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TRANSCRIPT
Recombinant Antibody Production in Therapeutic Antibody Projects
Keshav Vasanthavada
Senior Marketing Specialist, GenScript
April 7, 2016
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Presentation Outline
Introduction
Recombinant Ab Production
GenScript Capabilities & Services
Case Studies
Summary & Conclusion
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Introduction
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About GenScript
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GenScript Products & Services
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Gene
Peptide
Protein
Antibody
Discovery Biology
Cell Line
Introduction
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Expression Systems
Introduction
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Production Track Record
Track Record • >6000 batches of high quality protein • >1500 batches of high quality rAbs • >92% success rate for all projects • Successfully delivered a variety of protein targets
We deliver guaranteed and fully customizable, high quality recombinant antibody and protein services with fast turnaround timelines and at very competitive prices
Introduction
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GenScript Ab Drug Discovery Services
Introduction
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Recombinant Ab (rAb) Production
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Why rAbs?
Traditional mAb technology first developed in 1975 by Kӧhler and Milstein.
Mature technology, very robust and cost-effective.
Issues include
• Ethical concerns
• Non-specificity
• Instability of hybridomas causing loss of Ab genes
• Potential immunogenicity (HAMA response)
• Irreproducibility
• Series of Nature articles reported on the “reproducibility crisis” in biomedical sciences caused by Abs. Nearly $350M/year wasted on irreproducible Abs.
Scientists at leading organizations including >100 co-signatories proposed shifting entirely towards rAbs.
Recombinant Ab Production
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Non-animal technology
More control over the process
Speed – production time in weeks
HTP processing
Can be constructed against virtually any antigen
Immunogenicity not an issue
rAbs can be produced in several formats: Fabs, scFv, diabodies
Amenable to fusion with drugs and toxins
Sequence-defined making them more reliable and reproducible
Conducive to optimization and engineering
There are various ways in which a researcher can utilize rAb technology – rAb technology is not intended to entirely replace conventional technologies. In most cases rAb technology supplements existing technologies.
Advantages of rAbs
Recombinant Ab Production
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rAb Production
Hybridoma
Display Transgenic
Animal
Other
Entry Point into rAb Production
Recombinant Ab Production
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GenScript Capabilities & Services
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Production Technologies
Transient Expression 50ug to gram-level expression
Rapid - as early as 6 weeks
Starting from $499
Stable Cell Pool 300mg to gram-level expression
As early as 8 weeks
Competitive pricing
Stable Clonal Cell Line
Multiple gram-level expression
Between 14-17 weeks
Suitable for large scale consistent production of your rAb
Options for rAb Production
GenScript Capabilities & Services
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Transient Expression Licensed NRC Technologies
GenScript Capabilities & Services
HEK293 • Highly stable 293EBNA1 clone.
• Afford the advantages of serum-free production.
• Rapid and high yield recombinant production.
CHO-pTT • Provides comparable protein expression level
to those of HEK293 system.
• Rapid production of recombinant proteins that require post-translation modification for research or pharmaceutical use.
• Licensed from NRC, Canada
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Stable Cell Pool
Use of GenScript Technology results in high-producing cell pools.
Cell pools achieved within 8 weeks
GenScript Capabilities & Services
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GenScript CHO-GS Stable Cell Line
• GS based selection system
• High productivities without amplification
• Speed – no need for rounds of gene amplification
• Scalable
GS
• CHO derived host cell line for expression
• Adapted to suspension and AFCDM
• Selected for superior growth and expression CHO
• Fully optimized process
• Product concentrations of >5g/L achieved
• High probability of monoclonality
Cell Line Development
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2
4
6
Pool Single cellclone
Batchculture
Fed-batchround 1
Fed-batchround 2
Tite
r (g
/L)
mAb Expression level
mAb Expression level
High volumetric
productivity
Cost
GenScript has created cell lines producing >5g/L of recombinant antibody with production rates in the range 50-100 pg/cell/day.
GenScript Capabilities & Services
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General Schematic of rAb Production
GenScript Capabilities & Services
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Discovery Preclinical
Development GLP Workup GMP Workup
Industry Pipeline
Target Validation Screening
Preparation Lead Generation
Lead Optimization &
Characterization Lead Selection
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No. Examples
1 HTP expression and purification of recombinant proteins & antibodies: Quantity: 1-5ml, purification: 96 well plates Protein A column
2 Lab scale transient expression in shaking flasks (100ml-10L): Transient expression in mammalian cells, mostly HEK293
3 Mid scale expression in wave bags (10L-25L): Transient (HEK293) or Stable pool production (CHO cells)
4 Large scale wave bags or bioreactor (varies): Transient or Stable pool production (CHO cells, >25L), or Stable cell line production (CHO-GS, >10L, deliver cell line)
5 Characterization services with LC-MS as the core technology: Critical for verification and the release of bio products, more important in the latter stage of R&D, absolutely required in clinical stage
GenScript Capabilities & Services
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rAb Service Guide
Purpose Service Production
Type Volume
Antibody quantity
Timeline Guiding Price per unit QC standard
Screening HTP Gene to Antibody
Production HTP Transient
Expression 1ml-5ml 40μg-200μg 2-3 weeks ~$300 per variant*
• Titer: ELISA • Purity: SDS-PAGE -85% • Quantitation: UV A280
Candidate validation
MamPower™ guaranteed
recombinant antibody production
Transient Expression in Shaking flask
0.1L-1L 4mg-100mg 3-4 weeks $30-$500/mg antibody# • Titer: ELISA • Purity: SDS-PAGE -90% • Quantitation: UV A280
In vitro efficacy
MamPower™ guaranteed
recombinant antibody production
Transient Expression in Shaking flask
1L-10L 40mg-1g 4-5 weeks $12-75/mg antibody#
• Titer: ELISA • Purity: SDS-PAGE -95%;
HPLC -95% • Quantitation: UV A280 • Endotoxin:<1EU/mg
Large scale recombinant antibody
production
In vivo efficacy
Large scale recombinant antibody
production
Transient Expression in
Wavebag 10L-25L 400mg-2.5g 6-8 weeks $10-30/mg antibody#
• Titer: ELISA • Purity: SDS-PAGE -95%/99%;
HPLC -95%/99% • Quantitation: UV A280 • Endotoxin:<1EU/mg or <0.1EU/mg
Custom Services Wavebag cell
pool 10L-25L 1g-12.5g
12-14 weeks
$3.2-22/mg antibody#
Safety test with GLP
Custom Services Bioreactor cell
pool 25L-100L 2.5g-50g
12-16 weeks
$2.4-16/mg antibody# • Titer: ELISA • Purity: SDS-PAGE -95%/99%;
HPLC -95%/99% • Quantitation: UV A280 • Endotoxin:<1EU/mg or <0.1EU/mg • Identification: MS
Gram Level Bioproduction cell line
service
Bioreactor cell line
10L-100L 10g-100g 24-28 weeks
$1.5-6/mg antibody#
*Not including gene synthesis #Including gene synthesis
With solid expertise in recombinant antibody (rAb) production techniques, GenScript provides a comprehensive rAb service portfolio. Choose from services that deliver microgram to gram quantities of pure rAb for each stage of your Ab drug discovery program
GenScript Capabilities & Services
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MamPower™ Guaranteed Services
Key features of MamPower™ guaranteed recombinant antibody production services • Gene synthesis is included in the package – no additional cost to you.
• Guarantee recombinant antibody amount, purity and endotoxin control – no cost to you if we don't deliver as promised.
• Flexible scale from 5mg to 500mg.
• Competitive price.
• Fast turnaround time - as little as 5 weeks.
Customer provides
Amount Purity & endotoxin level
options Timeline Deliverables
Price starting
from
Antibody sequence
5mg • ≥95% by SDS-PAGE 5-6 weeks • LC and HC in pUC57 vector
• Optimized gene sequence report
• Purified antibody at listed amount and purity
• QC data
$2,999
10mg 25mg 50mg
100mg
• ≥95% by SDS-PAGE • Endotoxin ≤10EU/mg • Concentration ≥1mg/ml
6-8 weeks
250mg 500mg
• ≥95% by HPLC • Endotoxin ≤1EU/mg
8-10 weeks
GenScript Capabilities & Services
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HTP Gene to Antibody Service Workflow
• Ideal for Antibody Screening and Optimization Campaigns
• Minimum 24 Abs required
• 50ug or 1mg packages
• 6-8 weeks
• Starting at $499/Ab (including gene synthesis)
http://www.genscript.com/high-throughput-antibody-purification.html
GenScript Capabilities & Services
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Case Studies
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Expression level was low (2.5 mg/L)
Case Study 1: Low Expression
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384390
Expression level was significantly increased (25 mg/L)
OptimumGeneTM Codon Optimization
Optimized Antibody Sequence
Optimized Transfection and
Purification
Case Studies
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Case Study 2: No Expression
Western blot
Fig.1. SDS-PAGE and Western blot analysis of Antibody 1
Lane M: Marker Lane 1~3: Cell culture supernatant from day 2, 4 and 6 post-transfection R (293) Lane 4~6: Cell culture supernatant from day 2, 4 and 6 post-transfection R(CHO) Lane 7~9: Cell culture supernatant from day 2, 4 and 6 post-transfection N (293) Lane 10~12: Cell culture supernatant from day 2, 4 and 6 post-transfection N (CHO) Lane P: Mouse IgG1, Kappa (Sigma, Cat.No.M9269) as positive control
The target Antibody has no expression (left panel) Upon sequence revision, the expression level of target antibody reached ~15 mg/L.
Target: Antibody 1 (MW ~ 150 kDa)
Case Studies
Western blot
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Client has access to a proprietary technology for
identifying and isolating antigen-specific B-cells.
Using a group of patients [4, healthy immunocompetent]
who contracted and cleared HIV infection:
• Identified B-cells specific for the desired epitope
• Isolated and sequenced the unique antibody sequences
• Cloned, purified and confirmed reactivity of antibodies
Engaged GenScript to continue pre-clinical development of a panel of positive Abs.
Client focused on biology and developability of lead Abs to ensure efficacy while minimizing downstream risks.
Case Study 3: Client HTP Ab Production
Case Studies
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Case Study 3 Specifics Screening & Candidate Validation
Customer in-silico optimized
sequences for production and
development of Abs 4, patient-derived anti-HIV
neutralizing Abs
HTP GtA, 1mg HTP Biophysical
Characterization [thermal stability, aggregation
propensity]
Identify 10-12 that are active and with
desirable biophysical properties
Finally 3-10 Abs
Few 100 mgs for: • Viscosity screening • Colloidal stability • Solubility • Stability • Screening assay • Filterability
Biotech Client
MamPowerTM and customized rAb services
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Few 100mgs required for:
• In vitro neutralization assays [current strategies to measure the neutralization activity of anti-HIV-1 mAbs involve well-established TZM-bl cell-based assays]
Few grams required for:
• In vivo protective activity of anti-HIV-1 Ab in mouse and macaques
Safety test
Case Study 3 Specifics In vitro & In vivo efficacy & Safety
MamPowerTM, Large scale rAb
Large scale rAb, Custom services cell pool
Cell pool, Cell line
Case Studies
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Summary & Conclusion
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GenScript Capability
His
VL VH
VH
VL
ScFv1
ScFv2
BiScFv
Linker
C-Fc tag protein N-Fc tag protein
Summary & Conclusion
• >1500 batches of high quality rAbs
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Quotations & Ordering Workflow
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Why Choose GenScript
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Antibody Handbook
http://www.genscript.com/rAb_handbook.html
Topics include • Antibody structure, isotypes and formats • Antibody fragments and advantages • Antibody drug discovery process • Humanization and Affinity maturation • rAb Production • rAb purification and characterization and
more
Summary & Conclusion
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Upcoming Webinars
April 12, 2016 / 2:00 PM EST
Genetic variants: tips & techniques for creating novel synthetic genes – Laura Geuss, Ph.D.
April 21, 2016 / 9:00 AM EST
Anti-idiotype antibody generation and application in antibody drug discovery - Liusong Yin, Ph.D.
May 10, 2016 / 9:00 AM EST
Antibody generation: challenges and solutions – Glen Marszalowicz, Ph.D.
April 28, 2016/ 9:00 AM EST
CRISPR gRNA libraries: design options, applications, and how they can accelerate your genetic screening studies – Peter Dai, Ph.D.
May 18, 2016/ 9:00 AM EST
Recombinant protein production in baculovirus/insect expression system – Yong Liu, Ph.D.
To register for other webinars in the GenScript Webinar Series visit http://www.genscript.com/webinars.html
Summary & Conclusion
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Thank You!
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