Glycosylation 2 Flashcards
What is gaucher disease?
Lysosomes lack glucocerebrosidase and hence you get a build up of glycolipids in lysome
How is gauchers disease treated?
providing patient with enzyme that is lacking from lysosome
What do humans have at the end of the glycan chains?
NeuAc (sialic acid)
What do mammals have at the end of the glycan chains?
Nu5Gc
Why are mammalian glycans not overly immunogenic?
We consume them regularly in meat and dairy
CHO cells produce glycans that are bioactive in humans. True or false?
True
How do you create CHO cell lines that all produce your POI?
Use DHFR on expression vector and do not put any glycine, hydroxanthine and thymidine in the media. Only cells with plasmids will survive
Do CHO cells lack Dihydrofolate reductase?
most do, but not all
What is the function of DHFR?
converts serine to glycine
and produces nt
What does adding methotrexate do to DHFR deficient CHO cells?
educes activity of DHFR and hence only those with low plasmid expression will survive
Name 3 mammalian cell other lines other than CHO?
PER.C6 - human embryonic retina
HEK293 - human embryonic kidney
NS0 - mouse myeloma
What is cGMP?
current good manufacturing products
What is genzyme and what happened when one of its cell lines got infected?
Genzyme is a company that produces Glucocerebrosidase. Infected cell line with virus & had to turn off production. Patients had to go without the enzyme because it was the only company that produced it.
What are the draw backs of animal cell cultures?
- Medium may become infected wth virus’ or prions
- Expensive fermentation
- Type of glycosylation depends on fermentation process
When was an alternative to Glucocerebrosidase produced in plant cells approved?
2012
Where is the plant expressed alternative to mammalian cell expressed glucocerebrosidase produced and why?
Israel
Jewish incest
Why in the case of glucocerebrosidase was the plant glycosylation beneficial?
No terminal NeuAC so ends with mannose.
Macrophages have a system for taking up things with terminal mannose residues
Hence enzyme targetted to macrophages
How many genes had to be removed from pichia pastoris to humanise the strain?
5 genes
How many genes had to be introduced from pichia pastoris to humanise the strain?
14
coding for glycosyltransferases, epimerases, precursor synthases and transporters
Does it matter where the genes introduced to pichia pastoris were localised?
Yes, they all had to be localised to specific compartments of the secretory pathway
What was the proof of principle protein produced in humanised pichia pastoris?
anti-CD20 antibodies (normally made in CHO cells)
These result in killing of B cells by ADCC
Where are IgG antibodies glycosylated?
The Fc region
Does the presence of fucose affect the structure of glycosylated IgG?
Yes
Without fucose the antibody binds higher to the receptor
What might the Fc receptor bind to cause?
Bind FcRIII on NK cells to cause cells displaying antigen to be killed (ADCC)
What is the largest class of biopharmaceuticals?
mAbs
CHO cells can be treated not to produce fucose. How?
By overexpression of two enzymes that divert pathways that normally adds fucose
What two enzymes are expressed in CHO cells to prevent fucosylation?
N-acetylglocuaminyltransferase III
Golgi alpha-mannosidase II
What pathway does EPO signal through?
JAK-STAT
How more glycans can be introduced to EPO to increase the half life but not affect function?
2
By mutating positions to ASN
Could plants be humanised?
Yes
The DNA in tobacco plant makes EPO but has the wrong Glycans. What could you do to combat this?
KO enzymes tha tmake plant specific glycans
Introduce 3 mammalian enzymes and build the rest of the glycan
