As GEN enters a new year, one that coincides with our 40th anniversary year, we are filled with optimism and hope for a post-pandemic future. To help us preview this future, we asked opinion leaders, all from outstanding technology companies, to discuss a range of new initiatives. Following is a discussion informed by Michael M. Meagher, PhD, Mani Krishnan, and Claire Davies, PhD.

Michael M. Meagher, PhD, Mani Krishnan, and Claire Davies, PhDThe path to developing monoclonal antibody (mAb) drugs has been clearly defined and refined over the years. The same cannot be said for gene therapies. Although gene therapies were first developed decades ago, only in recent years have advances brought this modality close to realizing its full potential.

Unfortunately, significant uncharted territory and unique challenges remain. Lacking established (and qualified) processes, consistent analytics, and clear regulatory guidelines, gene therapy developers must create their own paths to commercialization.

This article amounts to a journey through these issues. Along the way, it consults with three scientists who can offer unique perspectives on the challenges of gene therapy and how these challenges can be overcome.

Not a conventional biologic

Protein therapeutics are manufactured using well-established cell lines. A batch of protein or mAb is fairly homogeneous, and the entire batch can be analyzed to confirm its character and quality, according to Michael Meagher, PhD, vice president of therapeutics production and quality at St. Jude Children’s Research Hospital. That is not the case with the viral vectors used to deliver gene therapies and genetically modified cell therapies. Two to four plasmids must transfect each cell and create a capsid protein with a structure that is suitable for encapsulating the transgene and generating the viral vector.

“With viral vectors, there are no stable cell lines that produce the same vector.
Every cell—every cell—must be transfected by a series of plasmid complexes to produce the viral vector,” explains Mani Krishnan, vice president and general manager of CE & BioPharma at SCIEX. “Imagine the probability of things not working and the level of induced heterogeneity.”

The key to improving the manufacture of gene therapies is to develop consistent and scalable platforms with increased manufacturing yields, maintains Claire Davies, PhD, associate vice president of bioanalytics, Sanofi. “The low manufacturing yields and low production volumes can be tackled with advances in transfection technology and vector design to improve cell line stability and viral packaging,” she says. “Meanwhile, the development of higher-cell-density processes, improved downstream purification processes, and better critical raw material control will improve process consistency and yield.”

Another current hurdle to achieving robust processes is the lack of rapid analytical methods. “Without speedy assays, the ability to fully understand all of the relevant process parameters and how they impact product quality attributes is limited, which prevents the development of robust processes,” Krishnan asserts. The difficulties mount when the viral vectors are used to modify cells.

Such is the case with chimeric antigen receptor (CAR) T-cell therapies, according to Meagher. Whether the gene delivery vehicle is an adeno-associated virus (AAV) or a lentivirus (LV) vector, or whether the vehicle is meant to carry gene editing elements consisting of messenger RNA, guide RNA, or protein, the process and generated product are both highly complex and can be characterized only to a certain degree.

Because they are so complex, it is essential to observe how genetically modified cells behave over time. The situation becomes even more complicated with genetically modified hematopoietic stem cells (HSCs) because these cells are “forever cells” that stay with the patient.

“If we are treating a two-month-old infant, we won’t really know how the treatment turns out until that child lives for 70 years,” Meagher stresses. “Research and development efforts must address this entire continuum, and we need to be assessing not only the long-term survivability of patients, but also the long-term impacts of these therapies.”

The need for speed

The promise of gene therapy is substantial. There are over 7,000 genetic diseases that could potentially be cured using gene therapy. However, while the promise, interest, and investment have been high, the technology for manufacturing and analyzing gene therapy products is still evolving. Many of the processes used for manufacturing gene therapy products were borrowed from traditional antibody processes based on adherent cells that result in very low titers. Likewise, many of the analytical methods used for gene therapies are traditional virus assays.

“While these assays are tried and tested, it takes significant time to obtain the results,” Krishnan observes. “Current assays for potency, efficacy, and other properties, for instance, are cell based and can take up to three weeks.

“Similarly, infection studies (TCID50) following downstream purification are important for confirming the capsid protein structure, but they can take up to a week. To develop a robust pharmaceutical manufacturing process, it is important to have assays in real time. To develop truly optimized processes and make effective decisions about whether to forward-progress a batch, rapid results are needed.”

The lack of optimized processes and timely testing are huge contributors to the high cost of gene therapies. Compounding the problem is the variability of the results obtained using many of the current analytical methods. Some of these methods have positive/negative variability of 50%.

“Having robust, close-to-real-time, easy-to-use, reproducible assays is crucial for further advancement of gene therapies,” Krishnan continues. “Rapid assays that provide robust responses in a reasonable period of time will enable the development of gene therapies with long-term sustainable impacts for patients and change the game completely.”

At St. Jude, which works with AAV and lentiviral vectors for gene therapies and CAR T-cell and HSC (CD34+) treatments, cell-based potency assays present challenges, according to Meagher. “Technologies for determining efficacy are currently limited with respect to the availability of predictive potency models,” he explains. “We would also really love to have biophysical characterization methods that can indicate functionality because they would be invaluable in accelerating process development, particularly for lentiviral vectors.”

Other important assays include characterization of post-translational modifications on the capsids, quantification of nonproduct DNA derived from the plasmids used in the manufacturing process, and determination of the empty-to-full capsid ratio. “The ability to conduct effective analyses in a timely fashion has a huge impact on development timelines,” Meagher notes.

Advances in automation and data analysis have the potential to reduce analysis times, according to Davies. Fully integrated systems that prepare samples in multiwell plates,
automatically place them into the instrument, run the analysis, and then seamlessly transfer the resulting data from the acquisition to the analysis software would simplify analyses while increasing consistency and accuracy. Ultimately, she would like to have the ability to fully integrate multiple devices into an automated workflow, with the ability to conditionally adjust instrument settings based on instrument sensor feedback.

Davies adds, “If we could combine new separation systems with multivalve configurations to allow for switching between separation modes and combine this with automated data processing and trending after the data is exported in a common data format, it would facilitate the development of fully automated workflows.”

The sample size dilemma

The lower titers of viral vector production processes create challenges with respect to sample volumes required for testing. In one study, Sanofi found that a standard batch of mAb produced for early-phase clinical trials generated 2,003 vials, of which 664 were required for testing, leaving 1,339 vials for the clinical study. Gene therapy batch sizes are much smaller, however, and if they are intended to treat systemic indications, the volume needed for testing will be higher.

In an example Davies cites, 185 out of 220 manufactured vials were needed for testing, leaving just 35 vials for use in the clinical study. “Even for rare diseases,” she points out, “it can be difficult to ensure sufficient material is available for clinical trials.”

Low production volumes and protein concentrations on the order of just 0.01 mg/mL are a challenge that is complicated, once again, by less mature analytical methods and limited product understanding. “Low production volumes require strategic approaches to reducing testing volumes,” says Davies, “and analytical assays for gene therapies need to be more sensitive due to the low protein concentration and the general safety requirements.”

For instance, screening of lentivirus vectors for replication-competent variants also currently requires large quantities (10–100 L per batch) to show there is <1 replication-competent lentivirus (RCL) per patient dose as per U.S. Food and Drug Administration (FDA) guidance, given average patient doses and titers, Davies attests. Similar issues exist for replication-competent AAV, and thus there clearly is a need to dramatically improve the sensitivity of the cell-based assays used for this application.

Davies notes that there are also opportunities to develop lean and strategic testing/control paradigms and stability testing designs that reduce the number of release tests using platform technologies and product knowledge. Development of novel nondestructive methods as alternatives to compendial methods will also reduce testing volume requirements. She adds, however, that any solutions that reduce the quantity of sample required must facilitate quality control (QC).

“One approach we are investigating at Sanofi is the development of multi-attribute methods that will allow us to obtain several important outputs from one analysis, thus reducing the total quantity of product required for testing,” Davies comments. Examples include methods for multiple capsid-specific and DNA-specific attributes.

Both Sanofi and St. Jude Children’s Research Hospital have developed high-sensitivity and high-resolution methods based on capillary electrophoresis (CE) for the determination of AAV capsid protein purity that require dramatically reduced sample quantities yet are QC-friendly.

Evolving regulatory guidance and constant communication

Although there is clearly not the established regulatory guidance for gene therapies that exists for recombinant protein biologics, regulatory authorities have expressed support for the evolving gene therapy field. The FDA continues to develop guidance documents to support the development and commercialization of these novel, life-changing medicines.

Most recently, the agency published guidance on endotoxins in investigational drugs. Whether gene therapy products are based on virus vectors or whether they incorporate gene editing technologies such as CRISPR-Cas9, the focus is on preventing off-target effects, according to Meagher. “The agency,” he notes, “encourages technologies that allow for fast and efficient off-target identification and quantification.”

Meagher adds that the field is moving at warp speed: “It is terrific that there is so much competition. The best technologies will win, which will only benefit patients. The FDA is learning along with gene therapy developers and has gone out of its way to create opportunities where developers can meet with agency experts to discuss the best approaches to ensure the safest products. They want to help companies figure out the best way forward.”

Looking to the future

“To realize the full potential of gene therapies for patients worldwide, suppliers such as SCIEX will continue working with drug developers to keep innovating and driving the technology forward to deliver the fast, precise, robust, and efficient analytics required to get gene therapies from concept to clinic,” states Krishnan. “Hopefully soon, there will come a point when these processes and analytics become sufficiently qualified and well established in conjunction with clear regulatory guidance to ensure that biopharmaceutical developers have a well laid out path to develop and commercialize gene therapies for the multitude of patients needing a disease-modifying therapy or cure.”

Meagher offers a research and development perspective: “We are most interested in showing therapeutic safety and in expediting clinical studies. Markets will improve as fast and reliable analytical technologies are developed, especially those methods that can bridge cell-based potency/efficacy assays. Cell-based potency assays will be a part of gene therapy release assays for the long term.

“When we have rapid assays that correlate to cell-based assays, the cost and time of product development will be reduced, which is always the goal. The greatest task is to make these therapies affordable to every patient worldwide!”

“On the production and analytical side, innovative, strategic, and digital approaches in gene and cell therapy development will advance our process and product understanding, delivering affordable, personalized therapies to patients,” declares Davies. “Not only will gene therapy methods continue to improve and get faster, but the development of orthogonal tools will help improve our understanding of the product quality attributes and their potential impact on patient safety and efficacy.”


Michael M. Meagher, PhD, is vice president of therapeutics production and quality at St. Jude Children’s Research Hospital. Mani Krishnan is vice president and general manager of the CE & BioPharma Business Unit at SCIEX. Claire Davies, PhD, is associate vice president of bioanalytics at Sanofi


To help us preview the future, we asked opinion leaders, all from outstanding technology companies, to discuss a range of new initiatives. The full list of articles is below.

Leroy Hood: Reflections on a Legendary Career
Uncharted Territory: Top Challenges Facing Gene Therapy Development
Envisioning Future Trends in Regenerative Medicine
Engineering Biology—Accelerating Transition
Bioprocessing in a Post-COVID-19 World
Sustainability and the Synthetic Biology Revolution
Sowing the Seeds of Agricultural Biotechnology
Neuroscience Widens Its Investigations of Disease Mechanisms

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