Glycosylation of proteins in cell culture is affected by many factors. The host cell line determines the complement of glycosylation enzymes and resulting glycoprotein structures. Culture conditions like batch versus continuous processing and extracellular environment can also impact glycosylation. Understanding glycosylation is important because protein properties like pharmacokinetics, bioactivity, and antigenicity are influenced by glycans.

1. Glycosylation of Proteins in Cell Culture
• Carbohydrates (glycans) are attached to proteins
as co-translational and post-translational
modifications (glycosylation)

2. 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.

3. Overview
1. 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

4. 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

5. 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

6. beta-interferon Immunoglobulin (monoclonal
antibodies, Mabs)
Protein N-glycan sites
Erythropoietin (EPO)
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

7. Approved Monoclonal Ab’s (2008)

8. Monoclonal Antibodies in Cancer Treatment
Majidi et al 2009

9. The list of glycosylated
biopharmaceuticals in
rapidly growing.
Proper glycosylation is
essential for the
function of these
biotherapeutics.
Kawasaki et al 2008

10. 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)

11. 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

12. 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)

13. Glycosylation is Important in Development:
Cell fate choices dependent on the Notch receptor require
appropriate glycan expression
A- Aberrant wing morphology results from mutation in glycosylation of the Notch receptor
B- normal neural development,
C-mutant with altered glycosylation
Essentials of Glycobiology
Second Edition Chapter 24, Figure 3

14. 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
Fv region
which
binds the
antigen
IgG oligosaccharide
affects the conformation Fab region which
of the Fab region and binds effector
affects how the antibody molecules and
binds to other molecules cells
which results in an
immunue response

15. Glycosylated vs Nonglycosylated
• Glycosylation can add up to 100% mass to a protein
• Example: EPO – erythropoetin
39 kDa
18 kDa

16. 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

17. 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- Peptide
linked fucose and xylose residues N-acetylglucosamine
Mannose
Galactose
• CHO cells are most commonly used for recombinant protein Fucose
production N-glycolylneuraminic acid
– Close to human glycosylation N-acetylneuraminic acid
Xylose
• Important to use Mammalian cells for glycoprotein
production
Transgenic Transgenic
Bacteria Yeast Insect Plants Animal Cells Human

18. Two Primary types of glycosylation are
differentiated by the type of linkage to the
protein
Serine
Asparagine

19. Differences in Oligosacchride Structures in N-linked or O-linked
Glycans
N-acetylglucosamine
Mannose
Galactose
N-acetylneuraminic acid
Fucose
N-acetylgalactosamine
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.

20. 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

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

22. 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)

23. Mucins

24. N-linked Glycosylation
• 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
Figure 12-51 (Alberts)

25. 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)

26. 3 Types of N-linked Glycans
Core region
Complex n-Linked Glycan:
Core with Terminal
Can be heterogeneous
-3 terminal branches
“Sequon” -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

27. Protein Glycosylation in RER
Polypeptide
enters ER
lumen
Proteins and lipid-glycan are
generated separately then
glycan transferred on to the
protein structure from the
lipid.
9 Mannose
Oligosaccharyl
transferase enzyme
Man- Man
transfers precursor
Man
oligosaccharide from Man- Man
dolichol to Asn GlcNAc-GlcNAc-Man
Man-Man-Man-Glc-Glc-Glc
2 N- 3 glucose
Acetylglucosamine
Figure 12-52 (Alberts)

28. Production of the Lipid-Glycan
The molecule is flipped from the ER
membrane to the ER lumen.
Cytoplasm Lumen
Sugar residues are added sequentially to the Additional sugars are added via dolichol
lipid to give a Man5-Glc3 structure (using phosphate. Finally, the oligosaccharide
nucleotides sugars. (14 residues) is transferred to a specific
Asn in the lumen (Man9-Glc3)

29. Dolichol Cycle
-synthesis of the sugar chain on
the lipid, dolichol
FLIPPASE ENZYME
Flips oligosaccharide to
internal
lumen of ER membrane
Oligosaccharide is
transferred from dolichol-
phosphate to the protein at a
sequon (Asn-X-Thr/Ser)

30. The Processing Reactions: the introduction of structure variation in the glycan
Begins after the glycan is added to the protein.
l n
k
j m
jProcessing begins – removal of glucoses ER Lumen
kMannosidase I removes 1 mannose
lGolgi mannosidase I removes 3 mannose Golgi Lumen
mN-acetylglucosamine transferase I adds GlcNAc
nMannosidase II to removes 2 mannose

31. Role of N-linked Glycosylation in Protein folding
If the glycoprotein is not correctly folded, glucose will
be readded and sent back through the calnexin cycle
To the GOLGI for processing
and modification of the glycan
and protein
-Binds glycoprotein to help with folding
-Recognizes glucose residues and
glucosidase cleaves off

32. N-Linked Glycosylation PathwayOligomannose
Asn Xaa Ser/Thr
Type
Dol Endoplasmic
Reticulum
P
a-Glc I a-Glc II a-Glc II a-Man I
P NH2
Oligosaccharide
transferase
Man
Glc Golgi
Glc
Glc
SialT GalT GnTII Man II GnTI
FucT
Complex
type
Man Hybrid
Processing Reactions
type

33. Fig 11
Production of tri- and tetra-antennary structures
GnT IV GnT V
Asn
GnT V
Asn GnT IV Asn
M3Gn2 M3Gn4
Asn
M3Gn3

34. Fig 12
Reaction network for N-linked glycosylation
Leads to great diversity in structures
M9 (From Umana and Bailey, 1997)
4x ManI 1-4
M5
GTI 5
GalT13 M Gn GnTIII 20 27
M5 GnGn b GalT M
M5 GnG 5 5
GalT 6 GnTIII 28 GnGnbG
M4 GnG 14 M4 Gn 21 M4 GnGnb GalT M4
GalT 7 GnTIII GnGnbG
M3 GnG 29
22 M3 GnGnb GalT M3
15 M3 Gn
8 30 GnGnbG
16 GnTIII
M3 Gn2G GalT M Gn 23 M Gn Gnb GalT M3 Gn2GnbG
3 2 3 2
GnTIV10 9 GnTV
18GalT 17 GalT
M3 Gn3G M3 Gn3 M3 Gn3’ M3 Gn3’G
12 GnTIII
GnTIII GnTV
25 b GnTIV 24 GalT
M3 Gn3Gn 11 M3 Gn3 ’Gnb M3 Gn3’Gnb
M3 Gn4 31
GalT GalT GnTIII
32 19 26 GalT
M3 Gn3Gn bG M3 Gn4G M3 b
b
33 M3 Gn4Gn G

35. Cell-associated factors that affect product
glycosylation in cell culture
• 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

36. 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

37. 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

38. 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

39. 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

40. Adherent Cells Suspension
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

41. 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

42. 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

43. 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

44. Effect of Dissolved Oxygen on
Sialylation of EPO
100
% sialylated structues
95
90
85
80
75
70
65
60
3% 10% 50% 100% 200%
DO concentration (% air saturation)

45. 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

46. 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