Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

Abstract

Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly. (J Am Soc Mass Spectrom 2009, 20, 2258 -2264) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry M ass spectrometry is one of the most indispensable techniques for the characterization of recombinant monoclonal antibodies because most of the modifications that result in heterogeneity and degradation are involved in molecular weight differences [1,2]. Various modifications are determined by analyzing recombinant monoclonal antibodies at different levels, depending on the molecular weight differences of the modifications. Modifications, such as N-terminal glutamine and glutamate cyclization [3][4][5][6][7][8][9], different types of the conserved N-linked oligosaccharides [5,[7][8][9][10][11][12][13], amino acid truncation and insertion [8,11], cysteinylation [14], C-terminal lysine processing [5,7,11,15,16], fragmentation [12,15,17], glycation [18], oxidation [19,20], and nitration [21] can be directly determined by measurements of the molecular weights of intact antibodies, antibody light chain and heavy chain, and Fab and Fc fragments after papain or lys-C digestion [14]. On the other hand, analysis at the peptide level is normally required to determine the sites of modifications and modifications with small molecular weight differences, such as deamidation [22,23] and amidation [11], which results in a molecular weight difference of only 1 Da.Mass spectrometry is commonly coupled with reversedphase high-performance liquid chromatography (RP-HPLC), which desalts the samples and separates different components based on hydrophobicity. The molecular weights of intact antibodies [12,15] and their light chains and heavy chains can be readily measured by on...

A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates

“…[94–97] Lazar et al [95] applied a similar principal for the analysis of immunoconjugates. In this case, covalently linked immunoconjugates were distinguished from the intact monoclonal antibody under SEC-MS conditions, providing utility that could not be achieved by either offline sample preparation or reversed phase desalting.…”

“…In this case, covalently linked immunoconjugates were distinguished from the intact monoclonal antibody under SEC-MS conditions, providing utility that could not be achieved by either offline sample preparation or reversed phase desalting. Liu et al [94] developed a similar approach for the analysis of reduced and alkylated monoclonal antibodies with acetonitrile and TFA in the mobile phase, thus allowing for rapid desalting (Figures 5b and 5c). [94,97] …”

Fragmentation of monoclonal antibodies

“…42 When combined with reduction of the sample, denaturing SEC (dSEC) should, theoretically, detect the same fragments as reducing SDS PAGE, although with significantly lower resolution.…”

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity

“…Strong organic acids can effectively dissociate the heavy and light chains of reduced antibodies and allow chromatographic separation of the chains by size-exclusion, reversephase, or cation-exchange columns. 28,29 Unfortunately, these methods also required the use of trifluoroacetic acid/acetonitrile or sodium chloride gradients for proper elution, both of which are incompatible with capillary isoelectric focusing. Furthermore, the antibodies are often modified (alkylated) prior to chromatography.…”