Innovative Strategies for Bispecific Antibody Production

Innovative bsAb production platforms overcome challenges like chain mispairing, delivering >98% heterodimer purity across diverse formats.

WHITE PAPER

Innovative Strategies for Overcoming Bispecific Antibody Production Challenges

Zhumei Feng, Jiansheng Wu Protein Sciences Department, WuXi Biologics CRO Services

Abstract Bispecific antibodies (bsAbs) represent a significant advancement in biologic therapeutics, offering enhanced therapeutic efficacy through dual antigen targeting. However, bsAb development poses key challenges, such as the need to screen a large number of pairings to identify the optimal ones at early discovery, finding the best bsAb formats to meet the biology needs, and addressing low yield and purity during production. At WuXi Biologics’ Protein Sciences (PS) Department, we address these challenges through innovative strategies and solutions. Our high-throughput platform enables rapid and cost-effective production of many bsAb combos. Our ultra-high-titer CHO production platform, coupled with tailored engineering and Intact Mass-driven purification, ensures exceptional titer and purity across various formats, including CrossMab, scFv-Fab, bispecific VHH, and more. This white paper outlines our strategies and showcases how we overcome bsAb production challenges and consistently deliver high-purity bsAbs. Introduction The transition from monoclonal antibodies (mAbs) to bsAbs represents a major breakthrough in biologic therapeutics. By targeting two distinct antigens, bsAbs offer novel mechanisms of action that enhance therapeutic efficacy, such as engaging immune cells, bridging both targets, or disrupting multiple signal transduction pathways. 1 Despite their promise, bsAb development faces unique challenges, including the need for meticulous screening of bsAb pairings and overcoming production hurdles like chain mispairing, which compromise bsAb quality and functionality. 2,3 Our PS Department leverages extensive drug development expertise to address these challenges, delivering bsAbs with exceptional yield and purity.

wuxibiologics.com 1

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.

wuxibiologics.com 2

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

wuxibiologics.com 3

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.

wuxibiologics.com 4

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.

wuxibiologics.com 5

Optimizing the HC/LC chain ratio can significantly enhance production efficiency and purity. 8 When needed, we can conduct chain ratio studies to achieve higher expression titers and improved heterodimer purity.

Figure 10. This case study focuses on a scFv-Fab with three chains: a LC, a HC1 and a HC2. The initial transfection ratio of HC1:HC2:LC at 2:1:1 resulted in low titer and limited heterodimer percentage. By increasing HC1 ratio, we significantly improved heterodimer purity from 12% to 72%.

Conclusion Bispecific antibodies continue to drive therapeutic innovations by offering highly targeted and effective treatments. Leveraging extensive drug development expertise, we employ innovative strategies tailored to various bsAb formats to achieve superior purity and quality in bsAb production. These approaches ensure the consistent delivery of high-quality bsAbs, contributing to the advancement of drug development.

References

1. Herrera M., Pretelli G., Desai J., Garralda E., Siu L. L., Steiner T. M., Au L. Bispecific antibodies: advancing precision oncology. Trends Cancer . 2024;10(10):893-919. doi: 10.1016/j.trecan.2024.07.002. 2. Ma J., Mo Y., Tang M., Shen J., Qi Y., Zhao W., Huang Y., Xu Y., Qian C. Bispecific Antibodies: From Research to Clinical Application. Front Immunol . 2021;12:626616. doi: 10.3389/fimmu.2021.626616. 3. Sun Y., Yu X., Wang X., Yuan K., Wang G., Hu L., Zhang G., Pei W., Wang L., Sun C., Yang P. Bispecific antibodies in cancer therapy: Target selection and regulatory requirements. Acta Pharm Sin B . 2023;13(9):3583-3597. doi: 10.1016/j.apsb.2023.05.023. 4. Hofmann T., Krah S., Sellmann C., Zielonka S., Doerner A. Greatest Hits-Innovative Technologies for High Throughput Identification of Bispecific Antibodies. Int J Mol Sci . 2020;21(18):6551. doi: 10.3390/ijms21186551. 5. Chen S. W., Zhang W. Current trends and challenges in the downstream purification of bispecific antibodies. Antib Ther . 2021;4(2): 73-88. doi: 10.1093/abt/tbab007. 6. Ridgway J. B., Presta L. G., Carter P. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng . 1996;9(7):617-21. doi: 10.1093/protein/9.7.617. 7. Mullin M., McClory J., Haynes W., Grace J., Robertson N., van Heeke G. Applications and challenges in designing VHH-based bispecific antibodies: leveraging machine learning solutions. mAbs . 2024;16(1):2341443. doi: 10.1080/19420862.2024.2341443. 8. Haryadi R., Ho S., Kok Y. J., Pu H. X., Zheng L., Pereira N. A., Li B., Bi X., Goh L. T., Yang Y., Song Z. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells. PLoS One . 2015;10(2):e0116878. doi: 10. 1371/journal.pone.0116878.

About WuXi Biologics WuXi Biologics is a leading contract research, development, and manufacturing organization (CRDMO) that provides end-to-end capabilities to healthcare organizations worldwide. With operations in China, the United States, Ireland, Germany, and Singapore, we enable our partners to effectively and efficiently bring biologics and vaccines to patients worldwide through our comprehensive and high-quality drug development model.

The world’s leading global single-source platform from concept to commercialization wuxibiologics.com PS_Marketing@wuxibiologics.com

wuxibiologics.com 6

Page 1 Page 2 Page 3 Page 4 Page 5 Page 6

www.wuxibiologics.com

Powered by