Although KiH technology reduces HC mispairing, the production of KiH bsAbs still results in byproducts (Figure 2). Our PS Department employs Intact Mass analysis throughout the purification process to accurately evaluate byproducts such as half antibodies, homodimers, and LC mispairings.
Sample Info
Target
Main Byproducts
Post ProA Quality
AC
M
KDa
100 150 250
75
50 37 25 20 15 10
Purification
Intact Mass
Target
1) Mixed Mode Chromatogram
2A12-2B2 SEC-HPLC Purity: 95.5%
Homodimer
2A12
2B11
2A9 2B2
Target
2) CEX Chromatogram
load
KDa
2B2-2B4
M
2A5
SEC-HPLC Purity: 98.4%
2A7
2A3
100 50 150 75 250
2A5
2C8-2C12
25 37 15 10 20
2A7 2B2-2B4 2C8-2C12
Figure 3. This case study presents the production of a common LC bsAb. After affinity purification, the half antibody and homodimer were observed using SEC-HPLC and SDS-PAGE. Mix mode was used as the first polishing step to remove half antibody. However, Intact Mass indicated that the homodimer still existed. CEX was applied as the second polishing step to eliminate the homodimer and trace amount of half antibody.
To address LC mispairing, formats like CrossMab offer effective solutions, as this format reduces LC mispairing by exchanging domains in the Fab region. 6
Sample Info
Target
Main Byproducts
LC1
LC2
HC1 HC2
HC2LC2
HC2LC2 homodimer
LC mispairing
Purification
Pool1
HIC Chromatogram
Zoom in
Pool2
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