Key Analytical Methods for Ensuring Safety & Efficacy in Therapeutics – Part II : Viral Vectors

May 22nd, 2025

As your end-to-end CDMO partner specializing in viral vectors, Exothera is committed to supporting the development and manufacturing of high-quality viral-based therapeutics. Ensuring the integrity, safety and efficacy of these products requires a robust and tailored analytical approach, from starting materials to final release. In today’s blog, we’ll explore key analytical challenges and solutions associated with viral vectors manufacturing, navigating from the inside of the products towards their outside.

Having the right message inside

One can think of selecting the best routes of administration for a vector to increase its therapeutic success, but what if the message that is delivered is incorrect? Therefore, the identity of genomic material is key. A quick check with digital droplet PCR can provide dual information, not only about the identity of the genomic information but also about the quantity of the product per milliliter.
However, some of the viral capsids can also contain additional wrong information which remains undetected by the targeted PCR approach. Therefore, the combination of multiple measurements produced by orthogonal methods is key to success. Combining PCR – which provides a count of particles filed with the correct message – with other physical titration methods of the viral particles (either performed with ELISA, MADLS, OD260, or HPLC) will provide an estimation of the percentage of product containing the correct genome.

Restriction enzyme assays can give a general overview of the presence of the correct sequences, as they produce a specific pattern of DNA fragments that are observable by agarose gel or capillary electrophoresis.

As optimal, for more detailed information, long-read sequencing provides a nucleotide-level view of the actual sequence within the capsids, ensuring that not only the length but also the intended message is present in the final products.

Know the messenger

Different viral vectors will have different characteristics that might be relevant for their proper function. Adequate analytical methodology to observe key features is part of the key to success. As a general view, capsid identity can be confirmed by immunoassays (ELISA or western blot). For specific vectors, like AAV, it could be interesting to verify that the capsid components are present in the right proportion by the analysis of the VP ratio. Other methodologies can be applied instead, as capillary electrophoresis or mass spectrometry can be used to go deeper into the study of those proportions and the presence of post-translational modifications in the capsid proteins. 

Now, you have the right material, but… how much? Methods to quantify physical titers per unit of volume are necessary to prepare the right dosing. Therefore, one can use physical measurements (like spectrometry, or light scattering), titers based on the specific recognition of the capsids (ELISA), nucleic acid technologies (qPCR or dPCR) to measure viral genome equivalent titers, All these measurements can even be applied to the same vector in a complementary approach that will lead to a better understanding of the nature of the product.

Measuring the effect

 Let’s say that the right product is achieved on paper, but… will it work in real conditions? There, cell-based assays constitute useful models of what the viral vector will encounter after its administration into a host. And depending on the virus, it could be that infection and transduction are weighed differently, and they need to be separately assessed.

Hence, selecting the right model is critical to evaluate the response towards the vector. FACS or immunocytochemistry can be used to monitor either the presence of viral proteins within the cellular model or the response of the cells producing intracellular or surface markers characteristic of the response.

To calculate the potency of the product, these analyses can be properly designed to obtain a relative response against a certain reference material or to produce an absolute quantification of the infectious titer (as in a plaque assay or a TCID50 approach).

The environment counts

Generating a therapeutic vector requires the proper environment. Viruses need to be generated in the right cell line (that, of course, also needs some associated analytical testing). Unfortunately, residuals coming from those host cells can end up in the final product, and they need to be quantified. dPCR (for DNA), ELISA (for proteins), and gel electrophoresis (for general purity) are often used at this stage.

Ensuring that the plasmids used for transfection, or the master viral seed stocks contain the right information for the creation of the intended vector is good practice for viral vector manufacturing. That can be achieved by nucleic acid testing (PCR or ideally sequencing). A good design of the starting material will avoid or reduce unwanted packaging of material within the capsids and ensure correct production of the vector in terms of structure and quantity. For some viruses, it is important to perform a positive selection of stable clones that are monitored by genetic stability assays based on a combination of cell-based protocols with amplification by PCR.

Although product-related impurities might have already been detected or suspected during identity testing, the process-related ones can add certain safety concerns. Our DSP team works to reduce to the minimum the presence of those process-related impurities; however, some can still end up in the final vector. The methods to analyze them do not differ from the ones described earlier, but as with any residual quantification, specificity and sensitivity need to be maximized to allow proper quantification of their presence. The presence of entities that can self-replicate (recombinant competent virus), adventitious viruses, or other organisms growing during the production process (like mycoplasma, mycobacteria, bacteria and fungi) needs to be measured as well through the production process.

The general appearance of the product is also critical, including the evaluation of the physical characteristics of the product (color, appearance, pH, …), and the absence of undesired particulates in the solution. Finally, a product is only good for a period of time, and stability in the storage buffer is something that also needs to be assessed.

Building a Comprehensive Analytical Strategy

Exothera places at the service of our clients a synergic collaboration between the analytical and CMC teams to generate a testing panel that is adequate to each viral vector, ensuring the safety and efficacy of viral vector therapeutics. This comprehensive analytical strategy includes:

• Critical Quality Attributes (CQAs): These include identity, purity, potency, structural integrity, and visual appearance of the final product. These attributes are thoroughly characterized and consistently monitored throughout the manufacturing lifecycle.
• Release Specifications: Defined acceptance criteria supported by validated analytical methods, enabling the release of product batches that meet quality standards, including accurate dose quantification.
• Safety: Ensuring the absence of undesired impurities and sterility of the product for clinical use.
• Shelf-Life Studies: Stability testing under real-time and accelerated conditions provides essential data to establish storage conditions, define shelf life, and support toxicology and Phase I clinical studies.

At Exothera, We Understand What’s at Stake

With viral vectors at the core of many advanced therapies, analytical precision is critical to ensure safety, efficacy, and regulatory success. At Exothera, we combine deep expertise in viral vector process development and analytics to support your product through every stage, from early development to commercial readiness.

Let’s work together to optimize your viral vector program. Contact us today to learn how our analytical solutions can help bring your therapeutic to life.

Authors: Antonio Salgado, Senior Analytical Scientist, Fabien Baldacci, Analytical Science Manager and Camille Lemaigre, CMC & Regulatory Associate, Exothera 

About Exothera

Exothera is a specialized CDMO partner delivering preclinical and clinical process development, manufacturing optimization and CMC support for virus and RNA based therapeutics and vaccines.

With headquarters in Belgium, at the heart of Europe, we assist clients worldwide from start to scale, up to commercial manufacturing, including analytical testing and regulatory consultation. Thanks to our optimal technologies and experienced team, we help bring innovative therapies from bench to patient with quality, at speed and cost-effectively.