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 ¹³C₃,¹⁵N-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

Thyroid state in skeletal muscle of progeroid and naturally aged mice.

<p>T3 concentrations (A) and activities of D2 (B) and D3 (C) in muscle of 15-day-old WT and XAA (Csbm/m/Xpa-/-) mice (n = 3/group). T4 (D) and T3 (E) concentrations and activities of D2 (F) and D3 (G) in muscle of 18-week-old WT and MAA (Ercc1-/Δ-7) mice (n = 3/group). T4 (H) and T3 (I) concentrations and activities of D2 (J) and D3 (K) in muscle of 26-, 104-, and 130-week-old WT mice (n = 3-5/group). Values represent mean ± SE per group. * P < 0.05</p

Thyroid state in serum of progeroid and naturally aged mice.

<p>Serum T4 (A) and T3 (B) concentrations in 7-, 12-, 15-, and 18-day-old WT (squares) and XAA (Csbm/m/Xpa-/-) mice (circles) (n = 3/group). Serum T4 (C) and T3 (D) concentrations in 4-, and 18-week-old WT (black bars) and MAA (Ercc1-/Δ-7) (white bars) mice (n = 3/group). Serum T4 and T3 concentrations in 26-, 104-, and 130-week-old WT male mice (n = 3-4/group) (E). Serum TSH levels in 15-day old WT and XAA (Csbm/m/Xpa-/-) mice (F) and in 26-, 104-, and 130-week-old WT male mice (G). Values represent mean ± SE per group. * P < 0.05; ** P < 0.01; *** P < 0.001; # P = 0.054.</p

Thyroid state in brains of progeroid and naturally aged mice.

<p>Homogenates of whole brain or hemispheres were used. T4 (A) and T3 (B) concentrations in brains of 7-, 12-, 15-, and 18-day-old WT (squares) and XAA (Csbm/m/Xpa-/-) mice (n = 3/group). Activities of D2 (C) and D3 (D) brains of 7-, 12-, 15-, and 18-day-old WT and XAA (Csbm/m/Xpa-/-) mice (n = 3/group). T4 (E) and T3 (F) concentrations and D3 activity (G) in brains of 4-, and 18-week-old WT (black bars) and MAA (Ercc-/Δ-7) (white bars) mice (n = 3/group). It was not possible to measure D2 activity due to technical constraints. Values represent mean ± SE per group. * P < 0.05; ** P < 0.01; *** P < 0.001.</p

Schematic representation of the survival response.

<p>Several types of stress (e.g. DNA damage and aging) can trigger a differential response in various tissues. This response ensures decreased TH signalling in liver and kidney, while it preserves TH signalling in brain, muscle and heart.</p

Liver D1 and D3 activity in DEHP-treated WT mice.

<p>Activities of D1 (A) and D3 (B) in 10-wk-old WT animals after exposure or not to subtoxic doses of the pro-oxidant DEHP for 2, 12 and 39 weeks. Values represent mean ± SE per group (n = 5). * P < 0.05; ** P < 0.01.</p

Gene expression changes in livers from progeroid and normal aging mice.

<p>Gene expression of the T3-responsive genes Dio1 and Thrsp in 18-day-old XAA (Csbm/m/Xpa-/-) (A) and 16-week-old MAA (Ercc1-/Δ-7) mice (B). * P < 0.005; # P = 0.12. Expression profiling of a set of known T3-responsive genes in 15-day-old XAA (Csbm/m/Xpa-/-) (C) and 16-week-old MAA (Ercc1-/Δ-7) mice (D) mutants compared to age-matched controls. Values represent mean ± SE * P < 0.05; ** P < 0.005; $ P < 0.1; # P = 0.12.</p

Histological examination of haematoxylin/eosin-stained thyroid glands of 15-day-old WT and XAA (Csbm/m/Xpa-/-) (A) and 16-week-old WT and MAA (Ercc1-/Δ-7) (B) mice (all magnifications 10x).

<p>The thyroid follicles (denoted by asterisk) surrounded by thyrocytes (denoted by arrow) are similar between WT and progeria models.</p

Thyroid state in liver of progeroid and naturally aged mice.

<p>T4 (A) and T3 (B) concentrations in livers of 7-, 12-, 15-, and 18-day-old WT (squares) and XAA (Csbm/m/Xpa-/-) (circles) mice (n = 3/group; each time point of Csbm/m/Xpa-/- mice represents pooled tissues). Activities of D1 (C) and D3 (D) in livers of 7-, 12-, 15-, and 18-day-old WT and XAA (Csbm/m/Xpa-/-) mice (n = 3/group). T4 (E) and T3 (F) concentrations and D1 activity (G) in livers of 4-, and 18-week-old WT (black bars) and MAA (Ercc1-/Δ-7) (white bars) mice (n = 3/group). D3 mRNA expression in livers of 13-week-old and 130-week-old mice (H). D1 (black bars) (I) and D3 (grey bars) (J) activities in livers of 26-, 52-, and 104-week-old WT mice (n = 5/group). Values represent mean ± SE per group. * P < 0.05; ** P < 0.01; *** P < 0.001; # P = 0.051.</p