Innovative Strategies for Bispecific Antibody Production

Sample Info

Target

SEC-HPLC Purity from 20 mL Pilot Run

100

The preliminary developability assessment indicated good stability prior to scale-up.

50

0

T0

High conc. High Conc. 40°C,1D

Freeze-thaw Freeze-Thaw

T 0

Purification AC Results NR

SDS-PAGE

SEC-HPLC

SEC Chromatogram

M R

NR

R

kDa

L FT E

M L FT E

kDa

Target

250 150 75 100

250 150 75 100

HC

37 50 20 25 15

50

37 25 20 15 10

LC

10

Figure 7. This case study highlights the early-stage stability assessment and scalable production of a symmetric bsAb with Fc-fused scFvs. Initial micro-developability tests demonstrated that the bsAb maintained almost 100% purity under high concentration, thermal stress (40°C for 1 day), and freeze-thaw conditions. The subsequent scale-up production involved AC-SEC purification, yielding high-purity bsAbs as confirmed by SDS-PAGE and SEC-HPLC.

Nanobodies, or VHHs, are single-domain antibody fragments known for their high affinity, specificity, and thermostability. 7 Due to their small size, nanobodies are often used as building blocks for bsAbs. Our high-titer CHO expression systems effectively produce VHHs with various formats (Figure 8), all achieving high yields and superior quality.

Monomer

Dimer

Bispecific VHH-Fc

Building Blocks for BsAb

Figure 8. Scheme of various VHH formats, including monomer, dimer, bispecific, VHH-Fc, and VHH-related bsAbs.

MMC Chromatogram CEX Chromatogram

Intact Mass

SDS-PAGE

+ Scan (rt: 4.133-4.917 min, 48 scans) WBP2677_2_DGL336-DM-20230905.d Deconvoluted (Isotope Width=5.0)

M R NR

2.75

190 115 kDa

76153.63

2.5

2.25

VHH1

VHH2

1.75 2

70 80 50 30 25

1.5

1.25

0.75 1

0.5

0.25

15

0

Counts vs. Deconvoluted Mass (amu) 75800 76000 76200 76400 76600 76800 77000

Figure 9. This case study highlights the successful purification of a bsAb molecule with 2 different VHHs from CHO stable pool. Using MSS-mixed mode-CEX purification strategy, 8 g of bsAb was delivered within 9 weeks, achieving outstanding purity as confirmed by Intact Mass and SDS-PAGE.

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