In a detailed interview with BioPharma BoardRoom, Kerstin Pohl, Senior Global Marketing Manager for Gene Therapy & Nucleic Acid at SCIEX, delves into the analytical challenges gene therapy developers face as the field evolves in 2025. From addressing small sample sizes and diverse delivery mechanisms to pioneering advancements in mass spectrometry and capillary electrophoresis, SCIEX is at the forefront of driving innovation in biotherapeutics. Join us as Pohl shares how SCIEX’s cutting-edge technologies are shaping the future of quality, accessibility, and regulatory compliance in gene therapy.

In 2025, what analytical challenges do you anticipate gene therapy developers facing, and how is SCIEX addressing them?

An ongoing challenge is small sample sizes available for analysis. Especially for gene therapies targeting rare diseases, developers are urging regulatory bodies to rethink the required testing of drug substances and drug products to reduce redundancies and save precious samples. In some cases, more batches are needed to satisfy analytical testing requirements than used for treating patients.

SCIEX is committed to pushing the boundaries of sensitivity and providing high-quality analytical technology, helping to keep analytical variation low, reducing sample consumption. The triple quadrupole mass spectrometry (MS) solutions from SCIEX has been a leading solution in MS sensitivity for decades. SCIEX capillary electrophoresis (CE) systems provide ultra-high resolution, which results in sharper peaks [1]. The higher resolution can also be beneficial in gaining a deeper understanding of the sample, leading to more informed decision-making.

Another challenge can be the alignment needed for analytical testing. The gene therapy realm is highly diverse and talented scientists are constantly working on new potential delivery and/or drug substances. For instance, delivery mechanisms can be based on a range of viral vectors, virus-like particles, and non-viral carriers (lipid nanoparticles with various combinations of lipids, exosomes, polymers, etc.). Drug substances used in certain genetic medicines can range from short oligonucleotide therapeutic (i.e. antisense oligonucleotides or small interfering oligonucleotides) to large messenger RNAs, circular RNAs, self-replicating RNAs, and conjugates of various kinds, in addition to mixtures of RNAs, proteins and DNA. The potential in the future if sizable. 

Analytical assays are not all easily transferable from one therapeutic class to another and new potential drugs might require new approaches for analyzing them. We are committed to working closely with biotech and biopharma scientists on new methods to overcome analytical challenges with existing and new solutions.

How does SCIEX help improve the development of next-generation therapies?

A variety of analytical technologies and assays are needed to develop next-generation therapies. Some analytical challenges can be overcome by rethinking the use of existing technology, while others require new ones to break through the analytical boundaries. 

An example of leveraging existing technology in a new way is a method for the purity analysis of self-replicating RNA (srRNA) that Replicate Bioscience and SCIEX recently published [2]. Self-amplifying or srRNA has the potential for lower dosage, therefore enabling faster and more economical manufacturing per dose and prolonged expression due to the self-replication and expanded indications. However, these in vitro transcribed RNAs (IVT RNAs) can be significantly larger than other mRNAs, making standard methods unsuitable to characterize them. The developed method achieved high-resolution separation of a very large RNA of ~16,000 bases to determine its purity, leveraging existing kit-based CE technology from SCIEX. Another example is the work on the poly(A) critical quality attribute, published late last year by a research group from Regeneron [3], achieving outstanding single-base resolution of the 3’end of mRNAs with capillary gel electrophoresis with a PA 800 Plus system. 

Plasmid analysis is an area in which new products are helping development. Plasmid DNA can be used as a drug directly, but more commonly, it is an important starting material for IVT RNA, viral vectors, and protein therapeutics. Complex and often large plasmids are used for viral vectors and IVT RNAs. A new kit based on bare-fused silica capillaries, released in September 2024 ^[4], facilitates the analysis of plasmid DNA and linear double-stranded DNA material up to 20,000 base pairs. It enables rethinking of other technologies or methods, such as slab gels or older kit-based methods, to increase automatization, reproducibility and efficiency.

Advancements in MS fragmentation technology is crucial for the structural elucidation of analytes such as lipids used for lipid nanoparticles or viral vectors. Lipid components can contain impurities that are difficult to be identified with charged aerosol detection or collision-induced dissociation mass spectrometry. Viral vectors can contain a variety of post-translational modifications that are difficult to be fully elucidated in terms of identity and exact position. Advances in alternative fragmentation, such as electron activated dissociation (EAD) can help in streamlining efforts around identification of analytes, impurities and modifications ^{[5, 6].} 

What role do you see mass spectrometry and capillary electrophoresis playing in ensuring quality and regulatory compliance for emerging biotherapies?

For the development of biotherapeutics and other new modalities, a variety of assays are needed to ensure quality and safety during manufacturing. Capillary electrophoresis technology has been a standard for assessing purity and charge heterogeneity of traditional biotherapeutics, such as monoclonal antibodies, for decades. While there is overall more variability and complexity for CGT products, CE remains a highly relevant assay for assessing quality attributes, such as, plasmid DNA purity and identity.  

Additionally, CE is often the method of choice for viral vectors structural protein identity and purity. Its resolving power has proven to be superior to LC-based methods, for instance, for the separation of viral vector protein isoforms (VP3 variants) of different AAV serotypes ⁽⁷⁾. As an orthogonal technique, mass spectrometry is relevant for verifying viral protein identity. In addition, guidelines require products produced in living cells to be tested for residual host cell proteins (HCPs) (i.e., USP chapter <1047>). While traditionally, ligand-binding assays were commonly used to test for HCPs, mass spectrometry assays gained in popularity over the past decade, for reasons such as mass spectrometry assays have a shorter assay development timeline compared to ELISAs, when no off-the-shelves assays or antibodies exist. Another application area for mass spectrometry can be detecting and quantifying expressed proteins. The versatility of mass spectrometry in protein and peptide identification and quantitation without the need for antibodies is highly advantageous compared to other assay types.

As global demand for gene therapies grows, how does SCIEX plan to support accessibility for these life-saving treatments?

Analytical assays that reliably monitor stability under various conditions set the baseline for developing drug properties to allow for easier storage and distribution. This is especially important to enable accessibility on a global scale.  SCIEX has a track record in delivering reliable CE and MS instrumentation to support these efforts, including the recent launch of the SCIEX 7500+ system, a highly sensitive triple quadrupole solution with increased robustness. 

As the biopharma industry navigates the complexities of gene therapy development, insights from leaders like Kerstin Pohl underscore the critical role of innovation and collaboration in overcoming analytical challenges. SCIEX’s commitment to advancing precision technologies, enhancing quality, and addressing accessibility paves the way for a brighter future in biotherapeutics. At BioPharma BoardRoom, we are excited to witness how these innovations will shape the landscape of gene therapy and contribute to life-saving treatments globally in 2025 and beyond. 

(editor@biopharmaboardroom.com )

References:

[1] Mantri P, Juneja B, Henderson S, Koufos E, Moon Y, Dayeh DM, Di Grandi D, Fu Y, Muthusamy K, Ihnat PM, Palackal N, Pyles EA. Comparison of capillary electrophoresis-based methods for the analytical characterization of purity and stability of in vitro transcribed mRNA. J Pharm Biomed Anal. 2024 Oct 15;249:116352. doi: 10.1016/j.jpba.2024.116352. Epub 2024 Jul 14. PMID: 39029354.

[2] Addressing an unmet need: developing a new method for purity analysis of large-sized RNA drugs, SCIEX technical note, 2024. https://sciex.li/dwxzbp 

[3] Di Grandi D, Dayeh DM, Kaur K, Chen Y, Henderson S, Moon Y, Bhowmick A, Ihnat PM, Fu Y, Muthusamy K, Palackal N, Pyles EA. A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines. J Pharm Biomed Anal. 2023 Nov 30;236:115692. doi: 10.1016/j.jpba.2023.115692. Epub 2023 Sep 2. PMID: 37696189.

[4] DNA 20 kb Plasmid and linear kit, SCIEX product page. https://sciex.li/ek0qey 

[5] Automatic characterization of the lipid nanoparticle ionizable lipid MC3 and its impurities using Molecule Profiler software, SCIEX technical note, 2022.  https://sciex.li/xumk1y 

[6] A new approach for high-resolution full and empty AAV capsid analysis utilizing a high-throughput method for comprehensive AAV evaluation on a single CE platform, SCIEX technical note, 2022 https://sciex.li/mya1tw 

[7] Oyama H, Ishii K, Maruno T, Torisu T, Uchiyama S. Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry. Hum Gene Ther. 2021 Nov;32(21-22):1403-1416. doi: 10.1089/hum.2021.009. Epub 2021 Jul 16. PMID: 34082578; PMCID: PMC10112878.