The recent resurgence of industry interest in microbial fermentation for the production of biologics can be attributed to several factors, including advances in biochemistry and an increasing understanding of when glycosylation is undesirable. Additionally, the emergence of new protein formats and scaffolds that are well-suited for this production technology, along with their applicability to DNA plasmid production, has contributed to this trend. Successful optimization efforts can lead to scalable fermentation processes that not only achieve high titers and productivity but also demonstrate significant robustness and reliability.

The cost-effectiveness of microbial fermentation, when compared to cell culture, makes it highly attractive under the right circumstances. However, several challenges remain to be addressed. Bacteria and yeast typically lack the cellular machinery necessary to handle complex biological structures, making it crucial to identify the most suitable production system for each target molecule. Another significant challenge is the insufficient understanding of the expression system and process, which complicates the production of the appropriate quantity of protein with the desired quality. Fortunately, as the industry increasingly adopts more sensitive technologies, it is now possible to gain a deeper understanding of product quality at earlier stages of development than ever before. This advancement necessitates more development iterations to ensure that product quality attributes are achieved early in the process.

When scaling up microbial fermentation, process control can present challenges, particularly concerning oxygen transfer rates and heat management. For rapidly growing bacterial cultures, it is essential to ensure that sufficient oxygen transfer occurs throughout the culture system to maximize growth. This, in turn, necessitates adequate mixing and airflow rates. Heat management is crucial because microbial fermentation typically requires precise temperature control, usually within ±1 or 2 °C. Microbial fermentation generates more heat than cell culture, and all excess heat must be dissipated by the equipment to prevent uncontrolled temperature spikes. This can become a significant challenge when scaling up for production.

The use of antibiotics to select for bacteria capable of maintaining high expression levels of the desired product presents a unique challenge in bacterial fermentation. During downstream processing, it is essential to remove antibiotics, and regulatory agencies expect producers to demonstrate effective removal. This objective is typically achieved by employing very low initial concentrations of specific antibiotics and validating their removal through targeted analytical methods, as well as through the dilution effects observed during multiple purification steps. Furthermore, the development of host systems that do not require antibiotics while ensuring stable expression has become a significant goal in recombinant protein and plasmid production. Increasing plasmid production poses another challenge; producing more plasmid is not as straightforward as merely increasing the number of cells. Retaining the plasmid can impact cell growth, necessitating a balance to optimize both plasmid yield and growth rate, which can be difficult to achieve.

Another characteristic that influences the suitability of microbial fermentation for biopharmaceutical production is the glycosylation capabilities of the specific microorganism involved. Escherichia coli (E. coli) can perform only a limited number of post-translational modifications, whereas yeast can glycosylate proteins, albeit not in the same manner as mammalian cells. The type of post-translational modifications required for the product will determine the most appropriate host organism. While some modifications may be performed in vitro, the preferred approach is to utilize the cellular machinery of the host organism. Although it may be possible to enhance results through process parameter optimization, existing pathways cannot be entirely replaced. For these reasons, E. coli and yeast serve as effective and complementary production systems for biomolecules that do not require human glycosylation.

For the fermentation process, real-time monitoring of production parameters to enhance process control has become a crucial objective for the biopharmaceutical industry. The implementation of Process Analytical Technology (PAT) during fermentation runs can identify potentially problematic changes and facilitate manual or automated adjustments to return the process to the center of a validated operating range. By utilizing PAT to ensure success and consistency, both time and resources can be conserved while upholding the highest quality standards.

Optimizing a microbial fermentation process for protein production necessitates careful consideration of the type of organism employed. In certain process designs, the host organism can efficiently secrete the protein into the fermentation medium, allowing for relatively straightforward recovery using hollow fiber-based tangential flow filtration (TFF) techniques. Conversely, in other processes, such as those involving E. coli, the protein is produced in the cytoplasm, either as soluble protein or as insoluble inclusion bodies. These distinct production methods require tailored harvesting strategies, depending on the specific product being generated. Proteins retained in the cytoplasm necessitate complete cell lysis, which can be achieved through mechanical methods like high-pressure homogenization. In contrast, proteins produced as insoluble inclusion bodies require unfolding and refolding procedures to yield soluble, properly folded products. These additional steps increase the complexity of the process and may introduce more impurities or potentially damage the final product.

Cell line engineering is a promising strategy to tackle this challenge, focusing on promoting the formation of correctly folded molecules within cells. However, it necessitates a specific approach and a customized process for each product, which demands significant time and effort in host selection and early process development. To achieve the highest possible titer and yield, it is essential to select the most suitable organism, expression system, sequence of unit operations, and operating conditions from the project's inception.

The diversity of fermentation processes and the molecules they produce makes it challenging to establish universal microbial fermentation platform processes. Developing a versatile cell line and plasmid platform that can be effectively utilized based on the specific protein or product type would be a promising solution to address significant challenges in fermentation. Additionally, adopting a platform-like approach for downstream processing steps could help standardize certain aspects of these production processes. While platform solutions for the production of plasmid DNA are becoming increasingly common, these approaches still require flexibility to accommodate various plasmids and formats.

Consistency between batch runs is another crucial area of focus. Regulatory agencies have established clear guidelines for developing fermentation processes that meet quality requirements. This involves understanding the system and its functionality, as well as defining the design space within which fermentation can occur. A systematic approach is essential during process development and optimization, which includes experimental design studies to comprehend how the cell line and culture conditions interact to influence growth and product yield. Most process development and characterization work can be effectively modeled using small-scale fermenters under laboratory conditions. Achieving consistency between laboratory and plant-scale models is essential for fine-tuning processes to ensure robust performance across both scales. However, maintaining consistency across multiple parameters to ensure comparability can be challenging, particularly for critical factors such as dissolved oxygen concentration. Therefore, careful consideration of the type of tank used is vital for optimizing consistency between the models. Aligning small and large-scale models enables the investigation of deviations from normal conditions in plant operations at the laboratory scale, which accelerates the analysis of process consistency and deviations while also reducing costs. Furthermore, laboratory-scale models can be utilized to quickly identify raw materials and assess the risk that certain materials may introduce variability in plant-scale production runs.

Optimization of fermentation reactions should not focus solely on one aspect, such as yield; it should also encompass process robustness, consistency, and other critical attributes, including oxygen demand and oxygen transfer rate. Furthermore, the optimization process is influenced by the ultimate goal of the fermentation. Initiating process development without taking the final objective into account often results in scalability issues later on. Therefore, target yield expectations must be considered when developing cell lines and fermentation processes.

Duoning offers a one-stop solution for the production of biological products through microbial fermentation. This includes fermenters of various scales for upstream processes, hollow fiber-based filtration technology for harvesting, high-pressure homogenizers for cell disruption, chromatography resins for downstream processing, and bulk filling systems for drug substance storage. Duoning is committed to establishing a high-quality, responsive, and flexibly customizable bioprocess technology platform that adheres to compliance standards. Our goal is to meet user needs, facilitate a faster and more optimized development process, and enable high-yield, high-quality, and cost-effective large-scale production.