Size-exclusion chromatography-mass spectrometry with m-nitrobenzyl alcohol as post-column additive for direct characterization of size variants of monoclonal antibodies.
Size-exclusion chromatography (SEC) is commonly used to monitor low molecular weight fragments and aggregates present in recombinant monoclonal antibody (mAb) biopharmaceuticals. It has been previously demonstrated that SEC could be coupled with mass spectrometry (MS) to directly measure the molecular weights of these protein species to aid in their identification. However, the use of certain mobile phase modifiers led to compromised sensitivity in MS detection. In this work, we demonstrate that the use of m-nitrobenzyl alcohol (m-NBA) as a post-column additive in an SEC-MS method is able to improve the ionization of antibody light chain and heavy chain approximately 7-fold and 2-fold, respectively, and thus allows the MS detection of low-abundance size variants present in mAb biopharmaceuticals. Application of the 15-min reducing SEC-UV/MS method enabled the direct identification of size variants present in an IgG1 mAb sample. One high molecular weight species observed under reducing conditions was identified to be a thioether-linked heterodimer of light chain and heavy chain. Multiple lower molecular weight species were found to result from cleavage of the heavy chain at a number of sites throughout the conserved sequence. The reducing SEC-UV/MS method provides a straightforward approach for identification of size variants present in mAb and may be applicable generally to all types of mAb biopharmaceuticals.
Analytical Development, Biogen Idec, Inc. 14 Cambridge Center, Cambridge, MA 02142 United States.
Chong-Feng
CF
Xu
Analytical Development, Biogen Idec, Inc. 14 Cambridge Center, Cambridge, MA 02142 United States. Electronic address: li.zang@biogenidec.com.
Li
L
Zang
Analytical Development, Biogen Idec, Inc. 14 Cambridge Center, Cambridge, MA 02142 United States.
Andrew
A
Weiskopf
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000737:chemistry*; D001592:Benzyl Alcohols / Q000737:chemistry*; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001592:Benzyl Alcohols; D011994:Recombinant Proteins; C046886:3-nitrobenzyl alcohol
Aggregate; Antibody fragment; Liquid chromatography; Mass spectrometry; Size-exclusion chromatography