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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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
2 Feb 11th, 2016
Boehringer-Ingelheim RCV Biopharma Process Science Austria
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
3 Feb 11th, 2016
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
4 Feb 11th, 2016
Source: N. Ferrer-Miralles et al. (2009), Microbial Cell Factories
Classification of Biopharmaceuticals in Accordance to their Production System
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
5 Feb 11th, 2016
Overall Costs of Production for Biopharmaceuticals
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
6 Feb 11th, 2016
Biosimilars
Individual Medicine
Limited access to available treatment options
Top Challenges in Biopharmaceutical Production
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
7 Feb 11th, 2016
Reduce Costs of Goods
Fasten Time to Market
Increase Flexibility
Top Challenges in Biopharmaceutical Production
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
8 Feb 11th, 2016
Change of perspective from
Screening for
API /APC
Screening for High Titer
Production System
DSP Development
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
9 Feb 11th, 2016
Change of Perspective to
Screening for
API /APC
Screening for High Titer
Production System
DSP Development
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
10 Feb 11th, 2016
Reduce Costs of Goods
Fasten Time to Market
Increase Flexibility
Change of Perspective to
Screening for
API /APC
Screening for High Titer
Production System
DSP Development
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
11 Feb 11th, 2016
Vision
Analytics Analytics
Screening for High Titer
Production System
DSP Development
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
12 Feb 11th, 2016
Screening for High Titer Production
System
Upstream & Bioprocess
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
17 Feb 11th, 2016
Essential questions to address 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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
18 Feb 11th, 2016
Can we address essential questions at early stage?
• Cell Disintegration
• IB Recovery & Processing
• IB Solubilization
• Refolding /Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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)
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
21 Feb 11th, 2016
Can we address essential questions at early stage?
• Cell Disintegration
• IB Recovery & Processing
• IB Solubilization
• Refolding /Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
23 Feb 11th, 2016
IB Recovery & Processing
Micro Scale Bench Scale Wash 1 Wash 2 IBs Wash 1 Wash 2 IBs
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
25 Feb 11th, 2016
Can we address essential questions at early stage?
• Cell Disintegration
• IB Recovery & Processing
• IB Solubilization
• Refolding /Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
26 Feb 11th, 2016
A. Dürauer, S. Mayer, W. Sprinzl, A. Jungbauer, R. Hahn, Biotech. J. 4 (2009), 722 - 729
IB Solubilization
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
27 Feb 11th, 2016
IB Solubilization
DoE with screening of ! 3 pH values ! 3 urea concentrations ! Concentration of added
GuHCl
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
28 Feb 11th, 2016
Can we address essential questions at early stage?
• Cell Disintegration
• IB Recovery & Processing
• IB Solubilization
• Refolding /Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
30 Feb 11th, 2016
Can we address essential questions at early stage?
• Cell Disintegration
• IB Recovery & Processing
• IB Solubilization
• Refolding /Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
34 Feb 11th, 2016
Can we address essential questions at early stage?
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 !
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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%
Refolding & Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
38 Feb 11th, 2016
Summary - Product titer
IB Recovery & Processing
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
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
39 Feb 11th, 2016
Reject turbid samples for following analysis
Refolding & Oxidation Turbidity
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
40 Feb 11th, 2016
Combination of wavelength shift and scattering (RALLS) allows qualitative detection of successful refolding
Determine aggregation
Intrinsic fluorescence Refolding & Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
Refolding & Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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
Refolding & Oxidation
University of Natural Resources and Life Sciences Department of Biotechnology
9th Annual BioInnovation Leaders Summit A. Dürauer
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