Yanyu Peng, Liz Schanuel, Stephen Eisenberg, Misha Plam and Michael Stowell

AmideBio, LLC, 331 South 104th Street Louisville CO 80027, USA

BIOPURE PROCESS. AmideBio has developed a low cost

peptide production platform combining recombinant and

chemical methods for rapid SAR of complex or difficult to

manufacture peptides1. The process implements a library of

expression vectors optimized for bacterial or yeast expression

combined with an on column chemical cleavage process which

provides a highly orthogonal platform enabling the rapid high

purity production of a variety of peptides and proteins for drug

discovery. Here we describe the BioPure method and its

application to a variety of peptides from the amyloid peptide

Ab42 to the Kv1.3 specific channel blocker from Hadrurus

gertschi2.

B B I (S o y b e a n ) In h ib it io n A s s a y

3 U /m l try p s in , 0 .3 m M s u b s tra te in c u b a te 2 5o

C 3 h rs

-1 0 -9 -8 -7 -6 -5

0 .0

0 .2

0 .4

0 .6

0 .8

A m id e B io B B I_ 0 1

A m id e B io B B I_ 0 3

L o g [G ra m ]

Ab

s 4

05

nm

IC 5 0

1 1 .7 n g

IC 5 0

8 7 n g

S ig m a B B I

APPLICATION TO SINGLE CHAIN INSULIN. AmideBio has

implemented the BioPure process for the discovery of novel single

chain insulins3 (SCIs) suitable for the pump and patch market where

high concentration and long-term stability at elevated temperatures

are required. We have produced over different 75 analogues from a

library of more than 200 designed SCIs and have examined their

physical and biological properties.

APPLICATION TO GLUCAGON. AmideBio has implemented the

BioPure process for the discovery of solution stable glucagon

suitable for the treatment of hypoglycemia and for the patch and

pump market. Glucagon is inherently unstable in solution and

currently is only available in a lyophilized powder that must be

reconstituted prior to use in the case of hypoglycemia. Using a

structure based design approach we created a library of more than

100 potential candidates for testing. Using the Bio-Pure process we

manufactured 35 candidates for testing. Because solution stability it

the most critical aspect of this program we tested all analogues for

long term stability.

APPLICATION TO BOWMAN-BIRK INHIBITORS. Bowman-Birk

inhibitors are bi-functional inhibitors of both trypsin and

chymotrypsin. They have been shown to have potential therapeutic

application in cancer and a number of rare diseases4.

APPLICATION TO TOXIN THERAPEUTICS. A large number of

toxin molecules have been discovered with a diverse set of activities

and potential therapeutic and non-therapeutic applications including,

multiple sclerosis, psoriasis, rheumatoid arthritis, myasthenia gravis,

chronic pain, tumor diagnostics as well as pesticides. Because of

the large diversity of such toxins the ability to quickly and efficiently

produce various forms for screening will be important for realization

of any future therapeutic potential. AmideBio has implemented the

BioPure process to produce a variety of toxins with potential use in

autoimmune diseases.

1. The BioPure Process is covered under US Patent 8,796,431.

2. Chen et al. J. Biol Chem. 2012 Apr 20;287(17):13813-21.

3. The SCI molecules are covered under US Patent 9,006,176.

4. Grant et al. Multiple Sclerosis 2006; 12: 688697

5. Ming and Hellekant. "FEBS letters 355.1 (1994): 106-108.

www.amidebio.com

APPLICATION TO BRAZZEIN. Brazzein is a naturally occurring

peptide produced by the oublie berry (pentadiplandra brazzeana)

that is >2000 times sweeter than sugar on a mass basis. Brazzein

is a potential product for the $6B low calorie sweetener market.

Brazzein is all natural and heat stable with a “true” sugar taste that

is lacking in current sweeteners such as Stevia, Aspartame and

Sucralose5. AmideBio applied the BioPure process to brazzein to

determine the economic feasibility of manufacturing a food product.

TIME %A %B Flow

0.00 92 8 1

2.00 92 8 1

3.10 85 15 1

4.00 85 15 1

60.00 25 75 1

60.10 25 75 1

66.00 10 90 1

66.10 10 90 1

70.00 92 8 1

75.00 92 8 1

Ab42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

99.1% Ab42

Analysis Column: Ascentis® Express ES-Cyano, 2.7 Micron HPLC Column

Temp: 60OC, Buffer A: H2O + 0.05% TFA, Buffer B: ACN + 0.05% TFA

CONCLUSIONS. AmideBio has applied the BioPure process to a

wide range of difficult to manufacture peptides with excellent

success. The ability to rapidly manufacture high-purity peptides for a

variety of therapeutic and non-therapeutic applications facilitates

rapid SAR for a large number of current and future targets.

Rapid SAR of Peptide Therapeutics for

Diabetes and Beyond