High hydrostatic pressure between 500-4000 bar can disaggregate and refold proteins while maintaining native structure. This "PreEMT" technology enables refolding of inclusion bodies and aggregates from bacterial and mammalian expression into properly folded monomers. Studies show refolded interferon-beta and growth hormone had reduced aggregates and immunogenicity compared to commercially available versions. The technology is scalable and can improve safety and yields of protein therapeutics.

1. High Pressure Refolding of Protein
Aggregates: Applications for Reduced
Immunogenicity and Protein Production
Matthew Seefeldt Ph.D.
PepTalk 2010
www.barofold.com

2. PreEMT™ Technology
An “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
2

3. Potential Manufacturing Process Flows using PreEMT
Technology
Bacterial Expression Mammalian Expression
Centrifugation Conditioned Medium
Cell Column Chromatography
Lysis/Homogenization Capture Step
Centrifugation Viral Inactivation
PreEMT
PreEMT Refolding Column Chromatography
from Inclusion Bodies (1-2 Columns)
Column Chromatography
Viral Filtration
(1-2 Columns)
UF/DF BDS UF/DF BDS
Fill/finish DP Fill/finish DP

4. Acknowledgements
Amber Haynes Fradkin
John F. Carpenter
Ted W. Randolph
University of Colorado Center for Pharmaceutical
Biotechnology
4

5. 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)
5

6. Possible Basis for Aggregate-
Induced Immunogenicity
• Patterned Presentation of Microbial Structures
• Hapten Hypothesis (Dintzen et al.)
– >100kDa
– Valency >10
– 5-10nm spacing
(Rosenberg, FDA 2006) 6

7. 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
7

8. High Pressure Refolding of mGH
Aggregates
Insoluble aggregate Error
Monomer (%)
(%) (%)*
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
8

9. Particle Content by
MicroFlow Imaging
109
Monomer
108 High Pressure Treated
Number/ml/bin (m-4)
107
106 Initial
105
104
103
102 Freeze-Thaw Agg.
101
109
100
10 20 30 40 50 108
Number/ml/bin (m-4)
Mean particle diameter (micron) 107
106
105
Agitated Agg. 104
109
103
108
102
Number/ml/bin (m-4)
107
101
106
100
105 10 20 30 40 50
104 Mean particle diameter (micron)
103
102
101
100
10 20 30 40 50
Mean particle diameter (micron)
9

10. Reduced Immunogenicity
Monomer
10

11. 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
11

12. Murine IFN-beta
2.3 g/day IP for 15 days
>99% monomer by SEC Sera collected on day 21
INF-
>99% monomer by SEC
after high pressure
treatment (PreEMT™)
of insoluble aggregates.
Aggregated by vortexing for
5 min. 53% insoluble and 7% soluble
aggregates. 40% monomer by SEC
Seefeldt et al., 2009 12

13. 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.
13

14. Reduced Immunogenicity
of BaroFeronTM
Zeng, D, Recovery of Biomolecules XIII Meeting 14

15. Reduced Immunogenicity of
Commercial rhGH in Naïve Mice
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 15

16. PreEMT™ Technology
Easily scalable using existing technology
GMP PreEMT 10 L NC Hyperbaric
BaroFold PreEMT+
(3’ x 4’ x 4.5’) Wave 6000 / 135
Research to Production Scale
16

17. Advantages of PreEMT Technology
Protein 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.
17

18. Thank You
www.barofold.com
mseefeldt@barofold.com