Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells

Abstract

Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.

Extracellular vesicles as a platform for membrane‐associated therapeutic protein delivery

“…One of the major challenges in the development of membrane protein therapeutics is the need to recover sufficient amounts of recombinant proteins from classical expression systems, such as those based in mammalian cells, yeast, and bacteria [43,44]. Despite continuing efforts to develop membrane protein therapeutics, the high content of hydrophobic amino acids in such proteins makes it difficult to generate a sufficient quantity of functional membrane proteins to enable structural studies and functional applications.…”

Production of valuable compounds by molds and yeasts

“…129,130 Biopharmaceuticals comprise one-sixth of the pharmaceutical market and are the most rapidly growing segment. They are employed to make up for the deficiency of body proteins used for normal function.…”

Genetically modified microorganisms as producers of biologically active compounds

“…В периплазме E. coli гетерологичные белки, кроме того, меньше подвергаются протеолитической деграда-ции (Berlec, Strukelj, 2013). Например, секреция про-инсулина человека в периплазму повышала время его полужизни в 10 раз .…”