For decades, Escherichia coli (E. coli) has reliably produced hormones, enzymes, vaccines, monoclonal antibodies, and fusion proteins. With a fast reproduction rate and minimal nutrient requirements, E. coli enables rapid biologics production through a long-proven fermentation method offering simplicity, speed, and scalability. However, unlike mammalian systems, proteins expressed in E. coli lack post-translational modifications (PTMs) that improve stability and reduce immunogenicity. As next-generation biologics increasingly target applications that don't require PTMs, microbial fermentation is experiencing a resurgence.
This renewed focus brings both opportunity and challenge. While E. coli offers advantages in efficiency and cost-effectiveness, maximizing these benefits requires proper planning and specialized capabilities, and scientific teams that can work through these complexities. In this post, you can find a basic roadmap for embracing the new age of E. coli-based microbial coli-based biomanufacturing—from expression system design through fermentation and midstream processing—is essential to ensure reproducibility at any scale.
The compressed timeline of microbial fermentation—approximately 48 hours compared to two or more weeks for mammalian cell culture—leaves little time to analyze problems and implement corrections. This is why thoughtful host strain selection and expression system design at the outset are critical to long-term success.
Choosing the right strain requires understanding of how it will modify your expressed protein, and which yields the lowest product-related impurities. Analytical technology can reveal these differences, helping minimize downstream purification burdens. However, expression system design must balance yield with robustness. While some genetic modifications boost productivity, they can result in unwanted effects such as an increased shear sensitivity or reduced oxygen tolerance. At small scale these strains may look promising, but at larger volumes and in later phases such physiological changes can reduce overall product yield. The key is designing a system that delivers both high expression and the resilience needed for reliable manufacturing at any scale.
Robust and scalable fermentation development hinges on optimizing both growth and expression phases. During growth, adequate oxygen transfer is essential—microbial cultures consume oxygen rapidly, requiring approximately 100 times the gas flow rate of mammalian systems. Dissolved oxygen must be precisely controlled through increased agitation, enriching oxygen percentage in sparged gas, or raising gas flow rates. Equally important is controlled nutrient delivery: providing nutrients at the right time, rate, and amount maintains microbial metabolic health and balances cell growth with protein production.
The induction stage represents a critical transition where metabolic pathways switch from cell growth to product expression. Success requires optimizing nutrient concentration and timing to express target protein while limiting fermentation byproducts, adjusting temperature to control growth and promote stability, and balancing induction duration for yield and quality. Process analytical technology (PAT) enables tighter control through real-time data, ultimately delivering higher quality and greater reproducibility between manufactured lots.
Following fermentation, midstream steps separate the desired protein product from the host cells for purification. As upstream fermentation yields continue to improve, downstream purification has increasingly become the main bottleneck in microbial manufacturing. This makes optimizing midstream processes essential for maintaining overall production efficiency.
The choice of harvest method—whether to collect product as part of a cell paste or to lyse cells and release proteins prior to separation—is largely driven by which host strain is used and where the protein is expressed (intracellular, periplasmic, or extracellular). Understanding where the protein is produced, its stability profile, and optimal processing conditions—including speed, temperature, and pH—helps drive midstream optimization decisions that minimize impurity introduction and maximize downstream purification success.
Every biologic product is unique, requiring flexible platform processes optimized to meet specific needs. Some of the most notable outsourcing partners in our industry have set processes that work. However, rigid adherence to set protocols can limit development potential, stability, and titer yield for your particular product. Finding a CDMO partner that adapts their facility and processes to fit your product is key to ensuring your product’s design specifications are met.
As an experienced partner with over 35 years of experience in E. coli fermentation, AGC Biologics can do more than just help you scale. We carefully review each process from end-to-end, identify risks, and mitigate those risks to save timelines, reduce costs, and create robust, scalable processes that meet your needs. Having brought seven commercial products to market, we understand regulatory standards and have the experience to guide the most complex products from the clinic to widespread market availability.
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Want more research? Read our latest white paper, “How To Develop Reliable, Repeatable Microbial Processes At Any Scale”, about microbial fermentation best practices for drug production.
Incir, I., and Kaplan, O. Escherichia coli in the production of biopharmaceuticals. Biotechnic Appl Biochem. 2025;72:528-541. DOI:10.1002/bab.2664