baro fold pep talk 2010
TRANSCRIPT
High Pressure Refolding of Protein
Aggregates: Applications for Reduced
Immunogenicity and Protein Production
Matthew Seefeldt Ph.D.
PepTalk 2010
www.barofold.com
PreEMT™ TechnologyAn “Elegant Method”
• High hydrostatic pressure (500-4000 bar) disaggregates and properly
refolds proteins at conditions that maintain native protein structure
• Enables proprietary products with enhanced activity and safety
• Operated at scale in GMP environment
• Broadly applicable
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Potential Manufacturing Process Flows using PreEMT Technology
Centrifugation
Cell Lysis/Homogenization
Centrifugation
PreEMT Refolding from Inclusion Bodies
Column Chromatography (1-2 Columns)
UF/DF BDS
Fill/finish DP
Conditioned Medium
Column Chromatography Capture Step
Viral Inactivation
Column Chromatography (1-2 Columns)
Viral Filtration
UF/DF BDS
Fill/finish DP
Bacterial Expression Mammalian Expression
PreEMT
Acknowledgements
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Amber Haynes Fradkin
John F. Carpenter
Ted W. Randolph
University of Colorado Center for Pharmaceutical Biotechnology
Aggregates and Biologic Immunogenicity
• Previous studies demonstrates that aggregates in biologics can lead to immunogenicity against native self-proteins and the native therapeutic
– h Growth Hormone (Moore et al., 1980)
– rh Insulin (Ratner et. al., 1990)
– rh IFN-beta-1b (Runkel et. al., 1998)
– rh EPO (Gershon and Casadevall et al., 2002)
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Possible Basis for Aggregate-Induced Immunogenicity
• Patterned Presentation of Microbial Structures
• Hapten Hypothesis (Dintzen et al.)
– >100kDa
– Valency >10
– 5-10nm spacing
6(Rosenberg, FDA 2006)
Hypothesis
• High pressure treatment can decrease the presence of aggregates in final formulations, decreasing the immunogenicity of the final product.
• Experiment
– Recombinant murine growth hormone (rmGH)(ECP, LPS Free)
• >99% Monomeric (SEC-HPLC)
• Aggregated via Agitation, Freeze-Thaw
• High Pressure Refolded (2000 bar/4hr.)
– 2ug dose X 5 days/week X 3 weeks
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High Pressure Refolding of mGHAggregates
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Monomer (%)Insoluble aggregate
(%)
Error
(%)*
Monomer 100 0 ±1.8
HP Monomer 100 0 ±1.7
Agitated 46 54 ±1.5
HP Agitated 100 0 ±1.5
FT 76 24 ±1.2
HP FT 100 0 ±1.3
UV 90 10 ±1.3
Glass 0 100 ±0.5
Alhydrogel 0 100 ±0.3
Particle Content by MicroFlow Imaging
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Mean particle diameter (micron)
10 20 30 40 50
Num
ber/
ml/
bin
(m
-4)
100
101
102
103
104
105
106
107
108
109
Mean particle diameter (micron)
10 20 30 40 50
Num
ber/
ml/
bin
(m-4
)
100
101
102
103
104
105
106
107
108
109
Mean particle diameter (micron)
10 20 30 40 50N
um
ber/
ml/
bin
(m
-4)
100
101
102
103
104
105
106
107
108
109
High Pressure Treated
Initial
Monomer
Agitated Agg.
Freeze-Thaw Agg.
Reduced Immunogenicity
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Monomer
Conclusions - mGH
• High pressure treatment decreases aggregate content
– SEC-HPLC (Yields ~100%)
– Decreases particle sizes
• Monomer - <10 m
• Agitated Agg. - <20 m
• Freeze-Thaw Agg. - <30 m
• Immunogenicity was eliminated in High Pressure Monomer sample
• No correlation with immunogenicity and particle size in other samples
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Murine IFN-beta
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Aggregated by vortexing for
5 min. 53% insoluble and 7% soluble
aggregates. 40% monomer by SEC
>99% monomer by SEC
INF-
>99% monomer by SEC
after high pressure
treatment (PreEMT™)
of insoluble aggregates.
2.3 g/day IP for 15 days
Sera collected on day 21
Seefeldt et al., 2009
Applications to Human Therapeutics
• IFN-beta
– BetaseronTM contains ~40% agg. (Runkel et al.,1998)
– BaroFold implemented PreEMTTM to produce an >99% aggregate-free, HSA-free, intereferon-beta-1b.
• rhGH
– PreEMTTM Refolding studies were conducted on two commercial rhGHs.
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Reduced Immunogenicityof BaroFeronTM
14Zeng, D, Recovery of Biomolecules XIII Meeting
Reduced Immunogenicity of Commercial rhGH in Naïve Mice
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Group average anti-hGH IgG concentrations
determined from 4th week bleeds (peak antibody
production) for naive adult animal model. Error bars
shown are standard error.
Fradkin et al. 2008
PreEMT™ TechnologyEasily scalable using existing technology
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Research to Production Scale
BaroFold PreEMT+ GMP PreEMT 10 L(3’ x 4’ x 4.5’)
NC HyperbaricWave 6000 / 135
Advantages of PreEMT TechnologyProtein Therapeutics
• Improved product safety profiles
– Reduced soluble aggregates
– Lowered immunogenicity risks (breaking tolerance)
• Enabling
• Lower cost of goods
– Higher yields
– Faster refolding reduces floor time
– Can replace dilution tanks; reduced scale
• We are seeking collaborations to apply PreEMT technology on your proteins.
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