1 research outputs found
Cysteine-SILAC Mass Spectrometry Enabling the Identification and Quantitation of Scrambled Interchain Disulfide Bonds: Preservation of Native Heavy-Light Chain Pairing in Bispecific IgGs Generated by Controlled Fab-arm Exchange
Bispecific antibodies
(bsAbs) are one of the most versatile and
promising pharmaceutical innovations for countering heterogeneous
and refractory disease by virtue of their ability to bind two distinct
antigens. One critical quality attribute of bsAb formation requiring
investigation is the potential randomization of cognate heavy (H)
chain/light (L) chain pairing, which could occur to a varying extent
dependent on bsAb format and the production platform. To assess the
content of such HL-chain swapped reaction products with high sensitivity,
we developed cysteine-stable isotope labeling using amino acids in
cell culture (SILAC), a method that facilitates the detailed characterization
of disulfide-bridged peptides by mass spectrometry. For this analysis,
an antibody was metabolically labeled with <sup>13</sup>C<sub>3</sub>,<sup>15</sup>N-cysteine and incorporated into a comprehensive panel
of distinct bispecific molecules by controlled Fab-arm exchange (DuoBody
technology). This technology is a postproduction method for the generation
of bispecific therapeutic IgGs of which several have progressed into
the clinic. Herein, two parental antibodies, each containing a single
heavy chain domain mutation, are mixed and subjected to controlled
reducing conditions during which they exchange heavy–light
(HL) chain pairs to form bsAbs. Subsequently, reductant is removed
and all disulfide bridges are reoxidized to reform covalent inter-
and intrachain bonds. We conducted a multilevel (Top-Middle-Bottom-Up)
approach focusing on the characterization of both “left-arm”
and “right-arm” HL interchain disulfide peptides and
observed that native HL pairing was preserved in the whole panel of
bsAbs produced by controlled Fab-arm exchange