Generation Change in Biopharmaceuticals Production From ... · Employment Number (change from the prior year) 365 (+1.7%) ... • Oxidation • Freeze-thaw ... GBF’s 100 l Pilot

Generation Change in Biopharmaceuticals Production

From Fed-Batch to Hybrid Processes

Key Facts About the Global Pharmaceutical

Industry in 2013

219.04.20189. DBT Berlin, Roland Wagner

Data taken from The World Bank Group, 2015 ; WifOR calculation; WifOR illustration

330 Mrd. € gross value addd in

2013 – equivalent to the gross

domestic product of Austria in

2014.

4.8 million employees – equivalent

to the polulation of Ireland.

3.9 % annual growth rate of

employment from 2005 bis

2013.

68500 € gross value added

per employee.

Pharmaceutical industry covered

3.6 % of the global share of global

gross value added of the

manufacturing industry.

Approvals for Biopharmaceuticals on the EU

Are Higher than Ever

32018-02-14Roland Wagner

0

5

10

15

20

25

30

35

40

45

50

2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

Other Pharmaceuticals Biosimilars Originals

More than 250

biopharmaceuticals

are on the market.

Biotech-Report 2017

Germany‘s Biopharmaceutical Pipeline Is Well-Filled2/3 of active ingredients are monoclonal antibodies

42018-02-14Roland Wagner,Biotech-Report 2017

0

50

100

150

200

250

300

350

400

MonoclonalAntibodies

Vaccines Other Recom.Proteins

Gene Therapeutics

Phase I Phase II Phase III

Change from the prior year +3% +1% -6% +10%

Total amount 399 116 98 23

Biopharmaceutical

Companies in Germany

(change from the prior year)

Employment Number

(change from the prior year)

365 (+1.7%) 44100 (+8.1%)

2016

Pharmaceutical Location GermanyThe biggest in Europe (EU28 + Switzerland, 2015)

52018-02-14Roland Wagner,

EFPIA, IW Köln

16%

14%

9%

9%6%5%

5%4%

4%

4%

24%

Employees

Germany

France

Great Britain

Italy

Switzerland

Spain

Belgium

Poland

Ireland

Romania

Others

18%

13%

13%

9%8%

8%

7%

6%

5%3%

10%

Manufacturer

Switzerland

Germany

Italy

France

Ireland

Great Britain

Spain

Denmark

Belgium

Netherlands

Others

Number 2 in Europe, Number 4 in the World


Clinical Studies

USA Canada France Germany

USA 41 %

D 6 %

F 7 %

CA 7 %

vfa

A Seemingly Easy Way from Gene to Therapy


From Gene to Bedside

Development

Cell Line Cultivation

Product Purification

ProductionProcess

Molecule

design

Host cell

selection

Upstream

process

Downstream

process

Fill

finish

• Sequence

• Hydrophobicity

• Proteases

• Oxidative stress

• Media components

• Posttranslational

modifications

• Stirring

• Temperature

• Trace metals

• pH

• Air/liquid interface

• Osmolality

• Pressure

• Shear forces

• Mixing

• pH

• Air/liquid interface

• Light

• Agitation

• Shear forces

• Excipients

• Oxidation

• Freeze-thaw

• Light

Factors affecting protein stability

Pioneering Cell Culture Bioprocesses in 1967


Paul van Hemert with the so-called “Bilthoven Unit” with a Polio virus vaccine production bioreactor in

1967.

Filled with cells grown on microcarriers

(Anton van Wezel. 1967. Nature 216: 64).

From Basic Pilot Research to Manufacturing Sciences


2015: Rentschler’s 2× 3000 l TWIN

Facility for cell culture-based

biopharmaceutical manufacturing

1990: GBF’s 100 l Pilot Plant for cell

culture-based bioprocess research

Celldays Increased Within 20 Years By a Factor of 10

Titer Increased Within 20 Years by a Factor of 20


0 5 10 15

105

106

107

Via

ble

ce

ll c

on

ce

ntr

aito

n / m

l-1

Cultivation time / d

1985

0.2 g l-12005

5 g l-1

20 × 106 ml-1

2 × 106 ml-1

5 g l-1

0.2 g l-1

0 2 4 6 8 10 12 14 16 18

0

1

2

3

4

5

1985

2005

Pro

du

ct tite

r / (g

l-1)


High-End Cell Culture Processes Are Approaching

Natural Blood Cell Concentrations


Cell concentration in bioreactor:

1.25 × 108 ml-1

25 % packed cell volume

Ø = 10-20 µm

Cell concentration in blood:

5-10 × 109 ml-1

50 % packed cell volume

Ø = 7.5 µm

Boost Performed by Cell Culture TechnologistsA fantastic job over the last 20 years


● Drivers are

o Improving Safety to patients (CD media)

o Improved Control & Consistency

o Development Time Line Acceleration

● In principle for antibodies and Fc-fusion proteins

0

2.000

4.000

6.000

8.000

10.000

12.000

14.000

1990 1995 2000 2005 2010 2015 2020

Tit

er

/ (m

g l

-1)

Date / y

According to Thomas Ryll, 25th ESACT Meeting, Lausanne, 2017

Improved Cell Culture Production is Driven by


● Safety, Control and Robustness

o Enhanced patient safety

o Improved process robustness and

consistency

o Tailor-made product quality

(comparability & similarity)

● Efficiency, Potency and Cost

o Enhanced productivity and facility output

(titer and volumetric productivity)

o Excellent pharmacokinetics

o Reduced processing time

o Reduced process scale

o Ease downstream operation

o Ease technology transfer

o Overall cost reduction (cost per gram of

product produced)

Only 3 % of the Genes Are Different in

Humans and Rodents


Chinese hamster Cricetulus griseus

Industrial cell line provider

Graphodatsky AS et al. 2011. Mol Cytogen 4: 22

The CHOdyssee


Ovary of “outbread”

Chinese H amster

0.1 g

Trypsin

Fibroblast-like culture, near diploid

adherent culture for 10 months, near diploidy?

Modified morphology observed, unspecified

Cloning of cellspoorly described

1957

1958

CH O- oripseudo-diploid

Theodore Puck

24.09.1916 – 06.11.2005

University of Colorado,

Denver

Lawrence Chasin

Columbia University, NY

Wurm F. 2015. In Hauser H, Wagner R. Animal Cell Biotechnology in Biologics Production. WdG, Berlin

The CHrOmosomesThe world of quasispecies

162018-02-14Roland WagnerWurm F. 2015. In Hauser H, Wagner R. Animal Cell Biotechnology in Biologics Production. WdG, Berlin

Cricetulus griseusStarts in 1919 for typing pneumcococci

† 1957

Lifespan: 2-4 years

2n = 22 Chromosomen

predestinate it for research of

radiation cytogenetics

Typical Antibody Manufacturing Process Over the

Last 20 Years


Levine H et al. 2013. BioProcess Int 11(4s): 40-45

Inoculation Train

15 – 18 d

● Challenge:

Overall Process

Intensification

● Focus areas for

improvements

o Cell Banks

o Seed

Expansion

o N-1 Cell Mass

o Production

Culture

Seed Train 6 – 12 d

From Vial to Harvest 30- 50 d

12 – 18 d

Shake Flasks/

Rocker Bags

USP

DSP

Inoculation Train:

Thaw and expand cells

Cell Banks and Seeding Intensification


● Increasing starting cell mass and reducing initial expansion steps

o Rolling seed train

o High cell concentration in vials or bags

o Frozen seed train intermediates in bags

Heidemann R et al. 2002. Cytotechnology 38: 99-108; Heidemann R et al. 2010. Biotechnol Prog 26: 1154-1163; Tao YW et al. 2011. Biotechnol Prog 27: 824-829; Seth G et al. 2013.

Biotechnol Bioeng 110: 1376-1385; Clincke MF et al. 2013. Biotechnol Prog 29: 768 - 777

Basic Strategy Advantage Disadvantage

One Vial Only Batch • Batch to batch identical seed train• Identical population doublings

• Identical cell age

SLOW

• More WCB vials used

• More labor intensive

RELIABLE

Rolling Seed Train

(small scale bioreactors)

• Less labor intensive

• Less WCB vials used

• Reduced number of seed train stages

for each production run

FAST

• Each batch has a different seed

train history• Each batch has different cell age

• Cell line stability limits

• FAILURE-PRONE

Cell Banks and Seed IntensificationHigh cell number working cell bank


Thaw directly into Wave from 5-ml CryoTube vial (cell conc.100×106 ml-1)

Elimination of more variable seed expansion stages

9 days facility time saving after change over

Standard Vial Master Cell Bank + High Density Working Cell

Bank

20-L Wave system with floating perfusion filter

Tao Y et al. 2011. Biotechnol Prog 27: 824-829

Standard WCB

1 ml, 25×106 ml-1

HDWCB

4.5 ml, 100×106 ml-1

20-L Wave system with floating perfusion filter

Via

ble

Cell

Concentr

ation

/ (1

06

ml-1

)


Via

ble

Cell

Concentr

ation

/ (1

06

ml-1

)


Production Culture IntensificationFrom batch to hybrid processes


Batch Culture Simple Fed-Batch

Glucose, Glutamine

Complex Fed-Batch

Complex feed, includes hydrolysates,1 – 3 bolus feeds

High Efficiency Fed-Batch

CD feed,daily or continuous feeding, potentially multiple feed streams

Controlled Fed-Batch

Multiple CD feed streams, continuous or discrete addition controlled through feedback loops and advanced monitoring

No feeding

Pre 1990Legacy Processes

Current Best Practice

Future State

Below 1 g l-1

1 - 3 g l-1

(“2 g l-1 in 2 weeks”)

5 - 10 g l-1

> 10 g l-1

Hybrid Process

Continuously perfused process for 4 daysfollowed by a controlled fed-batch

● Shortened process time by 1 day

● Comparable cell conc. (<10×106 ml-1) & viability

● Doubled protein titer at harvest

Effects of Accelerated Feed Strategy

0

200

400

600

800

1000

1200

0 2 4 6 8 10 12 14

Pro

tAH

PLC

m/

(mg

l-1)

Cultivation time / dGMP 1 GMP 2 GMP 3 GMP 4

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14

Pro

tAH

PLC

/ (

mg

l-1)


GMP 5 GMP 6 GMP 7 GMP 8 GMP 9


Fed-Batch Production Improvements Over the Last 20 Years

Iso-Titer Lines in Efficiency / Intensity Space


Media / Feed Optimization

o CD formulas, balanced feeding

Host Cell Line that grows to high cell mass

o Adapted to process materials and format

Improved Process Control

o Feedback loop driven feeding: Feed per cell, Glc / Lac control

o Lactogenesis: HIPDOG, HIPCOP

Perfusion Applications:

- Perfusion supported Fed-Batch

- Concentrated Fed-Batch

- High Density Perfusion Culture

0 2 4 6 8

105

106

107

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Via

ble

ce

ll co

nce

ntr

atio

n / m

l-1


Lac

Glc

VCC

Glc

/La

c c

on

ce

ntr

atio

n / (

g l

-1)

Cellsp.

Productivity

[pg d-1]

Integral Cell Mass

[106 ml-1]

Titer

[g l-1]

Year

20 20 1 1990

30 70 2.5 - 2005

50 100 5 - 2010

50 200 10 > 2015

50 300 15 > 2017

Combining Benefits of Fed-Batch and Perfusion to

Hybrid Processes


Perfusion Fed-Batch

N-1 PerfusionSeed culture Perfusion followed by Fed-Batch

Hybrid Perfusion - Fed-BatchPerfusion during Fed-Batch growth phase to boost cell mass

Concentrated Fed-BatchPerfusion during Fed-Batch production phase using UF membrane to concentrate the product

Fusion

Process

Feature

Titer Media

Consumption

Complexity Available Manufacturing

Capacity

Cell Mass Volumetric

Productivity

Product

Residence Time

Benefit Fed-Batch Fed-Batch Fed-Batch Fed-Batch Perfusion Perfusion Perfusion

1st Concept: The N-1 Perfusion ProductionPerfusion by Microfiltration

242018-02-14Roland WagnerReduced production time & simultaneous increase of volumetric productivity

Referring to Ryll T. 2017. 25th ESACT Meeting

0 5 10 15 20

106

107

0

2

4

6

8

10Batch N-1 SeedPerfusion N-1 Seed

Harvest d17Harvest d12

Batc

h N

-1 T

iter

N ProductionN-1 Seed

Batc

h N

-1

Perfusi

on N

-1

Via

ble

ce

ll co

nce

ntr

atio

n / m

l-1


Perfusi

on N

-1 T

iter

Tite

r / g

N-1

Bioreactor

N

Bioreactor

Bioreactors drawn by Dethardt

Müller, modified

2nd Concept: The Hybrid PerfusionPerfusion by Microfiltration


● Perfusion during initial

growth phase followed

by fed-batch culture at

very high cell

concentrations

● HIPCOP: High-end pH

control of perfusion

● Increased celldays result

in highest titers

● Titers beyond 10 g l-1

were achieved during 12

days run

● Volumetric productivities

range at 1 g l-1 d-1 for

multiple cell lines with a

moderate cellular

productivity

0 2 4 6 8 10 12 14

106

107

108

Perfused Fed-Batch

Fed-Batch

Via

ble

ce

ll co

nce

ntr

atio

n / m

l-1


25

N

BioreactorBioreactor drawn by Dethardt

Müller, modified

3rd Concept: The N-1 Perfusion Hybrid ProcessPerfusion by Microfiltration


0 2 4 6 8 10 12 14

106

107

108

109

N-1 Perfused Hybrid

Perfused Fed-Batch

Via

ble

ce

ll co

nce

ntr

atio

n / m

l-1


N-1

BioreactorN

Bioreactor

● Start perfusion at the N-1 seed bioreactor state followed by a

short perfusion period at production scale and finished with a

fed-batch process

● Reduction of process time by simultaneous increase of

productivityBioreactors drawn by Dethardt

Müller, modified

Go the Extra Mile: The Concentrated Fed-Batch ConceptPerfusion by Ultrafiltration


● Ultrafiltration using the XCellTM ATF

module consisting of a 30-50 kDa

membrane

● Retains cells and proteins

● Concentrates proteins beyond 20 g

l-1 (XD® Process, DSM)

● Permeate allows waste removel

● Total cumulative protein content in

XCellTM ATF perfusion is ~6 times

higher than in the fed-batch culture

Pro

du

cttite

r/ m

g


Genzel Y, MPI, Magdeburg, Germany

N

Bioreactor

The Concentrated Fed-Batch Concept


Process Cell

concentration

[ml-1]

Titer

[g l-1]

Volumetric

productivity

[mg l-1 d-1]

Reference

Fed-Batch 5 × 107 8 430 Yang WC et al. 2016. J

Biotechnol 217:1-11

Concentrated

Fed-Batch

18 × 107 25 790 Yang WC et al. 2016. J

Biotechnol 217:1-11

XD® Process 15 × 107 27 1800 Douwenga R. 2013. Bioproc

Intl. Industry Yearbook: 99

Concentrated

Fed-Batch

9 × 107 37 2500 Xu S et al. 2017. Biotechnol

Prog 33: 867-878

130 75 40 20 10

Cell density / (106 ml-1)

Linz F. 2011. DSM

Impact of Continuously Perfused High Density CulturesIso-Volumetric Productivity Lines in Efficiency / Intensity Space


Xu S et al. 2017. Biotechnol Prog 33: 867-878

Via

ble

cell

concentr

atio

n/ (

(10

6m

l-1)

Volu

metr

icpro

ductivity

/ (

(g l

-1d

-1)


Eff

icie

ncy

IntensityRyll T. 2017. 25th ESACT Meeting, Lausanne

0,0

10,0

20,0

30,0

40,0

50,0

60,0

0 10 20 30 40 50 60 70 80 90 100 110 120

5 g l-1 d-1210.50.1 3 4

Cell

sp

ec

ific

pro

du

cti

vit

y /

(p

gd

-1)

Average cell mass per run day / (106 d-1)

Perfusion with

basal medium

Perfusion with basal

medium + feed

Supply of an Antibody Market -

Economy Determines the Process


● Case study to supply a large market (e.g. Alzheimer - a worse case)

o Potential patients: >10 million next 30 years.

o 50 % market penetration: 5 million patients

o Yearly demand per patient: 24 g

o 5 million patients: 120 000 kg

o 2500 kg with 20 000-l bioreactor per year

o 10 – 50× 20-k bioreactors required

Not a problem even with todays technology

One Step Further


Cricetulus griseus

† 1957

2n = 22 Chromosomes

CHO cell size: 15×15 µm

Max. cell concentration 8×108 ml-1

Doubling time: 20 h

Culture temperature: 37 °C

Will be born

2n = 36 Chromosomes

Cell size: 15×5 µm

Max. cell concentration: 5×109 ml-1

Doubling time: 6 h

Culture temperature: 26 °C

Leishmania tarentolae

Leishmaniasis Hosts


Man Homo sapiens

Kala Azar from Leishmania donovaniWhite-Spotted Wall Gecko Tarentula annularis

Leihmaniasis from Leishmania tarentulae

Phlebotomus

Carries promastigotes.

Poikilothermic insects

have an average

temperature of 26 °C.

Comparison of N-Glycan Patterns


CHO

● Complex type N-glycans with α-

linked galactose and fucose

residues.

● Very homogeneous.

● Only biantennary asialo N-glycans.

● In vitro sialylation possible.

GlcNAc Man Gal SA Fuc

Leishmania

Next Challenges Just Around the Corner


● Drug Safety and Potency

o Quality control

o ADC

● Processes for Gene & Cell Therapeutics

o Viral vectors

o Cells as products

● Artificial Cells

o Cell specific productivity

o Stable cell lines for continuous processing

● Process Intensification

o Highly concentrated media and feeds

o Harvest process

Documents

Generation Change in Biopharmaceuticals Production From ... · Employment Number (change from the prior year) 365 (+1.7%) ... • Oxidation • Freeze-thaw ... GBF’s 100 l Pilot