Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography.
OBJECTIVE
The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.


METHODS
IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).


RESULTS
Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact.


CONCLUSIONS
ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.
Kükrer
Başak
B
Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
Filipe
Vasco
V
van Duijn
Esther
E
Kasper
Piotr T
PT
Vreeken
Rob J
RJ
Heck
Albert J R
AJ
Jiskoot
Wim
W
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D031521:Fractionation, Field Flow; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis* / Q000737:chemistry; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G