Micro Developability for early-stage lead optimization

One-stop solution for early-stage antibody drug developability assessment using <1mg antibody. No need to outsource to multiple vendors.

High-Throughput Developability Assessment for Early-Stage Lead Optimization

Yile Chen, Lei Guo, Jiansheng Wu CRO Services, Global Biologics Research Department, WuXi Biologics NO.240 Hedan Road, Pudong New District, Shanghai, China (Contact: PS_BD@wuxibiologics.com)

Abstract Early-stage screening for therapeutic antibodies often prioritize binding affinity and function, while critical developability factors are frequently overlooked. This can lead to CMC and clinical challenges, such as high viscosity, poor PK, and stability issues. To minimize these risks, WuXi Biologics has developed the Micro Developability platform, a high-throughput (HTP) analytical suite that requires less than 1 mg of antibody per assay. This platform utilizes HTP in silico screening and early-stage developability testing to assess biophysical properties, PK, and manufacturability. By analyzing critical developability aspects with expert interpretation, these early assessments help optimize leads, mitigate downstream risks, and facilitate a seamless transition to CMC development.

A: Light chain of HmAb

Binding

0.0% 20.0% 40.0% 60.0% 80.0% 100.0% 0.0% 20.0% 40.0% 60.0% 80.0% 100.0%

Wash/ Elution

Automation

B: Heavy chain of HmAb

1 µg/mL

Intact MS

Enriched antibody

Peptide mapping

High-resolution detection. Detectable concentration <0.5 µg/mL

Serum Stability Test

Introduction

Case Study 3: Forced Degradation Assessment

Early-stage developability assessment is important to identify and mitigate risks before advancing molecules to CMC development. Our Micro Developability platform provides a comprehensive suite of HTP analyses, including in silico screening, poly-specificity and PK prediction, and manufacturability assessment. Each assay requires minimal sample consumption, supporting early-stage lead optimization.

In addition to evaluating PK profiles, thorough development assessment should include stability testing under diverse stress conditions, including low pH, high temperatures, and freeze-thaw cycles. This evalua - tion, known as forced degradation testing, is critical for identifying potential stability challenges.

Low pH Stress

Thermal40 Freeze/Thaw °C

In Silico Analysis • Sequence liability • Immunogenicity • Aggregation

Immunogenicity • CD134/CD137 T-helper-cell activation

T 0

End point

T 0

End point

T 0

End point

iCIEF

45.6% 97.5% 86.0% 98.4%

38.4% 96.1% 83.1% 97.9%

STAGE I

SEC

97.5%

95.8%

97.5%

95.0%

CE-SDS NR CE-SDS R

NA NA

STAGE II

Poly-Specificity /PK-Predicting

Manufacturability • Biophysical: DSF • Stability: thermo, F/T, pH • Mass spectrometry: Peptide mapping cIEF-MS

STAGE III

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• AC-SINS (self-interaction) • Baculovirus particle (BVP) /DNA/Insulin/ELISA • Human FcRn affinity • Serum stability (cleavage)

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CMC • Sequence liability • Immunogenicity • Off-target binding/poly-specificity • Poor stability

Total sample required: <1 mg for most assays

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After one week at 40°C, a ~7% decrease in the main species and an ~8% increase in the acidic species were observed compared to baseline (T 0 ), indicating that the charge profile is highly susceptible to thermal stress.

Case Study 1: In Silico Analysis

Liability

Aggregation

Immunogenicity

• Sequence similarity based • MHC based • Humanization

• Post-translational modification • CDR hotspots • Free cysteine • F C mutation • Amino acid usage frequency

• Structure prediction • Hydrophobicity based • Free cysteine based

Sequence liability and CDR hotspot, aggregation, and immunogenicity

Sequence Liability

Aggregation Score

Chain Heavy_2

Region xxx-xxx

Comment A hydrophobic region (LI) with a medium risk of aggregation was detected in H-CDR3.

· Oxidation · Deamidation · N-Linked Glycosylation · Pyroglutamate · Lysine Glycation · Tyrosine Sulfation · O-Linked Glycosylation · C-Terminal Lysine · Carbonylation · Hydroxylation · Isomerization

Immunogenicity Evaluation and Humanization Chain Region Amino Acid Usage Frequency The amino acids with low frequency are indicated using color schemes.

CDR Hotspot and Risk Assessment

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Amino acid Asn

Modification Deamidation

Chain Heavy_1

Position xx

Region H-CDR2

Comment

Asparagine at this position is buried. Low risk. Aspartic acid isomerization at this position may decrease stability and impact antigen binding.

Comment 9mer peptides in this region of variable domain were detected as immunogenicity risk region, which overlaps FW3, CDR3, FW4. Modification advice: Sxx->Yxx (CDR3). There is no risk region after modification.

Isomerization

Heavy_1

H-CDR1

Asp

Light_1

xx-xxx

Post-Translational Modifications and Assessment Tyrosine Sulfation Chain Heavy_1 Position xx Region H-CDR2 Amino acid Amino acid Tyr

ADCC/CDC Response Receptor FcRn Affinity No change

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Tyrosine sulfation at this position may impact antigen binding.

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AC-SINS Evaluation of self-association tendency

AC-SINS

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6;< BV/DNA/Insulin ELISA Evaluation of charge- based/non-specific binding

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BV/DNA/Insulin ELISA

FcRn Affinity

FcRn Affinity Evaluation of clearance

Conclusions

Serum Stability

Serum Stability Evaluation of half-life

Acknowledgment We thank the scientists in WuXi Biologics’ CRO Services Department for their support in developing the Micro Developability platform. We also thank the legal, public relations, and marketing department for their assistance with patent filings and preparation and revision of this poster. Our Micro Developability platform is specially designed to support early- to mid-stages of drug discovery, offering a suite of HTP, low-sample-consumption assays that can evaluate biophysical properties, PK, and manufacturability. Complemented by powerful in silico screening, this platform integrates wet-lab data into predictive models, providing actionable insights to optimize leads and accelerate biologics drug discovery.

Introduction of PK-predicting assays

https://www.news-medical.net/whitepaper/20211110/ FcRn-and-its-role-as-a-therapeutic-target.aspx

mAb at pH 6.0

mAb at pH 7.4

WuX i B iologics is a global leading Contract Researc h, Development discove r, develop and manufacture biologics from concept About WuXi Biologics For more information, visit us at wuxibiologics.com partners to

and Manufacturing Organization ( CRDMO ) o ffe ring end-to-end solutions to enable

to commercialization.

AC-SINS

DNA vs Insulin vs BVP

Human FcRn Affinity

To discuss this poster: PS_BD@wuxibiologics.com

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