The step elution method may be an approach that is scaled up further or could be used as a process development tool to determine the conductivity necessary for single elution steps of both empty and full capsids.

References

• US Food and Drug Administration (FDA) Cellular, Tissue, and Gene Therapies Advisory Committee (CTGTAC) Meeting #70. Briefing Document Toxicity Risks of Adeno-associated Virus (AAV) Vectors for Gene Therapy (GT) September 2-3, 2021.

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