direct quality control of glycoengineered erythropoietin ......galactose feeding increases...
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![Page 1: Direct Quality Control of Glycoengineered Erythropoietin ......Galactose feeding increases galactosylation Increase in glutamine/ammonia reduces galactosylation and sialylation Serum](https://reader033.vdocument.in/reader033/viewer/2022041702/5e41ea897d88303ad21df3e1/html5/thumbnails/1.jpg)
Biomolecular Mass Spectrometry and ProteomicsUniversity of Utrecht
Direct Quality Control of GlycoengineeredErythropoietin Variants
Tomislav Čaval
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Biotherapeutics
+80% of biotherapeutics are glycosylated
Walsh G. Biopharmacutical benchmarks 2018. Nature Biotechnology
Drug Generic nameSales in 2017
(Billions) Glycosylation
1 Humira Adalimumab $18.94 2 N-glycans
2 Enbrel Etanercept $8.34 6 N-glycans, 26 O-glycans
3 Rituxan Rituximab $7.78 2 N-glycans
4 Remicade Infliximab $7.77 2 N-glycans
5 Herceptin Trastuzumab $7.39 2 N-glycans
6 Avastin Bevacizumab $7.04 2 N-glycans
7 Lantus Insulin glargine $6.72 /
8 Eylea Aflibercept $5.93 12 N-glycans
9 Opdivo Nivolumab $5.79 2 N-glycans
10 Neulasta Pegfilgrastim $4.50 /
11 Stelara Ustekinumab $4.01 2 N-glycans
.. …. …. … …
20 Aranesep/Nesp Darbepoetin Alfa $2.62 5 N-glycans, 1 O-glycan
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Anti-inflammatory properties
serum half-life increase
Core fucose -ADCC
Control of glycosylation = control immune system activation/deactivation
Major issues : 1. Controlling the glycosylation2. Product characterization
Biotherapeutics
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Controlling the glycosylation
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Characterizing the glycosylation
Yang, Y.; Franc, V.; Heck, A. J. R. Trends Biotechnol. 2017
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Why Native MS?
Yang, Y.; Franc, V.; Heck, A. J. R. Trends Biotechnol. 2017
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Native MS of antibodies
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Erythropoietin
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Erythropoietin Glycoengineering (Controlling the glycosylation)
EPO CLONE Gene engineering Effect
C 01 mgat1 KO High mannose glycans
C 02 mgat3/4A/4B/5 cosmc KO Biantennary glycans + Tn antigen
C 03 mgat2 KO Loss of β2-Branch
C 04 KI st6gal1 in mgat4A/4B/5
st3gal4/6 -/-
Homogenous biantennary α2,6-NeuAc sialylated glycans
C 05 KI st6gal1 in mgat4A/4B/5
st3gal4/6 -/-
Biological replicate
C 06 mgat4A/4B/5 st3gal4/6 B3gnt2 KO
+ st6Gal-I KI
Homogenous biantennary α2,6-NeuAc sialylated glycans
C 07 B3gnt2/mgat4A/4B/5 KO Biantennary glycans with eliminated polyLacNAc
C 08 mgat4A/4B/5 KO Biantennary sialylated glycans
C 09 st3gal6 KO in
mgat4a/4b/5/ST3Gal4 -/-
Biantennary glycans with decreased sialylation
C 10 st3gal6 KO in
mgat4A/4B/5/st3gal4 -/-
Biological replicate
C 11 B3gnt2 KO in mgat4A/4B/5
st3gal4/6 -/-
Biantennary glycans with decreased sialylation and no
polyLacNAc
C 12 B3gnt2 KO in mgat4A/4B/5
st3galt4/6 -/-
Biological replicate
C 13 fut8 KO No fucosylation
C 14 B4galt3 KO No effect
C 15 B3gnt1 KO No effect
C 16 B3gnt2 KO Eliminated polyLacNAc
WT / Wild type
C 17 B4galt4 KO No effect
C 18 st3gal4/6 KO Decreased sialylation on N-glycans
C 19 st3gal4/6 KO Biological replicate
C 20 mgat3/4A/5 cosmc KO Eliminates β6-branch of N-glycans, O-glycans all in the
form of Tn
C 21 mgat 5 KO Eliminates β6-branch of N-glycans
C 22 mgat4A/4B/5/st3gal4/6 cosmc KO UNKNOWN (sample mislabeled), native data indicate
mgat4b
C 23 mgat4A/4B KO Eliminates β4-branch of N-glycans
C 24 mgat4B KO Eliminates β4-branch of N-glycans
1. Characterization of glycosyltransferases with native MS
2. Can we create homogeneous EPO
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Wild type EPO
Čaval et al. Nature Communications 2018Bern M., Caval T. et al. Journal of proteome research 2018.
PMI Intact Mass Deconvolution
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Wild type EPO (II)
Čaval et al. Nature Communications 2018
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Custom-engineered EPO: Mgat5 versus Mgat 4a/4b example
Mga
t4
a/4
b
Mga
t5
They should result in the same glycoforms (compositions should be identical)
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Custom-engineered EPO: Mgat5 versus Mgat 4a/4b example
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Sialylation knock out (MALDI)
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Sialylation knock out (EMR)
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Sialylation knock out (EMR)
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Can we profile quicker?
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Similarity based EPO profiling
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C 22 mgat4A/4B/5/st3gal4/6 cosmc KOC 24 mgat4B KO
Similarity based EPO profiling (II)
Čaval et al. Nature Communications 2018
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Conclusions (1/3)
Glycoengineering enables the study of structure to function relationship of glycans
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Conclusons
HO
O
OH
OHCO2-
O
HO
HO
O
OH
HONH
O
O
O
OH
OH
O
CO2-
O
HO
HO
OH
HONH
OHO
O
OAcNH
O
CO2-
O
HO
HO
O
HONH
O
CO2-
O
HO
HO
OH
HONH
O
O O
OH
OH
HOO C12H25
OH
HN
O
R
CO2-
O
HO
HO
OH
HONH
OHO
O
HONHAc
O
HO
O
OH
OHCO2-
O
HO
HO
O
O
HONH
O
CO2-
O
HO
HO
O
HONH
O
CO2-
O
HO
HO
OH
HONH
O
n = 55-200
ST6GALNAC6
ST6GALNAC5
ST6GALNAC4
ST6GALNAC3
ST8SIA1
ST8SIA5
ST3GAL1
ST3GAL2
ST3GAL4
ST3GAL5
ST3GAL6
ST6GALNAC3
ST6GALNAC2
ST6GALNAC1
ST6GALNAC4
ST6GAL2
ST8SIA3
ST8SIA4
ST8SIA2
ST3GAL1
ST3GAL2
ST3GAL3
ST3GAL4
ST3GAL6
CMPCMPNT
CO2-
O
HO
HO
OH
OH
HONH
O
a2,8-
a2,3-
a2,6-
a2,6-
a2,3-
a2,8-
a2,8-
NEU1
NEU2
NEU3
NEU4
Sialoglycoconjugate
production
Glycan
recycling
ST6GAL1
ST8SIA6
Golgi
KL
Native MS serves as a rapid profiling tool for selection of cell clones during cell line developments for recombinant therapeutics
Conclusions (2/3)
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Glucose limitation reduces site occupancy
Galactose feeding increases galactosylation
Increase in glutamine/ammonia reduces galactosylation and sialylation
Serum enhances fucosylation, but introduces non-human sialic acid
Manganese
Sodium-butyrate
glycerol
DMSO
Lipids
Nucleotide precursors
Dissolved oxygenpHCarbon dioxide
TemperatureMode of culture
Cell growth speedCell density
Expression rate
Conclusions
Native MS:
1. enables screening of consistency in glycoprotein products
2. especially suited for studying glycosylated biotherapteuics- differences between biosimilars and originator- divergence of a biologic through its product lifecycle
Conclusions (3/3)
DMSO Shear Stress Glycerol Manganese
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• University Utrecht• Albert Heck
• University of Copenhagen• Henrik Clausen
• Yang Zhang
• Weihua Tian
Acknowledgments