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Exercices Mabs M2 D2HP
Exercices Mabs M2 D2HP
M2 D2HP TU 19 Biotechnology
Exercices
Chronic inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, systemic lupus
erythematosus, psoriasis, Crohn's disease and multiple sclerosis all involve pro-inflammatory cytokines.
TNF- is a prime therapeutic target for the development of monoclonal antibodies (mAbs). This cytokine is
indeed overproduced during the natural history of these diseases, either in a membrane form, expressed on
the surface of the various producing cells, or in soluble form, secreted by these same cells. The direct and
deleterious involvement of TNF- has been clearly demonstrated in these diseases. Several formats of anti-
TNF- mAbs have been developed since the 1990s, the main molecules of which are shown in Figure 1.
Figure 1.
Question: Describe the particularities of each of these molecules, as well as their associated advantage
for their functionality (= activity(ies) of the molecule).
What differences and similarities can you cite in the production and purification processes used for
these molecules?
Anion exchange
Cation exchange
Tangential filtration(UF/DF)
Figure 2
Formulation
Here is the overall formulation of Remicade (Infliximab):
Saccharose, Polysorbate 80, mono and di-sodic phosphate
Please comment the use of each element.
Figure 4: SDS-PAGE gel electrophoresis of monoclonal antibody, 7.5 % polyacrylamide; Coomassie blue
staining.
Lane 1: peptides of known molecular weight. Lane 2: Start material. Lane 3: Flowthrough. Lane 4: Eluted
Mab. Lane 5: peptides of known molecular weight.
5) Unpurified
4) UF/DF
ophoresis
Figure (SDS-PAGE), capillary electrophoresis-SDS
6: Exclusion size chromatography biopharmaceuticals
analysis of a Protein A purified monoclonal antibody c
S), andrecombinantly
size exclusion
expressedchromatography (SEC)] , charge reviewed by Rudaz et
■
in Chinese hamster ovarian (CHO) cells.
s isoelectric
Separation focusing
mobile phase of(IEF) and phosphate
150 mM sodium cationic pH 7,exchange
flow rate of 0.8 mL/min, and UV detection
ography (CEX),
at 220 nm and thehydrophilic/ hydrophobic balance MASS SPECTR
reverse phase-high performance liquid chromatography Among all analytic
C)]. Most of separation techniquesused for protein-based tion, massspectrom
717
Figure 7: Glycosylation profile of infliximab
Samples were injected onto a UPLC BEH glycan column (2.1 mm £ 150 mm, 1.7 mm). The labeled N-
glycans were separated at a flow rate of 0.5 mL/min with mobile phase A (50 mM ammonium formate) and
mobile phase B (100% acetonitrile). The signal was detected by a fluorescence detector at an excitation
wavelength of 330 nm and an emission wavelength of 420 nm. From Lee and al. Mabs 2017
Figure 9: Representative TNF neutralisation assay using KLJ TNF responsive reporter gene cells showing
the differential loss of biological activity of freeze-dried infliximab in the two freeze drying formulations
SS-571 and SS-575. Each point is represented as a mean and standard deviation of four individual wells.
From Metcalfe et al. MABS. 2019; 1, 13–25. https://doi.org/10.1080/19420862.2018.1532766