PEPTIDES & PROTEINS inducible alcohol oxidase ( AOX1 ) promoter . This system is ideal for largescale recombinant protein production , including therapeutic proteins like insulin . Despite its advantages , the use of methanol for induction can present scaling challenges due to safety concerns , although alternative nonmethanol-based promoters exist .
In general , E . coli fermentation is easy to scale up and has a low risk of failure . This bacterial strain allows for the production of large quantities of recombinant proteins in a short period , at a low cost and using an environmentally sustainable process . The key to success is controlling the growth rate during the production phase . If this is not properly managed , there is a high risk of plasmid loss or the formation of inclusion bodies .
Yeast offers better genetic stability as a host because the recombinant gene is typically integrated into the chromosome . However , using eukaryotic cells for fermentation requires more complex processes , making scaling up more challenging . Oxygen limitation can severely impact fermentation yield , and the production of ethanol in S . cerevisiae can disrupt the process entirely .
Strict control over feed rates and the optimisation of the feeding profile is essential in yeast fermentations , and the fermentation kinetics must be carefully defined to ensure the proper secretion of correctly folded products .
After the upstream phase , which aims to achieve the highest concentration of the target molecule , the downstream process ( DSP ) begins . The DSP focuses on isolating and purifying the target molecule to meet the required drug substance quality criteria . This is accomplished in multiple stages , each enhancing the quality of the stream from the initial broth to the final API .
The first step of the DSP is to isolate the peptide in an aqueous solution or the supernatant phase of the broth . Yeasts like S . cerevisiae or P . pastoris are often preferred for expressing APIs , as they typically produce the target molecule extracellularly , meaning it is already present in the liquid phase .
In contrast , when bacteria like E . coli are used , the target peptide is often expressed intracellularly , requiring a cell disruption step . Regardless , a volume reduction step is often performed early on to save costs and time . This can be done using centrifugation or membrane filtration to concentrate the broth and remove some of its components .
Once the peptide is in solution , the first stage of purification focuses on removing impurities that could destabilise or degrade the product . The broth or lysate often contains various contaminants , including pyrogens , proteases and other highmolecular-weight proteins .
Common methods for purifying the product include filtration and chromatography techniques . Filtration is typically used early in the process and is cost-effective . By selecting the appropriate filter media , solid particles can be removed , or soluble compounds can be separated .
The membranes used in filtration vary in size , material , porosity and configuration , giving the process developer a wide range of options to select the most suitable one for the specific needs . In some cases , flocculation or sedimentation processes may be used to improve the efficiency of phase separation when dealing with complex mixtures like broths .
Figure 1 - Quantity of therapeutic peptides vs . number of amino acids
Note : Red dots indicate APIs produced through synthesis , while green dots represent APIs produced via recombinant DNA technology . The size of each dot correlates with the year of production , with larger dots representing more recent years
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