Advanced Analytical Characterization to Support Gene Therapy Development

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Drug development is rapidly evolving, with breakthroughs paving the way for new, highly specialized treatments. Those in the field of gene therapy, in particular, are witnessing unprecedented opportunities, with innovative treatments offering hope for previously incurable diseases.

However, the path from concept to market is fraught with challenges, particularly when it comes to comprehensively characterizing lipid nanoparticles (LNPs) and complex viral protein structures along with their post-translational modifications (PTMs). Early and detailed characterization in the drug development process is increasingly crucial for reducing risk and achieving the marketing approval of novel therapies.

Trends in Gene Therapy

Gene therapy is at the forefront of medical innovation, offering the potential for long-lasting treatment and even cures for genetic disorders. These therapies, which often involve correcting genetic defects at the molecular level, promise to transform the treatment landscape for many diseases.

For instance, in June 2024, a research team from Harvard Medical School and multiple institutions in China published the results of a study in which hearing in children with hereditary deafness was successfully restored using an adeno-associated virus (AAV)-based gene therapy.¹ The recent FDA approval of an investigational new drug (IND) application based on prime editing—a novel gene editing technology—for the first human trials further underscores the significant progress taking place in this field.²

Prime editing is a novel variation on CRISPR systems that utilizes a prime-editing guide RNA (pegRNA) to achieve precise genetic modifications with high editing efficiency without inducing DNA double-strand breaks. This approach can offer significant advantages over conventional CRISPR/Cas9 genome editing methods, including higher precision, more efficiency, and a reduced risk of dangerous adverse effects (AEs) due to the absence of DNA double-strand breaks, which can cause unintended genetic impairment.

In addition to gene therapy, transformative genomic medicines based on messenger RNAs (mRNAs) present opportunities. The best known are mRNA-based drugs in the context of COVID-19 vaccines, which employ LNPs to deliver an mRNA payload that triggers a protective immune response.

This type of drug is highly adjustable as a vaccine and is being investigated to target various diseases beyond preventing infectious diseases. The use of mRNA-LNPs is expanding in applications as diverse as protein replacement therapy, gene editing, and cancer treatment.^3,4

Challenges in Characterizing New Treatments

As gene therapies advance, they bring unique challenges. The high complexity of these new treatments requires advanced analytical techniques to ensure their quality, safety, and efficacy. An increasing number of product quality attributes (PQAs) and critical quality attributes (CQAs) must be assessed and monitored for the various therapeutic classes within genomic medicines.

A few years ago, a new class of lipid impurities was discovered in mRNA-LNP drugs.^5-7 These impurities can emerge from producing lipid raw material or when lipids used to generate new LNPs degrade over time.⁵

Ionizable lipids were identified as a culprit; however, novel modifications of lipids and greater combinatorial complexity in LNP design bear the risk of other sources of impurities, such as polyethylene glycol (PEG) lipids.^5,8 The discovered impurity likely results from oxidation and aldehyde breakdown products that are very reactive towards mRNA.

The covalent binding of the aldehyde breakdown impurity results in an mRNA-lipid adduct.⁶ Because these lipid adducts are relatively small compared with the entire mRNA, some analytical methods cannot detect them.⁵

If undetected and not controlled for, these adducts may lead to a loss in efficacy in the final mRNA-LNP drug.^5,6 Other mRNA-LNP components also require rigorous analysis and control. The integrity and size assessment of nucleic acids—specifically mRNA encapsulated within LNPs—are critical for helping to ensure the efficacy of RNA-based vaccines and therapeutic formulations.⁹

Robust, high-resolving analytical techniques are needed to confirm that the mRNA was properly transcribed and is adequately encapsulated and stable under defined storage conditions. While high-performance liquid chromatography (HPLC) is suitable for a range of oligonucleotide sizes, larger sized in vitro transcribed RNA products often require higher resolving power than that offered by HPLC methods.

Another CQA for mRNA-based therapeutics and vaccines is the length and distribution of the poly(A) tail in mRNA, as it significantly impacts the stability and translation efficiency of the mRNA.^10–12 However, accurate measurement and characterization of the poly(A) tail present challenges.¹¹ For example, current methods, such as ion-pair reversed-phase liquid chromatography (IPRP-LC), lack the resolution power to fully separate poly(A) tail distributions >100 nucleotides, while complex next-generation sequencing (NGS) requires advanced bioinformatics tools for implementation in QC testing.¹¹

The development of gene editing-based therapeutics raises new hurdles for existing CQAs. As an example, pegRNA presents challenges in purity analysis due to its long and highly complementary nature.¹³

Accurate and thorough characterization of these molecules is essential to avoid AEs and ensure therapeutic effectiveness.¹³ The characterization of PTMs in the Cas9 enzyme is also challenging.

PTMs such as acetylation, deamidation, and phosphorylation can significantly affect the function and stability of the protein by altering its structure and hydrophobicity. Precise PTM identification is vital to maintaining and improving Cas9 protein stability and functionality.¹⁴

In addition to developing novel gene therapy approaches, scientists continue to improve the assessment and control of a slightly more established gene therapy class based on viral vectors while expanding towards new, artificial serotypes and synthetic virus-like particles, which present their own analytical challenges. PTMs of viral vectors, such as AAVs, can impact the quality, efficacy, and safety of drug products.

Modifications—including glycosylation, phosphorylation, and acetylation—can affect the structural integrity and functionality of viral capsid proteins, which are crucial for stability, viral uptake, and transduction efficiency.¹⁵ However, the chromatographic separation of viral proteins is complicated due to their similar physical properties and the presence of low-abundance impurities.

Accurately mapping these PTMs requires advanced analytical techniques that provide meaningful answers despite limited sample quantities, such as differentiating between isomers and identifying fragile modifications and their locations. Moreover, ensuring the purity and integrity of the viral genome and monitoring host cell proteins and nucleic acids are critical to maintaining vector potency and limiting immunogenic responses. Overall, comprehensive and precise analytical methods are vital for the successful development and quality control of viral vector-based therapies.¹⁵

Innovative Analytical Solutions

To address these analytical challenges, new approaches have been developed, such as a novel fragmentation mechanism that uses electron-activated dissociation (EAD) with tandem mass spectrometry (MS/MS). This cutting-edge method allows for detailed characterization of lipids, related impurities, and PTMs of proteins, which are critical for drug developers as they push forward with first-of-their-kind treatments.

EAD MS/MS provides detailed insights into structural components that enable better understanding and control of CQAs of new lifesaving therapies.^15–17 To mitigate the risks posed by lipid impurities in mRNA-LNP drug products, lipid manufacturers should consider implementing thorough analytics on raw materials using state-of-the-art mass spectrometry (MS) tools, which allow for detailed structural analysis through EAD.

mRNA-LNP manufacturers should also consider testing obtained batches for impurities in addition to monitoring for mRNA-lipid adduct formation after formulation. These strategies help ensure early identification of lipid impurities and corrective measures that safeguard the efficacy and safety of mRNA-LNP therapeutics and vaccines.^5,6

The assessment of nucleic acid integrity and size, including that of the mRNA in mRNA-LNPs, can be achieved using capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) detection. State-of-the-art CGE systems offer higher resolution power than HPLC-based methods and can effectively quantify the purity percentage from naked mRNAs or from mRNAs derived from formulated LNPs.

Reliable separation of impurities and understanding of purity are vital for informing drug development and for maintaining product quality in manufacturing processes.⁹ The poly(A) tail lengths of mRNAs can be precisely determined and quantified, with a single-nucleotide resolution over a size range of at least 9 to 156 nucleotides, using a CGE with UV (CGE-UV) detection method.

Such a workflow offers excellent assay repeatability, with a streamlined and QC-friendly approach for poly(A) tail analysis that is essential for the late-stage development and quality monitoring of mRNA drugs (for an example, see the workflow in Figure 1).¹¹

Figure 1. Workflow for mRNA poly(A) tail analysis by CGE-UV. Here, the mRNA sample is first digested with RNase T1. The poly(A) tails released are then purified with oligo dT conjugated magnetic beads and separated on the PA 800 Plus system using the ssDNA 100-R kit. The electropherogram in the right panel demonstrates single-nucleotide resolution of >120 nucleotides of a typical poly(A) tail profile obtained from a digested mRNA sample. Credit: SCIEX.

A CGE-based method can also be applied to overcome the challenges related to secondary structure formation associated with pegRNA purity analysis. It was found that in addition to using denaturation agents, leveraging tight liquid-based temperature control during the separation of pegRNA at 50°C resulted in a single sharp peak for pegRNAs of different sizes. This technique thoroughly disrupts the hydrogen bonds in pegRNA molecules, maintaining their denatured state during analysis. This high-resolution approach enables comprehensive purity analysis of these complex molecules.¹³

Techniques such as LC coupled with MS/MS (LC-MS/MS) were demonstrated to achieve comprehensive characterization of engineered Cas9 proteins.¹⁴ The application of MS/MS can provide comprehensive sequence coverage of the Cas9 protein, which is critical for confirming amino acid sequences and PTMs.¹⁴

Furthermore, EAD was applied for improved fragment coverage and, thus, confident identification of long peptides. In addition, differentiation between isobaric modifications from deamidation events (aspartate and isoasparate) was achieved with EAD.

Sequence variants containing leucine/isoleucine substitutions can be differentiated, and in combination with dedicated data processing software, this method allows for the sensitive and confident identification of low-abundant peptides, the elucidation of isobaric modifications, and the calculation of relative amounts of modified peptides.¹⁴

Analyzing PTMs of viral vectors such as AAVs is essential for ensuring the quality and efficacy of gene therapy products and next-generation vaccines. LC coupled to MS (LC-MS) and capillary electrophoresis (CE) are powerful techniques that facilitate the comprehensive analysis of viral capsid proteins and viral genomes.

Time-of-flight MS instrumentation provides high mass accuracy for intact viral proteins, enabling the determination of intact protein molecular weights and the identification of and relative quantities of PTMs such as acetylation and phosphorylation, which can affect viral potency and infectivity.¹⁵ Peptide mapping approaches enable sequence confirmation and the identification and exact localization of low-abundance PTMs.

Additionally, leveraging EAD MS/MS enables the differentiation of amino acid isomers and the localization of more fragile PTMs, such as phosphorylation, ensuring accurate assessments of viral vector attributes. CE can provide relevant orthogonal information to LC or LC-MS methods.

In addition to the assessment of protein form ratios in a highly reproducible manner, the use of sodium-dodecyl-sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) can achieve the separation of viral particle (VP) protein forms (often referred to as VP prime), which cannot be achieved with LC-based methodologies.^18,19 The integrity and purity of the viral vector genome are additional quality aspects of viral vectors that can be assessed with CE.

These methods address challenges in the characterization of viral vectors and the assessment of protein and genome integrity and purity. A comprehensive analytical approach using multiple analytical tools helps in understanding the diverse aspects of the PQAs and CQAs of these highly complex medicines, which is crucial for the optimization of their development and for their final release.¹⁵

Conclusion

Innovations such as prime editing, novel LNP formulations and synthetic viruses are transforming the therapeutic landscape. As these technologies continue to evolve, they hold the promise of delivering groundbreaking treatments for a variety of genetic disorders, heralding a new era in personalized medicine.

Alongside these innovations, analytical technology is evolving and must continue to evolve to enable the development and market introduction of safe and more effective gene therapies. The rapid advancements in gene therapy highlight the need for sophisticated analytical techniques to keep pace with innovation.

By adopting advanced characterization methods such as EAD MS/MS and CGE, drug developers can reduce the challenges posed by new treatments. As the field continues to evolve, these technologies will play a pivotal role in shaping the future of medicine, bringing hope to millions affected by genetic disorders.

In summary, the integration of advanced characterization techniques earlier in the drug development process—particularly through innovative methods—is essential for reducing risks and expediting the market entry of novel gene therapies. Detailed and accurate assessments ensure that potential impurities and modifications are identified, their impacts are understood, and countermeasures can be implemented, improving the quality of new treatments. These advancements can revolutionize the treatment of genetic diseases, offering new hope and improved outcomes for patients worldwide.

About the Author

Kerstin Pohl, MSc, Senior Global Manager – Gene Therapy & Nucleic Acid, SCIEX.

References

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