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1 Celonic GmbH, Jülichwww.celonic.deKickoff Meeting - Forsys

Celonic Group

Contract Development & ManufacturingBiopharmaceutical Drug Pipeline


Company Overview


• founded in 1998 in Jülich

• First spin-off of the „Research-Center Jülich“

• Located in the Technologiezentrum Jülich (TZJ)

• Privately owned company

• Jülich: ~ 500 sqm office and lab-space (R&D, GLP)

• Basel: ~ 1500 sqm office and lab-space (Process Development)

400 sqm GMP facility (clinical phases & market supply)

• Staff: 90% scientists and engineers, >40 employees

• Certified by the DQS (German association for Quality assurance)

according to DIN EN ISO 9001:2000 since 1999

• GLP certified by German authorities

• GMP certified by SwissMedic

Celonic Biopharmaceuticals


Business Areas

Hardware

Biotools

Contract

Manufacturing

Biopharma-ceuticals

Mammalian Cell Culture Technology


Business Area: Products

Fluidized Bed Bioreactors (E50 – E2500)

Electrochemiluminescence Technology

(M1M, BioVeris)

ELISA Kits

Biotools for Process Development

Products for Bioprocessing....


Fluidized Bed Reactor (FBR)

CirculationPump

FeedingPump

BasePump

Gas-mixstation

ControlUnit

Fluidized BedBioreactor E500

Probes(pO2, pH, Temp)

Gassing


Siran – Micro Carrier Hybridoma Cells growingon Siran – Micro Carrier

Siran®- Micro Carrier


23

435

050

100150200250300350400450500

Stirred vessel Fluidized bed reactor

prod

uctiv

ity (m

g/L/

d)

The production capacity of a E500 fluidized bedreactor is at least comparable to a 30 L stirred vessel



Cell densities of up to 1011 cells/500 ml bed volumecould be achieved.


Cell Density

0,0E+00

2,5E+07

5,0E+07

7,5E+07

1,0E+08

0 3 6 9 12 15Time [d]

Cel

ls p

er m

l car

rier



Comparable to vessel of

Product / run

Function

System

150 L30 L3 L

∼ 50-450 g∼ 10-90 g∼ 1-5 gmg

ProductionLarge scale

ProductionMid scale

ProcessdevelopmentTest-system

E2500*E500E50E10

Dedicated to be used for:• Therapeutic proteins for niche indications• Production of diagnostic antibodies• Production of proof of concept material• Production for research purposes

Product line from small to large-scale:

* Currently in development



Concept• Fermentor for the continuous, efficient cultivation of mammalian cells• Enables cultivation of adherent and suspension cells• Applicable for GMP production of recombinant proteins

Features• Optimal supply with nutrients and gas ⇒ suitable for very sensitive cell

types• High production capacity (up to 1011 cells/500 ml Siran® Micro Carrier)• Productivities up to 0.5 g / day• Easy installation, minimal utilisation of personnel and space• 500 ml WSF(conti) = approx. 30 L tank reactor (fed-batch)• Various sizes for many applications are available


Process Development and Production of TherapeuticProteins and Antibodies....

Business Area: Contract Manufacturing

Generation of Guideline Conform Cell-lines / Cell-banking (MCB/WCB)

Process Development Upstream and Downstream

Characterization / Quality Control of recombinant proteins

GMP-Production of Biopharmaceuticals (Basel)


Production of Biopharmaceuticals

Cell-line / USP development

DSPdevelopment

AnalyticalMethods

„Upscaling“

Tox-Production

GMP-Production

Regulatory conform:• ICH-guidelines, PTC• FDA (CBER/CDER)• EMEA• BfArM / PEI• (USP, EP)

„Spirit of GMP“ lab:• SOP´s• Documentation• Archiving procedures• Qualification of critical

equipment• Guideline conform procedures• Quality assurance

„Spirit of GMP“ / GLP GMP

Master / WorkingCell Bank

Jülich / Basel Basel


Contract Service – GMP facility Basel

Approved for production of APIs for clinicalphases and market supply

GMP-facility incl. State-of-the-art supply management approved bySwiss Medic (2004)


Contract Service – Bioreactors



Fermentation capacities:

Wave Bioreactors:• 2/10/20 L• 50 L• 1000 L

Stirred Tank Bioreactors:• 2/20 L, • 75 L and • 300 L• 2000 L (available 2010)

Bioengineering-Bioreactors equipped withCIP / SIP, to be operated in batch, fed-batch or perfusion mode



DSP capacities:

Equipment• Äkta Chromatography Systems

(Pilot, Purifier, Explorer, Prime)• BPG chromatography columns

(various sizes)• Micro, Ultra- and Tangential Flow

Filtration Units (Pall, Sartorius)• Buffer preparation room equipped

with Sartorius balances and controlunits

Capacity• Up to 2000 L


Research & Development

Business Areas

Biopharmaceuticals (preclinical stage)

Enzyme from aplysia punctata for treatment of cancer

Antibodies and fusion proteins for treatment of T-cell

mediated immune diseases (RA, Morbus Crohn)


Generation of high Generation of high producerproducer cellcell--linelineusingusing serumserum--freefree strategystrategy


Serum-free Cell-Line Establishment / Processes

Advantages of serum-free Processes:• Regulatory compliance (no serum or undefined animal components

are used; chemically defined or FDA-approved media

• Defined, more reproducable process

• Downstream Processing easier;

• Increased purity of product: no contamination with serum

components such as IgG, BSA,…

Hurdles in cell-line development:Regulatory conform single-cell derived cell-line must be established

by limited dilution, but usually host-cell lines are not acceptable to

limited dilution in serum-free media


Generation of Generation of stablestable high high producerproducer cellcell--lineslines

Selection LimitedDilution Production

PoC-material

Adaptation

Cell-BankingPSB

6 month 10 month 15 monthTime after vector

construction

Cla

ssic

alst

rate

gy


PoC-material

Adaptation

Cell-BankingPSB


construction

Cla

ssic

alst

rate

gy

Serum Serum-frei


Disadvantages of Adaptation Strategy:

1. Time and cost consuming• Cell-line development: ~6 month, Adaptation: ~ 3 Month• Costs: high; mainly due to personal work load

2. Disadvantages / Risks involved in adaptation procedures:

• Increased development time:

• Risk of drop in productivity during adaptation

• Risk of product change during adaptation (e.g. glycosylation pattern)

• Prolongation of population doubling time (PDT)

• Decrease in maximum cell density

Standard Approach


Serum-free cloning and expression technology (SEFEX)

Features:

• Use of well characterized and documented cell-lines based on CHO-

K1 / CHO-DG44 host cells adapted to FDA conform serum-free media

• Serum-free media was developed in order to support:

• High transfection efficience in serum-free media

• Cloning and subcloning without use of serum or feeder-cells at

any time

• Highly efficient expression system

Serum-free cell line development


Advantages

• Fast track: ~3-6 month less compared to classical approach

• No risk in drop of productivity or change of product quality afteradaptation

• Suitability for large-scale has been shown:

• Expressions rates up to 20 pg/c/d for fusion proteins and antibodies

• Cell densities up to 1x107 cells/ml

• Low population doubling time (~24 h)

• Proper level of glycosylation, High level of sialylation

Serum-free cloning and expression technology (SEFEX)

Serum-free cell line development



PoC-material

Adaptation

Cell-BankingPSB


construction

Cla

ssic

alst

rate

gy


PoC-material

Adaptation

Cell-BankingPSB


construction

Cla

ssic

alst

rate

gy

Serum Serum-frei

6 month lessSelection

LimitedDilution

ProductionPoC-material

Cell-BankingPSB

Seru

m-fr

ee10 month less(no product change via adaptation)

6 month lessSelection

LimitedDilution

ProductionPoC-material

Cell-BankingPSB

Seru

m-fr

ee10 month less(no product change via adaptation)

Serum-frei

Generation of Generation of stablestable high high producerproducer cellcell--lineslines


Business Areas

CEMAX Technology for sitedirected gene insertion


StrategiesStrategies forfor high high expressionexpression yieldsyields

• Increasing copy number of integrated expression cassettes

– Multicopy integration– Amplification of integrated single copies (DHFR approach)

Problems: Stability, time consuming

• Integration of a single copy into a transcriptional activelocus

Problems: random integration of expression cassette leads to high number of clones which have to be screened (~2000 clones)


StrategyStrategy forfor targetedtargeted genegene insertioninsertion

1. Generation of a placeholder cell-line having theexpression cassette flanked by recognition sitedplaced into an expression hot spot

2. Exchange of expression cassette by the gene of interest using Meganuclease technology


MeganucleaseMeganuclease technologytechnology

CuttingSpecificity :

1 cut per 70 billion base pairs

..CTAGGATGTAGGGATAACAGGGTAATCCTAGGA..

..GATCCTACATCCCTATTGTCCCATTAGGATCCT..

..CTAGGATG CCTAGGA..

..GATCCTAC GGATCCT..ATCCCTATTGTCCCATTATAGGGATAACAGGGTAAT

Act

ive

Site

Natural Meganucleases are:

• Found in Protozoa, Yeast, Phages, Archeas, …

• DNA endonucleases with very large sites «more than 18 base pairs!»

• More than a 100 known sequences and 300 candidates.

• Small proteins (average size of 230 aa.)

=> Open double-strand DNA and increase homologousrecombination up to 1000 fold


Site-specific DNA Integration

CEMAX cell-line

Producer cell-line

expression Reporter gene

Locus of Interest – hot spot

Recombination

Insertion of new geneby gene replacement

Proof of concept: GFP


Flp

Genomic acceptor construct in “place holder” cell

Extrachromosomaldonor construct

PSV40Δ

ATG FRT HygromycinSV40 pA

PSV40

BGH pA FRTmut3 hSEAPCHO-Genom

FRT Neomycin SV40 pAP CMVenhanced pA

FRTmut3EnhancedGFP

pFRT2/CMV/EGFP

PSV40Δ

ATG FRTmut3

CHO-GenomFRT Neomycin SV40 pA

P CMVenhanced pA

Enhanced GFP

pOG44

eGFP

X

G418 R

XExchange of Exchange of expressionexpression cassettescassettes


Proof of Concept: Exchange Of Place Holder Gene byeGFP

A B

■ After the exchange of an enzymatic reporter gene by a gene encoding for a green fluorescent protein (GFP) all cells produce the green protein.


Approach 5: 8 µg HN /4 µg exchange vector

Exchange of hSEAP against eGFP


CEMAX‘ Advantages At A Glance

■ Cell-line development within 4 weeks

■ Guarantee of stable expression and high productivitybecause of single, known integration site

■ Serum-free process development ensures regulatorycompliance

■ Smooth production of highly glycosylated proteins

■ Suitability for large scale

■ Proprietary system


Cell Line Development Time

Start Cloning of GOI into CEMAX expression vector and preculture of CEMAX cells

Week 1 Transfection, inoculation of 96-wells

Week 2 Start of selection process

Week 3 Analysis of several clones for expression of GOI

Week 4 10 pcd cell-line in 24-well scale

Week 5 Transfer of positive clone(s) into 6-well scale

Week 6 10 pcd cell-line in 6-well scale

Week 7 T25

Week 8 T75

Week 9 Spinnerflask

Week 10 1 L-culture

Week 12 >100 mg available

No more extensive selection and screeningof at least severalthousand clones


Arbeiten im Rahmen des Projektes „FORSYS“

38 Celonic GmbH, Jülichwww.celonic.deKickoff Meeting - ForsysArbeitsbesprechung F+E13.08.08

1. scTRAIL

2. αEGFR-scFv

3. αEGFRscFv-TRAIL

Produktion von 3 Modellproteinen


VerwendungVerwendung derder ModellproteineModellproteine

Proteine und Konjugate mit Nanopartikeln:

1. Tumor/Organ Level:Lungentumorperfusionmodell• Biodistribution• Pharmakokinetik

2. Organismus Level: therapeutische Wirkung in derMaus (Xenografts tumor model)

Bedarf der Proteine• ca. 25 mg pro Protein


• Ligand für DR4 oder DR5

• proapoptotisch

• bindet als funktionelles Trimer an seine Rezeptoren => Trimerisierungder Rezeptoren

Molekül Design:

• single chain als Homotrimerexprimieren

• Linker: 4X(GGGS)

• His-Tag für Reinigung

• Cys-Tag für Kopplung an Nanopartikel oder Chromogene

scTRAIL

scTRAIL

Cys tag

His tag


• Bindet an EGF-Rezeptor als Tumormarker

Molekül Design:

• Expression als klassischer single chain Antikörper

• His-Tag zur Reinigung

• Cys-Tag zur Kopplung an Nanobead und Chromogene

αEGFR-scFv

Anti EGFR-scFV

Cys tag

His tag


Fusionsprotein aus αEGFRscFv-TRAIL zur gezielten Apoptose von Krebszellen

Molekül Design

• Vgl mit anderen Molekülen

αEGFRscFv-TRAIL

Anti EGFR-scFV/TRAIL Fusionsprotein

Cys tagHis tag


HerstellungHerstellung derder ZelllinienZelllinien

• Verwendung der CEMAX-Technologie• Klonierung in CEMAX-Austauschvektoren• Transfektion der CHO-K1 basierten Zelllinie• Selektion mit Zeocin und G418• Klonselektion auf der Basis eines zu etablierenden

ELISA– Quantifizierung mittels anti-His-TAG-AK– Produkt-spezifischer ELISA?

• Expansion und Anlegen einer Zellbank


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PCMV PromotorIg leader

His taglinker

scTR

AIL

linker

l inke

r

IRES

ZeoRB GH

f1 oripSV40

Ne oR

pUC

ori AmpR

scTRAIL8245 bp

VektorVektor

Features:• Enhanced CMV Promoter

/ Intron A• BGH poly A• IRES

Selektion:• Zeocin• G418


ReinigungReinigung

• Produktion der Proteine im Wirbelschichtreaktoroder Wave Bioreaktor

• Ziel: 50 mg pro Protein

• Etablierung einer Reinigungsstrategie

• Affinitätschromatographie über His-Tag (IMAC)

• Ionenaustauscher

• SEC

• Ziel: Reinheit > 90%; 25 mg pro Protein


CharakterisierungCharakterisierung derder ProteineProteine

• SDS PAGE (red.; non-red)• Native PAGE• IEF• Western Blot• ELISA / BCA• Sterilität• Endotoxin• Potency?


FormulierungFormulierung

• Offene Punkte• Formulierung (PBS?)• Konzentration• Stabilität der Proteine in Lösung• Lagertemperatur (+4 °C)• Freeze / Thaw Tests

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