Key Facts Flashcards
What is the immunogenic part of Fc region of anitbody?
N-glycans (sugar decoration)
What is the immogenic part of proteins produced by Pichia pastoris?
N-glycans (sugar decoration)
Good thing about glycosylation in Pichia pastoris?
More similar to humans that S. cerevisiae
Minimal mannosylation
What is Fc?
The antigen-binding region of an antibody (IgG)
What is CMV?
cytomegalovirus promoter (good strong constitutive promoter)
How do transfection of plasmid into mammalian cells?
lipofectamin (liposome protein) + DNA -> coats DNA
complex absorbed into cell
What do after initial transfection for transient cell line creation?
Select?
Incubate for 2-8 days then purify out product
What do after initial transfection for stable cell line creation?
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
use of transient cell lines
high throughput screening of potential early-stage drug candidates
use of stable cell lines
greater production of candidate drug (next step in development)
How to retrieve protein from periplasmic expression?
osmotic shock - releases recombinant protein
What is FLAG?
antibody tag - elute with FLAG tag peptide (soluble form)/low pH buffer
Antibody-specific affinity column (no tags)
protein on column - A, G or L
binds at high pH
elutes at low pH
Different protein column types for antibody-specific affinity column and uses
A = Fc region of monoclonal antibodies
L = kappa light chain
G - no example given
Use of different ELISAs for antibody purification
Capture ELISA - number of antibodies
Competition ELISA - antibody competition for epitopes, which is better
…etc.
How do you know that antibodies expressed are what want?
Protein gels and western blot
- sizes match what want (IgG, fvSC…etc.)
What affects antibody-antigen binding?
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
Cation Chromatography outline
if pH < pI (i.e. alkali-neutral antibody)
resin is +ve
antibody is -ve
elute by raising the pH/salt
Anion Chromatography outline
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)
Difference between flowthrough and bind & elute chromatography
flow through - impurities stick to resin
bind & elute - impurities washed through
benefits of stable cell line Case Study
increased yield of murine mAb by almost 1000 times
Pseudomonas aeruginosa
Why antibody engineer?
reduce immunogenicity increase expressibility (smaller/less complex is easier)
Why switch isotypes?
IgG is most therapeutically important
What is isotype?
e.g. IgG, IgM, IgA…etc
why reformat?
e.g. to create bispecific
why change subtype?
suited to different responses (disease)
e.g. IgG 1, IgG 2 …etc
chimeric antibody
change every part of antibody except binding area (Fab)
humanized anitbody
change everything to human but CDRs (antigen binding sites) of Fab region
What are the difference in the molecules produced by in vitro technologies for fully humanized anitbodies?
Ribosome - max. scFv
Phage - max. Fab
Yeast surface display - max. IgG
Common phage vector
pHEN2
Usually bacteriophage used for phage display
N13K07 helper phage
How purify in phage display?
ELISA
- those that display antibody fragment & produce it (contain plasmid)
- select for higher affinity (reduce number of antigens)
how to you get genotype-pheotype link with phage display?
several rounds of ELISA - bacteriophage will contain the plasmid AND display the antibody fragment
phage display antibody library approach
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
Special advantage of selection in phage display
high expression & high affinity clone
Niave libaries
“normal” antibody presence
- no special response (so no high affinity binders)
- immature (IgM req. -> IgG)
Immunised libraries
response to an antigen
- specialised reponse (good&bad) > high affinity binders can be found
- mature IgG
Novel way to improve protein folding in recombinant expression
Origami E.coli
- cytoplasm is less reducing, so disulphide bonds can be formed there
Plus side of ribosome display
libraries can be large because no loss through bacterial transformation of plasmids
