High-Growth Microbial Fermentation for the Manufacture of Biologics

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Challenges in fermentation can be addressed through equipment changes, facility design, and process development.

Biologics have revolutionized drug delivery, offering innovative therapies for a range of diseases, including cancers, autoimmune and other genetic disorders, and infectious diseases. To meet booming demand—the market is projected to reach $27 billion by 2027 (1)—biopharmaceutical manufacturers are increasingly turning to high-growth microbial fermentation as a biologics production platform. This includes the use of expression systems such as Escherichia coli (E. coli) and the yeast, Pichia pastoris, that are programmed to generate recombinant proteins (e.g., human growth hormone), peptides, enzymes, antibody fragments, and the plasmid DNA (pDNA) and messenger RNA (mRNA) needed for vaccines.

Many companies, from biotech startups with early-phase biologics to companies with products in commercial production, have made this switch from mammalian cell culture to microbial fermentation, largely because microbial expression systems are more cost-effective, amenable to new modalities such as mRNA and cell/gene therapies, and faster—production runs can be completed in days instead of weeks.

Despite the attraction of speed and cost-effectiveness, fermentation comes with its own challenges, including maintaining sufficient oxygen transfer and removing metabolic heat. In this article, the authors explore how these challenges can be addressed through equipment changes, facility design, and process development.

Equipment advancements for high-growth microbial fermentation

Microbial fermentation has been used for decades to produce biologics, including insulin and other recombinant proteins. Microbes are much less vulnerable to mechanical shear than mammalian cells and able to withstand more vigorous mixing, which can be advantageous for increasing the efficiency of mass transfer. Faster cell growth, however, demands higher oxygen transfer rates (OTR) and leads to the generation of excessive metabolic heat. Fermentation units must have sufficient cooling capacity to avoid adverse effects on microbial cultures and product yield and quality.

For these reasons, microbial fermenters need to be fitted with radial impellers to ensure adequate gas dispersion within the broth and oxygen enrichment to maximize cell growth, as well as adequate heat transfer surface area and coolant flow to remove heat generated from metabolic growth and vessel agitation.

Cooling stainless-steel fermenters. Large-scale commercial production of biologics generally requires stainless-steel fermenters, some as large as 20,000 L. Adequate jacket cooling typically becomes a problem when fermenter volume surpasses 1000 L, at which point further equipment modifications are usually required. Large fermentation tanks with supplementary heat transfer capabilities have been used for many years, but the fit/finish of these vessels may not live up to the exacting standards of contemporary biotech. More recent heat transfer innovations, such as internal helical coils and vertical tube bundles, are compliant with cell-culture guidelines and are designed for the excessive production of metabolic heat. They can, however, be difficult to clean.

There are suppliers that provide vertical panel upgrades with internal cooling coils that resemble traditional vessel baffles for increased heat transfer and to sidestep the cleaning challenges of older equipment, which can be applied to fermenters as large as 17,000 L.

The benefits of single-use fermenters. Single-use fermenters (SUF) are ideal for smaller-scale (20 L–2000 L) biologics manufacturing and needed during development, early phase clinical trials, the treatment of rare diseases, or for personalized medicine applications where one batch is produced per patient. They are a flexible and less-expensive alternative to the high costs of stainless steel. SUFs allow more rapid transition from batch to batch, while removing the need for clean-in-place (CIP) and sterilization-in-place (SIP) procedures, reducing the risk of cross-contamination, and lowering the cost of manufacture.

Microbial SUFs have sufficient mixing and cooling capacity to perform as well as stainless-steel fermenters of the same volume for high OTR processes exceeding 300 mmol/L-h, which is typical of E. coli and P. pastoris.


Harvesting tanks. End-of-fermentation broth is collected in harvesting vessels, some as large as 12,000 L. The fermentation broth is processed to separate cells from liquid, using centrifugation or tangential flow filtration (TFF) on a microfiltration skid, followed by homogenization if the product is within the cells. The resulting mid-stream in-process material is then transferred to downstream processing equipment. Sometimes, it is necessary to perform depth filtration during mid-stream processing to clarify the product-containing liquid before it enters downstream processing.

Bioanalytical testing. Analytical testing supports process development and ensures quality throughout scale-up and, ultimately, during manufacturing. Equipment and technologies are needed to characterize and measure drug products, ranging from proteins to mRNA. This includes testing such as product titer, enzymatic activity, electrophoresis for assessing the integrity of products like pDNA and RNA, peptides, and proteins, pDNA/mRNA sequencing, and tests for impurities, such as bioburden, host cell protein, and endotoxins.

Automation and digitalization. Automation and robotics can streamline and enhance process development and production runs. Here are a few areas where automation and advanced digital tools can play a significant role in reducing the manual labor burden while ensuring the safety and quality of biologics:

  • automatic pipetting to analyze resident screenings during analytical testing
  • small-scale multiple chromatography runs to test for ideal resins, salts, buffers, and pH, as well as column lifetime coupled with biostatistical analysis to analyze the results and set ideal operating conditions
  • process analytical technology (PAT) to collect in-line, real-time data to regulate and control key culture parameters (e.g., oxygen, temperature) and critical indicators (e.g., product titer)
  • various studies to measure the removal of impurities.

Facility design

From small-scale production to commercial batches. The design of a biologics manufacturing facility must take numerous considerations into account, including the scale of production. Flexible design and equipment choices can make it possible to scale from small batches, such as those used for personalized medicines, to commercial production of metric tons of purified recombinant proteins annually. Combining both SUFs (for smaller-scale production) and stainless-steel fermenters (for commercial production) in the same facility allows scale-up to occur internally and enables a company to take a promising drug candidate from a clinical program to commercialization without needing to outsource.

Enabling tech transfers. When a company has a biologic candidate moving through clinical trials and either doesn't want to handle large-scale internal production or has constraints due to supplier capacity, it might turn to a contract development and manufacturing organization (CDMO) for their expertise in high-growth microbial fermentation. The first part of these technical handovers begins with a familiarization process, during which information about the current process parameters is shared with the CDMO. Typically, the existing process would be optimized or altered as needed by the CDMO, or in some cases, a new one developed. This can be approached with an initial review to identify areas in the process that could be enhanced for commercial production, which includes making changes to the facility's fit. Afterward, a thorough gap analysis further refines these changes, ensuring that all necessary adjustments have been considered. The remaining gaps are bridged with dedicated process development and facility tailoring, aiding proper scaling.

Having a range of fermenter sizes also supports tech transfers of high-growth cell cultures at any phase of development, including lab scale (typically 250 mL–30 L), clinical trials (typically 100 L–1000 L), or all the way up to commercial campaigns (up to 17,000 L). Having this range allows a CDMO to do comparability studies of a supplier’s process at any scale. Ideally, scale-up tests are performed at the same time as bioanalytical testing for qualification and validation to reduce the time to commercialization.

Process development

Upstream process development. Established cell lines need to be tested for fermentation conditions that will be ideal to scale a process and provide the best yields of a purified recombinant protein, including broth composition, mixing speed, oxygen levels, pH, and temperature. These tests also need to land on the range of operational parameters that will achieve appropriate drug substance quality.

Downstream purification and isolation. Following lysis and removal of the cultured cells, the biologic drug substance is typically purified through rounds of chromatography plus, on occasion, different modalities, then concentrated and buffer exchanged with TFF to produce final bulk drug substance. There are fewer downstream processing parameters that are scale-dependent. Still, these downstream processes also need to be optimized with studies to:

  • test centrifugation and homogenization
  • screen for ideal purification conditions with robotic chromatography, as well as for the cost effectiveness of using each chromatography resin at the commercial scale
  • assess the removal of impurities at each purification stage.


Microbial fermentation is an attractive alternative to mammalian cell culture, as long as the required protein is non-glycosylated. While it cannot replace mammalian cells for certain proteins that require full, complex human or human-like glycosylation, it is a cost effective, scalable, and flexible option for non-glycosylated proteins and pDNA production.


  1. Technavio. Biologics CDMO Market by Type, Product Type, and Geography–Forecast and Analysis 2023-2027. p. 163. December 2023. (accessed, February 2023). https://www.technavio.com/report/biologics-cdmo-market-industry-analysis?&search=biologics

About the authors

Cameron Graham is senior process engineer, Biologics at BIOVECTRA. Gregor Awang is specialist, Biologics Development and Innovation at BIOVECTRA.