FIGURE 1 Illustrations of disulfide bond structure. (A)
Intra-chain disulfide bond connects two beta-sheets in Ig domain (Li,
Prabakaran et al. 2016). (B) Intra-chain and inter-chain disulfide bonds
in a IgG monomer; (C) “knob-into-holes (Kih)” IgG-like bispecific
antibody (BsAb) stability can be improved by having additional disulfide
bonds (red line) formed in the Kih region; (D) Disulfide bond can
improve correctly pairing IgG-like BsAb light chain and heavy chain: in
one arm, the cysteine residues in CH and
CL that originally formed disulfide were mutated into
valine, and introduced an addition disulfide bond (marked as red) during
the mutation; the other arm kept unmutated; (E) Dual Affinity
Retargeting Molecules (DART), light chain variable region from one
antibody is linked to the heavy chain variable region from the other
antibody through small peptide (black dash line), and the two variable
regions are linked through disulfide bond (red solid line).
FIGURE 2 (A) Protein A chromatography profile from mAb3
purification process (B) AEX (Flow through mode) from mAb1 purification
process; (C) CEX (bind and elute mode) from mAb1 purification process.
Blue line represented the load sample that had no significant level
disulfide bond reduction, red line represented the load sample that had
disulfide bond reduction. The tail part for each step were normalized
and enlarged for comparison purpose.
FIGURE 3 Qualifications of product quality attributes for mAb1
and mAb2 during downstream processing. (A) During the low pH viral
inactivation step, for mAb1, the HMW% for intact mAb pool and reduced
mAb pool increased at similar level (0.5~0.6%); for
mAb2, the HMW% decreased more for intact mAb pool (-1.7%) than reduced
mAb pool (-0.3%); (B) For both mAb1 and mAb2, reduced mAb pool purity
increased during downstream process, showed the possibilities that
reduced mAb can re-oxidize.
FIGURE 4 Stability of antibody samples with varied initial
disulfide reduction levels under the conditions of room temperature (25
ºC), high temperature (40 ºC), and light exposure at 25 ºC for total 14
days. A, Aggregation level as a function of time for all studied
conditions; B, Zoom-in profile of aggregation level as a function of
time for 25 C and 40 C.
FIGURE 5 Different approaches have been applied in mAb
manufacturing process to prevent disulfide bond reduction and recover
reduced mAb.
FIGURE 6 Comprehensive evaluation of using redox wash system in
the platform Protein A chromatography (Tan, Ehamparanathan et al. 2020).
The study was performed using three mAb harvest cell cultures according
to the design including three arms. The protein A pools from each run
was tested for product quality attributes. (A) Comprehensive study: Arm
1, control; Arm 2, combined wash step; (B) Intact mAb impurity; (C)
Aggregates (HMW%).