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Glycosylation

Glycosylation sites

N (Asn, asparagine) are potential N-glycosylation sites when they are part of the N-glycosylation motif NXS/T, where X is any amino acid except proline. Glycans are linked to the nitrogen atom N (N-linked glycosylation)[1].

S (Ser, serine) and T (Thr, threonine) are potential glycosylation sites but the motifs are not very conserved. Glycans are linked to the oxygen atom O (O-linked glycosylation).

N-glycosylation of immunoglobulins (IG) or antibodies

Glycosylated IgG

All currently approved therapeutic antibodies on the market are IgG or derivatives.

IgG contain one conserved N-linked glycosylation site in each CH2 domain of their C-REGION. This site (asparagine, Asn N84.4) is located at the DE-TURN. They may also have secondary sites in the Fab regions but those are not conserved.

The IMGT Collier de Perles of CH2-DOMAIN is represented below for human IGHG1 (1hzh in IMGT/3Dstructure-DB) on one layer (left panel) or on two layers (right panel).

Collier 1D & 2D

Positions at which hydrophobic amino acids are found in more than 50% of analysed IG and TR sequences are shown in blue and prolines (P) are shown in yellow. The glycosyaltion site (84.4) is indicated in red.


The 3D structure of the human IgG b12 antibody with its 2 carbohydrate chains is shown below as an example (1hzh in IMGT/3Dstructure-DB, Jmol version 12.2.15).


3D structure

The heavy chains are represented in blue and purple, the light chains are in orange and green, respectively. The 2 N-glycans are illustrated in ball-and-stick representation.


Oligosaccharides found in human IgG

The human glycans are mainly classified as 'biantennary complex' structure with a core fucose and are often terminated with N-acetylneuraminic acid (NANA), a sialic acid.

The figure below shows the largest N-linked oligosaccharide structure found in human IgG. The third N-acetylglucosamine (GlCNac) bisecting arm represents around 10% of human IgGs glycoforms [2].



Carbohydrate

Gal: galactose, Man: mannose, Fuc: fucose, GlCNac: N-acetylglucosamine, NANA: N-acetylneuraminic acid and NGNA: N-glycolylneuraminic acid. Symbol nomenclature is from the Nomenclature Committee [3]


Description of the carbohydrate

The 4 most abundant glycans in mAb biopharmaceuticals are shown below. The Fc oligosaccharides are terminated by 0, 1 or 2 galactoses and are called G0, G1 or G2 respectively.


Carbohydrate

The conserved heptasaccharide core is composed of 2 N-acetylglucosamine (GlcNAc), 3 mannose (Man) and 2 other GlcNAc residues that are β-1,2 linked to α-6 Man and α-3 Man, forming two arms.

For G1F, Gal can be on the α1,3-arm or on the α1,6-arm.

Additional fucose (Fuc), galactose (Gal), N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) residues may be present or not, particularly depending on the expression system.

 

Current monoclonal antibodies production systems

Mammalian cell expression systems are the favorite methods for the commercial production of monoclonal antibodies because their protein glycosylation machinery closely resembles that in human.

The current marketed antibodies are mainly expressed in CHO (Chinese Hamster Ovary), SP2/0 (mouse myeloma cells), NS0 (Non-Secreting mouse myeloma cells) and hybridomas.

 

Glycosylation patterns of recombinant antibodies produced in human, CHO and NS0 cells

The figure bellow illustrates the main differences regarding the glycosylation patterns of antibodies produced in human cells, CHO and NS0.


Human vs. CHO and NSO

Fc glycosylation of monoclonal antibodies expressed in human cells

Monoclonal antibodies expressed in human cells are glycosylated on the IGHG CH2 N84.4 asparagin. The N-glycans are of the complex biantennary form.

There is a total of 36 possible isoforms, in 3 major classes G0, G1, G2, according to the number of terminal galactose.

Classes Glycan name Nb Fucosylated Afucosylated
G0agalactosylatedG0A04+B+F-B+F+B-F-B-Fasialylated
G1monogalactosylatedG1A04*2+B+F-B+F+B-F-B-Fasialylated
G1A14*2+B+F-B+F+B-F-B-Fmonosialylated
G2digalactosylatedG2A04+B+F-B+F+B-F-B-Fasialylated
G2A14*2+B+F-B+F+B-F-B-Fmonosialylated
G2A24+B+F-B+F+B-F-B-Fdisialylated

G0, G1, G2: with 0, 1 or 2 galactose; G0 = agalactosylated, G1 = monogalactosylated, G2 = digalactosylated
A0, A1, A2: with 0, 1 or 2 sialic acid; A0 = asialylated (or 'neutral'), A1 = monosialylated, A2 = disialylated. The percentage of A0, A1 et A2 inside each class is difficult to evaluate.
Monosialylation and monogalactosylation may occur on either the α1-3 or α1-6 arm of the biantennary structures.
+B, -B: with or without a 'bisecting' N-acetyl glucosamine, GlcNAc (between the two antennae of the glycan and attached to the position 4 of the branching mannose); +B = bisected
+F, -F: with or without a core fucose; +F = fucosylated, -F = afucosylated.

N-Glycan structures of IG expressed in human cells

The possible different structures from the table above are represented. Links when provided are to UniCarbKB.

Glycan name Fucosylated Afucosylated
G0A0 G0A0
UC 1495
G0A0_1
UC 1099
G0A0_2 G0A0_3
UC 1623
G1A0 G1A0_1
UC 1499
G1A0_1_1
UC 1599
G1A0_1_2 G1A0_1_3
UC 1489
G1A0_2
UC 1497
G1A0_2_1
UC 1493
G1A0_2_2 G1A0_2_3
UC 2073
G1A1 G1A1_1 G1A1_1_1 G1A1_1_2 G1A1_1_3
G1A1_2 G1A1_2_1 G1A1_2_2 G1A1_2_3
G2A0 G2A0
UC 1501
G2A0_1
UC 1603
G2A0_2 G2A0_3
UC 1521
G2A1 G2A1_1 G2A1_1_1
UC 1119
G2A1_1_2 G2A1_1_3
G2A1_2 G2A1_2_1
UC 1973
G2A1_2_2 G2A1_2_3
G2A2 G2A2 G2A2_1
UC 1527
G2A2_2 G2A2_3

Other N-glycan structures

G1F-GlcNac G1-GlcNac
G1F-GlcNac
UC 2099
G1-GlcNac
UC 3135

Mannose

Man9 Man8 Man7 Man6
Man9
UC 1645
Man8
UC 1643
Man7
UC 1641
Man6

Correspondance between nomenclatures for the IG glycans expressed in human cells

Correspondance between the GA and HN nomenclatures is given for the 36 possible structures of IG N-glycans expressed in human cells.

The GA nomenclature is based on the number of galactose (G) (0, 1 or 2) and sialic acid (A) (0, 1 or 2) at the terminal ends of the arms of the IG N-glycans. The presence or absence of the 'bisecting' N-acetyl glucosamine (B) and of the core fucose (F) are indicated by the letter B or F preceded by a + or a -.

The HN nomenclature is based on the number of carbohydrates in the glycan composition (H: number of hexoses, N: number of N-acetyl glucosamine), with, if present, S number of sialic acids. The presence of the core fucose is indicated by the letter F. The number between square brackets represents the total number of monosaccharides in each structure.

The graphical representation of the 36 possible structures of IG N-glycans expressed in human cells are shown above.

Glycan name
subclass
Fucosylated Afucosylated
+1N for 'bisecting' N-acetyl glucosamine
+ F for fucose
+1F for fucose+1N for 'bisecting' N-acetyl glucosamine
G0A0G0A0+B+FG0A0-B+FG0A0+B-FG0A0-B-Fasialylated
H3N4 [7]H3N5F [9]H3N4F [8], G0,H3N5 [8]H3N4, G0(-F)
G1A0G1A0+B+FG1A0-B+FG1A0+B-FG1A0-B-Fasialylated
H4N4 [8]H4N5F [10]H4N4F [9]H4N5 [9]H4N4 [8]G1 on α1-3 arm
H4N5F [10]H4N4F [9]H4N5 [9]H4N4 [8]G1 on α1-6 arm
G1A1G1A1+B+FG1A1-B+FG1A1+B-FG1A1-B-Fmonosialylated
H4N4S1 [9]H4N5S1F [11]H4N4S1F [10]H4N5S1 [10]H4N4S1 [9]G1 and A1 (S1) on α1,3 arm
H4N5S1F [11]H4N4S1F [10]H4N5S1 [10]H4N4S1 [9]G1 and A1 (S1) on α1,6 arm
G2A0G2A0+B+FG2A0-B+FG2A0+B-FG2A0-B-Fasialylated
H5N4 [9]H5N5F [11]H5N4F [10]H5N5 [10]H5N4 [9]
G2A1G2A1+B+FG2A1-B+FG2A1+B-FG2A1-B-Fmonosialylated
H5N4S1 [10]H5N5S1F [12]H5N4S1F [11]H5N5S1 [11]H5N4S1 [10]A1 (S1) on α1,3 arm.
S1 bond may be α2,3 or α2,6
H5N5S1F12]H5N4S1F [11]H5N5S1 [11]H5N4S1 [10]A1 (S1) on α1-6 arm.
S1 bond may be α2,3 or α2,6
G2A2G2A2+B+FG2A2-B+FG2A2+B-FG2A2-B-Fdisialylated
H5N4S2 [11]H5N5S2F [13]H5N4S2F [12]H5N5S2 [12]H5N4S2 [11]S1 and S2 bonds may be α2,3 or α2,6


Influence of the glycosylation modification

Glycosylation of IgG antibodies have been shown to have a large effect on the immunogeneicity, solubility and half-life.




References:
[1] Mellquist, J.L. et al. (1998) Biochemistry 37, 6833-6837.
[2] Raju, T.S. (2003) BioProcess International, 5398-9.
[3] Varki, A. (2009) Proteomics, 44-53.

External links:
Glycomics. Databases and tools at Expasy.