Platzhalter Bild

“Current Best Practise

in Biomanufacturing

and the

Critical Role of Innovation”

International Vellore Symposium “Bioprocess Industry-Academia

Interaction”

July 2011

Dr. Uwe Gottschalk, VP Purification Technologies, Sartorius Stedim

Biotech

What

are

the

hot Topics?

7th Annual Survey of Biopharmaceutical Manufacturing. Eric S. Langer, BioPlan

Associates Inc.

Existing Facilities to Meet Current Challenges

Data adapted from: F. Wurm

Production of recombinant Protein Therapeuticsin Cultivated Mammalian Cells. Nature Biotechnology 22, 1-6 (2004)

The

USDP/DSP Interface in a World of High Titers

Jim Davis, Lonza

Economics of Monoclonal Antibody Production: The relationship between upstream titer and downstream costs; IBC San Diego March 2008

DSP is

Mass

not

Volume

driven

Current

Best Practise

in DSP

Increasing biomass and contaminant levels

Protein A pool volumes and step cost

DNA & HCP levels post Capturing

Polishing load volumes and conductivity

Pathogen clearance as a moving target

High Titer

Implications:

Chromatography Technologies for DSP

Polishing

(Membranes)

• Highly porous structure

• Pore size: 3 –

5μm

• Convective Flow

• Minimal buffer useCapturing/IP

(Resins)

• Bead size distribution: 15 -160 μm

• Average pore size: 15 -

40 nm

• Diffusion limited flow

• High capacity

Capture Costs: Why bother?

Jim Davis, Lonza

Economics of Monoclonal Antibody Production: The relationship between upstream titer and downstream costs; IBC San Diego March 2008

Emerging capture

technologies expected to have limited market potential in upcoming years

Source: Sartorius

MaturityMaturity RiskRiskDescriptionDescription

•

CIM, BIA Separations, methacrylate

based monoliths•

Similar to membrane adsorbers•

Purification of large biomolecules

(viruses, plasmid DNA, conjugates), good resolution

•

Upfront/DSM work on single use technology for MABs•

Already used in depletion of valuable biomolecules

from particle containing feedstreams

at large scale (milk, juice, etc.)

•

Membrane adsorber

technology•

Own development and IP, depletion of valuable biomolecules

from particle containing feedstreams

•

Currently low priority, combines cell harvest and capture chromatography step

•

instAction, Prometic

(mimetic ligands), BAC, GEHC pipeline, Repligen

Protein A

•

Used at large scale in plasma fractionation (precipitation)

and APIs (crystallization)•

Not developed for mABs

•

Polybatics•

Disruptive technology•

Single use alternative to Protein A•

Platform character

TechnologyTechnology

Monoliths

Expanded bed ad-

sorption

Direct Capture MA

Ligands

Precipitation/Crystallization/

Extractionn

Affinity

nano-

particles

RelevanceRelevance

•

No significant market in upcoming years expected –

Potential for Niches (e.g. Vaccines, DNA)

•

No immediate commercialization possible

•

Potential for disruptive technology

Capt

ure

and

inte

rmed

iate

pur

ific

atio

n

Already used In development Start of development Low risk Moderate risk High risk

• Product

precipitation

batch/continuous

• Impurity

precipitation

(followed

by

non-Protein

A process)

• Alternative Capturing

(Protein A Mimetics, Mixed Mode, CEX)

Issues: Selectivity, Scale

up, Reproducibility, Comparability

Alternatives to Protein A Capture

D. Low BioManufacturing

Paris 2007

Protein A pool volumes and step cost

addresses:

• Simulated

Moving

Bed

(SMB) and related:

» Tarpon

(„single

use

flow

path“)

» Novasep

» Chromacon

» Chromatan

» ...______________________________________________________________________

• Expanded

Bed

Chromatography

» DSM/Upfront

(„single

use

flow

path“)

Issues: Complexity, Scale

up, Reproducibility, Comparability

Alternative Protein A Chromatography

Formats:Goal: Intensified

Use/Volume

Reduction

Limitation: Oleosin yields < 1kg/ha

2000: Oleosin

Platform 2005: TMV Nanoparticles

Immunoabsorbent

nanoparticles

based on a tobacco mosaic virus displaying protein AS. Werner et al. PNAS 103, 17678 -

17683

Polyester Granule100-300 nm

Grage, K. and Rehm, B.H.A. (2008) Bioconj. Chemistry, 19(1):254-62.

Polyester Synthase

2010: Bio Polyester Platform

Alternative Protein A Formats:Goal: Low Cost

–

Real Single Use

CEX

TFF

Q Membrane2 g/ml

Dilution

HCP < 1000 ng/mg

VF

Mix Mode

Fig 7b. TFF based process

Dilution

HCP < 1000 ng/mg

CEX HCP < 10ng/mg

Contaminant precipitation

Q Membrane20 g/ml

Fig 7a. precipitation based process

VF

HCP BDL

Dilution

CEX

TFF

Q Membrane2 g/ml

Dilution

HCP < 1000 ng/mg

VF

Mix Mode

Fig 7b. TFF based process

Dilution

HCP < 1000 ng/mg

CEX

TFF

Q Membrane2 g/ml

Dilution

HCP < 1000 ng/mg

VF

Mix Mode

Fig 7b. TFF based process

Dilution

HCP < 1000 ng/mg

CEX

TFF

Q Membrane2 g/ml

Dilution

HCP < 1000 ng/mg

VFVF

Mix Mode

Fig 7b. TFF based process

Dilution

HCP < 1000 ng/mg

CEX HCP < 10ng/mg

Contaminant precipitation

Q Membrane20 g/ml

Fig 7a. precipitation based process

VF

HCP BDL

Dilution

CEX HCP < 10ng/mg

Contaminant precipitation

Q Membrane20 g/ml

Fig 7a. precipitation based process

VFVF

HCP BDL

Dilution

Two Birds –

one Stone: Contaminant Precipitation at Pfizer and Medarex

Protein A pool volumes and step cost

DNA & HCP levels post Capturing

addresses:

Precipitation of Process-Derived Impurities in Non-Protein APurification Schemes for MAb; J. Wang et al. BioPharm

Intl. 10/2009, 2-9

Process Scale Precipitation of Impurities in Mammalian Cell Culture Broth; J. Glynn

et al. In: Gottschalk U (ed) Process-scale Purification of Antibodies. Wiley, NY.

Chromatography Technologies for DSP

Polishing

(Membranes)

• Highly porous structure

• Pore size: 3 –

5μm

• Convective Flow

• Minimal buffer useCapturing/IP

(Resins)

• Bead size distribution: 15 -160 μm

• Average pore size: 15 -

40 nm

• Diffusion limited flow

• High capacity

Pete Gagnon

2007

Convective Media

Q,S

Capture

Polishing

Low salt

High salt

Q,S

STIC

Polishing in flowthrough:

viruses, DNA, Host cell proteins, endotoxins, aggregates

Purification: large proteins (Factor VIII), viruses (vaccines), phages...

HIC

Selection

Guide Convective

Media

March 7, 2011 Page 9

Sartobind STIC®

-

Next Generation of Membrane Adsorbers Shares same cellulose base membrane as Q: >3 μm pore size

0.5 μmHere is the binding capacity

Binding capacity is distributed more evenly

Sartobind Q

Grafted

Quaternary ammonium

Sartobind STIC

Direct derivatisation

+ ligand density

+ pore accessibility

Primary amine (Sartobind STIC PA)

I. Tatárova, I., R. Fáber, R. Denoyel, M. Polakovic, J. Chromatography A 1216 (2009) 941

Host cell protein removal from up 10 kg mAb

per L (pH 8, 500 ppm HCP load, 10 MV/min

Application Note Sartorius-Stedim Biotech: 85032-540-18, 05/2011

Sartobind

STIC

Sartobind Q Sartobind STIC

Binding Capacity

(g/m²)

BSA 2 mS/cm @ 0 mM NaCl 9 19

BSA 20 mS/cm @ 200 mM NaCl 1 10

DNA 7 mS/cm @ 50 mM NaCl 2 6

Removal of ΦX174 (LRV)

LRV 1.4 mS/cm @ 0 mM NaCl 3,7 5,1

LRV 6.7 mS/cm @ 50 mM NaCl 0,1 5,1

LRV 16.8 mS/cm @ 150 mM NaCl 0,1 4,8

Removal of MVM (LRV) at Wuxi ApptecTrial 1 16.8 mS/cm @ 150 mM

NaCl 2.10 3,82

Trial 2 16.8 mS/cm @ 150 mM

NaCl 1.81 >4,96

Source: 2nd Annual Survey of the Bioprocessing Market for Single-Use SolutionsAspen Brook Consulting, 2010

Current Challenge in UF

•

Process efficiency should be HIGH

•

Membrane cleaning should be EASY

•

Final Mab

concentration may reach 20%

•

Maximum system pressure is LIMITED

E ECO

Pumping Requirements 8 L/m2/min 2 L/m2/min

Viscosity High Low

MAb

Concentration >15% <10%

Flux vs. Concentration for MabSartorius ECO & E cassetteCrossflow Rate: 360 L/m2-Hr

0

10

20

30

40

50

60

70

80

90

100

10 100 1000Concentration (g/l)

Per

mea

te F

Lux

(LM

H)

ECO

E

Select the Right Products

Yes

we

can!

Przybycien, Pujar, Steele: Current

Opinion

in Biotechnology

2004, 15 469-478

Upcoming

Alternatives

Disruptive Technologies from Inception to Maturation

Konstantinov, K. Towards fully continuous bioprocessing: What can we learn from Pharma? Cell Culture Engineering XII, Banff, Canada (2010)

Trend in New Drug Production Scales

Flexible and Disposable

New Facilities

to meet

Future Challenges

Uwe.Gottschalk@sartorius- stedim.com

Thank

you!