Dr. Yuning Chen discusses strategies involved in recombinant antibody production and introduces a novel antibody expression platform to facilitate antibody library generation. Antibodies are bio-macromolecules that bind to their corresponding antigens with exceptional specificity and affinity. They are the foundation of modern biopharmaceuticals and are widely used as therapeutics, diagnostic agents, and tools for biomedical research. Antibodies are derived from methods such as hybridoma, phage-display, and B cell sorting, and produced mainly via recombinant expression. In this talk, Dr. Yuning Chen from Sino Biological reviews strategies involved in recombinant antibody production, particularly aspects such as construct design, culture condition optimization, and purification methods to provide the audience with a gateway to the field of recombinant antibody expression, technology, and optimization. He also introduces a novel high-throughput antibody expression platform to facilitate antibody library generation and the identification of suitable candidates for further development. Key Topics Include: - What are antibodies and what is their role in modern biotechnology? - Recombinant antibody expression: host systems and general workflow - Strategies for recombinant antibody construct design - Methods for optimal recombinant antibody expression and purification - Applications of a recombinant antibody generation platform for research and therapeutic antibody production

1. Welcome!
Recombinant Antibody Production:
Current Methods and a Novel
Antibody Generation Platform
Yuning Chen, PhD
R&D Manager
Sino Biological

2. Recombinant Antibody Production
Current Methods and a Novel Antibody Generation Platform
Yuning Chen, Ph.D.
R&D Manager
Sino Biological Inc.

3. Sino Biological Inc.
CONTENT
Company Overview
1
Antibody and Its Derivatives
2
Strategies: Recombinant Ab Expression
3
Novel HTP Expression Platform
4

4. Sino Biological Inc.
Company Overview
01

5. Company Overview
Sino Biological Inc.
Subsidiary, Guangzhou
Global Presence
Subsidiary in PA,
USA
Subsidiary in
Frankfurt, Germany
Headquarter, Beijing
Subsidiary, Shanghai
Subsidiary, Taizhou & Suzhou
Subsidiary, Japan

6. Sino Biological Inc.
Our People
500+ Employees & Growing
30% of Staff with Masters or PhD Degrees
Primary Facility
66,000 sq ft Lab/Office Space
25,000 sq ft GMP Facility
Core Business
Reagents: Recombinant Proteins, Antibodies,
cDNA Clones, ELISA Kits…
Custom Services: Recombinant Expression,
Antibody Development…
Core Competencies
Certified
by:
ISO9001
ISO13485
CNAS
Company Overview

7. A One-stop Reagent Provider
Products: From Genes to Proteins and Antibodies, All in Stock
32,000+ Genes
14,000+ Antibodies 600+ ELISA Kits
6,500+ Proteins
Sino Biological Inc.
End-Users in 90+ Countries
Major Categories Product Citation Count User Distribution
• >1700 citations in 2020
• Citations for SARS COV2 related products (as of 5-31-2021):
 22 published papers in “Cell”
 15 published papers in “Science”
 8 published papers in “Nature”
Company Overview

8. Featured CRO Services
Recombinant Protein Production Services
• Mammalian Cells, Insect Cells, E. coli Available
Recombinant Antibody Production Services
• High-throughput Antibody Production Service
• Large-scale Antibody Production Service
Sino Biological Inc.
Antibody Development Platforms
Recombinant Production Platforms
E. coli System
HEK293/CHO
Transient System
Baculovirus-insect
System
CHO/HEK293 Stable Cell
Line Development
Mouse mAb Development
(Hybridoma)
Rabbit mAb Development
(Phage Display)
Single B-cell Based Platform Rabbit pAb Development
Antibody Development Services
• Fast Mouse mAb Development
• Rabbit mAb Development
• Fast Rabbit pAb Development
• Anti-idiotype Antibody Development
Company Overview

9. Sino Biological Inc.
Antibody and Derivatives
02

10. Sino Biological Inc.
Concept and Physiology
Antibody and Derivatives
Highly Specific Interaction
Strong Affinity
Antibody Antigen
Unique Formats
Abundance: IgG (80%)**> IgM (10%)> IgA> IgD&IgE
Antibodies in Circulation (Humans and Other Mammals)

11. Sino Biological Inc.
History
Antibody and Derivatives
https://wellcome.org/reports/expanding-access-
monoclonal-antibodies
1770~1890
Smallpox inoculation
Immunized animal
serum cured
diphtheria
1900
Side-chain theory+
Ab-Ag binding+
complement
activation
1948
B cell in Ab
generation
1957
Clonal selection
theory
1959~1973
Molecular
structure of Ab
established
1975
Hybridoma
1984
Chimeric Ab
1985~1990
Phage display &
Ab engineering
1994
Transgenic
mice & full
human Ab
1997
Yeast display
2000’s
B cell sorting
2020 and beyond
The rise of AI
The story continues…
1986
1st mAb
approved
(OKT3, CD3)
1994
1st Fab
approved
2000
1st ADC
approved
2002
1st full-human
Ab approved
(Humira, TNF)
2005
1st bi-specific
Ab approved
2014
1st BiTE
approved
Till 2022
>135 Ab
therapeutics
approved or in
regulatory
review
1997
Rituximab (CD20)
1998
Trastuzumab (HER2)
2015
Nivolumab (PD1)
Pembrolizumab(PD1)
2021
Sotrovimab
Regdanvimab
Casirivimab + Imdevimab
for COVID-19
2020
Ansuvimab
Atoltivimab+ Maftivimab+
Odesivimab-ebgn
for ebola infection

12. Sino Biological Inc.
Applications
DOI: 10.2147/BCTT.S175360
Diagnostics
Therapeutics
Biomedical
Research
Antibody and Derivatives
WB, anti-COV2 NP IF, anti-EGFR FCM, anti-CD31 ELISA, anti-HER2
IHC, anti-Claudin 18.2

13. Sino Biological Inc.
Formats
Antibody and Derivatives
FragmentedAb
Fab scFv VHH
Chimeric Ab
Ab Fusion Proteins
scFv-Fc VHH-Fc
Bi-specific Ab
Full-length IgG
DOI: 10.3389/fimmu.2019.02454
(1) Extraction of hybridoma mRNA (2) Sequencing
(3) de novo transcriptome assembly (4) Search for Igh /IgI/Igk sequence
DOI: 10.1016/j.molimm.2015.01.003

14. Sino Biological Inc.
Strategies for
Recombinant Ab Expression
03

15. HEK293/CHO
Insect cell
E. coli
Yeast
• Low cost, rapid expression, easy to scale-up
• No PTM*, inclusion bodies, MW limitation
• Low cost, rapid expression, easy to scale-up
• PTM* with high mannose content glycans
• PTM*, high cell density, advanced folding,
soluble proteins
• Partial glycosylation, costly
• Correct PTM*, folding, and soluble
proteins
• Long culture duration, costly
*PTM = Post translational modification
Sino Biological Inc.
Concept and Hosts
Recombinant Antibody Expression
E.coli or yeast serve as hosts for VHH
DOI: 10.1186/s12934-019-1094-0
• secreted molecules
• complex high-order structure (multiple S-S)
• correct folding (V) for Ag interaction
• correct folding (Fc) for FcRn
interaction/effector function
• glycosylation for effector functions
Suitable host cells for rAb

16. Sino Biological Inc.
Core Process
Recombinant Antibody Expression
https://plueckthun.bioc.uzh.ch/antibody/Introduction/IGGinteractions/index.html
Ab
Sequences
• Hybridoma
• Phage-display
• B cell sorting
• AI-based calculations
Expression
Vector(s)
Transient
Transfection
(PEI
Mediated)
HEK293FT DG44
SBI
Competitor
Culture Purification
Activity
Assessment
Stable Cell Line
(CHO)
Establishment
Optimize
Culture and
Purification
Protocol
Large-scale
Manufacturing
Ab
Discovery
• Affinity purification
• Other chromatography methods
Vector #1: LC
Vector #2: HC
+

17. Sino Biological Inc.
Construct Design
Recombinant Antibody Expression
Name Sequence
Human OSM MGVLLTQRTLLSLVLALLFPSMASM
Mouse Ig Kappa METDTLLLWVLLLWVPGSTGD
Mouse Ig Heavy MGWSCIILFLVATATGVHS
BM40 MRAWIFFLLCLAGRALA
Secrecon MWWRLWWLLLLLLLLWPMVWA
Human IgKVIII MDMRVPAQLLGLLLLWLRGARC
CD33 MPLLLLLPLLWAGALA
Human Chymotrypsinogen MAFLWLLSCWALLGTTFG
Human trypsinogen-2 MNLLLILTFVAAAVA
Human IL-2 MYRMQLLSCIALSLALVTNS
Albumin (HSA) MKWVTFISLLFSSAYS
Human insulin MALWMRLLPLLALLALWGPDPAAA
• commonly used for Ab expression
• full-Ab SPs are also suitable for scFv, VHH…
• SP can impact the yield of rAb
• select a SP with good cleavage potential for protein-Ab fusions
SignalP 6.0: https://services.healthtech.dtu.dk/service.php?SignalP
Glycan
N297
Fuc
Fucosylated Ab
aFucosylated Ab
HEK293
FUT 8-/-
HEK293
“Miniaturization”–scFv
• Linker
• Purification Tag
• “Accessories”
Signal Peptide, SP
(leader sequence)
DOI:10.1371/journal.pone.0116878.g003
Length of the linker impacts scFv activity
DOI: 10.1080/09168451.2016.1156482
Optimize construct to improve rAb expression
scFv-His-Avi
• >95% purity (PAGE)
• Low yield: <0.5 mg/L
• Low biotin ratio: <0.2
• Aggregation: >90% oligomer (SEC)
scFv-His-spacer-Avi
• >95% purity (PAGE)
•Improved yield: ~6 mg/L
• Improved biotin ratio: 0.85
• Aggregation: ~10% oligomer (SEC)
scFv-Fc-Avi
• >95% purity (PAGE)
•Improved yield: >25 mg/L
• Improved biotin ratio: 0.9
• Aggregation: ~5% oligomer (SEC)

18. Sino Biological Inc.
Fc Modifications
Recombinant Antibody Expression
Orthogonal conjugation site: Cys MT
DOI: 10.1073/pnas.0904191106; DOI: 10.1021/bc900509s
CH1 CH3
CH2: L242C, K334C
CH3: P343C, A431C
Distance between
ß-carbon< 4.7 Å
Enhance stability: Cys MT
DOI: 10.1074/jbc.RA118.005367; DOI: 10.1186/s13036-021-00272-7
Reduce degradation: hinge region H229-Y
DOI: 10.1074/jbc.M111.255026; DOI:10.1038/s41598-018-35489-y

19. Sino Biological Inc.
Recombinant Antibody Expression
Culture Conditions
Ab
Sequences
Expression
Vector(s)
Transient
Transfection
(PEI
Mediated)
HEK293FT DG44
SBI
Competitor
Culture
Culture Optimization
Enhance Ab expression
• selected 20 low expression Ab (<20 mg/L)
• optimization by testing a combination of different
culture media and transfection reagents
• two media recopies use (M1 and M2)
• two transfection reagents formulas used (T1 and T2)
• same culture volume, duration, and temperature
Additives
Weak GFP+
Strong GFP+

20. Protein A Protein G Protein L
Species of Origin Staphylococcus aureus Streptococcus spp. (Group C and G) Peptostreptococcus magnus
Native Size/Sizes 40~60 kDa 40~65 kDa 76 kDa
Binding Regions IgG IgG, HSA Ig
Ig Binding Regions Fc of IgG Fc of IgG VL-kappa
Sino Biological Inc.
Recombinant Antibody Expression
Purification
Custom Bi-specific Ab
Host: HEK293
Attributes:
1. Format: Fab+ HC with a Knobs-in-holes design
2. Purity: >95% by SDS-PAGE and SEC
https://plueckthun.bioc.uzh.ch/antibody/Introduction/IGGinteractions/index.html
https://www.protpi.ch/Calculator/ProteinTool
Affinity Purification of Ig
Prot pi Calculator
Protein A
Ion Exchange
Gel Filtration

21. Sino Biological Inc.
Recombinant IgM
W/out J chain-hexamer
Purification Strategies
W/ J chain- pentamer
Transfection Strategies
Method 1
Method 2
116.0
66.2
45.0
35.0
25.0
18.4
116.0
66.2
45.0
35.0
25.0
18.4
Pentamer
Pentamer
116.0
66.2
45.0
35.0
25.0
18.4
Hexamer
IgM with Kappa LC
1. Protein-L Affinity
Pentamer
Removal of
lower MW
contaminants
2. Gel Filtration
Pentamer
Pentamer enriched
25% before gel filtration
99% after gel filtration
Other IgM Ab
1. Ion Exchange
2. Gel Filtration
Pentamer
Ab reached >95% purity
Recombinant Antibody Expression

22. Sino Biological Inc.
HTP Ab Expression Platform
04

23. Sino Biological Inc.
High-throughput Expression Platform
DOI: 10.1016/j.cell.2020.05.025
Antibody/Protein
Sequence Library
Analysis
HT Primer
Design+
Synthesis
Primer Library
HT Vector
Synthesis*
Vector Matrix
Transfection
HEK293
Culture Array*
One-step*
n
>90% Purity
Validation
Custom
Antibodies/Proteins Scale-up
Protein A
Ni-affinity
B cells from COVID-19
Patients
NGS
Antibody
Sequence
Library
~600 Abs
Expression (0.3mg)
ELISA (S1/RBD)
14 Neutralizing Abs
Scale-up
10~300mg
Structure Analysis
Animal Studies
High-throughput
Screenings
Recombinant Antibody Expression
Custom VHH library generation
* Library size: 74 Ab
* Format: VHH-Fc
* Success rate: 100%
* Purity: >90% (PAGE and SEC)

24. Sino Biological Inc.
Summary
Gene
Synthesis
Transient
Expression
Cloning
Affinity Purification
Purity
Aggregation
Endotoxin
Quality Control
1. Antibodies are essential molecular sentinels that are highly specific towards
their corresponding antigens. They are widely used in life science research
and as therapeutics.
2. Antibodies are derived from various methods (immunization campaign,
patient B mRNA sequencing, in silicon design…) and their large-scale
production is achieved via recombinant expression.
3. Antibodies can be engineered to a variety of formats and mammalian cells
are the most suitable hosts for their expression.
4. The expression of a recombinant antibody can be optimized at various
steps, from the initial construct design to the selection of culture methods
and purification strategies.
5. High-throughput full-length IgG and VHH-Fc fusion antibody expression is
achieved using transient HEK293 transfection with a high success rate.

25. Sino Biological Inc.
THANK YOU
Sino Biological US Inc. (USA)
Address: 1400 Liberty Ridge Drive, Suite 101, Wayne, PA 19087
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, 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
www.sinobiological.com
Get a Quote for Your
Recombinant Production
Recombinant Antibody
Production Services

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