baro fold pep talk 2010

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

2

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

4

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)

5

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

7

High Pressure Refolding of mGHAggregates

8

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

9

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

10

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

12

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.

17

Thank Youwww.barofold.com

mseefeldt@barofold.com