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Demystifying Synthetic Biology IV: Fermentation Bioprocess Development

Learn more about synbio and the tools and technologies advancing this field in Part I, Part II, and Part III of this blog series.

Fermentation is one of humanity’s oldest and most pivotal technological developments. Through fermentation, societies began adapting their diets nearly ten millennia ago, to include new and innovative foods such as alcohol and leavened bread, with the rise of alcohol brewing starting in 7000 B.C., and baking leavened bread beginning around 1500 B.C. When paired with new technologies, like synthetic biology, modern fermentation practices hold new opportunities beyond preserving and making foods, such as producing industrially, medically, and nutritionally important compounds. Industrial fermentation for manufacturing of these products can potentially be performed anywhere globally, assuming the technical challenges of engineering a commercially-viable strain and process are overcome. This technology offers a sustainable solution to meet the demand for high-quality products of a growing global population.

In a modern synthetic biology lab, microbes can be engineered to produce specific natural, non-native, or even entirely novel compounds by fermentation. Small-scale fermentation of these organisms allows researchers to test and optimize production before scaling up to pilot and manufacturing quantities. In this blog, we explore what factors determine a successful fermentation run, how fermentation is leveraged for strain development, and the myth that fermentation for synthetic biology products cannot be scaled.

Fermentation factors

In nature, the fermentation process occurs when microbes like yeast or bacteria break down a sugar source for energy under certain conditions that lead to a byproduct, such as alcohol. Humankind harnessed this process millennia ago for brewing beer in what’s widely considered our first foray into biotechnology. Beer was produced at scale in ancient Egypt about 5,000 years ago, with over 22,000 liters produced at a single brewery, laying the groundwork for modern-day industrial fermentation.

Despite being a natural process, fermentation can be regulated and optimized for greater output by regulating just a few key variables – temperature, acidity, oxygen, and nutrition source or feedstock – to optimize growth and production.

Temperature and acidity (pH) in particular have a significant impact on the growth of the organism which is required for fermentation. All microbes have a preferred temperature range in which they grow most efficiently. Outside of this range, they either become stressed and die, or they enter into a viable but metabolically dormant state. Similarly, microbes have a range of pH tolerance in which they can grow effectively. If acidity levels increase in their environment, microbes become overwhelmed by protons from the acid and prioritize pumping them out to stay alive. This is a common problem, as many microbes produce acidic compounds as byproducts during fermentation, leading to a natural increase in acidity that can negatively impact the growth of the organism.

Oxygen, carbon, and nitrogen sources – energy for the organism – are also critical for powering optimal, stress-free growth that allows the production of a targeted compound. Many microbes, including yeast, rely on oxygen to catalyze chemical reactions and power their growth. Microbes also require a carbon source – typically a sugar, like glucose – to drive growth and product formation. Identifying the appropriate sugar to use, how much of it to use, and when to add it to the culture must be identified early on in the fermentation process development. However, oxygen and sugar are not always essential components, depending on the organism. A select few can use other gasses like carbon monoxide and hydrogen to fuel their growth and produce industrially relevant compounds, as LanzaTech’s synthetic biology approach using waste gas as a feedstock for fermentation has successfully demonstrated.

In synthetic biology, fermentation plays an essential role from strain development to product development. A successful fermentation – where an organism grows and makes its target product – must be within the permissive range of each of the core variables of temperature, pH, oxygen transfer, and feedstock. When it comes to optimization of fermentation – where an organism grows and makes its target product to the best of its ability – another factor comes into play: volume. The volume of the fermentation varies drastically from R&D to product development to large-scale manufacturing, and must be considered during early process development.

Fermentation for strain development

Synthetic biology R&D may generate dozens or even hundreds of candidate strains and each must be evaluated for their potential growth and product profiles. Small-scale fermentation helps researchers screen these candidates, occasionally using high-throughput tools and software to iterate strain development, identifying those that require another round in the Design-Build-Test-Learn (DBTL) cycle and those that should progress to process development.

Small-scale fermentations are typically in volumes ranging from a few hundred microliters up to 50 milliliters. At these small volumes, the key variables of temperature, pH, oxygen transfer, and feedstock are highly constrained, making the fermentation process difficult to control. Micro-fermentation bioreactors may use microfluidics to regulate some of these variables, and specifically can offer better accuracy and precision on pH control than microtiter plates and flasks. For example, it is easier to add base to maintain the pH in a bioreactor, just as you would in a benchtop fermenter. However, it should be noted that the most optimal operational constraints for these variables are typically determined at a larger scale post-screening.

Dozens of strains can be screened simultaneously using high-throughput microtiter plates, allowing researchers to assess the growth profile and get an idea of how effectively the engineered microbe produces the compound of interest under less-than-ideal conditions. Once suitable candidate strains have been identified, they can be transferred from the DBTL cycle and taken forward into benchtop-scale process development. At the benchtop scale, each variable can be controlled and optimized to determine the most successful fermentation conditions in a bioreactor.

Optimizing fermentation

Benchtop scale fermentation offers the most control over the fermentation process. Benchtop bioreactors – ranging from ~150 mL to 5L in volume – are large enough to tweak each major variable, but small enough to retain tight control over the process. Common additives to fermentation cultures include acid and base solutions to regulate the pH, feedstock strategies to maximize growth and production, and oxygen to maintain an optimal dissolved oxygen transfer rate during the process.

Process optimization is performed at this benchtop scale, however, it does not reflect nor capture the limitations of pilot and manufacturing scales which are on the order of hundreds to tens of thousands of liters in volume. Transitioning to the manufacturing scale presents an entirely new challenge: the larger the fermentation volume, the more difficult it is to precisely control each variable. Gradients in temperature, pH, oxygen, and even feedstock develop across the huge volume of the fermentation culture because mixing thousands of liters efficiently is challenging – though not impossible.

Process optimization is performed with this end process in mind to pre-empt these challenges at an industrial scale. The limitations of a manufacturing-scale bioreactor’s mixing ability and oxygen input can be factored in when designing the experiments, as can understanding the typical temperature and pH gradients that form during fermentation. Researchers often use the same materials, such as growth medium components and even water, to ensure that process development conforms to the limitations and potential variations that may be encountered when scaling up, reducing the risk of failure at this stage.

Ultimately, the simpler the process, the easier it is to scale. In traditional biotech fermentation, researchers try to fit the constraints of the operating space at the manufacturing scale to their organism’s ability to tolerate the gradients that form in the key fermentation variables. Synthetic biology offers another route via strain engineering, where metabolic pathways resulting in unwanted byproducts can be altered, and strains can be selected or engineered based on their tolerance of pH and other conditions in the DBTL cycle. Rather than be limited by the fermentation process, synthetic biology has enabled us to engineer the organism to adapt to those limitations.

A common – and entirely unfounded – criticism of synthetic biology is that it cannot be scaled by fermentation. While synthetic biology faces additional hurdles such as genetic stability and product toxicity, the synthetic biology approach to fermentation has proven itself commercially successful. In 2003, DuPont developed an engineered strain of E. coli for 1,3-propanediol and is now expanding its flagship plant for the production of plant-based Bio-PDOTM. Genomatica developed a strain of E. coli for 1,4-butanediol production which was scaled successfully from 30L to a massive 600,000L commercial fermentation. In the food sector, Motif FoodWorks announced HEMAMI™, an ingredient produced by fermentation that gives an umami flavor to plant-based meat products. These examples are just a few recent demonstrations of the successful scaling of products made with synthetic biology, and in the coming years, we will see countless more.

Next Time …

In the next and final blog of our Demystifying Synthetic Biology series, we’ll delve into the world of downstream processing where we’ll explore how the product itself is key to determining the type of purifying process required. We’ll look at the tools and technologies required to separate, purify, and augment fermentation products and how strain development in synthetic biology can benefit from beginning with this end-process in mind.

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Richard Sherwin

Head of Commercialization

Richard is an industry veteran with more than 30 years of experience in the KSM, API, and intermediate markets. He is responsible for leading the commercialization and revenue generation for Antheia’s robust pipeline of products. Richard brings an exceptional track record of leading international sales teams, driving revenue growth, building strategic partnerships, and delivering innovative products to market, including ANDA and NDA developments. Richard led commercial efforts at some of the leading global pharmaceutical companies and most recently, built his own consultancy business advising a range of clients, including $1B divisions of major multinationals.

Appropriate regulatory submissions will be prepared and submitted to support Antheia’s customers who need to reference and access necessary process-related information.

Yihui Zhu, PhD

Head of Fermentation

Yihui leads the fermentation team at Antheia. With over 25 years of hands-on experience in the field, he brings in-depth knowledge and expertise in microbial metabolism and fermentation process development. He is also skilled in developing comprehensive fermentation data collection, analysis, and visualization systems. Prior to joining Antheia, he served as a fermentation lead at Intrexon and Codexis where he successfully built fermentation labs and teams and led multiple biofuel and biochemical projects to reach stretch milestones and tech transfer. Yihui is passionate about the potential of fermentation and is dedicated to advancing the field through innovative research and development.

Yen-Hsiang Wang, PhD

Head of Strategy, Partnerships, and Finance

Yen-Hsiang leads strategy, partnerships and finance at Antheia. He completed his M.S. and Ph.D. in Bioengineering at Stanford, with extensive research experience in synthetic biology, metabolic engineering and computational modeling. Before joining Antheia, he worked at McKinsey and Tencent with a strong focus in corporate strategy and big data/advanced analytics. At Tencent, he served as Director of Strategy and Business Development for the AI Lab, leading corporate initiatives in healthcare AI/ML applications and commercialization. He also served in AI4H (Artificial Intelligence for Health), a collaboration between WHO and ITU, to establish global standards for AI in healthcare.

Audrey Wang

Head of Financial Planning and Analysis

Audrey leads financial planning and analysis at Antheia. With an MBA from Washington University in St. Louis, Audrey is passionate about leveraging financial analysis, digital technology, and data analytics to guide companies in making optimal investments and strategic business decisions. Audrey has a decade of experience in helping companies solve unique problems and creating long-term impact with unconventional approaches. Before joining Antheia, she was at Vir Biotechnology and Merck where she led various FP&A workstreams, including investment valuation, asset prioritization, and manufacturing sites operation finance support. Audrey completed CFA Level II and passed the U.S. CPA exam in 2011.

Antonij Tjahjadi, CPA

Head of Accounting

Antonij Tjahjadi leads accounting at Antheia and holds active CPA license. He joined Antheia with more than 20 years of experience in corporate accounting, bringing deep expertise in ramping up accounting operations for start-up companies, SEC reporting/technical accounting, and SOX implementation efforts. Before joining Antheia, he held various leading roles in both public and private company settings, including directing accounting functions at Ambys Medicines, where he successfully implemented Netsuite with Point Purchasing integration and set up various accounting policies and processes, and played a key role in the initial public offering of Nutanix, Inc.

Ken Takeoka

Head of Biology

Ken leads the Biology team at Antheia, which incorporates both strain and protein engineering functions. He has more than 16 years of experience in the synthetic biology field, working with leading companies, including Amyris and Novartis. One of his passions is molecular biology tool development and he previously worked to build the foundation for the automated strain engineering pipeline at Amyris. At Novartis, he modernized the molecular biology techniques and established a platform to model mechanisms of antibiotic resistance in a range of organisms.

Suzanne Sato

Head of Downstream Processing

Suzy leads downstream chemistry processes at Antheia. She has 19 years of experience in process development, including route development through synthetic chemistry and scale-up of small molecule APIs for GPCR targets under cGMP for Phase I-III trials. Before joining Antheia, Suzy led a full DSP team at Amyris where she successfully pivoted developments from biofuels hydrocarbon products to pharmaceutical intermediate, flavor, fragrance and nutraceutical products. She led a team that scaled 11 products and took five products to commercial manufacturing.

Farrah Pulce, PMP

Head of Project Management

Farrah leads program and project management at Antheia. She has over 20 years of experience leading program and project management, operations, and engineering for companies across the CPG, aerospace, and automotive industries. Prior to joining Antheia, Farrah implemented and led the sustaining program management team at Impossible Foods. She also led product operations, project management, and cost optimization at Blue Bottle Coffee and Tyson Foods to develop and commercialize new products. As a certified project management professional (PMP), Farrah has a proven record of successful project delivery, improving project management practices, and building collaborative teams.

Jordyn Lee

Head of Communications

Jordyn leads communications and external affairs at Antheia. She brings a decade of multidisciplinary communications experience in helping companies make complex science and technology accessible to broad audiences, all while maintaining technical accuracy and integrity. She has a passion for visionary storytelling and translating impact across the entire communications ecosystem – her work has spanned from public relations to corporate communications to marketing. Jordyn has served as an advisor to a number of different life sciences companies and most recently led corporate communications at Amyris.

Ben Kotopka, PhD

Head of Data Science

As Head of Data Science at Antheia, Ben manages in-house software development and external partnerships for storing and interpreting research data, executing bioinformatics analyses, and streamlining business processes. Prior to Antheia, Ben worked as an academic researcher at the intersection of machine learning, bioinformatics, and synthetic biology. Following this, as an entrepreneur and consultant, he developed and deployed data science solutions for biotechnology applications ranging from metabolomics-driven compound discovery to MRI segmentation.

Guerin Kob

Head of Supply Chain

Guerin is responsible for leading the design, development, management and improvement of Antheia’s end-to-end global supply chain. He has over 15 years of experience leading high-performing supply chain and procurement teams at leading biotechnology and specialty chemical companies, with extensive experience in process development and end-to-end supply chain optimization. Prior to joining Antheia, Guerin served as Senior Director of Global Supply Chain for Sumitomo Chemical’s biotechnology division with Valent Biosciences, where he led the end-to end supply chain including procurement, logistics and distribution, integrated business planning, materials management, customer service, and supply planning functions globally.

Pavel Aronov, PhD

Head of Bioanalytics

Pavel leads the Bioanalytics team at Antheia. He has 20 years of experience in analytical and clinical chemistry, mass spectrometry, chromatography, and metabolomics. Pavel built and led the original Chemistry and Analytics team at Impossible Foods enabling strain development, fermentation, DSP, regulatory, QC, and scale-up of leghemoglobin biomanufacturing. During his academic career at UC Davis and Stanford University Pavel developed a vitamin D assay used by all major clinical diagnostics laboratories and pioneered metabolomics studies to investigate kidney disease and microbiome.

Jesse Ahrendt

Head of Quality Assurance and Regulatory Affairs

Jesse has more than 25 years of experience in regulatory affairs, quality systems, manufacturing quality, and regulated industries, ranging from early- to late-stage pharmaceuticals, biomanufacturing, consumer care, and medical devices. He has supported global product launches and the underlying quality supply chain components in industries that require strict adherence to internationally accepted quality standards. Before Antheia, he led quality efforts at Zymergen and Sandoz, and supported many global pharmaceutical companies during his time in Biotech Consulting at NSF International, all to bring quality to the forefront in manufacturing, standardize global processes, and support customer regulatory requirements.

Heidi Pucel

Chief People Officer

Heidi is a results-driven human resources executive and HR business partner who leverages decades of experience in empowering, motivating, and inspiring to drive transformation within high-performing and rapidly-growing workforces. A certified executive coach and passionate advocate for people-oriented solutions, Pucel serves as a partner to executive teams to design programs that support employee development, engagement, and recruitment and retention. Pucel most recently served as Chief People Officer for Countsy, where she worked as an interim HR executive for clients in the biotechnology and software industries, such as Ceribell and Tune Therapeutics.

Zack McGahey

Chief Operating Officer

Zack is a leading executive in operations management, specializing in bioprocess engineering and manufacturing management. He has over 20 years of experience leading manufacturing functions for companies across the pharmaceutical, synthetic biology, diagnostics, and automotive industries. Before joining Antheia, Zack was VP of manufacturing and capex project management at Zymergen. He also gained experience managing commercial scale facilities operations for Tesla, where he was responsible for managing 10 million square feet of factory, lab and warehouse space during the Model 3 ramp.

Kristy Hawkins, PhD

Co-Founder & CSO

Kristy has over 20 years of experience in the field of synthetic biology, focusing on yeast metabolic engineering for the production of small molecules. She did the founding work on the benzylisoquinoline alkaloid pathway during her graduate studies and gained valuable industry experience at Amyris and Lygos. Kristy is an expert in tool development, high-throughput screening, and host strain and heterologous pathway engineering.

Christina Smolke, PhD

Co-Founder & CEO

Christina is a pioneer in synthetic biology and metabolic engineering, where she has over 20 years of experience. As Professor of Bioengineering and Chemical Engineering at Stanford University, her laboratory led the breakthrough research to engineer baker’s yeast to produce some of the most complex and valuable medicines known. Under her leadership, Antheia’s synthetic biology platform enables new possibilities for drug discovery and efficient, sustainable, transparent, and on-demand drug manufacturing at scale. Her vision and accomplishments have garnered numerous awards, including the Chan-Zuckerberg Biohub Investigator, NIH Director’s Pioneer Award, Nature’s 10, Novozymes Award for Excellence in Biochemical Engineering, and TR35 Award.

U.S. Secretary of State Antony J. Blinken visits Antheia to discuss biotechnology innovation

Appropriate regulatory submissions will be prepared and submitted to support Antheia’s customers who need to reference and access necessary process-related information.