POST TRANSLATION MODIFICATION

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Somayeh bakhshalizadeh

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POST-TRANSLATION MODIFICATION OF MONOCLONAL ANTIBADYSuperviser :Dr.Nasiri

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2Monoclonal antibodystructure of monoclonal antibodies5 major classes of secreted antibodyPost-translational ModificationProtein Misfolding and Aggregation GlycosylationPyro-glutamateDeamidationIsomerisationOxidationVariants Involving CysteinesSulphation

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Monoclonal antibodyMonoclonal 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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1.1 Central Concepts

HeavyHeavyHeavyLightLightDisulfide BondLight

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Two pairs of identical heavy and light tertiary proteins

The chains are joined by disulfide bonds

The structure of monoclonal antibodiesHeavy

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VHVLCH1CLCH2CH3

FcFab

Fab

5The structure of monoclonal antibodiesConserved 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

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CH3CH2

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CH3CH2CH1

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CH3CH2CH1VH1

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CH3CH2CH1VH1VL

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CH3CH2CH1VH1CLVL

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CH3CH2CH1VH1CLVL

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HingeCH3CH2CH1VH1VLCL

Elbow

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Hinge

FvFb

FabCH3CH2CH1VH1VLCL

Fc

Elbow

Carbohydrate

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5 major classes of secreted antibody

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Post-translational Modification

16What 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 - ....

18 Post-translational ModificationWhat purpose ?- regulation of functionsignal transductioncellular regulationDegradationstructural/conformational rearrangementsWhy do we study it?

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18Types of post-translational modificationsA variety of changesPhysicalChemistry

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Fragmentation of Intact mAb

19The cleavage rates are sequence?- PH- temperaturecleavage of amino acidAsp-pro

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The Chemical Modification of Antibody

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Protein Misfolding and Aggregation GlycosylationPyro-glutamateDeamidationIsomerisationOxidationVariants Involving CysteinesSulphation

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Protein Misfolding and Aggregation

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The folding and initial glycosylation of most secretedproteins 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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22 GlycosylationGlycosylation 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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23 GlycosylationGlycosylation

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

Thr

SerCys

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24 GlycosylationGlycosylation 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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25 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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26 GlycosylationTerapeutic 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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27RitoximabThe 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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31Pyro-glutamateConversion 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-17 Da-18 Da

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32 Pyro-glutamate

Pyro-Glu

Gln

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33DeamidationDeamidation of asparagines is commonly observed and has an important role in regulating the heterogeneity and stability of recombinant mAbs.

Glutamines are also susceptible to deamidation but at a much lower rate unless subjected to particularly harsh conditions such as extreme pH.

pH, buffer type, and temperature are known factors that can affect the rate of Asn deamidation.

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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34 Deamidation

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

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IsomerisationNative 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: pH-dependent reaction

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36 OxidationExposure 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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37 Variants Involving CysteinesA disulfide bond can undergo reversible -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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38 Sulphation

Sulphation is a PTM predominantly associated with secretory and membrane proteins.

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.

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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