Abstract
During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultrahigh-performance liquid chromatography-triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multilevel structural characterization is important for the successful development of therapeutic antibodies.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7091-7100 |
| Number of pages | 10 |
| Journal | Analytical Chemistry |
| Volume | 88 |
| Issue number | 14 |
| DOIs | |
| State | Published - Jul 19 2016 |
Fingerprint
ASJC Scopus subject areas
- Analytical Chemistry
Cite this
Quantitation of Site-Specific Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs. / Yang, Nan; Goonatilleke, Elisha; Park, Dayoung; Song, Ting; Fan, Guorong; Lebrilla, Carlito B.
In: Analytical Chemistry, Vol. 88, No. 14, 19.07.2016, p. 7091-7100.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Quantitation of Site-Specific Glycosylation in Manufactured Recombinant Monoclonal Antibody Drugs
AU - Yang, Nan
AU - Goonatilleke, Elisha
AU - Park, Dayoung
AU - Song, Ting
AU - Fan, Guorong
AU - Lebrilla, Carlito B
PY - 2016/7/19
Y1 - 2016/7/19
N2 - During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultrahigh-performance liquid chromatography-triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multilevel structural characterization is important for the successful development of therapeutic antibodies.
AB - During the development of recombinant monoclonal antibody (rMAb) drugs, glycosylation receives particular focus because changes in the attached glycans can have a significant impact on the antibody effector functions. The vast heterogeneity of structures that exist across glycosylation sites hinders the in-depth analysis of glycan changes specific to an individual protein within a complex mixture. In this study, we established a sensitive and specific method for monitoring site-specific glycosylation in rMAbs using multiple reaction monitoring (MRM) on an ultrahigh-performance liquid chromatography-triple quadrupole MS (UHPLC-QqQ-MS). Our results showed that irrespective of the IgG subclass expressed in the drugs, the N-glycopeptide profiles are nearly the same but differ in abundances. In all rMAb drugs, a single subclass of IgG comprised over 97% of the total IgG content and showed over 97% N-glycan site occupancy. This study demonstrates the utility of an MRM-based method to rapidly characterize over 130 distinct glycopeptides and determine the extent of site occupancy within minutes. Such multilevel structural characterization is important for the successful development of therapeutic antibodies.
UR - http://www.scopus.com/inward/record.url?scp=84978887918&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84978887918&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.6b00963
DO - 10.1021/acs.analchem.6b00963
M3 - Article
VL - 88
SP - 7091
EP - 7100
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 14
ER -