Glycosylation of Proteins in Cell Culture

• Carbohydrates (glycans) are attached to proteins as co-translational and post-translational modifications (glycosylation)

Many biological molecules are Glycoproteins

Glycosylation affects the functional qualities of the protein. We must know how the structure relates to the function to create effective biotherapeutics.

Overview1. Why is glycosylation important in biotechnology.

2. Purpose of glycosylation in general.

3. Types of glycans: N-linked, O-linked.

4. Variation in glycosylation between cell types

5. Synthesis of N-linked glycans

6. Cell culture conditions that affect glycosylation

Understanding glycosylation in biological drugs is important for two main reasons:

• Glycan can affect many of the protein properties: pharmacokinetics (uptake and length of time in the body), bioactivity, secretion, in vivo clearance, solubility, recognition, and antigenicity

• Quantitative and qualitative aspects of glycosylation affected by production process in culture, including cell line, method of culture, extracellular environment, and protein itself

Why is the biotech industry concerned about Glycosylation?

• Batch to batch variability of glycosylation patterns affect product quality

• Too much variation in glycosylation leads to discarding of product

• Regulatory agencies (eg FDA, Health Canada) have regulations for amount of acceptable variability in glycosylation –deviations can lead to redoing clinical trials

• Change in glycosylation can lead to another company claiming a new patent

• Adverse reactions in patients to non-human glycosylation

beta-interferon Immunoglobulin (monoclonalantibodies, Mabs)

Erythropoietin (EPO)

Protein N-glycan sites

gp120 25

huCD36 9

huICAM-1 (CD54) 8

hu-tPA 3

hu-Epo 3

hu-IFN gamma 3

rhu ant-IL-8 (IgG) 2

hu-CSF 2

hu-IFN beta 1

Approved Monoclonal Ab’s (2008)

Monoclonal Antibodies in Cancer Treatment

Majidi et al 2009

The list of glycosylated biopharmaceuticals in

rapidly growing. Proper glycosylation is

essential for the function of these biotherapeutics.

Kawasaki et al 2008

FAQs about glycosylation

• 50% of eukaryotic proteins are glycosylated• N-linked (Asn) and O-linked (Ser/Thr) glycosylation • N-linked glycosylation is the more complex• 65% of sequons (attachment sites) are occupied• Macroheterogeneity = variation in occupancy of

sequons (eg. one site vs two site occupied)• Microheterogeneity = variation in structures of

glycans (eg. Biantennary vs triantennary at site)

Why is Glycosylation Important in Biotherapeutics?

Carbohydrate structures can affect the properties of the glycoprotein, including:

→ pharmacokinetics → bioactivity → secretion → in vivo clearance → solubility → recognition → antigenicity

General Function of Glycosylation• N-linked glycosylation prevalent in eukaryotes but not as

common in prokaryotes• Function of glycans not well defined for many GP’s:

– May be to aid protein folding and transport process– Prevent self adhesion of molecule (eg. beta-interferon)– Oligosaccharide can limit the approach of other

macromolecules • Eg. Inhibit digestion of glycoprotein by proteases (eg. high

concentration on cell surface)

– Regulatory roles• Eg. Notch: cell surface signalling receptor – important for proper cell

fate determination (O-glycosylated)

Chapter 24, Figure 3

Glycosylation is Important in Development: Cell fate choices dependent on the Notch receptor require

appropriate glycan expression

Essentials of Glycobiology Second Edition

A- Aberrant wing morphology results from mutation in glycosylation of the Notch receptorB- normal neural development, C-mutant with altered glycosylation

Purpose of Glycosylation (cont’d)• Activation of secondary pathways:

– Eg. IgG (monoclonal antibodies)• Differences in glycan structure can change the way the antibody

elicits a response when it binds to the antigen

IgG oligosaccharide affects the conformation of the Fab region and affects how the antibody binds to other molecules which results in an immunue response

Fab region which binds effector molecules and cells

Fv region which binds the antigen

Glycosylated vs Nonglycosylated• Glycosylation can add up to 100% mass to a protein• Example: EPO – erythropoetin

18 kDa

39 kDa

Glycosylation of protein in cell culture

• Mammalian vs prokaryotes, lower eukaryotes– Mammalian cells perform post-translational modifications and

achieve a product close to that produced in vivo

– Most Prokaryotes lack glycosylation machinery (exception: Campylobacter, N-linked glycosylation)

– Yeast, insect, and plant cells produce different glycan structures• glycan processing in golgi differs from mammalian cells

Organisms Differ in Glycosylation• Bacteria are incapable of glycosylating recombinant

mammalian proteins• Yeast have the tendency to hyper-mannosylate• Plant and Insect produced glycoproteins tend to have α 1,3-

linked fucose and xylose residues

• CHO cells are most commonly used for recombinant protein production– Close to human glycosylation

• Important to use Mammalian cells for glycoprotein production

Peptide

N-acetylglucosamine

Mannose

Galactose

Fucose

N-glycolylneuraminic acid

N-acetylneuraminic acid

Xylose

YeastTransgenic

Plants InsectTransgenic

Animal Cells HumanBacteria

Asparagine

Serine

Two Primary types of glycosylation are differentiated by the type of linkage to the protein

N-linked or O-linked oligosaccharide chains on proteins can have many different patterns of sugar residues at the same sequon. This is called Microheterogeneity.

Differences in Oligosacchride Structures in N-linked or O-linked Glycans

N-acetylglucosamineMannoseGalactoseN-acetylneuraminic acidFucoseN-acetylgalactosamine

O-glycans• Glycan is bound via an O-glycosidic bond of GalNAc to a Ser/Thr (O-

glycosylation)

• Classified as one of 8 core structures

• Any Ser/Thr residue is a potential site for O-glycosylation, no consensus sequence identified

• Addition of the glycan occurs on a fully folded protein

Core Structures Can Have Many Additions (Rose and Voynow 2006)

O-linked Glycans

• Huge variety of structures: from very short to very long chains

• Are important in mucins (major component of mucus), with very long chains

• Are often found altered in cancer cells• Important in blood cell types (A, B, etc)

Mucins

N-linked Glycosylation

Figure 12-51 (Alberts)

• N-linked precursor added to most proteins in RER membranes

• Only Asn in Asn-X-Ser/Thr become glycosylated

• Core region survives extensive oligosaccharide trimming in Golgi

Presence of Sequon (Asn-X-Ser/Thr) does not guarantee glycosylation

1. Spatial arrangement of the peptide during translation process may expose or hide the tripeptide sequence

2. Glycosylation depends on X: (sequon Asn-X-Ser/Thr)

glycosylation high when X = Ser, Phe,

intermediate for Leu, Glu,

very low for Asp, Trp, and Pro

3. Availability and correct assembly of precursors (eg. nucleotide sugars)

4. Level of expression of the oligosaccharyltransferase enzyme(s)

5. Disulfide bond formation within protein (makes site inaccessible to precursor addition)

3 Types of N-linked Glycans

“Sequon”

Core region

Complex n-Linked Glycan: Core with Terminal Can be heterogeneous -3 terminal branches -2 or 4 also common

High Mannose N-linked Glycan:• Not trimmed to core and more mannose are added on• 2 to 6 Additional mannose added onto core

Hybrid N-Linked Glycan:Hybrid of high mannose and complex One Mannose Branch One GlcNAc and Gal branch

Protein Glycosylation in RER

Proteins and lipid-glycan are generated separately then glycan transferred on to the protein structure from the lipid.

Polypeptide enters ER

lumen

Oligosaccharyl transferase enzyme transfers precursor

oligosaccharide from dolichol to Asn GlcNAc-GlcNAc-Man

ManMan- Man

Man- Man

Man-Man-Man-Glc-Glc-Glc

2 N-Acetylglucosamine

9 Mannose

3 glucose

Figure 12-52 (Alberts)

The molecule is flipped from the ER membrane to the ER lumen.

Additional sugars are added via dolichol phosphate. Finally, the oligosaccharide (14 residues) is transferred to a specific Asn in the lumen (Man9-Glc3)

Sugar residues are added sequentially to the lipid to give a Man5-Glc3 structure (using nucleotides sugars.

Cytoplasm Lumen

Production of the Lipid-Glycan

Dolichol Cycle-synthesis of the sugar chain on

the lipid, dolichol

FLIPPASE ENZYMEFlips oligosaccharide to

internallumen of ER membrane

Oligosaccharide is transferred from dolichol-

phosphate to the protein at a sequon (Asn-X-Thr/Ser)

jProcessing begins – removal of glucoseskMannosidase I removes 1 mannose

lGolgi mannosidase I removes 3 mannosemN-acetylglucosamine transferase I adds GlcNAc nMannosidase II to removes 2 mannose

jk

l

m

n

ER Lumen

Golgi Lumen

The Processing Reactions: the introduction of structure variation in the glycanBegins after the glycan is added to the protein.

Role of N-linked Glycosylation in Protein folding

-Binds glycoprotein to help with folding-Recognizes glucose residues and glucosidase cleaves off

To the GOLGI for processing and modification of the glycan and

protein

If the glycoprotein is not correctly folded, glucose will be readded and sent back through the calnexin cycle

Asn Xaa Ser/ThrDol

PP

NH2

Golgi

Hybrid type

Man

Complex type

Oligosaccharide transferase

Glc

GlcGlc

EndoplasmicReticulum

a-Glc I a-Glc II a-Glc II a-Man I

N-Linked Glycosylation Pathway

Man

OligomannoseType

GnTISialT

FucT

GalT GnTII Man II

Processing Reactions

Asn

Asn

Asn Asn

M3Gn3

M3Gn4M3Gn2

Production of tri- and tetra-antennary structures

GnT V

GnT IV

GnT IV

GnT V

Fig 11

M9

M5

M5 Gn

M4 Gn

M3 Gn2

M3 Gn3 M3 Gn3’

M3 Gn4

M3 Gn3Gnb

M3 Gn3GnbG

M3 Gn3G

M3 Gn4G

M3 Gn3’G

M3 Gn3’Gnb M3 Gn3

’Gnb

M3 Gn4Gnb M3 Gn4GnbG

M3 Gn

M5 GnG

M4 GnG

M3 GnG

M3 Gn2G

4x ManI

GTIGalT

GalT

GalT

GalT

M5 GnGnb M5 GnGnbG

M4 GnGnb M4 GnGnbG

M3 GnGnb M3 GnGnbG

M3 Gn2 Gnb M3 Gn2GnbG

GalT

GalT

GalT

GalT

GnTIII

GnTIII

GnTIII

GnTIII

GalT

GalT GalT

GalT

GalT

GalT

GnTIIIGnTIIIGnTIV

GnTIV

GnTIII

GnTV

GnTV

1-4

5

6

7

8

910

11

12

13

14

15

16

1718

19

20

21

22

23

2425

26

27

28

29

30

31

32

33

Reaction network for N-linked glycosylationLeads to great diversity in structures

(From Umana and Bailey, 1997)

Fig 12

• host cell line- complement of processing enzymes • mode of culture

- suspension/ attached- batch/ continuous• specific protein productivity- changes rate of transit through Golgi• extracellular degradative enzymes- release of sialidases by cells

Cell-associated factors that affect product

glycosylation in cell culture

Factors affecting protein glycosylation (N-linked)

1. Host cell• glycan structures on the same proteins can vary

between species and even different tissues

• due to:– differences in relative activities of glycan processing

enzymes (glycosidases and glycosyltransferases)

– differences in the monosaccharide precursors

CHO and BHK• Structure of sialic acid from CHO and BHK differ from human sialic acid

(also in rodents, pigs, sheep, cows, and new world monkeys)– NGNA – N-glycoyl-neuraminic acid (humans don’t produce this)– NANA – N-acetyl-neuraminic acid (most common sialic acid)

• Presence of a2,3 terminal sialic acid addition compared to a2,6 terminal sialic acid (in humans)

• Absence of a functional a1,3 fucosyltransferase• Absence of N-acetylglucosaminyltransferase III (Gn TIII)

– differences do not lead to immunogenic responses to glycoproteins– no adverse physiological effect due to structural differences

Hamster vs Mouse cells

• Mouse cells express: a1,3 galactosyltransferase:

generating Gala1,3-Galb1,4-GlcNAc (not found in humans)

– gene is present in CHO and BHK but not expressed

• Limits use of murine cells in therapeutic glycoprotein production

2. Culture environment

• Specific conditions of the culture can affect glycosylation independently of the cell line

• During the process of a batch culture, nutrient consumption and product accumulation can change the culture environment– gradually decreasing the extent of protein glycosylation

• may lead to variable glycoform heterogeneity and batch-to-batch variation

3. Mode of culture

• adaptation from anchorage dependent growth to suspension culture may also affect the glycsosylation process

• presence or absence of serum also has a significant affect on glycosylation– presence of hormones and growth factors, high

activities of sialidase and fucosidase

Adherent Cells Suspension

4. Protein productivity • differences in growth rate, specific productivity,

and cell density among the bioreactors may cause variation in the pattern of N-linked glycan structures

• rate of protein expression may also affect glycosylation

5. Glucose availability • glucose limitation results in incomplete protein glycosylation

– synthesis of abnormal dolichyl precursor oligosaccharides– sequences that are normally glycosylated remain empty

6. Ammonia• accumulated ammonia is inhibitory to cell growth and to protein

glycosylation– increase in pH of the normally acidic distal golgi– increase in the UDP-GNAc pool (reduces sialylation)

7. pH • maximum glycosylation of a protein occurs between pH 6.9-8.2

8. Oxygen limitations• Limiting nutrient because of it’s low solubility in media

1. reduced dissolved oxygen (DO) may lead to reduction in UDP-Gal– reduced oxidative phosphorylation of UDP-Gal

– reduced UDP-Gal transport from the cytosol to the golgi

2. formation of premature disulfide bonds in the nascent protein

Effect of Dissolved Oxygen on Sialylation of EPO

6065707580859095

100

3% 10% 50% 100% 200%

DO concentration (% air saturation)

% s

ialy

late

d s

tru

ctu

es

9. Growth factors, vitamins and hormones• up- and down-regulation of specific glycosyltransferases in

conjunction with hormonal induction of cell differentiation

• changes due to induction or repression or induction of the enzymes involved in protein glycosylation

10. Extracellular degradation of glycoproteins• glycosidases may be released to the extracellular

environment by secretion or by cell lysis• activity of glycosidases depends on medium pH,

temperature, residence time of glycoprotein, and level of extracellular activity