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Technical Account Manager
Sino Biological
Amy Sheng, PhD
Antibody Discovery
by Single B Cell
Screening on Beacon®
Sharing of Antibody
Discovery of Single B
Cell Based on Beacon®
Sino Biological
Amy Sheng, Ph.D.
Technical Account Manager
Sino Biological US Inc.
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
B Cell Differentiation and Development
01
Sino Biological
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
Introduction of Antibody Discovery Platform
02
Sino Biological
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
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
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
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®
Introduction of Beacon®
03
Sino Biological
Sino Biological
Beacon® System
Flow
Diffusion
Sino Biological
An Example of Cell Culture
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
Cell Separation Load Assay Unload
Samples
Incubation
Antibody Magnetic
Particle
Target
Cell
Immunomagnetic
Beads Capture
Sino Biological
Cell Separation Load Assay Unload
Sino Biological
Cell Separation Load Assay Unload
Antigen
Antibody
Secondary
antibody
Avidin
beads
Cell
Sino Biological
Cell Separation Load Assay Unload
Blocking
Unblocking
Antibody
Secondary
Antibody
Avidin
beads
Avidin
beads
Ligand
Antigen
Sino Biological
Cell Separation Load Assay Unload
Cell
Cell
Cell
Ligand
Blocking
Unblocking
Sino Biological
Cell Separation Load Assay Unload
Sino Biological
Case of Antibody Screening
Based on Beacon®
04
Sino Biological
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
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
Load
11978
Antibody Drug Binding
772
Human IgG Binding
151
Competition
Blocking 54
Unblocking 97
Antigen/hIgG Binding
Competition
Sino Biological
Case Study
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
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
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
Screening of Monkeypox A29 Antibody
Target Load Antigen+
A29 11390 321
Western Blot Assay ELISA Assay
The EC50=23.98 ng/ml
Sino Biological
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
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
Guangzhou, China
PA & TX, USA
Frankfurt,
Germany
Headquarters:
Beijing, China
Shanghai, China
Taizhou & Suzhou, China
Kawasaki-shi, Japan
Global Presence
Sino Biological
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

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Antibody Discovery by Single B Cell Screening on Beacon®

  • 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®
  • 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