Premium BsAb: Strategies for Improved Purity & Yield

LC-MS driven purification for high-purity bispecific antibodies ranging from 10 mg to grams in 4-6 weeks

Optimizing Bispecific Antibody Production for Higher Titer & Purity Across Diverse Formats Gaozhan Zhang, Li Zhang, Dawei Zhang, Mengjie Lu, Zhumei Feng, Jiansheng Wu

WuXi Biologics, No.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_Marketing@wuxibiologics.com)

The production of BsAbs faces challenges throughout the entire process, starting from expression and extending to purification. Our integrated BsAb production strategies address these challenges at every stage, including transfection, cell culture, purification, and analytical bioassays. These strategies guarantee high yields and improved purity across a wide range of BsAb formats. Introduction Abstract Bispecific antibodies (BsAbs) hold immense therapeutic potential but face production challenges such as low titers and impurities, including homodimers and mispairings. At WuXi Biologics, our CRO Services team leverages advanced molecule design, high-titer CHO expression, chain ratio optimization, and Intact MS-driven purification to generate grams of BsAbs with >98% heterodimer purity across diverse BsAb formats. These innovations enhance BsAb production e ff iciency and facilitate more e ff icient therapeutic development.

Fig.5 Intact MS Identified the Presence of LC Mispairing, LC Missing and Half Antibody

Target:

LC mispairing

LC2

Target

LC1

LC2

Half antibody

HC2

HC1

R NR M L FT E M L FT E

250 kDa 75 100 150

LC mispairing

LC missing

37 50 20 25 15

Fig.1 Di ff erent Formats of BsAb

Fig.6 CEX and HIC Purification Achieved >98% BsAb Purity Confirmed by Intact MS and SEC-HPLC Achieving >98% Heterodimer Purity Using Intact MS-Driven Purification

TCR

CommonLC

CrossMab

WuXiBody dy

Di ff erentLC

Step1: CEX

Step2: HIC

Final: SEC-HPLC

Pool

Charged Pairing

Others

FcFusion

ScFvFab

Fig.2 Premium BsAb Production Strategies

Molecule Design • Formats provided by clients or designed by CRO • Any formats

Expression

Step1: CEX

Step2: HIC

Final ： Intact MS

• Chain ratio optimization • Achieve acceptable target purity in starting material

HICfraction

M L

CEXfraction

kDa

Target

250 150 75 100

250 150 100

PNGaseF

Molecule Design

Expression

M L

CEX fraction

75 50 37 25 20

37 50 20 25

Purification

Analytics

Purification

15 10

• Remove impurities: 1/2 Abs, homodimers, mispaired species • Basedondi ff erences in charge and hydrophobicity

• LC-MS detects the target and byproducts • SDS-PAGE, SEC-HPLC, endotoxin

Fig.7 Early Stability Assessment and Scalable Production of a Symmetric BsAb with Fc-Fused ScFvs

SEC-HPLC Purity of 20 mL Pilot Run

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

0 20 40 60 80 100

Results

Optimizing Chain Ratio for Improved Titer and BsAb Quality

Fig.3 Chain Ratio Optimization Results in Higher BsAb Yield and Improved Purity

Highconc.

40°C 1D Freeze-thaw

SDS-PAGE post ProA

SEC Purification

Final SDS-PAGE

Challenges

Improving Strategy

Superior Product Quality

M R

L FT E

M L FT E

kDa

Optimize chain ratio

Higher Protein Yield Better Purity

Low transient expression titer Low SEC-HPLC purity a ft er one-step purification

250 150 75 100 kDa

250 150 75 100

37 50 20 25 15 10

Titer and Product Quality Improvement

37 50 20 25 15 10

HC2

Increase transfection ratio of HC1: HC1 : HC2: LC = 2 :1: 1 HC1 : HC2: LC = 4 :1: 1 HC1 : HC2: LC = 6 :1: 1

HC1

Method All experiments were conducted at WuXi Biologics CRO Services. Codon optimization, plasmid construction, and WuXian Transient™ expression followed SOPs. Culture supernatant was purified via tailored chromatography platforms and analyzed by SDS-PAGE, SEC-HPLC, Intact MS, and endotoxin assays. Conclusions WuXi Biologics has optimized BsAb strategies for scalable production with high titer and purity. Chain ratio tuning during transfection enhances yield and quality. Intact MS guides purification to consistently achieve >98% heterodimer purity. Acknowledgment Thanks to WuXi Biologics' CRO, legal, PR, and marketing teams for their support in BsAb platform development and patent work. About WuXi Biologics CRO Services WuXi Biologics is a leading global CRDMO, with the R represented by our premium-quality CRO Services. With expertise in diverse drug modalities, we deliver comprehensive research services, including antibody discovery, protein production and engineering, and in vitro/in vivo testing, to seamlessly advance our clients’ molecules from concept to CMC development.

Amount (mg) Purity by SEC-HPLC (%) HC1:HC2:LC=1:1:2 HC1:HC2:LC=2:1:1 HC1:HC2:LC=4:1:1 HC1:HC2:LC=6:1:1

Fig.4 Intact MS Confirmed the Existence of Homodimers While SEC-HPLC and SDS-PAGE Suggest High Purity Intact MS for the Detection of Homodimer, LC Mispairing, LC Missing, and Half Antibody

SEC-HPLC

SDS-PAGE

Intact MS

145381.27

145462.02

Drug Development Expertise Empowering Research Services for Biologics For more information see us at https://www.wuxibiologics.com/cro-services

Premium BsAb_Poster_202510_v2

To discuss this poster: PS_Marketing@wuxibiologics.com

Page 1