Identifying Optimal BsAb Pairings in Early Discovery Identifying optimal bsAb pairings is a pivotal step during early discovery. With many parental antibodies and formats available, identifying the optimal pairings requires the production and screening of numerous parental antibody combinations, which has been time-consuming and cost prohibitive. 4 To streamline this process, we have developed Quick ‘n’ Clean, a high-throughput, small-scale bsAb production platform that enables rapid and cost-effective production of high-quality bsAbs suitable for in vitro assays. Case studies demonstrate its high quality (99% heterodimer, >95% monomer, <0.1 EU/mg endotoxin):
Monomer Purity (%)
Yield (mg)
Endotoxin (EU/mg)
ScFv
12 15
90% 92% 94% 96% 98% 100%
0.2
98.1%
0 3 6 9
7.8 mg
0.1
0.032 EU/mg
0.0
Ni-Anti DYKDDDDK-SEC Flag His
Figure 1. This case study highlights the effectiveness of Quick ‘n’ Clean in producing 25 scFv-Fab in 20 mL CHO cells. Results showed an average monomer purity of 98.1%, an average yield of 7.8 mg, and low endotoxin levels averaging 0.032 EU/mg.
Various BsAb Formats and Production Challenges BsAbs are developed in diverse formats, each designed for specific therapeutic applications and associated with distinct production challenges. This section explores both IgG-like and non-IgG-like bsAbs, focusing on how our tailored strategies overcome their unique production challenges. IgG-Like BsAbs IgG-like bsAbs retain the structure of conventional IgG antibodies but are engineered to bind two different antigens. However, a major production challenge of IgG-like bsAbs is the heavy chain (HC) and HC mispairing, which lead to byproducts that compromise both purity and efficacy. 5 One key method to address HC mispairing is the “knobs-into-holes” (KiH) technology. It involves engineering a “knob” on one heavy chain to fit into a complementary “hole” on the other, facilitating the correct assembly of heterodimers and improving bsAb yield and purity. 2,3
Common KiH Formats
Common LC
CrossMab
WuXiBody™
Hetero LC
Main Byproducts of KiH Formats
Homodimer
Mispaired LC
Half Antibody
Figure 2. Schematic overview of common KiH bsAb formats: Common LC, CrossMab, WuXiBody™, and Hetero LC, along with associated byproducts: homodimers, LC mispairing species and half antibodies.
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