Amy Sheng, PhD provides an overview of antibody screening platforms and presents applications and case studies using the Beacon® platform for antibody discovery. Single B cell screening is a powerful and efficient strategy for generating antigen-specific monoclonal antibodies. Distinguished with fluorescence-activated B cell sorting, the Beacon® platform is based on plasma cell screening, making it easier to obtain antibody genes. The Beacon® single-cell optofluidic system combines a unique optoelectro positioning (OEP) technology with novel microfluidic technology. It can be used to accurately select single cells on a chip, perform multiple single-cell assays, and export target cells based on specific results. The Beacon® optofluidic platform preserves the diversity of B cells, generating high-quality positive hits at an early stage of discovery and avoiding the loss of “good clones”. Key Topics Include: - B cell differentiation and development - Pros and cons of mainstream antibody screening platforms - Mechanisms, applications, and case studies using the Beacon® platform for antibody screening - Sino Biological’s capacity using the Beacon® platform

1. Welcome!
Technical Account Manager
Sino Biological
Amy Sheng, PhD
Antibody Discovery
by Single B Cell
Screening on Beacon®

2. Sharing of Antibody
Discovery of Single B
Cell Based on Beacon®
Sino Biological
Amy Sheng, Ph.D.
Technical Account Manager
Sino Biological US Inc.

3. CONTENT
B Cell Differentiation and Development
1
Introduction of Antibody Discovery Platform
2
Introduction of Beacon ®
3
Case of Antibody Screening Based on Beacon®
4
Sino Biological

4. B Cell Differentiation and Development
01
Sino Biological

5. B Cell Differentiation and Development
Central development Peripheral development
Immature B Mature B
Pro-B
Common
lymphoid
progenitor
Germminal center
Centroblast
Centrocyte
Memory B
Plasma
Pre-B
Sino Biological

6. Introduction of Antibody Discovery Platform
02
Sino Biological

7. Hybridoma Technology
 Mature and stable
 Functional and affinity screening can be carried out
 Low cost
 Low fusion efficiency：1/1000
 Time consuming
 Species limits
Cell fusion
Primary B
cell
(HGPRT pos ) Myeloma B
cell
(HGPRT neg )
HAT
selection
Hybridoma
Single B cell
culture
ELISA
Sino Biological

8. Phage Display Techniques
 Broad spectrum
 Full human antibodies are
available
 Low cost
 Susceptible to antibody
repertoire
 Limited affinity
 Non-natural antibody pairs
Tissue RNA cDNA
SCFV
Phagemid
Transformation
Infection
Library
Sino Biological

9. Single B Cell Technology
 Broad spectrum
 Natural antibody pair
 High diversity
 Short time
 High affinity
 Complicated
 Rely on equipment
Laser beam
VHDJHCY1 VKJKCK
Sino Biological

10. Species
Sorting——Human, mouse,
rabbit…
Beacon ——Mouse
Cell
Sorting ——Memory B cell
Beacon ——Plasma cells
Functional Assay
Sorting——No
Beacon——Cell binding,
blocking
Throughput
Sorting——10000cell/s
Beacon ——4 chip, 80k cell
T
1 2
3 4
Sino Biological
Comparison of Fluorescence-activated Cell Sorting and Beacon®

11. Introduction of Beacon®
03
Sino Biological

12. Sino Biological
Beacon® System

13. Flow
Diffusion
Sino Biological
An Example of Cell Culture

14. 01
Cell Separation
02
Load
03
Assay
04
Unload
Immunization and
ASC Enrichment
Nanopen Load
OEP Gravity
In-Channel
Fluorescent”Bloom”
Assay
OEP Nanopen
Export
VH/VL Recovery, Cloning,
Expression
30-60 Minutes 30-60 Minutes 3-4 Minutes per
Nanopen
Import Export
Sino Biological
Technical Route

15. Cell Separation Load Assay Unload
Samples
Incubation
Antibody Magnetic
Particle
Target
Cell
Immunomagnetic
Beads Capture
Sino Biological

16. Cell Separation Load Assay Unload
Sino Biological

17. Cell Separation Load Assay Unload
Antigen
Antibody
Secondary
antibody
Avidin
beads
Cell
Sino Biological

18. Cell Separation Load Assay Unload
Blocking
Unblocking
Antibody
Secondary
Antibody
Avidin
beads
Avidin
beads
Ligand
Antigen
Sino Biological

19. Cell Separation Load Assay Unload
Cell
Cell
Cell
Ligand
Blocking
Unblocking
Sino Biological

20. Cell Separation Load Assay Unload
Sino Biological

21. Case of Antibody Screening
Based on Beacon®
04
Sino Biological

22. Screening of Anti-idiotype Antibody（ Anti-ID Abs）
An antibody that uniquely binds the idiotype of another antibody
Pharmacokinetics and pharmacodynamics analysis
Immunogenicity analysis: positive control antibody
Antibody drug
Anti-ID Abs
Target protein
Antibody drug
Anti-ID Abs
Target protein
Blocking Unblocking
Block antigen antibody binding
Detection of free antibody drugs
Does not block antigen antibody binding
Detection of total antibody drug (Free, half bound,
bound)
Sino Biological

23. 01
02
03
04
Immunization （2weeks）
Beacon（1day）
Antibody gene amplificat（1week）
Expression, purification, detection
（2weeks）
Antibody drug binding
Human IgG binding
Target protein competition
Sino Biological
Technical Route for Anti-ID Abs Development

24. Load
11978
Antibody Drug Binding
772
Human IgG Binding
151
Competition
Blocking 54
Unblocking 97
Antigen/hIgG Binding
Competition
Sino Biological
Case Study

25. Screening of Modified Antibodies
Phosphorylated polypeptide Non-phosphorylated polypeptides
Target Load Phosphorylated+ Phosphorylated+Non-phosphorylated-
SinoA 12400 109 63
Protein modification refers to group modification of protein after translation, including acetylation,
ubiquitination, methylation, phosphorylation…
Sino Biological

26. Screening of CD47 Antibody
CD47 Binding
Domain of SIRPα Blocks the DON’T EAT ME signal from CD47
but does not bind to red blood cells
Delivers an EAT ME signal to
macrophages through FcγRs:
Differentiated from lgG4 antibodies
SIRPαFc triggers activation of FcγRs
on macrophages
Human lgG1 Fc
FcγR
SIRPαFc
CD47
Sino Biological

27. Sample ID IC50(ug/mL)
CD47-M0002-1 0.273
CD47-M0065 0.074
CD47-M0137-1 0.198
CD47-M0091-1 0.183
CD47-M0075-1 0.155
CD47-M0120 0.103
CD47-M0126-2 0.134
Control-1 0.033
Control-2 0.204
Sample ID KD (M) kon(1/Ms) kdis(1/s)
Control-1 5.97E-10 2.62E+05 1.56E-04
Control-2 1.92E-09 2.50E+05 4.81E-04
CD47-M0002-1 3.68E-10 7.42E+05 2.73E-04
CD47-M0065 1.11E-09 5.45E+05 6.08E-04
CD47-M0091-1 4.52E-10 3.87E+05 1.75E-04
CD47-M0075-1 1.44E-09 4.41E+05 6.34E-03
CD47-M0120 1.60E-09 5.75E+05 9.18E-04
CD47-M0126-2 6.90E-10 4.40E+05 3.04E-04
Control-1 Control-2 M0002-1 M0065
M0091-1 M0075-1 M0120 M0126-2
Blocking
Affinity
Binding with Cells
Target Cell on Beacon® PCR ELISA Blocking Weak Binding with RBC
CD47 331 255, 77.04% 108 73 2
Jurkat RBC
Sino Biological

28. Screening of Monkeypox A29 Antibody
Target Load Antigen+
A29 11390 321
Western Blot Assay ELISA Assay
The EC50=23.98 ng/ml
Sino Biological

29. 32,000+
genes
14,000+
antibodies
600+
ELISA kits
International Reagent Supplier and Service Provider
Citations
Recombinant
protein
expression
Recombinant
antibody
production
Monoclonal
antibody
development
Polyclonal
antibody
development
High-quality reagents & one-stop CRO services
>10,000 citations till Sep, 2022
6,500+
proteins
Company Overview
Certification
ISO9001:2015 and ISO13485:2016 certified
End-Users in 90+ Countries
User Distribution

30. The Global Leader in Recombinant Technology
Advantages Development & Production
Capacity of thousand per batch
Kg level per batch
5 expression systems
Routine high-throughput (HTP)
production and screening projects
Up to 1,500L bioreactor for scale-up
production
FucoFree cell line, stable cell Line,
insect cells, mammalian cells, and E.
coli
In-house platforms
Proprietary culture media,
transfection reagents, and expression
vectors
Recombinant protein
HTP antibody
Large-scale antibody
Chimeric antibody
Fab/scFv/VHH antibody
Bispecific antibody
Humanized antibody
Recombinant
Production
Sino Biological

31. Guangzhou, China
PA & TX, USA
Frankfurt,
Germany
Headquarters:
Beijing, China
Shanghai, China
Taizhou & Suzhou, China
Kawasaki-shi, Japan
Global Presence
Sino Biological

32. THANK YOU
Sino Biological US Inc. (USA)
Pennsylvania Office: 1400 Liberty Ridge Drive, Suite 101, Wayne, PA 19087
Texas Office: 10101 Southwest Freeway, Suite 375. Houston, TX 77074
Tel: +1-215-583-7898
Email: order_us@sinobiologicalus.com
Sino Biological Europe GmbH (Europe)
Address: Düsseldorfer Str. 40, 65760 Eschborn, Germany
Tel: +49(0)6196 9678656
Email: order_eu@sinobiologicaleu.com
Sino Biological Japan Inc. (Japan)
Address: Tech-Pot, 2F, West Wing, KSP, 3-2-1 Sakado, Takatsu-ku,
Kawasaki-shi, Kanagawa, 213-0012, Japan
Tel: 044-400-1330
Email: order@sinobiological.co.jp
Sino Biological, Inc. (Global)
Address: Building 9, Jing Dongbei Technology Park,
No.18 Kechuang 10th St, BDA, Beijing, 100176, P.R.China
Tel: +86-400-890-9989
Email: order@sinobiological.com
Custom Antibody
Services
Beacon® Single B Cell
Screening Platform