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2ème ANNEE MASTER

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?

Purification of a monoclonal antibody (Mab)

Figure 2 describes the different general steps for a Mab purification. Please comment each step.
 Concentrated clarified supernatant (≈1- 5g
mAb/L)

Affinity chromatography A protein

Viral inactivation pH < 3.9, 60mn

Anion exchange

Cation exchange

Nanofiltration (20 - 50 nm)

Tangential filtration(UF/DF)

Sterile filtration 0,22μm

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.

Some aspects of Quality control

Here are different examples of quality control, which results are reported in figures to
For each figure, please indicate what type of control it is (identity, purity, safety, or activity)
What is the specific question behind each presented assay? Then give the general steps of the
protocols, analyze the figures and add a short conclusion.
Figure 3: Peptide mapping. SB5 is a new batch of purified infliximab biosimilar that is analyzed together
with a known well characterized infliximab batch.
What does each pic represent?

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

Figure 5: Native gel electrophoresis of a Mab, 7.5 % polyacrylamide. Silver staining.

Lane 1 : Sample after protein A chromatography purification step; Lane 2: Sample after cationic exchange
chromatography purification step; Lane 3: Sample after anionic exchange chromatography purification
step; 4) Sample after filtration purification steps; lane 5: Sample before purification.

Chromatogramsand electropherograms of apuri ed humanized therapeutic antibody. The

S-PAGE, CE-SDS, and SEC), charge (IEF and CEX), and hydrophilic/ hydrophobic balanc
m and microvariants.

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

Functional activity assays

Functional activity can be tested at the molecular level, the cellular level or at the organism or tissue level.
Molecular Level: target binging assay using Surface plasmonic resonance (SPR): what parameters can be
calculated using this method?

Figure 8: Surface plasmonic resonance principle (Wikipedia)

Cellular Level: cell-based assay

TNF neutralization assay
Inhibitory activity of infliximab samples on the soluble TNF signaling pathway was measured through the
TNF neutralization assay using a 293-NF-κB-luc cell line. The 293-NF-κB-luc cell line was engineered to
contain NF-κB response element upstream to the luciferase reporter gene. Mediated by TNF binding to cell
surface receptor, signal cascade activating NF-κB in turn promoted the expression of the luciferase reporter
gene. Serially diluted samples were pre-incubated with TNF at ambient temperature in a white 96-well
plate. Following incubation, cells were transferred to wells in the assay plate, and were incubated for 24
hours. TNF neutralization potency was determined by a luminescent signal using the Steady-Glo®
Luciferase Assay System (Promega) on a microplate reader.

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

Organism Level: imagine an assay for an in vivo infliximab activity evaluation.