Micro Developability for early-stage lead optimization

High-throughput, high-titer CHO production of miniproteins with high yields and low endotoxin levels, surpassing traditional E. coli production methods.

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_Marketing@wuxibiologics.com ) Early-Stage Developability Assessments for Lead Optimization

Abstract

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 assessment, and manufacturability assessment. Each assay requires minimal sample consumption, supporting early-stage lead optimization. Introduction Early-stage screening for therapeutic antibodies o ft en prioritizes binding a ff inity and function, while critical developability factors are frequently overlooked, leading to CMC and clinical challenges. WuXi Biologics has developed the Micro Developability platform, a high throughput (HTP) analytical suite that requires less than 1 mg of antibody. This platform utilizes HTP in silico screening and early-stage developability testing to assess biophysical properties, pharmacokinetics (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.

mAb at pH 6.0

mAb at pH 7.4

https://www.news-medi- cal.net/whitepaper/20211110/F- cRn-and-its-role-as-a-therapeutic- target.aspx

Human FcRn A ff inity

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 μ g/mL

IntactMS

Enriched antibody

Peptide mapping

High-resolution detection. Detectable concentration <0.5 μ g/mL

Serum Stability Test

InSilico Analysis · Sequence liability · Immunogenicity · Aggregation

STAGE I

i l

Case Study 3: Forced Degradation Assessment

STAGE II

In addition to evaluating PK profiles, a thorough development assessment should include stability test- ing under various stress conditions, such as low pH, high temperatures, and freeze-thaw cycles. This evaluation is critical for identifying potential stability challenges.

Non-specificity /PK assessment

Manufacturability · Biophysical: DSF · Stability: thermal stress, F/T, pH stress

STAGE III

· AC-SINS (self-interaction) · BVP/DNA/Insulin ELISA · Human FcRn a ff inity · Serum stability (cleavage)

Low pH Stress

Thermal 40 Freeze/Thaw °C

· Sequence liability · O ff -target binding/poly-specificity · Poor stability CMC

· Mass spectrometry: · Peptide mapping · CIEF-MS

T 0

Endpoint

T 0

Endpoint

T 0

Endpoint

iCIEF

45.6% 97.5% 86.0% 98.4%

38.4% 96.1% 83.1% 97.9%

Total sample required: <1 mg for most assays

SEC

97.5%

95.8%

97.5%

95.0%

CE-SDSNR CE-SDSR

NA NA

Case Study 1: 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

A ft er one week incubation at 40°C, there was approximately a 7% decrease in the main peak and an 8% increase in the acidic peaks compared to those of T0 samples, indicating that the charge profile is highly susceptible to thermal stress.

Sequence liability and CDR hotspot, aggregation, and immunogenicity

Sequence Liability

Aggregation Score

Case Study 4: Antibody Developability Optimization Optimization of antibody developability achieves optimal molecular attributes for CMC development.

Chain Heavy_2

Region xxx-xxx

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

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

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

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 a 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 A ff inity No change

FC Variant

Tyrosine sulfation at this position may impact antigen binding.

Heavy_1: LxxxF, …

Case Study 2: Non-specificity/PK Assessment Assays To address factors contributing to poor PK profiles, we o ff er a panel of high-throughput, sensitive, and robust assays to assess the physicochemical properties and assess the PK behavior of monoclonal antibodies.

AC-SINS Evalua Ɵ on of self-associa Ɵ on tendency

BVP/DNA/Insulin ELISA Evalua Ɵ on of charge- based/non-speci fi c binding

AC-SINS

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. Conclusions Our Micro Developability platform is specially designed to support early- to mid-stages of drug discovery, o ff ering a suite of HTP, low-sample-consumption assays that can evaluate biophysical properties, PK, and manufacturability. Complemented by powerful insilico screening, this platform integrates wet-lab data into predictive models, providing actionable insights to optimize leads and accelerate biologics drug discovery. 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.

BVP/DNA/Insulin ELISA

FcRnA ffi nity Evalua Ɵ on of clearance

FcRnA ffi nity

Serum Stability Evalua Ɵ on of half- life

Serum Stability

Introduction of PK Assessment Assays

AC-SINS

Referenced to WXB Posi Ɵ ve Control (%)

100

DNA

Baculovirus

Insulin

0 10 20 30 40 50 60 70 80

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

0 0.4 2 10 50 250

mAb concentration( μ g/mL)

DNA Insulin BVP

DNA vs Insulin vs BVP

mAb2 mAb3

mAb1

Micro Dev_Poster_202510_v2

To discuss this poster: PS_Marketing@wuxibiologics.com

Page 1