Key Facts

Question 1

What is the immunogenic part of Fc region of anitbody?

Answer 1

N-glycans (sugar decoration)

Question 2

What is the immogenic part of proteins produced by Pichia pastoris?

Answer 2

N-glycans (sugar decoration)

Question 3

Good thing about glycosylation in Pichia pastoris?

Answer 3

More similar to humans that S. cerevisiae

Minimal mannosylation

Question 4

What is Fc?

Answer 4

The antigen-binding region of an antibody (IgG)

Question 5

What is CMV?

Answer 5

cytomegalovirus promoter (good strong constitutive promoter)

Question 6

How do transfection of plasmid into mammalian cells?

Answer 6

lipofectamin (liposome protein) + DNA -> coats DNA

complex absorbed into cell

Question 7

What do after initial transfection for transient cell line creation?

Answer 7

Select?

Incubate for 2-8 days then purify out product

Question 8

What do after initial transfection for stable cell line creation?

Answer 8

incorporated into genome (stable inheritance)

make it incorporate into genome at higher number

take away essential protein function in native DNA

foreign DNA contains essential gene (e.g. glutamine synthetase)

ELISA screening for high producers

Question 9

use of transient cell lines

Answer 9

high throughput screening of potential early-stage drug candidates

Question 10

use of stable cell lines

Answer 10

greater production of candidate drug (next step in development)

Question 11

How to retrieve protein from periplasmic expression?

Answer 11

osmotic shock - releases recombinant protein

Question 12

What is FLAG?

Answer 12

antibody tag - elute with FLAG tag peptide (soluble form)/low pH buffer

Question 13

Antibody-specific affinity column (no tags)

Answer 13

protein on column - A, G or L

binds at high pH
elutes at low pH

Question 14

Different protein column types for antibody-specific affinity column and uses

Answer 14

A = Fc region of monoclonal antibodies

L = kappa light chain

G - no example given

Question 15

Use of different ELISAs for antibody purification

Answer 15

Capture ELISA - number of antibodies

Competition ELISA - antibody competition for epitopes, which is better

…etc.

Question 16

How do you know that antibodies expressed are what want?

Answer 16

Protein gels and western blot

- sizes match what want (IgG, fvSC…etc.)

Question 17

What affects antibody-antigen binding?

Answer 17

Valency - number of binding places (e.g. 2 bonds or 1…etc.)

Geometric arrangement - antibody density

Intrinsic affinity - how strong the anitbody-antigen bond is

Question 18

Cation Chromatography outline

Answer 18

if pH < pI (i.e. alkali-neutral antibody)

resin is +ve
antibody is -ve

elute by raising the pH/salt

Question 19

Anion Chromatography outline

Answer 19

if pH > pI (i.e. acidic-neutral antibody)

resin is -ve
antibody is +ve

elute by lowering the pH

used to remove endotoxins (-ve charge)

Question 20

Difference between flowthrough and bind & elute chromatography

Answer 20

flow through - impurities stick to resin

bind & elute - impurities washed through

Question 21

benefits of stable cell line Case Study

Answer 21

increased yield of murine mAb by almost 1000 times

Pseudomonas aeruginosa

Question 22

Why antibody engineer?

Answer 22

reduce immunogenicity
increase expressibility (smaller/less complex is easier)

Question 23

Why switch isotypes?

Answer 23

IgG is most therapeutically important

Question 24

What is isotype?

Answer 24

e.g. IgG, IgM, IgA…etc

Question 25

why reformat?

Answer 25

e.g. to create bispecific

Question 26

why change subtype?

Answer 26

suited to different responses (disease)

e.g. IgG 1, IgG 2 …etc

Question 27

chimeric antibody

Answer 27

change every part of antibody except binding area (Fab)

Question 28

humanized anitbody

Answer 28

change everything to human but CDRs (antigen binding sites) of Fab region

Question 29

What are the difference in the molecules produced by in vitro technologies for fully humanized anitbodies?

Answer 29

Ribosome - max. scFv
Phage - max. Fab
Yeast surface display - max. IgG

Question 30

Common phage vector

Answer 30

pHEN2

Question 31

Usually bacteriophage used for phage display

Answer 31

N13K07 helper phage

Question 32

How purify in phage display?

Answer 32

ELISA

• those that display antibody fragment & produce it (contain plasmid)
• select for higher affinity (reduce number of antigens)

Question 33

how to you get genotype-pheotype link with phage display?

Answer 33

several rounds of ELISA - bacteriophage will contain the plasmid AND display the antibody fragment

Question 34

phage display antibody library approach

Answer 34

surviving patients - from infection (blood antibodies - B lymphocytes) & want to find it

immunise an animal with target antigen > immune response &
want to isolate best binder

LOOKING FOR THE GENES FOR ANTIBODIES

Question 35

Special advantage of selection in phage display

Answer 35

high expression & high affinity clone

Question 36

Niave libaries

Answer 36

“normal” antibody presence

• no special response (so no high affinity binders)
• immature (IgM req. -> IgG)

Question 37

Immunised libraries

Answer 37

response to an antigen

• specialised reponse (good&bad) > high affinity binders can be found
• mature IgG

Question 38

Novel way to improve protein folding in recombinant expression

Answer 38

Origami E.coli

- cytoplasm is less reducing, so disulphide bonds can be formed there

Question 39

Plus side of ribosome display

Answer 39

libraries can be large because no loss through bacterial transformation of plasmids