SEC/MS Analysis of a Bispecific Antibody

Introduction

More potent formats of monoclonal antibodies (mAbs), such as bispecific antibodies (bsAbs), are on the rise in the area of biotherapeutics. bsAbs recognize two different epitopes. This dual specificity increases the potency of these molecules compared to mAbs and expands the range of possible applications. bsAbs can be used to redirect T cells to tumor cells, block two different signaling pathways simultaneously, dually target different disease mediators, and deliver payloads to targeted sites. At this time, more than 50 bsAb products are currently undergoing clinical evaluation.

Characterization of bsAbs is essential to ensuring product safety and efficacy. Size exclusion chromatography (SEC) coupled with mass spectrometry (MS) is increasingly being used to identify the accurate molecular  mass of biomolecules, including bsAbs. SEC/MS, however, requires the use of mobile phases that do not contain high concentrations of non-volatile salts and the use of columns that do not exhibit particle shedding  which will interfere with the MS signal response.

In this application note, a bispecific T cell engager (BiTE^®) consisting of two single-chain variable fragments (scFvs) recombinantly  linked by a nonimmunogenic five-amino-acid chain (Figure 1) was analyzed by SEC/MS using a TSKgel^® UP-SW3000, 2 μm column.