Innovative Strategies for Bispecific Antibody Production

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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