Accelerating the Development of High-Quality Cell and Gene Therapies with Comprehensive, In-house Analytical Support

Hui Chen, Ph.D., Vice President of Analytical & Quality Control and Gene Therapy at Porton Advanced, sat down with Pharma’s Almanac Editor in Chief David Alvaro, Ph.D., to discuss the company’s unique analytical capabilities for supporting viral vector and plasmid DNA manufacturing.

David Alvaro (DA): Could you give me a general overview of the scope of analytical development capabilities that Porton Advanced offers?

Hui Chen (HC): We parse the analytical work between two separate departments: an analytical development group and a quality control (QC) group. The analytical development group focuses on method development, optimization, and validation. Once a new method has been validated, it is transferred to the QC department. Our QC colleagues use validated methods to perform the quality control of our final products, such as viral vectors, oncolytic viruses, plasmid DNA, and even mRNA and cell-based products, for worldwide markets.

Within the analytical development department, we have several different groups based on the technology and functions involved. For example, we have a physical chemical group and a bioassay group. In the bioassay group, we’re also divided into groups focused on ELISA, cell-based assays, FACS assays, and molecular analyses, including qPCR and ddPCR. With our full range of capabilities, Porton Advanced can cover nearly 95% of all assays for viral vectors and plasmids.

The QC department has the same groups with the same capabilities, plus a microbiology group that focuses on safety analysis for products and in-house materials, such as water-for-injection, and conducts GMP environmental monitoring and other activities related to assuring sterility and safety.

For all viral vectors, we must outsource mycoplasma culture testing and sequencing tests (if applicable); for lentivirus, determination of replication competency and adventitious virus testing. Other than these three assays, we perform all analytical work in-house.

DA: For a viral vector project, can you tell me how Porton Advanced develops an analytical strategy early on for a client that ensures that the project is supported through its entirety?

HC: We start by discussing with the customer the intended application for the product and their specific needs with respect to final product titer and volume. The indication is very important, because it has a direct impact on the nature of the product. For instance, and AAV-based ophthalmic gene therapy will be very different from an AAV-based therapy to treat a liver disease in terms of the dose and the acceptable impurity levels. Titer considerations include not only the capsid titer, but also the viral genome titer and the transfection titer. There are also different requirements for different vector types. On the basis of the specifics of the project as provided by the customer, we select the critical assays that must be included on the quality control list

We also discuss the customer’s existing process in detail, because in order to establish effective in-process controls (IPCs), it is essential to understand the process. With this information, we build an IPC assay list of analytical tests that will help ensure achievement of a stable process.

Once we determine the assays that will be required, we develop or optimize those assays for the particular customer process. Typically, more than 80% of the assays we use are based on our internal platform assays, with some degree of optimization for the specific molecule. As a result, assay development time is often decreased significantly. For assays that must be developed from scratch, it typically takes about four weeks to develop an optimized method. Starting with our platform methods, that time is reduced to one to two weeks.

A method validation protocol is then developed for the optimized methods, and, once approval is received from the customer, validation is completed and the method is transferred to QC for final GMP product quality control or release testing.

DA: What individual tests or aspects of the analytical development for viral vectors are the most challenging or in the most need of innovation because they are time-consuming, expensive, or create bottlenecks?

HC: One of the most challenging tests is the RCL (replication competent lentivirus) assay for determining whether LV vectors can replicate. Replication-competent LVs are virus particles capable of infecting cells and replicating to produce additional infectious particle and thus present a safety concern. The RCL assay is a crucial test and is expected by the regulatory authorities.

The difficulty is that this test requires the use of live virus as positive control samples. While the virus is weak, it still means the assay must be performed in a biosafety level (BSL) P3 laboratory. In addition, the cells that are required for the assays are complex. Without access to a P3 laboratory, we must develop the assay without the positive control samples, which is quite a challenge.

DA: Are there specific analytical challenges as projects move from early to late-phase development and commercialization?

HC: The biggest challenges are faced during the early stages of process development. At this point, the process conditions are not yet established, and often neither are the product specifications. The process isn’t optimized, either, so there are many impurities, and samples can be quite complex. Once we have developed effective methods, they generally transfer well to GMP use, because the samples involved at the clinical and commercial phases are quite pure.

DA: What is the role of automation and inline testing in the cell and gene therapy space?

HC: There is a mix of automation and manual work. For instance, we have an automated sample extractor for collecting samples for qPCR analysis. We also have plans to implement more automated solutions for plasmid DNA (pDNA) analysis, such as automated pipetting and so on. The goal is to minimize manual labor and human interactions to save time and reduce the likelihood of error.

Automation of analyses for viral vectors and pDNA is important because there are many different assays — typically 20 or more — to be performed. Automation helps save time. It is also possible because we have access to sufficient sample quantities.

DA: How divergent are the regulatory requirements from the different global agencies for this kind of analytical testing?

HC: China classifies all viral vectors as drug substances, including LV vectors used to modify cells, even though in this case they are intermediates. That is not the case in the United States and Europe, where these LV vectors are considered raw materials.

Assay requirements at the IND phase also tend to be stricter for the Chinese authority compared with those of both the FDA and the EMA. The specific assays are basically the same, but with slight differences. For instance, the requirements for sterility testing involve a microbial screen, but what microbes must be screened for differs from one agency to the next.

Validation requirements are also different. To submit an IND in China, the methods must be validated. For the FDA and EMA, however, an IND application only needs to have methods that are qualified. Qualification is much simpler and focuses on specificity and the sensitivity. Validation requires demonstration of specificity, sensitivity, accuracy and robustness and is a much more complex process.

DA: Does your experience working under the stricter Chinese system influence the recommendations you make for clients?

HC: Normally, we recommend that clients meet the maximum requirements of the authority. We develop our product criteria on the basis of those requirements. For customers from China and those with experience developing products for the Chinese market, we can typically move directly forward with our service offerings. We also have big pharma clients that provide their specific requirements, and we deliver on them.

For customers that don’t have experience, we give them suggestions depending on the market(s) they are targeting. We also have strategic consulting partners that can help these customers with their IND applications for the NMPA and the U.S. FDA.

Of course, we always have to keep the customer’s budget in mind when recommending whether the minimum or strictest requirements should be met. For instance, achieving the strictest purity levels for AAV vectors involves extensive downstream processing and loss of product and thus a much higher cost. We work to find the right balance that assures quality and safety and meets at least minimum regulatory requirements — ideally more than that — in a cost-effective manner.

DA: With respect to changes, how do you manage the adoption of new, better methods once a filing has been submitted? Do you have a strategy for demonstrating the equivalence of different methods?

HC: That is one benefit of having platform assays. When a new technique comes out, we will still use the platform assay in parallel with the new method. When we believe the new method is ready for adoption, we will propose its use to the authority and introduce the method to our customers and show them the value it brings.

DA: What assays/analytical technologies for novel therapies have changed the most in the past couple of years? What do you anticipate for the future?

HC: The COVID-19 pandemic has had a big influence over the last year. The approval of mRNA vaccines has attracted a lot of financing into the mRNA field, and some of that money has been directed at analytical method development. Although mRNA technology has been in development for decades, the delivery systems used today are still not mature. The use of mass spectrometry (MS) has been a real enabler of technology advances in this area. That success is having a lot of influence, and we are seeing greater use of MS in the cell and gene therapy field, as well.

For cell therapy, flow cytometry has been an important tool in development laboratories that is now advanced to the point where it can be applied in a GMP setting.

DA: What do you believe is unique or differentiating about the analytical capabilities of Porton Advanced compared with other CDMOs that support cell and gene therapy manufacturers?

HC: Porton Advanced has biological, physical, and chemical assays grouped in one department. Many CDMOs, for instance, don’t have mass spectrometry capabilities within their gene and cell therapy departments. We have worked to develop in-house expertise in applying the whole gamut of assays to viral vectors, plasmid DNA, and so on.

Having an integrated assay group also makes it possible to really get to know and understand the customers’ products, because there is in-house access to such a wide range of characterization technologies. The need to outsource these activities to different parties and then collect all the data, on the other hand, typically limits the number of different tests that are performed. In addition, all the small details and other information that are gained through the process of developing and conducting assays would be missed. For CDMOs that outsource analytical development and testing, therefore, it is very difficult to really see the entire picture and fully understand their customers’ molecules.

Because we have integrated and comprehensive in-house analytical capabilities, Porton Advanced is able to decrease method development times and fully characterize drug substances and drug products to ensure that they are produced with the highest level of quality and safety in less time and using less material at lower cost. We help our customers make their products cheaper and faster but with a tighter quality profile.

Hui Chen, Ph.D.

Dr. Hui Chen has more than 10 years of experience in analytical development and quality control for biologics and cell and gene therapy. She has experience with new drug and biosimilar application procedures to authorities (U.S. FDA, EMA, and CDE). She got her Ph.D. in pharmaceutics at KU Leuven, Belgium, and obtained her MBA at Rotterdam School of Management, Erasmus University, the Netherlands, and an exchange MBA at Duke University. At Porton Advanced, Dr. Chen leads the gene therapy department and the analytical development department as Vice President.