Perfecting microbial manufacturing processes means taming its greatest asset: speed. This rapid process for creating biologics also means a race against time, where success hinges on conquering three key challenges: designing a robust microbial strain, optimizing fermentation for industrial scale, and ensuring consistent high-yield product yield.
Microbial fermentation, a long-established and highly efficient method for manufacturing biologics, is seeing a resurgence of interest for producing a new generation of therapies. While this production method predates many others and is prized for its speed, it also presents unique difficulties that require specialized capabilities to ensure process reproducibility.
The primary challenge lies in the speed of the process itself. While a process may appear stable in the lab, scaling up introduces new variables. With a typical microbial production run lasting only about 48 hours, the window to analyze and correct any process shortcomings is incredibly brief.
Successfully navigating these rapid timelines hinges on overcoming three common difficulties. First, is the development of a robust strain that maximizes expression while minimizing product-related impurities. Second, the fermentation process must be optimized to ensure efficacy and limit process-related impurities during scale-up. Finally, midstream development must be supported by predictive small-scale studies to ensure consistently high process yields.
Expression System Design and Election
Host cell selection and expression system design are critical in ensuring a successful process. These are among microbial development’s most significant challenges, as researchers must anticipate the impact of expression system design choices years into the future. Thus, while it is important to design a system that yields high expression, it is also critical to understand the microbial strain’s capabilities and limitations, which could further impact product-related impurities.
Analytical technology can be leveraged to support the selection of host strains. Specifically, researchers can examine how the host will modify the expressed proteins of interest and determine which strain results in the lowest amount of product-related impurities, minimizing the burden of developing future downstream purification steps.
Expression system design should also take into consideration the impact to future manufacturing processes. For instance, some modifications to the host strain that allow for higher theoretical product yield can be at the expense of process robustness. While at the small scale the strain may look promising, these modifications can change the cell physiology to cause unwanted characteristics like increased shear sensitivity, which can reduce the ability to transfer oxygen into the system at the larger scale and can lead to an overall decrease in product yield.
Fermentation Process Development
Developing robust and scalable microbial fermentation depends on a careful balance of optimizing the growth and expression phases. This is achieved through efficient oxygen transfer, as well as fine-tuning of nutrient delivery and induction of protein expression. Oxygen transfer must be sufficiently robust for the desired growth rate controlled by nutrient delivery, and the induction strategy must select an appropriate concentration, duration and temperature to maximize product quality.
Oxygen Transfer
Microbial systems consume oxygen rapidly, often needing about 100x the gas flow of mammalian cultures. Thus, oxygen must be supplied quickly and precisely to support cell division and protein production.
Typically, three strategies are used to maintain dissolved oxygen concentration: increasing agitation, increasing the percentage of pure oxygen in the gas being sparged into the tank, and increasing gas flow rates. Considerations like sensitivity to shear stress need to be considered when selecting which strategies to apply, and in what order.
Nutrient Feed Strategy
Nutrient feed rate must be calculated across production scales to control cell growth rate, keeping the cells in a consistent metabolic state to ensure they do not outgrow the system or underperform during protein expression. Fed batch production is an effective way to increase cell density and product yield, but implementing a feed successfully requires fine-tuning of its timing, rate and contents.
Feed too many nutrients too fast and the metabolic pathways could favor greater cell growth and expansion, leading to less specific productivity. Additionally, too aggressive a feed rate leads to a buildup of metabolic byproducts that are not favorable for product stability — for example, the metabolic bioproduct acetate is not conducive to microbial cell health and results in diminishing productivity, product quality and/or stability. Provide too meager a nutrient feed, and cell density does not build up adequately, leading to less overall product at the end of the process.
Induction of Protein Expression
For protein-based therapeutics, the induction stage of fermentation is a critical point in which a high cell density has been achieved and control conditions must be optimized to switch metabolic pathways from favoring cell growth to driving product expression. Feed rates are typically optimized to provide sufficient nutrients while also creating a carbon-source-limited environment to minimize the accumulation of unfavorable fermentation byproducts. Temperature may also be adjusted, both to control cell growth and to create an environment that promotes product stability. Finally, induction stage length must be determined by balancing overall product yield with product quality.
Midstream Process Development
Once fermentation is complete, midstream steps are necessary for separation of cells from fermentation byproducts, execution of lysis, and clarification of product from cellular waste. Preparation for midstream development starts with cell line selection, since specific cell line characteristics will influence how the product is extracted. Partnering with a CDMO like AGC Biologics, which has extensive experience, can help address issues with cell lines that may cause problems during lysis or separation, release certain byproducts during extraction, and more. Using this knowledge to guide strain selection from the beginning naturally leads to more efficient midstream development.
At the end of fermentation, multiple paths are possible. Typically, protein-based products are expressed inside the cell, after which the cell paste is collected and suspended in a buffer that is healthier for the proteins during lysis and the harvest phase in general. Less frequently, cellular lysis can occur before initial separation from byproducts to increase product yield. However, this strategy needs to consider a potential increase in impurities that will have to be cleared in subsequent purification steps.
Conclusion
Drug sponsors spend years developing molecules, creating therapeutic pathways, and producing API to predict efficacy. It’s easy to underestimate the challenge of shifting from academic precision to industrial repeatability, as the former focuses on perfection for one molecule, while the latter tests process consistency over hundreds of manufacturing runs.
Finding an experienced partner who understands speed is key to a project’s viability. A CDMO supporting rapid development and process robustness is essential for microbial success. AGC Biologics, with its track record of transferring processes and adaptability within tight timelines, can foresee pitfalls and build reliable manufacturing processes.
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