recombinant antibody production in therapeutic antibody ... · make research easy 10 non-animal...

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


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


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.



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



rAb Production

Hybridoma

Display Transgenic

Animal

Other

Entry Point into rAb Production


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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



Transient Expression Licensed NRC Technologies


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


Stable Cell Pool

Use of GenScript Technology results in high-producing cell pools.

Cell pools achieved within 8 weeks



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

0

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.



General Schematic of rAb Production



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



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


http://www.genscript.com/high-throughput-antibody-purification.html


http://www.genscript.com/recombinant-antibody-production.html








http://www.genscript.com/large_scale_recombinant_antibody.html






https://www.genscript.com/online_request/index.php?act=protein_quotation

https://www.genscript.com/online_request/index.php?act=protein_quotation

http://www.genscript.com/production-stable-cell-lines.html







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



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)











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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


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


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


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

Case Studies


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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GenScript Capability

His

VL VH

VH

VL

ScFv1

ScFv2

BiScFv

Linker

C-Fc tag protein N-Fc tag protein


• >1500 batches of high quality rAbs


Quotations & Ordering Workflow



Why Choose GenScript



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





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

[email protected]


http://www.genscript.com/webinars.html



mailto:[email protected]

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