By Antoine Awad, chief operating officer, Synlogic, Inc.

The rise of high-throughput molecular biology and DNA sequencing, in parallel with the increased sophistication of computational models, has enabled the field of synthetic biology, where precision genetic engineering is used to program bacterial cells in much the same way we program computers to perform different functions. In 2014, our co-founders, Jim Collins and Tim Lu, recognized world experts in synthetic biology, pitched the idea to Atlas Ventures of forming the first company that would apply the principles of synthetic biology to the creation and development of biotherapeutics. The idea was that this approach would allow us to address significant medical needs using a completely new approach based on our drug candidates, which we call synthetic biotics. Within eight years, Synlogic opened five INDs with the FDA, dosed more than 350 patients, and built a clinical-stage pipeline focused on metabolic and immunological diseases. This includes achieving proof of concept in one program (in phenylketonuria, or PKU), and proof of mechanism in another (hyperoxaluria, or HOX). From the beginning, we knew that as pioneers, manufacturing would present challenges and also would be a critical success factor.

Our drug candidates to date have used the same starter strain, or chassis, a well-studied probiotic called E. coli Nissle 1917. As live potential biotherapeutics, these present unique challenges. As is the case with many biotechnology companies, especially those advancing innovative therapeutic approaches, we evaluated the benefits of outsourcing manufacturing to third parties that have specialized expertise in producing medicines based on synthetic biology. We started discussions early in our development programs and assessed all our options to determine the optimal pathway that would deliver the levels of quality and precision that are essential in development of drugs based on synthetic biology.

Using E. coli Nissle (ECN) has advantages in a history of robust safety data validated in more than 100 years of clinical research. A challenge, however, when producing ECN as synthetic biotics is the need to strike a balance between increasing cell densities and inducing target enzymes. A disproportionate focus on one of these parameters can have an adverse effect on the other. While the technology to grow cells is very effective, the cells need to be kept alive and able to maintain high viability, which is imperative to their proper function in disease targeting. Fermenting bacteria for protein production is common, but expertise in maintaining high cell viability is both essential and rare.

To help address these challenges, we reached out to contract development and manufacturing organizations (CDMOs) with specialized expertise. While many CDMOs were using fermentation techniques for industrial purposes, these technologies would not meet good manufacturing practice (GMP) standards and FDA compliance guidelines for production of biotherapeutics. Many also do not have both fermentation and lyophilization (freeze drying) capabilities under one roof. The ones that do often have limited lyophilization capacity that does not align with fermentation scaling. Among the limited number of CDMOs that will work with live bacteria, most have long lead times and high costs, especially following demands on production associated with the COVID-19 pandemic.

Given the limited options for third-party support available, Synlogic invested in manufacturing to meet our needs at every phase of development and to keep a vigilant focus on product viability. Our drugs include cells that must remain metabolically active; over time, they will die unless they are formulated into a stable powder. To minimize the duration of our processing time, we decided to co-locate fermentation and downstream processing and lyophilization to prevent cell death and maintain high drug viability. We also implemented a lyophilization step that enhances the shelf life of our therapies and allows for more patient-friendly presentation as an oral powder.

In operations involving fermenters, lyophilizers, and analytical instruments in quality control settings, automation is critical to make processes efficient and minimize production costs. For example, a fermenter for E. coli Nissle must run between 16 and 22 hours. Without automated capabilities, this process would require manufacturing operators to be on-site around the clock. Automated technologies also play a central role in helping us meet both demand and quality control (QC) requirements at every stage of the product life cycle.

Our ambr 15 and ambr 250 high-throughput automated bioreactors or fermenters are used in process development, process optimization, and scale down models. With these systems, we can test different conditions and process parameters in a short timeframe and at low volumes, which gives us a quicker path to an established process while reducing costs per experiment. We have another high-throughput automatic analyzer that enables screening and analysis of fermentation metabolites. With this production system in place, we can better understand what is required to keep cells healthy, growing, and active. The technology also allows us to be faster and more confident in our decision-making and potentially reduce cycle time.

We also implemented a range of single-use technologies throughout our facility as well as customized processes to address specific challenges in manufacturing our biotherapeutics. Single-use technology allows us to switch between programs faster by minimizing required cleaning and risk of cross-contamination. It also reduces the facility footprint, thus decreasing the necessary up-front capital investment. We also established a cleanroom that incorporates procedures and layouts that reduce the risk of microbial contamination and product cross-contamination through an air pressure cascade, segregation of product operations, and cleaning requirements.

One of the major challenges with any new technology or therapeutic approach is the ability to rapidly scale manufacturing as needed from early-stage research through to commercialization. Recognizing our needs in terms of scaling up as well as the challenges in considering both in-house capabilities and engagement of CDMOs, we quickly recognized the potential benefits of a hybrid approach.

Our physical cleanrooms come with a menu of services that can be handled by CDMOs, including inventory control, warehousing, environmental monitoring, and other support areas. Meanwhile, we built an internal infrastructure at Synlogic that is able to meet product needs based on available resources and our own highly experienced staff who are trained in GMPs. Our in-house capabilities include process development, analytical development, formulation, current GMP production, packaging and labeling, QC, and quality assurance. In a hybrid model, we have the flexibility to outsource some of the required tests and assays to labs/CROs when needed. The facility was also designed to handle our process needs with the ability to readily scale up and expand further as our development programs advance.

When planning a manufacturing strategy, it can be advantageous for biotechnology companies to co-establish research and CMC process development in the same facility, allowing for more efficient exchange of technical expertise. Generally, companies advancing a program into clinical development can often handle production needs related to Phase 1 or Phase 2 clinical trials internally when required scales are more modest.

It is important that companies consider investing in automated processes wherever feasible and recognize that scaling up can require larger equipment and potentially exponential increases in the need for raw materials and consumables, many of which can have long procurement times. Planning early is essential to address potential supply chain issues and avoid bottlenecks. It can also often be advantageous to consider collaborating with regulators and other stakeholders early in the development process. Early input from regulatory agency contacts and consultants can support smoother transitions as companies advance to later stage clinical development.

Whether companies decide to establish in-house manufacturing capabilities, outsource to CDMOs, or build a hybrid model, planning to meet production goals at every stage can require significant levels of innovation and flexibility. Teams must be prepared to address new challenges and make quick, thoughtful decisions throughout the product life cycle to be successful. These demands can be even more important in emerging areas of research such as synthetic biology that can require development of entirely new and previously untried strategies and technologies to keep manufacturing on track.

About the Author:

Antoine (Tony) Awad is chief operating officer at Synlogic. He has more than 18 years of experience in the biotech and pharma industry with substantial experience in the development and manufacturing of novel therapeutics from pre-IND studies through global commercialization. Prior to joining Synlogic, he was most recently at Abpro Therapeutics and served as senior vice president of CMC and operations, where he was responsible for the development of bi-specific antibodies for oncology and leading corporate operational functions. Previously, he was at L.E.A.F. Pharmaceuticals and Merrimack Pharmaceuticals. Awad is a graduate of Boston University and holds a bachelors degree in biochemistry and molecular biology and conducted graduate research at Boston University School of Dental Medicine.

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Manufacturing Biotherapeutics Based On Synthetic Biology Lessons Learned - BioProcess Online