Biotechnology complements chemical synthesis in manufacturing of small organic molecules, often providing improved functionality and higher product purity. How does a biotechnology manufacturing process for small organics look like? What is the typical process flow and unit operations at large scale? Let us unpack a commercial campaign and highlight relevant trends, bioprocess metrics and challenges.   

Figure 1: Block flow diagram of the manufacturing process.

Upstream processing

Upstream processing begins with inoculum preparation from a working cell bank (WCB). In this example, a genetically engineered bacterial strain was employed, leveraging metabolic engineering strategies such as targeted gene deletions, pathway overexpression, and cofactor balancing to enhance biosynthetic efficiency. The culture was expanded stepwise through a seed train, transferring from small inoculum volumes to progressively larger seed fermenters until reaching production scale. Main fermentation was operated in fed-batch mode, with feeding initiated upon depletion of initial carbon sources, indicated by a dissolved oxygen (DO) spike. The feeding strategy combined exponential feeding for rapid biomass growth with linear and later constant feeding to promote product formation. The production phase was triggered by a one-time temperature shift, otherwise temperature as well as pH were maintained constant, with pH controlled via automated ammonium hydroxide dosing. DO was regulated primarily through dynamic aeration, with only minor adjustments to agitation and pressure. Optimized feeding and DO control were critical to prevent accumulation of overflow metabolites such as acetate, which can significantly reduce process yield.

Figure 2: USP process profile. The charts illustrate the relative trends of bioprocess control parameters (dissolved oxygen - DO, temperature - T, pH, pressure, airflow, feed flow, stirring). On the right-hand side, product, biomass, and by-product (acetate) formation throughout the fermentation is displayed. Values and scale have been intentionally omitted to emphasize trend visualization and comparative patterns rather than precise figures. The picture displays the upper floor of the upstream manufacturing asset.

Harvesting and downstream processing (DSP)

After completion of the fermentation stage, the broth was subjected to cell lysis using a chemical agent. Cellular debris and high-molecular-weight impurities were removed through a series of filtration steps. The clarified broth was then concentrated via reverse osmosis followed by evaporation. The resulting concentrate underwent crystallization, and crystals were separated from the mother liquor by centrifugation. If the impurity profile did not meet specifications, the crystals were redissolved, filtered, and the crystallization–centrifugation cycle repeated. The process concluded with vacuum drying of the crystals and milling-sieving to achieve a uniform particle size in the final product (Figure 1).

Analytical control was maintained throughout the process, including in-process measurements, impurity profiling, and final product quality assessment using advanced instrumentation such as HPLC, FTIR, NIR, UV-VIS spectrophotometry, and moisture analyzers

Manufacturing performance and challenges

Even mature manufacturing processes can present operational challenges. During the showcased campaign, key issues included microbial contamination, oxygen transfer limitations leading to productivity deviations during fermentation, membrane fouling during harvesting, and extended drying times coupled with crystal handling difficulties in downstream processing (DSP).

These challenges were addressed through enhanced sterilization and cleaning protocols, optimized feeding strategies with dynamic feed control, adjustments to aeration and pressure, and equipment upgrades in case of dryer and advanced CIP (clean-in-place) systems for crystallizers.

The implemented improvements delivered an 18% increase in volumetric productivity during fermentation compared to the initial campaign stage. Overall process yield improved by 10% (Figure 3). A slight yield reduction in harvesting and DSP was observed, primarily due to stricter cleaning protocols introduced to ensure quality and maintain a success rate above 99% (Figure 3).

Figure 3: Manufacturing campaign performance. Values and scale have been intentionally omitted to emphasize trend visualization and percentual changes rather than precise figures. 

 

Summary

Biotechnological production of small molecules offers a sustainable alternative to chemical synthesis, delivering high purity and stability. Optimized fed-batch fermentation, combined with robust downstream processing, can achieve high yields and consistent quality. Trade-offs between yield and quality are inevitable and should be managed proactively. Continuous improvement, through dynamic process control with real-time analytics, preventive maintenance, enhanced cleaning protocols, and equipment upgrades, is critical to remain competitive in a rapidly evolving market.

Our offer

•               Over 40 years of experience with various bioprocesses
•          Large as well as small molecule biomanufacturing
•          Fully integrated CDMO services in the field of industrial biotechnology
•          Engagement at any stage of product/process development 
•          99% batch success rate
•          Excellent on-time in full delivery performance with over 60 processes transferred                to commercial scale in the past decade
•          Strong focus on continuous process improvement  

Acknowledgments

This work was funded by Arxada AG, Peter Merian-Strasse 80‎‏‏‎, 4052 Basel, Switzerland.

 

For further information and/or if you would like Arxada to support your project(s), get in touch with:

myproject@arxada.com