BOKU BILS 2016

University of Natural Resources and Life Sciences Department of Biotechnology

9th Annual BioInnovation Leaders Summit A. Dürauer

1 Feb 11th, 2016

HTS platform for process development – can we address essential questions at early stage?

A. Dürauer

February 10th, 2016 9th Annual BioInnovation Leaders Summit Berlin, Germany



2 Feb 11th, 2016

Boehringer-Ingelheim RCV Biopharma Process Science Austria



3 Feb 11th, 2016



4 Feb 11th, 2016

Source: N. Ferrer-Miralles et al. (2009), Microbial Cell Factories

Classification of Biopharmaceuticals in Accordance to their Production System



5 Feb 11th, 2016

Overall Costs of Production for Biopharmaceuticals



6 Feb 11th, 2016

Biosimilars

Individual Medicine

Limited access to available treatment options

Top Challenges in Biopharmaceutical Production



7 Feb 11th, 2016

Reduce Costs of Goods

Fasten Time to Market

Increase Flexibility

Top Challenges in Biopharmaceutical Production



8 Feb 11th, 2016

Change of perspective from

Screening for

API /APC

Screening for High Titer

Production System

DSP Development



9 Feb 11th, 2016

Change of Perspective to

Screening for

API /APC


Production System

DSP Development



10 Feb 11th, 2016

Reduce Costs of Goods

Fasten Time to Market

Increase Flexibility

Change of Perspective to

Screening for

API /APC


Production System

DSP Development



11 Feb 11th, 2016

Vision

Analytics Analytics


Production System

DSP Development



12 Feb 11th, 2016

Screening for High Titer Production

System

Upstream & Bioprocess



13 Feb 11th, 2016

Downstream Processing

DSP Development

Centrifugation

Filtration - Ultra / diafiltration

Chromatography

Cell desintegration -  Mechanical -  Chemical -  Enzymatic

Extraction

Precipitation Crystallization Flocculation

IB solubilization Refolding



14 Feb 11th, 2016

Generalized Workflow of DSP Fermentation

Broth

Cell separation

Product intracellular

Cell disintegration

Product as IBs

Solubilization

Refolding/Oxidation

Product soluble

Extraction

Product membrane associated

Extraction

Product extracellular

Capture

Intermediate Purfication

Polishing

Formulation



15 Feb 11th, 2016

Process development chain St

rain

de

velo

pmen

t /

scre

enin

g Sc

reen

ing

culti

vatio

n co

nditi

ons

Ferm

enta

tion

Brot

h

Cell s

epar

atio

n

Prod

uct

intra

cellu

lar

Cell

disin

tegr

atio

n

IB R

ecov

ery &

Pr

oces

sing

IB S

olub

ilizat

ion

Refo

ldin

g /

Oxid

atio

n

Capt

ure

Inte

rmed

iate

Purif

icatio

n

Polis

hing

Screening for Host System or

High Titer

Defined DSP



16 Feb 11th, 2016

Process development chain St

rain

de

velo

pmen

t /

scre

enin

g Sc

reen

ing

culti

vatio

n co

nditi

ons

Ferm

enta

tion

Brot

h

Cell s

epar

atio

n

Prod

uct

intra

cellu

lar

Cell

disin

tegr

atio

n

IB R

ecov

ery &

Pr

oces

sing

IB S

olub

ilizat

ion

Refo

ldin

g /

Oxid

atio

n

Capt

ure

Inte

rmed

iate

Purif

icatio

n

Polis

hing

DSP Development

Selected Bioprocess Conditions



17 Feb 11th, 2016

Essential questions to address St

rain

de

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enin

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reen

ing

culti

vatio

n co

nditi

ons

Ferm

enta

tion

Brot

h

Cell s

epar

atio

n

Prod

uct

intra

cellu

lar

Cell

disin

tegr

atio

n

IB R

ecov

ery &

Pr

oces

sing

IB S

olub

ilizat

ion

Refo

ldin

g /

Oxid

atio

n

Capt

ure

Inte

rmed

iate

Purif

icatio

n

Polis

hing



18 Feb 11th, 2016

Can we address essential questions at early stage?

•  Cell Disintegration

•  IB Recovery & Processing

•  IB Solubilization

•  Refolding /Oxidation



19 Feb 11th, 2016

Cell disintegration

Analytics: -  Total protein release (UV, SDS-PAGE, 2D-DIGE) -  Product release (flourescence, Bradford) -  DNA release (picoGreen) -  Endotoxin release (LAL)



20 Feb 11th, 2016

Product release DNA release

Cell disintegration

Endotoxin release

For process development: Bead mill is preferable to enzymatic digestion

Product release DNA release Endotoxin release

mg

prod

uct /

g B

M



21 Feb 11th, 2016








22 Feb 11th, 2016

Homogenization via bead mill

Transfer of homogenate to new plate

Separation via centrifugation

Discard supernatant

Resuspension ~1:10 in Wash buffer

Separation via centrifugation

Discard supernatant

Transfer of IBs for analysis

Analysis of supernatant (DNA, OD, SDS-PAGE)

Analysis of supernatant /pellet (DNA, OD, SDS-PAGE)

IB Recovery & Processing

X time



23 Feb 11th, 2016


Micro Scale Bench Scale Wash 1 Wash 2 IBs Wash 1 Wash 2 IBs



24 Feb 11th, 2016

Wash 1

Wash 1

Wash 1

Wash 2

Wash 2

Wash 2

Wash 3

Wash 3

Wash 3

Homogenate IBs

Homogenate IBs

Homogenate IBs

Overall Protein Overall Protein

DNA content DNA content

Product

IB recovery & Processing

Product

?

Micro Scale Bench Scale



25 Feb 11th, 2016








26 Feb 11th, 2016

A. Dürauer, S. Mayer, W. Sprinzl, A. Jungbauer, R. Hahn, Biotech. J. 4 (2009), 722 - 729

IB Solubilization



27 Feb 11th, 2016

IB Solubilization

DoE with screening of !  3 pH values !  3 urea concentrations !  Concentration of added

GuHCl



28 Feb 11th, 2016








29 Feb 11th, 2016

Indication of aggregation / precipitation = unfavored conditions

Determine wavelength shift and scattering = Indication of right formation or aggregation

Dilution of solubilized IBs into refolding buffer

Turbidity measurement

Intrinsic Flourescence Emission Scan

Selective capture for refolded protein

cGE analysis of eluted protein

Refolding & Oxidation



30 Feb 11th, 2016








31 Feb 11th, 2016

Output St

rain

de

velo

pmen

t /

scre

enin

g Sc

reen

ing

culti

vatio

n co

nditi

ons

Ferm

enta

tion

Brot

h

Cell s

epar

atio

n

Prod

uct

intra

cellu

lar

Cell

disin

tegr

atio

n

IB R

ecov

ery &

Pr

oces

sing

IB S

olub

ilizat

ion

Refo

ldin

g /

Oxid

atio

n

Capt

ure

Inte

rmed

iate

Purif

icatio

n

Polis

hing

Screening for Host System or

High Titer

Defined DSP

Parallel screening of 32 fermentations Duration: 7 days



32 Feb 11th, 2016

Output St

rain

de

velo

pmen

t /

scre

enin

g Sc

reen

ing

culti

vatio

n co

nditi

ons

Ferm

enta

tion

Brot

h

Cell s

epar

atio

n

Prod

uct

intra

cellu

lar

Cell

disin

tegr

atio

n

IB R

ecov

ery &

Pr

oces

sing

IB S

olub

ilizat

ion

Refo

ldin

g /

Oxid

atio

n

Capt

ure

Inte

rmed

iate

Purif

icatio

n

Polis

hing

DSP Development

Selected Bioprocess Conditions

Parallel screening 24 x solubilization, 96 x refolding and 24 x capture conditions Duration: 5 days



33 Feb 11th, 2016

Benefits

Protein [mg] Time

Refolding screening

Äkta

Janus BioTx

Tecan

Bench 96

4

60 d

21 d

4 d

!  Time and material consumption significantly reduced !  Broader knowledge of process



34 Feb 11th, 2016


Stra

in

deve

lopm

ent /

sc

reen

ing

Scre

enin

g cu

ltiva

tion

cond

ition

s

Ferm

enta

tion

Brot

h

Cell s

epar

atio

n

Prod

uct

intra

cellu

lar

Cell

disin

tegr

atio

n

IB R

ecov

ery &

Pr

oces

sing

IB p

repa

ratio

n /

Solu

biliz

atio

n

Refo

ldin

g /

Oxid

atio

n

Capt

ure

Inte

rmed

iate

Purif

icatio

n

Polis

hing

Yes, we can !



35 Feb 11th, 2016 10.02.2016 35

Acknowledgements

Lars Demmel Nicole Weiner

Helga Zambiasi Marian Koglbauer

Matthias Berkemeyer Cécile Brocard Georg Klima

Boehringer-Ingelheim RCV Biopharma Process Science Austria

BOKU Vienna Department of Biotechnology

Cornelia Walther Martin Kellner



36 Feb 11th, 2016

IB solubilization

D

H L

F

d h

D

H L

F

d h

lF h

d‘

d

Scale Up Factor >500



37 Feb 11th, 2016

Selected refolding conditions

1 (DoE 1) 2 (DoE 2) 3 (DoE 3) 4 (DoE 4) 4 M UREA 0.1 M Arginine 0.1 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cysteine pH9.3

3 M UREA 0.5 M Arginine 0.4 M Tris 18 mM CHAPS 2 mM Cysteine 2 mM cysteine pH 7.8

3.5 M UREA 0.1 M Arginine 0.5 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cysteine pH 8.7

4 M UREA 0.05 M Tris 2 mM Cysteine 2 mM cystine pH 9.5

DoE 4 buffer chosen because of !  Least CoG !  No high dilution of refold required to reach target conductivity before CIEX !  No additives which need to be removed later in DSP

1 (DoE 1) 2 (DoE 2) 3 (DoE 3) 4 (DoE 4) 4 M UREA 0.1 M Arginine 0.1 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cystine pH9.3

3 M UREA 0.5 M Arginine 0.4 M Tris 18 mM CHAPS 2 mM Cysteine 2 mM cystine pH 7.8

3.5 M UREA 0.1 M Arginine 0.5 M Tris 0.18 mM Tween 20 2 mM Cysteine 2 mM cystine pH 8.7

4 M UREA 0.05 M Tris 2 mM Cysteine 2 mM cystine pH 9.5

Yield in bench scale ~100% ~30% ~90% ~85%




38 Feb 11th, 2016

Summary - Product titer


Small Scale Processing is Reproducible

g IB/g biomass mg Product/g IB wet

Small Scale 1

Small Scale 2

Bench Scale

Small Scale 1

Small Scale 2

Bench Scale

Factor Bench/Small

V554 0.408 0.33 0.136 17.6 17 126 7 V556 0.365 0.359 0.111 21.9 18.9 91.8 5 V561 0.259 0.279 0.124 19 21.3 81.4 4 V566 0.177 0.183 0.03 4.4 1.1 26.7 26

g IB/g biomass mg Product/g IB dry Small

Scale 1 Small

Scale 2 Bench Small Scale

1 Small Scale

2 Bench Factor

Bench/Small

V554 0.408 0.33 0.136 - 115.4 240.0 2 V556 0.365 0.359 0.111 - 199.9 266.2 1.4 V561 0.259 0.279 0.124 - 151.5 332.5 2 V566 0.177 0.183 0.03 - 7.65 92.4 11



39 Feb 11th, 2016

Reject turbid samples for following analysis

Refolding & Oxidation Turbidity



40 Feb 11th, 2016

Combination of wavelength shift and scattering (RALLS) allows qualitative detection of successful refolding

Determine aggregation

Intrinsic fluorescence Refolding & Oxidation



41 Feb 11th, 2016

Confirmation of capture to affinity resin So

lubiliz

ed IB

Stan

dard

Stan

dard

Stan

dard

Re

foldin

g

FT

Elua

te

Refol

ding

FT

Elua

te

Refol

ding

FT

Elua

te

Refol

ding

FT

Elua

te

Refol

ding

FT

Elua

te

Refolded protein

90% of refolded protein binds to affinity resin, 60% of bound protein is eluted and can be used for cGE measurements




42 Feb 11th, 2016

!  Fully automated screening of 96 refolding conditions !  Automated preparation of refolding buffers from stocks !  Variation of pH, salt concentration, stabilizing agents, redox system

and additives !  DoE based !  Less than 4 mg of solubilized protein required per screening

!  Result: Refolding yield & quality




43 Feb 11th, 2016

IB Solubilization

!  Fully automated screening of 24 solubilization conditions !  Automated preparation of solubilization buffers from stocks !  Variation of pH, chaotrope concentration, reducing agents, additives !  DoE based !  Less than 4 mg of Ibs required per screening

!  Result: Solubilization yield & kinetics

Presentations & Public Speaking

BOKU BILS 2016