Adenovirus Vectors, Cyclodextrins and the Patient Experience: A Deep Dive

Adenovirus vectors (AdVs) are biopharma’s star tools for the development of safe, efficacious vaccines and gene therapies. Adapted from one of the common-cold-causing viruses that cause millions of headaches (literally—pun intended) during every cold and flu season, these tiny trojan horses have been prized for their efficiency and adaptability for over four decades (1).

Their broad tropism and superior transduction capacity mean AdVs are able to deliver genetic material to a wide array of dividing and non-dividing cell types, while their non-integrative nature, high cargo capacity, and the ability to elicit a strong immune response add up to one powerful force for biopharmaceutical success (2,3). But, as always, there is a catch.

To take advantage of the many benefits offered by AdVs, formulators must first address their serious and varied, tendencies towards instability. Light, heat, physical shear stress, and more can impact the integrity of AdVs capsids (protein shells), potentially leading to DNA release and/or lowered effectiveness, leaving patients with less effective treatments (1).

Enter cyclodextrins, these multifunctional powerhouse excipients offer an effective solution for ensuring AdVs making it through the production process intact and ready to change or save lives. Below we expand on the potential of cyclodextrins to optimize the use of AdVs in biopharma formulation and the knock-on effects this could have for improving patient experience.

The “anatomy” of adenovirus vectors

First, some essential background. AdVs are widely used in vaccine and gene therapy development, effectively serving as vehicles to deliver genetic material into host cells.

Their unique structural and functional characteristics gleaned from their origins as non-enveloped DNA viruses make AdVs naturally capable of “infecting” a wide variety of cell types (2). At its core, an adenovirus vector is a modified version of a wild-type adenovirus (1).

The viral genome is housed within a protein capsid, which is made up of several proteins, including fiber proteins, which act as the key to unlock the cell's membrane by binding to specific receptors on the cell surface (1,4). This process, known as transduction, allows the vector to enter the cell and release its genetic payload (1).

When harnessed for therapeutic use, key parts of the adenovirus genome are removed and replaced with the desired gene (or genes). This can be material intended to correct a genetic defect, or a gene that codes for a specific antigen to stimulate an immune response for a vaccine.

By removing the viral genes responsible for replication, these vectors are made replication-deficient, which ensures they cannot cause an active viral infection (1). For close to 40 years, the high transduction efficiency and broad tropism (ability to infect a wide range of cells) offered by AdVs have made them versatile tools for targeting various tissues and cell types in different diseases while remaining outside the host's chromosomes (1).

This non-integration reduces the risk of insertional mutagenesis—a potential side effect in which the vector's genetic material integrates into the host's DNA and disrupts normal gene function (1). All this does not, however, mean AdVs are completely problem-free.

The same capacity for strong immune responses that makes them good vaccine candidates can also lead to systemic inflammation, limit the duration of gene expression, and prevent repeated administration due to patients’ development of neutralizing antibodies (2). There is also an issue of preexisting immunity, as many people have been previously exposed to common adenovirus serotypes, such as those that cause the common cold (2).

What’s more, though the neutralized virus cells are unable to replicate or cause infection, high doses of AdVs can still present a toxicity risk, especially for immunocompromised individuals (2). Challenging as they are, these drawbacks do not diminish the immense value AdVs offer as treatment tools, with their large genetic payload and robust immune activation benefits.

The question for biopharma formulators therefore becomes: how should production parameters be modified to fully mine AdVs’ many advantages?

Surviving the biopharma production line

The biopharmaceutical production process is an assembly of potential stressors for delicate biological materials like adenovirus vectors. From initial cell culture to final storage, these vectors must endure a series of physical and chemical challenges that can compromise their integrity and effectiveness.

Managing these potential pitfalls is critical to ensuring vaccine and gene therapy products are ready and able to serve patients. The production journey begins with cell culture and harvest.

During this stage, vectors are subjected to shear stress from mechanical agitation and pumps, as well as potential fluctuations in temperature and pH level (5). These stressors can damage the vector's protective capsid, leading to a loss of infectivity.

The next hurdle is purification. This multi-step process involves using techniques such as chromatography to separate the vectors from cellular debris and impurities (6).

Here, the vectors encounter more shear forces and sudden changes in pH and buffer composition (6). Exposure to various chemicals at this point can also cause the vectors to aggregate or lead to capsid disruption and the release of their DNA, rendering them useless (6).

Once purified, AdVs vectors are moved to the formulation stage, where they are combined with excipients, such as polysorbates 20 and 80, that will facilitate the delivery of the therapy to the patient. A key challenge here is the potential for freeze-thaw cycles, which can cause aggregation and a significant loss of infectivity (6).

Maintaining the correct pH and ensuring excipient compatibility are also vital to prevent destabilization (6). Finally, during storage and transport, the formulated vectors must be protected from temperature excursions, light, and agitation, all of which can lead to degradation and a loss of potency.

This can be especially challenging in environments with limited or fluctuating access to electricity, where maintaining the ideal temperature range for AdVs storage (2°-8° C) is impossible (1). Ultimately, the key to helping adenovirus vectors survive the production process intact is selecting the right excipient.

A high-performance surfactant can act as a shield throughout the biopharma production process, protecting AdVs from the various stressors encountered in their journey from a bioreactor to the patient. But in this vital stabilizing function, not all biopharma excipients are created equal.

Cyclodextrins versus polysorbates: Finding a stabilizing force

With decades of evidence pointing to their effectiveness and safety, polysorbate 80 (PS 80) and polysorbate 20 (PS 20) are considered the surfactants of choice for most biopharma formulations (2). Their usefulness as guards against protein adsorption and aggregation stems from their ability to compete with proteins at interface points like during ultrafiltration/diafiltration (UF/DF), reducing the interfacial exposure of the proteins.

Despite their popularity, however, polysorbates are far from the perfect choice for every biologic formulation. Like the proteins they are intended to protect, polysorbates can also be degraded via oxidation and hydrolysis, producing peroxides and fatty acids that could negatively impact the quality and stability of the final therapy.

Polysorbates can also be non-specifically absorbed onto UF/DF filter membranes, leading to the formation of micelles bigger than the membrane’s molecular weight cut-off, which in turn causes inconsistencies and membrane blockage. With all this in mind, researchers at Roquette set out to answer the question: “Could there be a more effective alternative to polysorbates capable of shielding AdVs, without the drawbacks?”

The resulting study investigated the use of hydroxypropyl beta-cyclodextrin (HPβCD) as an alternative stabilizer to protect adenovirus vectors from degradation caused by heat and light during manufacturing and storage. Researchers selected adenovirus serotype 5 (AdV5) as a model virus vector due to its wide application and well-known characteristics, and combined it into three distinct formulations; a control containing only a citrate buffer and ethanol, one featuring 0.02% polysorbate 80, and one containing 5% HPβCD (KLEPTOSE HPB).

These formulations were subjected to various heat and light stressors, with samples stored at 25° C for a total of 21 weeks and exposed to white light and UV light according to ICH Q1B guidelines (7). At the end of the testing cycle, the samples were collected and frozen before the AdV5 potency titres were quantified using a 50% tissue culture infectious dose (TCID50) assay.

Results showed that the formulation containing HPβCD maintained a TCID50 significantly higher than that of the polysorbate sample over 21 weeks of storage at 25° C (Figure 1). While the polysorbate 80 formulation initially showed some protection, its potency declined more rapidly from week two onwards, reaching the level of the control by week 16, and eventually showing a 2-log reduction in TCID50 by week 21.

When exposed to white or UV light alone, the HPβCD formulation showed 100% potency retention, while the other formulations experienced a significant loss of an almost 1-log reduction in TCID50 (Figure 2). Under stress from both white light and UV, the control sample plummeted 2-logs in infectability, while the polysorbate and HPβCD showed a far less dramatic reduction in potency.

These compelling results suggest that HPβCD is an effective alternative to traditional polysorbates for protecting AdVs against thermal and photo-induced degradation. This is all the more significant since HPβCD is a compendial excipient widely considered safe and non-hazardous, meaning it can be easily slotted into existing biopharma manufacturing processes to improve stability, extend shelf life and potentially save lives.

The patient perspective: Why optimizing AdVs matters

Optimizing the stability of AdVs is not just a win for biopharma manufacturing efficiency, it has significant implications for patient care too. Ensuring the vector remains intact and potent throughout its journey from the lab to the patient delivers undeniable benefits, improving both the effectiveness and accessibility of vital vaccines and gene therapies.

First and most obviously, maintaining vector integrity means delivering consistent potency, leading to more reliable therapeutic outcomes (8). A stable vector is also less likely to degrade into non-infectious fragments, which can otherwise trigger unwanted immune responses or toxicity, thereby reducing the risk of side effects such as inflammation or cytokine storms (9,10).

Heighted stability also supports more durable gene expression from AdVs, potentially reducing the need for repeat dosing and boosting the likelihood of patient compliance (8). Beyond these direct clinical benefits, optimizing AdVs can dramatically improve access and convenience for patients.

Formulations with enhanced stability, such as lyophilized (freeze-dried) vectors, can be stored and transported at higher temperatures, lowering dependence on complex cold chain logistics and making it easier to distribute lifesaving treatments to remote or resource-limited regions (8). Longer-term stability also enables pre-epidemic stockpiling of vaccines, ensuring they are available for rapid deployment during a health crisis (8).

Finally, advances in stability are crucial for continued innovation. Just as the development of “gutless” AdVs helped reduce inflammatory responses in patients by removing almost all viral genes, optimizing the efficacy of current AdVs helps pave the way for future improvements in vector design (1).

A more robust, stable platform provides the foundation for new generations of safer, more effective therapies for a growing global patient cohort, rightly demanding therapies that put them first.

A vector for change

Adenovirus vectors’ journey from lab-bench curiosity to a cornerstone of modern biopharmaceutical development has been paved with relentless innovation. From addressing issues of pre-existing immunity with chimeric vectors and non-human serotypes to expanding the genetic payload of “gutless” designs, this field of study has evolved at a breakneck pace (11) and will continue to do so.

Advances like these, coupled with sophisticated genetic engineering for the treatment of challenging diseases such as cancer, demonstrate that we have only begun to scratch the surface of these versatile vectors’ therapeutic potential. The smart application of excipients such as cyclodextrins will play a vital role in turning promising research into practical results.

Offering a more effective shield for AdVs as they go through the rigors of biopharma production lines, cyclodextrins ensure that every vial, without exception, contains a potent and effective dose. This seemingly simple step has profound implications, guaranteeing consistent therapeutic outcomes and reducing the risk of side effects from degraded vectors.

Every innovation in biopharma, no matter how small, must be viewed through the lens of the patient. The overarching goal of any development is to improve the patient experience, whether it's by boosting efficacy or making life-saving vaccines more accessible.

By focusing on the fundamentals like stability and safety, biopharma brands can build a future in which groundbreaking therapies reach those who need them most and solidify AdVs’ place as a true vector for change in global health.

About the Author

Michael LeBlanc is Global Biopharma market manager at Roquette Health and Pharma Solutions.

References

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