Modern process- och cellinjeutveckling – att nyttja automatisering

och mikrobioreaktorer

Ingrid Lange, Team Leader Cell Culture Services, Cobra Biologics

Lyckad Läkemedelsutveckling 2013-05-30

Outline

• Platforms strategy

• MaxXpress

• Microbioreactors for efficient cell line and process

development

– Implementation

– Case studies

Sodertalje: Mammalian Cell / mAb

Now: Modern facility, with non- GMP and GMP 250L SUB

Comprehensive analytics and cell line development

capability

Future: 1,000L disposable SUB for commercial capacity

Keele: Virus and Small Scale Microbial

Now: 14 year track record of developing processes and GMP

manufacturing for pioneering medicines

Future: Commercial virus capacity 250L disposable SUB

Matfors: Fill/ Finish and Large Scale Microbial

Now: 20 year track record for clinical and commercial supply with

600L microbial GMP capacity

New aseptic fill/ finish facility including lyophilisation

Future: Expansion space for fill/finish or bio-production

Cobra Biologics

• Successfully completed >320 batches

• Worked with >100 customers

• Located across 19 countries on 6

continents

Over 14 years of experience in

producing biopharmaceuticals for

worldwide pre-clinical and clinical trials

Track Record

DNA Proteins Virus Fill &

finish

Efficient process including disposables

Cost effective delivery

Platform Strategy for early stage clinical

development programmes

Utilization of single use materials increases flexibility and decrease turn around time as

well as risk of cross contamination between projects

Customer Project

Cell line development

Cello, plates, shake flask, microbioreactors

Process Development Shake flasks, microbioreactors, 2-5L Glas STR,

Wave, 50L SUB

Tox Supply

50-250L SUB

GMP

Production

250-1000L SUB

MaxXpress

UCOE helps maintain an Open Chromatin Environment

Silent DNA, compact

chromatin structure

DNA accessible for

transcription, Open

chromatin structure

+ UCOE

MaxXpress - Rapid production of protein from pool

Material can be produced within 2-4 weeks from transfection

• Provides confidence that desired product can be produced

• Early product characterisation

• DSP process development

• Yield often orders of magnitude higher than transient transfection

• No need for repeat transfections if request for re-supply

Protein Titer mg/L

antibody 100-1000

EPO 800

hGH 350

IgG-fusion 500

Soluble ligands 100-400

Quality in cell line development for production of

biopharmaceuticals

Clone selection

Titer Glyco-profile

Charge distribution

Aggregation rate

Potency Specific

productivity

Growth profile

Expression stability

CELL PROTEIN

• Plate handling robot (Stäubli)

• Up to three incubators and a cold

storage unit

• Two “pipetting robots”

• High resolution microscope

• Software, managing the complete

process from clone screening to

expansion to 6 well plates

• Database with full traceability for

each clone

Cello Robotic System for Cell Line Development

Cello Robotic System manufactured by TAP Biosystems

Cell Culture Process Development

Equipment

• Shake flasks or Culti Flasks for screens

• Small scale 2L/5L reactors for batch/fed batch/perfusion process development

• 50L SUB for scale up studies and production of pre clinical supplies

• Wave bioreactors (1-25L) for seed expansion or production of pre-clinical

supplies

Scalable and robust process development – To give a consistent product and titer at various scales

– Need to understand critical process parameters ideally early on

– Factorial design of experiment for efficient process development

– Process suitable for GMP manufacturing

• Shorten timelines from transfection to Tox supply

• Speed up the development work • More information from one round of process development

• Reduce costs by reduced work

• Improve quality of process development • Bioreactor process parameters evaluated early in a project

• Limitations in comparability between shake flasks and bioreactors

Driver for automation in Process Development

Driver for automation in Process Development

Customer Project

Cell line development

Cello, plates, shake flask, microbioreactors

Process Development Shake flasks, microbioreactors, 2-5L Glas STR,

Wave, 50L SUB

Tox Supply

50-250L SUB

GMP

Production

250-1000L SUB

High troughput system

for process development

of several clones and

process conditions in

parallell

Final production clone

Select

Process

Micro Bioreactor System

Ambr Micro Bioreactor System

• 24 single use bioreactors

– 24 experiments in parallell

– Individually controlled pH and DO

– Stirring and temperature control

– Automated sampling and additions

• Screening of process parameters

– Media, nutrition feeds, clones

• Process parameter optimization

– pH, DO, temperature etc

• Benefits

– Suitable for design of experiment setups

– Quick and easy to set up

– No cleaning

– Small footprint in the lab

Ambr Micro Bioreactor System

Manufactured by TAP Biosystems

Results using Ambr Micro Bioreactor system

• Max viable cell densities in Ambr, 5L glass bioreactor and 250 L SUB are

comparable

• Performance in Ambr is reproducable

Via

bilit

y [

%]

Via

ble

Ce

ll D

en

sit

y [M

VC

/mL

]

Culture time [days]

5L BR

Ambr

Selection of clones during cell line development

based on bioreactor performance

• Combination of clone screen and feed screen in one Ambr run

• Fed batch evaluation in stability studies of 8 clones (early, mid and late generation)

• More valid results, comparable to bioreactor

• Less manual sampling and additions compared to shake flasks

• Base Media screen, – chemically defined commercial media

– Performed in Culti flasks (12 ml)

• 1st = Feed Screen, – chemically defined commercial feeds

– Performed in Ambr using one base media

• 2nd = Feed optimisation – DoE set-up for selected feed using

Ambr

• 3rd = Feed w amino acid supplement

Case study; Titre optimisation

7-fold titer increase over 3 Ambr experiments in less than 2 months

• Alter glycoprofile by additives and process settings

• 24 different set ups in Ambr

• Selected samples taken further to Potency assay

Case study; Product Potency optimisation

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 1.10 1.11 1.12 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11

Titre

1.1 1.3 1.7 1.8 1.10 1.11 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11

Potency

One run of 24 microbioreactors could identify the pH and additives most benificial for high potency

Efficient process development with DoE approach

in microbioreactor system

• One run in the microbioreactor system can include a screen of up to 5 parameters in a DoE design

• The same DoE set up would require 4 rounds of 6x5L traditional bench scale

12 weeks in 6x5L compared to 2 weeks in 24 microbioreactor system

Process verification in BR

6x 5L C

lean

6x 5L 6x 5L 6x 5L

Cle

an

Cle

an

Cle

an

24x

MBR 2x 5L

2x 5L

2v

2v 2v

12v

Conclusions

• Cobra offers comprehensive services from cell line development to

fill/finish but this talk has focused on upstream strategies

• Platform strategy with short timelines requires reliable expression

system, high throughput technology and short lead times for

protein analyses

• Implementation of microbioreactor system enables more efficient

cell line and process development

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