Post-translational modification of monoclonal antibodies

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2. Somayeh bakhshalizadeh
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POST-TRANSLATION MODIFICATION
OF MONOCLONALANTIBADY
Superviser :Dr.Nasiri

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Contents
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 Monoclonal antibody
 structure of monoclonal antibodies
 5 major classes of secreted antibody
 Post-translational Modification
 Protein Misfolding and Aggregation
 Glycosylation
 Pyro-glutamate
 Deamidation
 Isomerisation
 Oxidation
 Variants Involving Cysteines
 Sulphation

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Monoclonal antibody
 Monoclonal antibodies (mAb) are antibodies that are identical
because they were produced by one type of immune cell, all
clones of a single parent cell.
 Given (almost) any substance, it is possible to create
monoclonal antibodies that specifically bind to that substance;
they can then serve to detect or purify that substance.
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Heavy
Disulfide
Bond
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 Two pairs of identical
heavy and light tertiary
proteins
 The chains are joined by
disulfide bonds
The structure of monoclonal antibodies
Heavy

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CH2CH3
Fc
Fab
Fab
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The structure of monoclonal antibodies
• Conserved subunits
create the majority of
structure in all mAbs.
• The variable sub-units
enable specific binding.
• Antigen binding occurs
at either Fab region.
• The Fc region recruits
the immune system.

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CH3
6

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CH3
CH2
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CH3
CH2
CH1
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CH3
CH2
CH1
VH1
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CH3
CH2
CH1
VH1
VL
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CH3
CH2
CH1
VH1
CLVL
11

13. CH3
CH2
CH1
VH1
CLVL
1213

14. Hinge
CH3
CH2
CH1
VH1
VLCL
Elbow
1314

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Hinge
Fv
Fb
Fab
CH3
CH2
CH1
VH1
VLCL
Fc
Elbow
Carbohydrate
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5 major classes of secreted antibody
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Post-translational Modification
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 What is it ?
These modifications can include the addition or replacement of
functional groups, or structural changes such as folding,
cleavage, and racemization.
 What groups ? How much ? Where ?
- Sugar
- Sulphate
- ....

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Post-translational Modification
• What purpose ?
- regulation of function
- signal transduction
- cellular regulation
- Degradation
- structural/conformational rearrangements
• Why do we study it?
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Types of post-translational modifications
A variety of
changes
Physical Chemistry

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Fragmentation of Intact mAb
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The cleavage rates are sequence?
- PH
- temperature
cleavage of amino acid
Asp-pro

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The Chemical Modification of Antibody
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Protein Misfolding and Aggregation
Glycosylation
Pyro-glutamate
Deamidation
Isomerisation
Oxidation
Variants Involving Cysteines
Sulphation

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Protein Misfolding and Aggregation
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 The folding and initial glycosylation of most secreted
proteins take place in the endoplasmic reticulum (ER) lumen.
 Misfolded proteins can accumulate as intracellular aggregates and
induce dilation of the ER .
 Molecular chaperones such as heavy chain-binding protein (BiP)
facilitate protein folding at high concentrations by binding of unfolded
protein chains, prevention of aggregation, and/or support of refoldin.
 In addition to aggregates causing manufacturing problems, the
administration of proteins with low-level aggregate contamination can
lead to an immune response in the patient resulting in inhibitory
antibodies to the therapeutic proteine.

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Glycosylation
 Glycosylation is a major PTM affecting
protein folding, conformation, localization
and activity.
 One of the factors that may affect the
stability of monoclonal antibodies is
glycosylation in the area FC.
 IgG1 mAbs that contain a single N-linked
glycosylation site on Asn297 of the heavy
chain.

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Glycosylation
Glycosylation at Asn-Ala-Cys has also
been reported.
Thr Ser Cys
Thr
Ser
Cys

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Glycosylation
 Glycosylation of proteins is a ubiquitous type of post-translational
modification in living systems.
 Variations in oligosaccharide structures are associated with many
normal and pathological events such as cellular growth, host-pathogen
interaction, differentiation, migration, cell trafficking, or tumor
invasion.
 The structures of asparagine-linked oligosaccharides in the conserved
CH2 region of the constant Fc domain of human immunoglobulin-g
(IgG1) have been shown to affect the pharmacokinetics, antibody-
dependent cellular cytotoxicity and complement-dependent cytotoxicity.
 In the last decade, many recombinant antibody molecules have been
licensed for the treatment of a variety of cancers and chronic diseases.

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Glycosylation
 Herceptin, also known as Trastuzumab, marketed by Genentech Inc. is
one example of therapeutic IgG1 antibody.
 Unfortunately, only 25 30% of patients with HER2/neu positive breast
cancer respond to this antibody .
 Therefore search for the potential biomarkers that could predict the
efficacy of clinical outcomes is needed.
 It was noted that instructions for resuspension of Remicade specifed
0.9% saline, pH 7.2, conditions that did not result in the formation of
complexes similarly, neither did Herceptin or Avastin; however, these
mAbs exhibit other instabilities at higher pH values .
 For example, Asn 30 of Herceptin deamidates at pH > 5.0, which
lowers product bioactivity .

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Glycosylation
o Terapeutic antibody cetuximab has
an Nlinked glycan at Asn88 and 299
of the heavy chain variable region
and an unoccupied N-linked motif at
Asn41 of the light chain variable
region.

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Ritoximab
 The molecular weight of rituximab is 144,544 Da and isconstituted of 1328 aa
.
 Rituximab contains a conserved N-glycosylation site at Asn297 of both heavy
chains and is occupied by biantennary glycan structures.
 Rituximab mechanisms of action comprise the binding of its Fab domain to
CD20+B-lymphocytes for the induction of apoptosis, either directly or
throughout the recruitment of immune effector functions by its Fc domain.

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Pyro-glutamate
 Conversion from N-terminal Gln to pyro-Glu
is usually near complete in mAbs (> 95%).
 The rate of Glu to pyro-Glu conversion in
vitro near physiological pH and temperature
is comparable to that in vivo .
 light chain Glu to pyro-Glu rates increased to
levels near that of the heavy chain. This
indicates that Glu cyclization can be affected
by mAb structur.
 The Gln conversion to pyro-Glu renders
antibodies more acidic, whereas the
conversion of Glu to pyro-Glu results in a
basic shift.
Pyroglutamat
Glu
Gln
Pyro-glutamate
-17 Da
-18 Da

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Pyro-glutamate
Pyro-GluGln

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Deamidation
o Deamidation of asparagines is commonly observed and has an
important role in regulating the heterogeneity and stability of
recombinant mAbs.
o Glutamines are also susceptible to deamidation but at a much lower
rate unless subjected to particularly harsh conditions such as
extreme pH.
o pH, buffer type, and temperature are known factors that can affect
the rate of Asn deamidation.
o Deamidation in conversion of –NH2 to –OH .
The most rapid
conversion rates
occurring
The residue C-terminal to the Asn is a Gly or Ser

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Deamidation
Under mildly acidic conditions, it involves direct hydrolysis of Asn to
produce mainly Asp.

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Isomerisation
 Native Asp first converts to a cyclic imide intermediate, and then either
hydrolyzes back to Asp or isomerizes to iso-Asp.
 In addition, Harris, et al., determined that isomerization of Asp 102 in a
heavy chain CDR3 region of IgG1 Herceptin reduced its potency to 70%,
causing serious implications on drug efficacy .
 It is likely due to the fact that isomerization results in insertion of an
additional methylene group into the backbone, which can influence
protein stability and structure.
 A decrease of antigen binding of several antibodies has also correlated
with isomerization of Asp residues.
The sequences most sensitive to isomerization include:
Asp-Gly
Asp-Ser
His-Asp
pH-dependent reaction

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Oxidation
 Exposure of proteins to attack by free radicals in the presence of oxygen can
result in the oxidation of amino acid side-chain groups, peptide backbone
fragmentation, unfolding, changes in hydrophobicity, and altered susceptibility to
proteolytic enzymes.
 All amino acids can undergo oxidation to some degree under certain conditions,
those most susceptible to oxidation are the sulfur-containing residues (Cys, Met),
the aromatics (Phe, Trp, Tyr), and His.
 Oxidation of biotherapeutic proteins can alter their physical and biological
properties, affecting their potency and stability characteristics.
 As with Met oxidation, it is important to understand the potential for Trp
oxidation in mAbs .
 Extreme levels of Trp oxidation can be visualized via changes in solution color.
+16 Da
+32Da

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Variants Involving Cysteines
 A disulfide bond can undergo reversible β-elimination to initially form one
dehydroalanine and one persulfide on constituent chains.
 Continued degradation of the persulfide converts into a Cys residue (free
sulfhydryl) representing a point of no return for the native disulfide.
 Free sulfhydryls may alternatively become covalently modified by a free Cys
in the solution, which is referred as “cysteinylation”.
 Subsequent hydrolysis of dehydroalanine residues may contribute
substantially to fragmentation of the antibody hinge region to produce Fab
and Fab-Fc components and is accelerated by heat and increasing alkaline
pH.
 Optimizing Cys feed strategies can minimize these trisulfide variants,
eventually leading to lower heterogeneity for the target molecule.

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Sulphation
o Sulphation is a PTM predominantly associated with secretory and membrane
proteins.
o The attachment of a sulphate (SO3−) group to an oxygen atom of tyrosine, serine,
or threo-nine residues is effected by a sulfotransferases enzyme present in the
trans-Golgi network.
o Several hormone cell surface receptors are known to be tyrosine sulphated, and
sulphation is required for high affinity ligand binding and subsequent receptor
activation.

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