Modern methods of producing medicinal products Flashcards
What are benefits of using recombinant products?
they are safer when producing vaccines for example its better to use a piece of the virus than the attenuated version of the virus and this means that production can be done on a large scale
what is the large scale production system?
this uses huge 300L production vessels. and batch and continuous culture techniques. the fermenter uses an impellor which is for mixing and oxygenation. a fermenter train has to be used this is when you use a serious of increasing culture sizes to reach a good optical density to inoculate your culture into the ferment er. the trains are all going at the same time so that production doesnt have to stop
what are the parts of the vector that is used to make bacterial recombinant products?
selectable marker ORI a multiple cloning site which then will hold the DNA to be transcribed the ribosomal binding site the promotor transcription terminator
what needs to be a feature of the promotr?
it needs to be a strong promotor that you can control
what is the transcription terminator for?
for if the promotor is particularly strong
why is it good to control the promotor?
turn the gene off while the culture grows
then when the culture is dense
turn the gene on so that the toxic product is produced in a high quantity before the culture dies
what is the problem with the lac promotor?
it is leaky even when the promotor is repressed
what does the arabinose promotor need to be on?
araC - act - arabinose
and no glucose signalled by CRP-cAMP complex
how does araC prevent trans when no arabinose?
binds to the DNA differently by binding to one of its previous binding sites and DNA downstream to cause bending of the DNA. meaning that there is no way that the RNA polymerise can bind - strongly off
when may you go for a low copy number plasmid tightly controlled too?
when the product of the plasmid is toxic to the cell
what can help companies stop spending loads on antiBs to make sure the plasmid is still in the cells?
using a low copy number plasmid as this is more likely to be retained in the cell as it is more stable.
what does no enough HGH cause?
reduced bone growth, loss of muscle, increased fat this is nomrally made in the PG
what is there a risk of getting HGH from the pituiatary?
prion disease
what might have to be done to make a sequece usable in ecoli
chaning the dna sequene but still gettin gthe same aa sequence
what prmotor is used to make HGH?
the tryptophan promotor which is only on when tryp isnt present
what is the structure of the insulin protein translation initiatlly?
its made of the leader seuqence the sequence for one part of the pp and then a cleaved sequene then the other part of hte pp this will eventually be joined by a disulfide bond done when the chain folds together
how is insulin made in e.coli?
two plasmid one for each bit using the lac promotor and then creating the right condition for the disulfide bond
whars the yeast method to make insulin?
clone the whole gene in
in the yeast the protein folds spontaneously and forms the DS bonds and proteolytic cleavage also happens spontaneously
what are the advntgaes of using yeast to make insulin?
- better for protein processing, like having the glycosylate products
what yeast are used
pichia pastoris
sacc. cere
where is glycosylation added?
to asparagine residues at the ER and Golgi
what does glycosylation effect?
protein folding
immune recognition
clearane in the body
what is the shuttle vector like?
ori and 2u(yeast)
ab marker and ura3 (yeast marker)
what do you use instread of an antibiotic marker in yeast?
ura3 - allows for uracil porduction so you can kill the yeast that cant make this
what is the gal1 promotor?
a promotor that increased expression 1000x fold in the presence ofgalactose, repressed by glucose - good for use in yeast
whats the aox 1 promtoor?
induced by methanol, when on 30% of the cell protein will be the product from this promotor
how is the hep b vaccine made?
its a subunit vaccine HbSAg vaccine mae in yeast because in e.coli it does not adopt its natural conformation
what are the advnatgss of subunit vccines?
cheaper to make the antigen in large quantities these vaccines are also safer to make as no acual viriuses are used. and this is good when some biruses like hep b cant be made in culutrue
what is hpv vaccine made of?
capsid proteins that are added in with sturctural paritcuales of the hpv so that the antigens are presented correctly - a viral mimic
how is gardasil made?
in yeast made of the L1 protein
how is cervarix made?
in insect culutre
what does making the vaccine in this way mean for the stregnth fo the baccine?
better as the vaccine conc is high in the blood
what needs to be done to antibodies to make them effective?
they need to be glycosylated
what is the problem with using pichia to make monoclonal ABS?
they dont have the sam glycos system as us theres is more simple they have less of a varied sugar compoisiton and only glycos are ser and threonine
what can we done to make pichia glycos like us?
engineer our glycos system into pichia so that the proteins made by it are like the ones in mammalian cells
why is it better to use non mammalian cell lined?
cheaper
harder to grow them in large qunatity
greatr control over the cell line
and you can genetically manipulate these cell lines easily
how can we make interfreron?
using e.coli its not glycosylated like by mammalian cells but works
how caan we make osp A borelia vaccine?
using ecoli a good antibiral and good anti cancer as its anti proiferative
what can glucagon made by yeast be used for?
hypoglycaemia and mnuscle relaxant
