Intact Mass
SEC-HPLC
SDS-PAGE
M R
NR
kDa
1
x10 2
…
x10
…
Pool1, Target
LC mispairing Pool2,
Purity: 100% RT: 7.96 min
250 150 100
146739.39
147813.29
1
4
75
0.8
3
50
0.6
37
2
0.4
20 25 15 10
1
110154.99
0.2
156997.15
134868.78
159810.09
143444.74
139125.83
151440.91
134865.83
155408.73
0
0
Counts (%) vs. Deconvoluted Mass (amu) 100000 110000 120000 130000 140000 150000 160000
135000
140000
145000
150000
155000
160000
Counts (%) vs. Deconvoluted Mass (amu)
Figure 4. This case study presents the purification of a CrossMab using the Premium BsAb platform. Intact Mass analysis was used to identify the target from two pools during HIC purification, with further verification by SEC-HPLC and SDS-PAGE. This comprehensive QC approach ensured the delivery of correctly assembled bsAbs with high purity while effectively removing half antibodies, homodimers, and mispaired species.
Non-IgG-Like BsAbs Non-IgG-like bsAbs, such as single-chain variable fragment (scFv)-based formats and bispecific nanobodies, deviate from traditional IgG structures. These formats offer unique advantages but also present distinct production challenges. 2,3 ScFv-based bsAbs, such as bispecific T-cell engager (BiTE), exhibit remarkable structural versatility and clinical efficacy. 1 However, producing scFvs as building blocks for bsAbs presents challenges such as suboptimal molecular design and poor stability. To address these issues, our PS Department employs advanced engineering and production strategies, such as optimizing scFv structures and introducing disulfide bonds to enhance structural integrity, thereby improving their purity and stability. We also produce diverse bsAb formats using scFv as the building block.
57% RT: 10.1 min
32% RT: 10.2 min
80% RT: 10.0 min
77% RT: 10.2 min
390 mg/L
290 mg/L
350 mg/L
130 mg/L
Linker 2:
Linker 1:
Figure 5. This case study compares four scFv configurations when reformatting IgGs into scFvs. Among the four molecules, VL-VH orientation achieved higher titers, while Linker 2 demonstrated largely improved purity.
Figure 6. This case study demonstrates the successful stabilization of a scFv by introducing disulfide bonds to the VH-VL interface, which prevents dissociation and further aggregation. SEC-HPLC analysis showed that the modified structure maintains 99% purity at 5 mg/mL, even after extended storage at 4°C.
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