Quorum sensing inhibitors: an overview.

The US Food and Drug Administration's 'quality by design' approach is likely to transform the manufacture of biologics.

Quality by design for biopharmaceuticals

Roadmap for implementation of quality by design (QbD) for biotechnology products

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.

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Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview

E. coli's hardiness, versatility, broad palate and ease of handling have made it the most intensively studied and best understood organism on the planet. However, research on E.coli has primarily examined it as a model organism, one that is abstracted from any natural history. But E. coli is far more than just a microbial lab rat. Rather, it is a highly diverse organism with a complex, multi-faceted niche in the wild. Recent studies of 'wild' E. coli have, for example, revealed a great deal about its presence in the environment, its diversity and genomic evolution, as well as its role in the human microbiome and disease. These findings have shed light on aspects of its biology and ecology that pose far-reaching questions and illustrate how an appreciation of E. coli's natural history can expand its value as a model organism.

/pdf/the-unexhausted-potential-of-e-coli-37a0smwe1d.pdf

The unexhausted potential of E. coli

Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements.

The concept of "design space" has been proposed in the ICH Q8 guideline and is gaining momentum in its application in the biotech industry It has been defined as "the multidimensional combination and interaction of input variables (eg, material attributes) and process parameters that have been demonstrated to provide assurance of quality" This paper presents a stepwise approach for defining process design space for a biologic product A case study, involving P pastoris fermentation, is presented to facilitate this First, risk analysis via Failure Modes and Effects Analysis (FMEA) is performed to identify parameters for process characterization Second, small-scale models are created and qualified prior to their use in these experimental studies Third, studies are designed using Design of Experiments (DOE) in order for the data to be amenable for use in defining the process design space Fourth, the studies are executed and the results analyzed for decisions on the criticality of the parameters as well as on establishing process design space For the application under consideration, it is shown that the fermentation unit operation is very robust with a wide design space and no critical operating parameters The approach presented here is not specific to the illustrated case study It can be extended to other biotech unit operations and processes that can be scaled down and characterized at small scale

https://aiche.onlinelibrary.wiley.com/doi/pdf/10.1021/bp070338y

Defining Process Design Space for Biotech Products: Case Study of Pichia pastoris Fermentation

This study focuses on the feasibility of producing a therapeutic Fc fusion protein in Pichia pastoris (P. pastoris) and presents an optimization design of experiment (DOE) strategy in a well-defined experimental space. The parameters examined in this study include pH, temperature, salt supplementation, and batch glycerol concentration. The effects of these process conditions were captured by statistical analysis focusing on growth rate and titer responses. Batch medium and fermentation conditions were also investigated prior to the DOE study in order to provide a favorable condition to enable the production of this Fc fusion protein. The results showed that approximately 373 mg/L of the Fc fusion protein could be produced. The pH was found to be particularly critical for the production of this Fc fusion protein. It was significantly higher than the conventional, recommended pH for P. pastoris fermentation. The development of this process shows that protein production in P. pastoris is protein specific, and there is not a set of pre-defined conditions that can work well for all types of proteins. Thorough process development would need to be performed for every type of protein in order for large-scale production in P. pastoris to be feasible.

Xiaoming Yang

Papers

Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements.

Defining Process Design Space for Biotech Products: Case Study of Pichia pastoris Fermentation

Enhancing the Production of Fc Fusion Protein in Fed‐Batch Fermentation of Pichia pastoris by Design of Experiments