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Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.
Biological and analytical advances enable modern fermentation processes to deliver safe and effective next-generation medicines.
The pharmaceutical industry is no stranger to fermentation. Synthetic biology has been used for the production of both biologic and chemical APIs for decades. While it has not been the predominant method in either sector, the greater sustainability and ability to access novel structures provided by fermentation processes are driving greater interest, particularly for small molecules.
Synthetic biology “will propel companies that understand the right strategy for the distinct value propositions it offers at each step in the production life cycle,” said Gihan Hewage, previously a Lux Research analyst, in a press release (1). “Companies with products in the ideation and early development stages will differentiate most from flexible production and the ability to create novel products, whereas products in later stages of development can leverage the environmental and marketing benefits of the technology,” he added.
Cell culture, while often more suited for the production of biologics, such as proteins and monoclonal antibodies, is more expensive and often more complex. Microbial fermentation tends to be, according to Jose Luis Barredo, biotechnology business director with AMRI, more straightforward and scalable. One key reason is that the organisms have faster and more stable growth patterns and lower intrinsic metabolic burden, notes Filippo Giancarlo Martinelli, head of global industrial partnerships development at Abolis.
In addition, fermentation often involves the conversion of cheap and readily available raw materials (sugar or other cheap substrates) into high-value products with high yields/titers, ensuring the commercially viable manufacture of high-quality products, observes Nikolay Krumov, senior project leader, upstream development microbial at Lonza.
Processes typically proceed at room temperature and ambient pressure in water, avoiding the use of harmful or toxic solvents or chemical raw materials and production of dangerous waste, according to Martinelli. Operators benefit from safer work environments, and drug makers avoid issues associated with the use and production of hazardous materials. Fermentation facilities and equipment may also be less costly to build and install.
Synthetic biology can in some cases also provide access to molecules that cannot be produced via traditional synthetic organic chemistry. “While this concept is not novel per se, over the last several decades, the development of improved tools and understanding have also enabled greatly increased yields and decreased development costs and risks,” Martinelli states. He points to steroids, analgesics, opioids, and some anti-cancer drugs. Similarly, he observes that some phytochemicals that cannot be practically extracted from plant sources may also be produced via fermentation.
There are also decades of fundamental research and proven manufacturing success supporting the growing interest and application of synthetic biology and fermentation for chemical API production, Krumov asserts. “The expression systems are well known, studied, and characterized, and the processes are well understood and controllable, facilitated by the development and improvement of precise monitoring tools,” he explains.
The key to realizing all of these benefits, however, Martinelli stresses, is to recognize when synthetic biology provides the optimum solutions—either by providing access to novel structures or higher yields/selectivities—and when organic chemistry is the best approach.
AMRI breaks down microorganisms useful for fermentation into two classes: natural, classic producers and engineered host strains (genetically modified organisms). Classic microorganisms widely used in biotechnology processes include bacteria (Streptomyces sp., Mycobacterium sp., Paracoccus sp.) and fungi (Aspergillus sp., Penicillium sp., Acremonium sp., Tolypocladium inflatum, Claviceps purpurea, Blakeslea sp., etc.).
A focused set of yeasts, such as Saccharomyces cerevisiae and Pichia pastoris (P. Pastoris) and bacteria such as Escherichia coli (E. Coli), make up the second group, according to Barredo. “These host strains are selected based on suitability for genetic modifications and utility to efficiently produce APIs and compounds, including many products not practical via fermentation previously,” says Peter C. Michels, head of global fermentation at AMRI.
The majority of developmental and commercial processes in the pharma industry involve E. coli and P. pastoris (Komagataella phaffii), Krumov adds. “E. coli is probably the most studied and well-known microorganism, a sort of work horse for the biotechnology industry. It provides simple and fast strain development, short fermentation times, high cell densities, and double-digit titers. P. pastoris achieves ultra-high cell densities grown on simple and cheap carbon sources and also secretes the product into the culture medium, delivering a highly pure product with decreased recovery efforts,” he explains.
New species, Krumov notes, including such requiring biosafety level 2 handling, are constantly evaluated and investigated, and some find commercial realization.
A primary driver of recent interest in fermentation is advances in molecular biology and synthetic biology. “These advances allow producer strains to be engineered with greater understanding and effectiveness, which is therefore enabling the development of new processes,” Michels states. They can also be adapted and improved using established fermentation process knowledge and infrastructure that has been built over decades.
The use of synthetic biology, including directed evolution of biosynthetic pathways, offers new, more specific ways of modifying pharmaceutical production, according to Michels. “These new innovations also allow us to get more out of classical fermentation processes. We’ve seen really dramatic advances in metabolic engineering across a broad spectrum, such as directed evolution, that allow us to simply and systematically evolve the biosynthetic pathways and achieve a myriad of advantages—the overproduction of APIs, reduction of related impurities, shortening the fermentation process and making it more cost effective, producing new compounds, improving process safety, and more,” he comments.
Metabolic engineering, therefore, is playing a much greater role because it can be applied to many different goals for continuously improving practical processes. Martinelli agrees that metabolic engineering and genetic engineering play a key role in the development of novel biosynthesis processes because they are crucial to yield enhancement as well as diversity generation. “By rewiring the microbial network, it is possible to drastically divert the flow of material through the cell metabolism and thus achieve production of the target molecule at the desired scale and cost,” he says.
For Lonza, genetic engineering has been most important in its efforts to develop processes for the production of biotherapeutics. “Technical advances in genome editing, next-generation sequencing, and genomics are increasingly important to support the design of productive and scalable production strains,” asserts Joachim Klein, head of strain development and cell banking for Lonza’s microbial development services.
As well as the development and selection of high-performing, stable, and productive strains, high-quality and high-throughput screening—in combination with the development of effective analytical methods for assessing product quality, such as such as post-translational modifications of candidates generated using candidates identified in the screening process—have also been important contributors to the greater application of fermentation in API manufacturing, according to Klein.
Advances in analytical technology, particularly mass spectrometry, nuclear magnetic resonance (NMR), and other analytical techniques that increasingly help to elucidate, in much greater detail, exactly what is going on inside the organisms, have a significant impact in driving much greater understanding and the more efficient design of practical production processes, Michels agrees.
Advances in biologic and analytical technologies have occurred in parallel with similar advances in informatics and other digital technologies. Dual development has been necessary to efficiently process the large quantities of data produced and ensure data integrity.
“Informatics and digital technologies are valuable in genomics, metabolomics, and transcriptomics analyses. They help us to know ‘in silico’ the modifications in the genome of the microorganisms necessary for the construction of an improved strain,” Barredo states. Special software, Krumov adds, are applied in the elaboration of process models, statistical analysis and predictions, parameter screening, and process optimization, and to establish more precise and agile process control.
In addition, with the growing complexity of target molecules/metabolites to be produced, it is becoming more difficult to design and implement a full genetic engineering campaign and the corresponding large-scale metabolic network rewiring needed to achieve the target yield, according to Martinelli. “Bioinformatics plays a key role in not only the discovery of the relevant enzymatic and cellular regulatory steps, but also in the design of the cloning tools required to implement such changes and the dosing and fine tuning of the changes needed, taking into account the implications of the different omics disciplines, such as transcriptomics, proteomics, metabolomics, and fluxomics,” he asserts.
“Without digital technologies,” Martinelli continues, “it would not be possible to operate at the current level of complexity at the interface between expert scientists and robots.” In fact, to make retro-biosynthesis more feasible, Abolis has developed a proprietary computer algorithm in which artificial intelligence is applied to bioinformatics for gene and pathway selection and manipulation towards the biosynthesis of target molecules.
Advances in process analytical and control technologies are also enabling improved fermentation processes. For instance, new in-process controls (IPCs) are being developed to control the evolution of raw materials, enzymatic activities, product, etc., according to Barredo.
“In addition to more traditional temperature, pH, dissolved-oxygen, carbon dioxide, and oxygen-demand sensors, biosensors are helping to control the fermentation process, and monitoring systems for submerged fermentations have taken a considerable step forward,” Barredo says. He also stresses that the availability of reliable sensors that are able to provide real-time, non-sampling-based process monitoring of physiologically relevant parameters such as cell mass, substrate, and metabolite concentration allows for automatic control of fermentation processes based on suitable process models and control strategies.
In particular, Michels notes AMRI has found a much broader array of practical approaches using NMR, in particular development insights and quality control, and most notably in combination with the use of in-process controls and mass spectrometry.
While much of the substantial capital investments made in the pharmaceutical industry for classical fermentation can be adapted for new synthetic biology processes, there are some new developments in equipment worth noting. Krumov points to new fermentation system concepts and designs that enable greater automation; intuitive, precise, and advanced parameter programming; and online monitoring of essential quality attributes. “New primary recovery equipment (separators, filters, membranes) also ensures better performance, higher productivity, and greater process robustness, facilitating better purification of the products and higher yields,” he adds.
Mobile applications, according to Michels, allow remote monitoring and control of fermentation parameters even from a smart phone and provide much better information, while parallel fermenter systems provide faster and improved development arrays for modified strains and multiple parameters. He also notes that the wider variety of impellers available today afford improved oxygen availability and mass transfer, particularly for the new engineered host strains.
Going forward, the focus will largely be on downstream processing, which according to Martinelli, represents a challenge when scaling up production of small-molecule intermediates and APIs produced via fermentation.
All of these rapid advances in molecular biology and engineering tools will impact fermentation in the pharmaceutical industry over the next several years. “We anticipate that continued exponential growth in molecular biology techniques—actually editing and designing new producer strains at lower cost and higher volumes—will have a significant impact on the design of new biosynthetic pathways in a synergistic way with chemical synthesis in the years ahead, not only in pharma, but in food, agriculture, enzymes for biocatalysis, and more” Barredo asserts.
“A longer-term vision,” Barredo adds, “might include using new competitive fermentation processes based on the circular economy, where food wastes are used as raw materials to design new, sustainable processes that contribute to the safety and quality of the global environment.”
Modern fermentation process development is, according to Krumov, a long, profound process that leverages the knowledge and experience of generations of bioprocess engineers in combination with current cutting-edge technologies and digital tools, to deliver the safest and most effective next-generation medicines. “The steadily increasing worldwide demand for microbial fermentation production capacities is an indicator of the qualities of this biosynthetic approach and recognition of its potential,” he asserts. Martinelli adds that this technology not only brings innovation to create new entities and new treatments, but to limit the environmental impact of their production.
One way to reduce development timelines, Krumov says, is through intensification. “For instance, intensification of process development will reduce the necessary gene-to-vial time through application of current design-of-experiment approaches, soft sensors, and data-based process modeling,” he says. Ensuring and confirming a product’s quality at the very early project stages will also be important, according to Krumov. Where possible, avoiding the use of complex compounds will further facilitate the development of robust, reproducible, and cost-effective fermentation-based manufacturing solutions.
1. Lux Research, “Lux Research Predicts Synthetic Biology Has High Potential as an Alternative Production Method for Chemicals,” Press Release, April 2, 2020.
Cynthia A. Challener, PhD, is a contributing editor to Pharmaceutical Technology.
Vol. 44, No. 11
When referring to this article, please cite it as C. Challener, “Fermentation Finds Fans in Small-Molecule API Synthesis,” Pharmaceutical Technology 44 (11) 2020.