Dear Sunil

endotoxin is an issue in case you need to use your ricombinant protein as drugs (eg vaccine ) fon humam or animal immunization because lps is reactogenic and cause seriuors adverse reactions if injected.

while in thw last years were developer some selective resin (eg endotrap) suitable for small scale removal this are still not applied in large scale prpbably due to the high cost and other approaches are used to reduce endotoxin extraction during the cell lysis or remove it ( eg using anoinic exchange) during purication.

https://sites.ualberta.ca/~csps/JPPS10_3/MS_996/MS_996.html

recently lucigen release a e.coli strain (clean e.coli) that is reported to do not produce emdotoxin. i tested it in small scale and it work well but of course to use it for production you need to Pay royalties.

bad smell is something that you can solve be designing you fermentation process using filter' and directing the fermentation gas

respect to the protocol that you can use in lab scale there are other limitations that are principally associated to the fact the an industrial process has to be simple, higly reproducible and you need to limit the cost of goods and so r example you need to carefully evqluate if you have to Pay royalties ti use an patetend Technologies or if you can optimize a different process that skip it.

some examples:

- limited temperature controll.

in laboratory scale we often perform bacterial growth at 37c and afterwards we shift the temperature at 25 or 17 to increase protein solubility. this is very difficult to perform with high volumes in industrial scale up refrigeration is conplex and expensive.

- use if antibiotic for plamid manteinance have to be limited and certain antibiotic as ampicillin are not admitted

- some purification step generally used in lab scale as imac and sec are generally not performed in large scale because long, expensive (sec) or need to pay royalties or perform steps ( as tag removal with proteases) that are very difficult to perform in reproducible way in high volumes. and therefore you need to re- optimize e purification.

this are some example and not all thr company has the same strategies but it is something that you have to take in account.

ciao

Manuele

Sunil Choe If your recombinant protein has a histidine tag you can purify by binding it to Ni-NTA resin and washing it repeatedly with ice cold 0.1 % Triton X, followed by a buffer without the detergent (Triton X) and then elute the protein with imidazole.

Bad smell is not really the problem with the purified protein, it may occur due to contamination with microorganisms.

Regarding endotoxin, there are different unit operations like chromatography and tangential flow filtration, which significantly reduce or eliminate the level of endotoxin in your protein solution.