Choosing Analytical Technologies At The Right Stage Of Development
By Seth Levy, Ph.D., Modalis Therapeutics
Analytical development has become increasingly technology-rich and for almost every product quality attribute there are many approaches one can take. Some methods are more sensitive than others, some are more automated than others, while other approaches may be more information rich. The question we will tackle here is not which technology is “better” but the concept of how different analytical methods will shape the development strategy and when to alter an analytical approach in the context of gene therapy products.
A newer or more sensitive method is not automatically the right method at every stage of development. If a new technology measures the same attribute but does not change the process decision or control strategy, adopting it too early may add complexity, cost, and risk without adding value. In preclinical and early-phase development, the right method may often be the one that provides sufficient information and is fast, available, and fit-for-purpose. As the program matures, the analytical strategy must mature with it, so the right method answers the right question at the right time.
Residual Host Cell DNA
Residual host cell DNA is a familiar example. ddPCR offers real advantages over qPCR: absolute quantification, improved tolerance to certain matrix effects, no need for a standard curve, and strong precision at low copy levels. Those features can become important as a program moves toward a later-stage control strategy, comparability studies, and release testing.
Despite these known attributes, for many programs, qPCR remains appropriate through IND. It is familiar, broadly commercially available, high-throughput, sufficiently sensitive, and well understood by development teams and regulators. If the questions are whether the process is directionally clearing host cell DNA and whether residual DNA is being controlled to support early clinical development, qPCR can still answer those questions.
When a qPCR assay is impacted by matrix interference, if low-level quantitation impacts development strategy, or if the sponsor needs greater confidence in quantitation, ddPCR may be the better choice. If ddPCR does not change the clearance interpretation or a development decision, it may be better to save changing methods for later in the product life cycle.
Host Cell DNA Fragment Sizing
The FDA CMC guidance from 20201 recommends limiting the residual host cell DNA size to below 200 base pairs to avoid the potential transfer of oncogenic sequences. The determination of the residual host cell gene fragment size is another area where technology selection can be a challenge if the team does not first define the analytical question. Are a broad size range and distribution of residual DNA fragments critical, or does the presence of a specific cell-line-derived sequence(s) require targeted detection?
CGE-LIF or other fragment-sizing approaches can provide useful information about size distribution while ddPCR-based sizing approaches can provide sensitive and targeted information, particularly when the question is tied to specific host cell contaminants or discrete amplicon-based assessments. One approach is not always more relevant or superior to the other. The right method depends on whether the program needs a broader physical sizing profile or a targeted, sensitive readout of specific residual DNA species.
Empty/Full And Aggregation
For early DOE work with small-scale purified upstream samples, in-process samples, and production runs with limited material, SEC-MALS or mass photometry can be highly practical. Mass photometry has a lower barrier to entry than SEC-MALS with a lower cost and less technical expertise needed, but both offer higher throughput compared to AUC while providing sufficient information to compare conditions, identify trends, and decide which process parameters deserve additional attention. In these development settings sample volume, turnaround time, and operational simplicity matter.
AUC becomes more valuable as programs scale up and a more definitive understanding of product heterogeneity is required, and there is no denying AUC is considered the gold standard for elucidating the empty-full ratio. When the team requires better resolution of empty, partial, and full capsids, or when higher-order species may influence product understanding, AUC can provide a more complete characterization picture. AUC also brings practical constraints: sample requirements, throughput, specialized expertise, and data interpretation burden. Another powerful option for later in a program’s life cycle is charge detection mass spectrometry (CDMS), but this method also requires specialized equipment and training.
This same logic applies to aggregation. The first question should be, “What type of aggregates do we expect, and what decision will the test results drive?” Historical product and process knowledge should shape the method strategy. If the question is early-stage DS/DP release support, DLS may be sufficient. If the question is deeper product characterization or comparability, the analytical package may need to expand and a matrix of assays including SEC-MALS, AUC, and DLS, may be required.
Genome Integrity
Genome integrity is a good example of how the “best” technology can shift depending on the purpose of the assay. Older technologies like alkaline gel electrophoresis can still serve a purpose to help screen genome integrity and may still be used as a release assay for early-stage assets.
During early transgene development and process development, long-read sequencing such as nanopore may provide valuable insight if base-calling is not the primary need. In the screening phase of a program, you may be more concerned with identifying structural issues like snapback genomes, truncation hotspots, unexpectedly packaged species, etc. Used this way, long-read sequencing is an emerging discovery and product understanding tool, though this does not automatically make long-read sequencing the best release method.
Duplex or triplex ddPCR may be more QC-friendly if early characterization has already identified any relevant issues and de-risked any problematic sequences. A targeted ddPCR approach can be easier to validate, transfer, and execute routinely. It will not provide the same details in discovery as long-read sequencing, but release testing is intended to confirm that the known critical risks remain controlled. Sequencing may help define the problem, while ddPCR may be a practical way to monitor it.
Capsid Protein Characterization
Capsid protein characterization follows a similar life cycle pattern. SDS-based methods are often the simplest and most practical choice for preclinical and early-phase work. They can support basic VP profile understanding, provide a familiar readout, and help the team determine whether major protein composition concerns are present.
As the product matures from early to late phase, the questions about your product that you want to answer may become more specific. Are VP isoforms consistent across lots? Perhaps a more quantitative approach such as capillary electrophoresis SDS (CE-SDS) is an appropriate method. Are post-translational modifications emerging as a stability concern or are process changes altering the capsid protein profile? In those cases, LC-MS intact mass can be a valuable supplemental late-phase characterization tool by providing additional information such as VP stoichiometry, VP sequence identity, post-translational modifications, etc.
Build The Analytical Strategy Around Decisions, Not Technology
In early development, methods should enable learning. They should support DOEs, candidate selection, process scale-up, and IND-enabling decisions. In later development, methods should increasingly support comparability, validation, release, and life cycle management. Somewhere between those stages, newer methods may need to be bridged against current gold standards or established approaches, so the sponsor must understand not only whether the data is different but whether it is better for the question being asked.
The most effective CMC analytical strategy answers specific questions with the appropriate methods at the appropriate time, knows when an orthogonal method is needed, and how a different technology changes product and process development.
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About The Author:
Seth Levy is vice president of manufacturing at Modalis Therapeutics. His experience spans both sides of the outsourcing agreement. He has led teams in MSAT development for the manufacture of viral vectors at a CDMO and for sponsor companies. He received his Ph.D. in molecular and cellular pathology from the University of Alabama at Birmingham.