Comparative sedimentation equilibrium analysis of two IgG1 glycoforms: IgGCri and IgGWid

Khalil Abu Hammad; Vlad Dinu; Thomas E. MacCalman; Jacob Pattem; Margaret Goodall; Richard B. Gillis; Roy Jefferis; Stephen E. Harding

doi:10.1007/s00249-023-01656-x

Comparative sedimentation equilibrium analysis of two IgG1 glycoforms: IgGCri and IgGWid

Khalil Abu Hammad, Vlad Dinu, Thomas E. MacCalman, Jacob Pattem, Margaret Goodall, Richard B. Gillis, Roy Jefferis, Stephen E. Harding^*

^*Corresponding author for this work

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Abstract

The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution.

Original language	English
Pages (from-to)	439-443
Number of pages	5
Journal	European Biophysics Journal
Volume	52
Issue number	4-5
Early online date	17 May 2023
DOIs	https://doi.org/10.1007/s00249-023-01656-x
Publication status	Published - Jul 2023

Keywords

Fc/Fab glycans
SEDFIT-MSTAR
c(s)-distribution
c(M)-distribution
MULTISIG

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@article{804cf895075844d18fb4e5ff1edb5ca3,

title = "Comparative sedimentation equilibrium analysis of two IgG1 glycoforms: IgGCri and IgGWid",

abstract = "The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution.",

keywords = "Fc/Fab glycans, SEDFIT-MSTAR, c(s)-distribution, c(M)-distribution, MULTISIG",

author = "{Abu Hammad}, Khalil and Vlad Dinu and MacCalman, {Thomas E.} and Jacob Pattem and Margaret Goodall and Gillis, {Richard B.} and Roy Jefferis and Harding, {Stephen E.}",

year = "2023",

month = jul,

doi = "10.1007/s00249-023-01656-x",

language = "English",

volume = "52",

pages = "439--443",

journal = "European Biophysics Journal",

issn = "0175-7571",

publisher = "Springer",

number = "4-5",

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TY - JOUR

T1 - Comparative sedimentation equilibrium analysis of two IgG1 glycoforms

T2 - IgGCri and IgGWid

AU - Abu Hammad, Khalil

AU - Dinu, Vlad

AU - MacCalman, Thomas E.

AU - Pattem, Jacob

AU - Goodall, Margaret

AU - Gillis, Richard B.

AU - Jefferis, Roy

AU - Harding, Stephen E.

PY - 2023/7

Y1 - 2023/7

N2 - The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution.

AB - The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution.

KW - Fc/Fab glycans

KW - SEDFIT-MSTAR

KW - c(s)-distribution

KW - c(M)-distribution

KW - MULTISIG

U2 - 10.1007/s00249-023-01656-x

DO - 10.1007/s00249-023-01656-x

M3 - Article

SN - 0175-7571

VL - 52

SP - 439

EP - 443

JO - European Biophysics Journal

JF - European Biophysics Journal

IS - 4-5

ER -

Comparative sedimentation equilibrium analysis of two IgG1 glycoforms: IgGCri and IgGWid

Abstract

Keywords

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