Key Facts Flashcards
(39 cards)
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
