Exploring a New Enzymatic Tool for AAV Production

Balancing Optimal Nuclease Activity with Effective DNA Digestion

The utilization of enzymatic tools for the processing of genetically engineered materials is certainly not a new concept. Among other activities, these enzymes are used to purify, modify, activate, and ligate the end product. Like most other strategies, there are some biochemical compromises that must be made when using certain enzymatic tools.

The concentration of salt, cofactors, detergents, reducing agents, incubation time, and temperature must all be controlled. Often, the trade-off is to accept a reduction in yield of the final product in favor of better control of the bioprocess workflow.

Frequently, scientists may refer to their “molecular toolbox,” which is usually comprised of a small array of enzymes that they employ for a variety of applications. However, once that molecular toolbox is full, it may be cumbersome to manage. So, when bioprocessing engineers are faced with a unique challenge, their conflict becomes whether they should reach for familiar biochemical tools, craft their own application-specific tools, or seek out an existing, yet unorthodox, approach?

Over the past several years, it seems that engineers always tackle problems in the aforementioned order. Only as a last resort will they explore the use of a new, unvetted tool. After all, a new tool requires familiarity to master and it must be approved for use by the appropriate regulatory agencies.

As viral therapy technologies have advanced, the need to develop reliable production strategies has grown. For example, many promising gene-therapy strategies use adeno-associated virus (AAV) as a vector. However, a challenge in the production of AAVs is residual DNA released from the host cell. FDA guidelines call for residual DNA to be reduced below 10 ng per dose. An effective strategy for reducing residual DNA is to use a nuclease.

However, some nucleases do not tolerate certain bioprocess conditions well, especially elevated salt concentrations. In the case of AAV, the capsids are particularly “sticky” and therefore, bind free DNA readily. To manage this problem, higher salt conditions can be implemented to dissociate DNA from the AAV capsids. Consequently, some engineers have been tackling the issues associated with balancing optimal nuclease activity with effective DNA digestion.

Some companies are looking at entirely new environments for extremophiles of all sorts. Traditionally, in enzymology, research has centered about thermophilic organisms; however, today enzymes are also derived from psychrophilic (cold-adapted) and halophilic (salt-tolerant) organisms, offering scientists new opportunities to modify and improve their processes and procedures.

Some halophilic enzymes have shown tolerance above 500 mM sodium chloride. In the case of AAV processing, this offers a tremendous opportunity to improve residual DNA clearance. Instead of overloading the process with traditional nucleases to overcome salt inhibition, more efficient clearance can occur by using enzymes that are better suited for the conditions of the process.

Beyond efficiency, there is also the notion of cost. Frequently, these “unique” enzymes that fall outside the traditional enzyme toolbox will be somewhat more expensive than their counterparts. In some cases, this may deter a process engineer from considering an alternative enzymatic strategy. While many alternative enzymes may not meet current regulatory and quality requirements, these products should still be evaluated in some way. Achievement of bioprocess ready enzymes is possible through collaboration with the vendor, directing that vendor on the requirements for utilization.

Enhanced purity specifications, endotoxin testing, and assurance of animal-free origin are all defining characteristics of a bioprocess-grade enzyme. Yet, these specifications are not normally required for traditional molecular biology applications like PCR, NGS, or sample prep. Still, many vendors of these enzymes are already capable of achieving specifications similar to those of bioprocessing industry-approved enzymes.

Here we describe a salt-tolerant nuclease that is being evaluated as an alternative to traditional nucleases for residual DNA clearance. Like most other general nucleases, the described enzyme digests most forms of nucleic acids (DNA, RNA, single-stranded, double-stranded, circular, etc.) into approximately 3–8 base pair fragments.

The hallmark of the enzyme is salt tolerance. Most nucleases in the molecular toolbox can tolerate sodium chloride up to about 150 mM (depending on other conditions), but other enzymes like salt active nuclease (SAN) in fact have optimal activity at around 500 mM sodium chloride. In Figure 1, we compare SAN (the high-quality “HQ” version) to two other broadly used endonucleases, which are compared at O, O.25 M, and 0.5 M NaCl as well as at 6 °C, 25 °C, and 37 °C.

Figure 1. Salt active nuclease (SAN, the high quality “HQ” version) is compared with two other commercially available endonucleases. At 0.25 M NaCl and 0.5 M NaCl, SAN outperforms other commercially available endonucleases.

Tolerance up to 500 mM is important, especially for AAV production. One study in particular has shown that by increasing sodium chloride up to 500 mM in upstream processes, substantial increases in titer yield are achievable.1

Regarding the use of SAN, efficiencies are realized not only through the increase in salt and the effect on virus production, but also in simplification of the workflow via the utilization of compatible biochemicals. It has also been posited that efficiencies can be achieved through the digestion of protein-bound DNA. In a recent study with a confidential collaborator (internal company data), it has been demonstrated that salt-tolerant nucleases are able to digest histone-bound DNA in combination with higher salt levels, leading to more efficient digestion of residual DNA.

Figure 2. Salt active nuclease (SAN) is compared with another commonly used endonuclease for residual DNA digestion in an AAV purification run. Ideal conditions for each enzyme were used for optimal activity.

Figure 2 demonstrates the additional efficiencies of using a high-salt buffer in combination with a salt-tolerant nuclease in an AAV production run. Other common endonucleases can achieve substantial reduction in residual DNA, but nuclease-resistant nucleic acids like chromatin are more difficult to digest.

The evaluation of novel enzymatic strategies for bioprocessing can be challenging, especially when a mountain of data and scientific literature favors existing tools. Nevertheless, manufacturers should not overlook or discount alternative enzymes simply because they are in investigational stages.

The time and effort spent to consult suppliers of novel tools and bioprocessing solutions can often be rationalized once process efficiencies are realized. In the case of SAN, significant reduction in the amount of enzyme needed for bioprocessing operations has led to significant cost savings not only through lower input costs, but also as a result of a reduction in the costs associated with handling the product.

1. M. Lock et al., “Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale,” Hum. Gene Ther. 21(10), 1259–1271 (2010), doi:10.1089/hum.2010.055.

Andrew Ward ([email protected]) is senior business development manager at ArcticZymes