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Biotechnology > Glycosylation 2 > Flashcards

Glycosylation 2 Flashcards

1
Q

What is gaucher disease?

A

Lysosomes lack glucocerebrosidase and hence you get a build up of glycolipids in lysome

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2
Q

How is gauchers disease treated?

A

providing patient with enzyme that is lacking from lysosome

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3
Q

What do humans have at the end of the glycan chains?

A

NeuAc (sialic acid)

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4
Q

What do mammals have at the end of the glycan chains?

A

Nu5Gc

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5
Q

Why are mammalian glycans not overly immunogenic?

A

We consume them regularly in meat and dairy

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6
Q

CHO cells produce glycans that are bioactive in humans. True or false?

A

True

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7
Q

How do you create CHO cell lines that all produce your POI?

A

Use DHFR on expression vector and do not put any glycine, hydroxanthine and thymidine in the media. Only cells with plasmids will survive

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8
Q

Do CHO cells lack Dihydrofolate reductase?

A

most do, but not all

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9
Q

What is the function of DHFR?

A

converts serine to glycine

and produces nt

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10
Q

What does adding methotrexate do to DHFR deficient CHO cells?

A

educes activity of DHFR and hence only those with low plasmid expression will survive

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11
Q

Name 3 mammalian cell other lines other than CHO?

A

PER.C6 - human embryonic retina
HEK293 - human embryonic kidney
NS0 - mouse myeloma

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12
Q

What is cGMP?

A

current good manufacturing products

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13
Q

What is genzyme and what happened when one of its cell lines got infected?

A

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.

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14
Q

What are the draw backs of animal cell cultures?

A
  • Medium may become infected wth virus’ or prions
  • Expensive fermentation
  • Type of glycosylation depends on fermentation process
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15
Q

When was an alternative to Glucocerebrosidase produced in plant cells approved?

A

2012

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16
Q

Where is the plant expressed alternative to mammalian cell expressed glucocerebrosidase produced and why?

A

Israel

Jewish incest

17
Q

Why in the case of glucocerebrosidase was the plant glycosylation beneficial?

A

No terminal NeuAC so ends with mannose.
Macrophages have a system for taking up things with terminal mannose residues
Hence enzyme targetted to macrophages

18
Q

How many genes had to be removed from pichia pastoris to humanise the strain?

A

5 genes

19
Q

How many genes had to be introduced from pichia pastoris to humanise the strain?

A

14

coding for glycosyltransferases, epimerases, precursor synthases and transporters

20
Q

Does it matter where the genes introduced to pichia pastoris were localised?

A

Yes, they all had to be localised to specific compartments of the secretory pathway

21
Q

What was the proof of principle protein produced in humanised pichia pastoris?

A

anti-CD20 antibodies (normally made in CHO cells)

These result in killing of B cells by ADCC

22
Q

Where are IgG antibodies glycosylated?

A

The Fc region

23
Q

Does the presence of fucose affect the structure of glycosylated IgG?

A

Yes

Without fucose the antibody binds higher to the receptor

24
Q

What might the Fc receptor bind to cause?

A

Bind FcRIII on NK cells to cause cells displaying antigen to be killed (ADCC)

25
Q

What is the largest class of biopharmaceuticals?

A

mAbs

26
Q

CHO cells can be treated not to produce fucose. How?

A

By overexpression of two enzymes that divert pathways that normally adds fucose

27
Q

What two enzymes are expressed in CHO cells to prevent fucosylation?

A

N-acetylglocuaminyltransferase III

Golgi alpha-mannosidase II

28
Q

What pathway does EPO signal through?

A

JAK-STAT

29
Q

How more glycans can be introduced to EPO to increase the half life but not affect function?

A

2

By mutating positions to ASN

30
Q

Could plants be humanised?

A

Yes

31
Q

The DNA in tobacco plant makes EPO but has the wrong Glycans. What could you do to combat this?

A

KO enzymes tha tmake plant specific glycans

Introduce 3 mammalian enzymes and build the rest of the glycan

Biotechnology (13 decks)

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