Enteris BioPharma
• Privately held, New Jersey based biotech company
• Owned by Victory Park Capital, a Chicago based investment firm
• Clinically validated oral formulation technology
• for peptides and challenging small molecules
• Extensive scientific know-how and R&D experience
• Proven GMP tablet manufacturing capabilities
2
Enteris BioPharma
• Enteris has effectively addressed both permeability and solubility
challenges with a simple, elegant and scalable solution
• Demonstrated a track record of clinical success across a range of
compounds and therapeutic indications
• Enteris offers robust IP protection, regulatory CMC support and
finished, solid dosage formulations for preclinical and early phase
clinical studies
3
Clinically Validated Oral
Delivery Technology
• Clinically validated oral peptide delivery technology
• Positive Phase 3 oral Calcitonin: Osteoporosis(1)
• Positive Phase 2 oral PTH: Osteoporosis(2)
• Positive Phase 2 oral Calcitonin: Osteopenia(3)
• Positive Phase 1 oral CR845: Neuropathic Pain(4)
• Sponsored preclinical peptide programs
• 15 ongoing or completed formulation programs
• (1) Tarsa Therapeutics, Inc. (JBMR 27, No.8, 2012, 1821-1829) • (2) Unigene Laboratories, Inc. (Bone 53, 2013, 160-166) (Clin Pharm 52, No. 6, 2013) • (3) Tarsa Therapeutics, Inc. (ASBMR, 2012) • (4) Cara Therapeutics, Inc. (data on file)
4
0
5
10
15
20
25
0 1000 2000 3000 4000 5000 6000
Actual Bioavailability Data
Peptides and Small Molecules
Molecular Weight (Da)
Ab
so
lute
Bio
ava
ilab
ility
(%
)
insulin PTH 1-34 calcitonin
proprietary peptide
proprietary peptides
kanamycin
CR-845
proprietary peptide
zanamivir
tigecycline
octreotide
Studies in beagle dogs
Except *Rat Study 5
tobramycin
*GLP-1 analog
Criteria for Selection of Peptides
ENTERIS PEPTIDE ASSESSMENT MATRIX
Molecular Weight (Da)
• Sequence of peptide
• Hydrodynamic radius (Å)
• Cyclic peptide?
Solubility
• Acid
• Water
• Buffers/salts
• FaSSIF/FeSSIF
Net Charge
• Anionic charges
• Cationic charges
• pI
Chemical Stability
• x-S-S-x
Resistant to Proteolysis?
Modified?
Physical Stability
• Aggregation rate
Projected Dose (mg)
Injectable Dose (mg)
Bioavailability (%) if known
• Rat
• Dog
6
Criteria for Selection of Small Molecules
ENTERIS SMALL MOLECULE ASSESSMENT MATRIX
Molecular Weight (Da)
BCS Class, if known
Describe the solid (salt form, hydrate, solvate, etc.
Solubility
• pH/Solubility curve available?
• Water
• Buffers/Solvents/Emulsions
• If insoluble, what is the most likely reason?
(hydrophobicity/crystal surface energy/etc.)
Are other salts or polymorphs available?
Is the API crystalline or amorphous?
• Has polymorph transition been observed
during processing or on stability
Moisture sensitive/hygroscopic
Is the API plastic, elastic or brittle?
Any known excipient incompatibilities?
Primary mechanism(s) of chemical degradation
Caco-2 Permeability
• Apical to Basolateral, Basolateral to Apical?
Identify known transporter interactions, (P-gp,
BCRP, MRP family, PepT1, OATP, etc.)
What is the primary clearance mechanism(s)?
(renal/Biliary/Metabolic) If metabolic, what
enzymes predominate?
Bioavailability of current oral formulation in
development?
Please describe any PO formulation attempted
thus far
7
Contents
• Introduction
• Mechanism
• Peptides
• Small molecules
• BCS Class III
• BCS Class II
• Safety
• Excipient Safety Profile
• LLC Toxicology Studies
• LLC Regulatory and Clinical
• Patents
• Business Development
• Manufacturing
8
Enteric Coat Prevents Tablet
from Opening in Stomach at Low pH
• Acid-stable enteric coating prevents
tablet release in stomach
• Less susceptible to food effects
or dilution with liquids
• API protected from degradation
by acid and pepsin
• Peptides
• Acid-labile small molecules
10
Enteric Coat Dissolves at
Neutral pH in the Small Intestine
• Water-soluble sub-coat acts as a
partition layer between the enteric
coat and the acidic tablet core
• Simultaneous release of API
and excipients
11
pH Modifier, Permeability
Enhancers and API Released
• Organic acid sequestered in coated beads
• Increases stability of tablet formulation
• Compatible with peptides and small
molecules
• Acts as protease inhibitor for peptides
• Calcium chelator
and membrane permeation enhancer
• pH-lowering agent that increases absorptive
flux
• Membrane wetting/charge dispersal agent
12
API Absorbed Across Intestinal
Wall via Paracellular Transport
• Lauroyl-L-carnitine (12-carbon fatty
acid)
• Modulates tight junctions in the intestinal
enterocytes and enhances paracellular
transport
• Acts as a solubilizing agent due to
surfactant properties
• Inhibits P-gp efflux transporters
13
Dog Model Predicts
Bioavailability in Humans
0 1 2 3 4 5
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Linear Regression for Cmax: Y = A + B * X R
2 =0.9801
Pla
sm
a C
ma
x (
pg/m
l)
Dose (mg) 10 100 1000 10000
10
100
1000 y=.995x - 0.7548 R=0.958
R 2 =0.918
Hum
an C
max (
pg/m
l)
Dog Cmax (pg/ml)
Enteris' dog model for oral delivery shows high degree of linearity with respect to dose
offering a wide range of dosing strategies. Comparability of PK results in dog and human
shows that Enteris’ dog model is an appropriate success predictor for human studies
16
Peptide Bioavailability
Formulated vs. Unformulated
Effect of Formulation on the Bioavailability of Various
Peptides in Pre-Clinical Animal Models
Program
Type
No. of
Amino
Acids
Study
Type
Ranges of Unformulated
Bioavailability (%) for 12
Peptides
Ranges of Formulated
Bioavailability* (%) for 12
Peptides
Sponsored 4 – 39
Rat 0.35 – 6.0 10.7 – 26.0
Dog <LOD** – 0.38 0.39 – 22.8
* in each case the percent bioavailability of the formulated peptide was higher than unformulated
** LOD = limit of detection
17
Bioavailability of 9 Amino Acid Peptide
Across Multiple Animal Models
Enteris Enteris Enteris
All D Amino Acid Peptide
18
Absorption of LHRH Analog in Dogs
as a Function of Enteric-Coating
Formulation B
Unformulated C
Formulation D
Formulation Enteric coat
(weight gain)
Tmax
(min)
Bioavailability
(% F)
A L30D-55
(10%) 111 3.0
B L30D-55
(15%) 116 4.6
D L30D-55/
FS30D (12%) 152 7.2
Unformulated L30D-55
(10%) 130 0.1
0
2000
4000
6000
8000
10000
12000
0 100 200 300 400 500
LH
RH
(p
g/m
L)
Time Relative to Tmax (minutes)
Capsule Formulation in Dogs
Formulation A
Formulation B
Formulation D
Unformulated
19
Bioavailability of Cara’s CR845 in
Preclinical & Phase I
0%
5%
10%
15%
20%
13% 13%
16%
Rat Dog Man
20
Phase I Oral CR845 Study
Time (hours)
CR
845
(ng/
mL)
0 4 8 12 16 20 240.1
1
10
100
3 mg
0.5 mg
1 mg
10 mg
N = 8/group
Mean + SEM
CR845 Demonstrated 16% Oral Bioavailability
21
0.00
0.50
1.00
1.50
2.00
2.50
3.00
rsCT Tablet Nasal Spray Placebo
p<0.001* p=0.014* p=ns*
1.5
0.8 0.5
Phase III Oral Calcitonin Study
Me
an
% C
ha
ng
e L
S-B
MD
Phase III Study for Oral sCT:
Primary Endpoint (Change in LS BMD) Achieved
23
Rationale for for BCS Class II, III and IV
Small Molecule Drugs
• Lauroyl-L-carnitine (12-carbon fatty acid)
• Modulates tight junctions in the intestinal enterocytes and enhances
paracellular transport
• Acts as a solubilizing agent due to surfactant properties
• Inhibits efflux transporters (P-gp)
• Citric Acid (Organic acid)
• Calcium chelator and membrane permeation enhancer
• pH-lowering agent that increases absorptive flux
• Membrane wetting/charge dispersal agent
25
• Currently approved only for IV infusion as a last resort antibiotic therapy
• 100mg loading dose, followed by 50mg every 12hrs, duration ranging
from 5 to 14 days
• Must be dosed in the clinic
• BCS Class III
• Very high solubility in water: >295 mg/mL at all pH ranging from 1 – 14
• Very poor permeability: Oral formulations explored to date exhibit a limit
of approx. 5 %F
• Technology is uniquely suited to enable oral formulation with suitable F
• Oral therapy offers out of clinic dosing
• Reduced overall healthcare costs
• Strategy: Initiate dosing by IV titration, then discharge with oral therapy
BCS Class III Molecule
Tigecycline Case Study
27
BCS Class III Molecule
Tigecycline Rat Study
Animals dosed via intraduodenal administration to simulate oral dosing
(0.64 mg/kg IV or 4.8 mg/kg ID)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
IV
ID 100mM CA, 26mM LLC
ID PBS
ID 400mM CA, 26mM LLC
Time (hrs)
Pla
sma
Tig
ecyc
lin
e (m
cg/m
L)
Pla
sm
a C
on
ce
ntr
ati
on
(m
cg
/mL
)
IV ID PBS ID 100mM CA, 26mM LLC ID 400mM CA, 26mM LLC
Cmax (mcg/mL) 1.79 0.10 0.71 1.11
Tmax (hr) 0.08 1.50 0.33 0.28
AUC(0-t) (mcg*hr/mL) 58.20 6.96 49.38 117.63
%F(0-t) -- 1.60 10.85 27.90 28
Summary of Rat Tigecycline Study
• %F for unformulated ID tigecycline: 1.60 to 2.76%
• %F of formulated ID tigecycline increased by 10 to 20 fold depending
on formulation
• 10.85% at 100mM CA / 26mM LLC
• 27.90% at 400mM CA / 26mM LLC
• Achieved high oral bioavailability for tigecycline where other
formulation technologies have failed, or only been marginally effective
29
Tigecycline Solid Dosage Form
Development Enteric Coated Capsule
• BCS class III molecule filled in capsules
• “Formulated”: API, CA, LLC and filler
• “Unformulated”: API and filler only
• Single ascending dose study in beagle dogs
• 4 single dose arms
• 15 mg formulated + unformulated (n=8 dogs each)
• 30 mg formulated (n=5 dogs) + unformulated (n=3 dogs)
• 45 mg formulated (n=5 dogs)
• 5 mg IV bolus (n=3 dogs)
30
BCS Class III Molecule (Tigecycline)
Beagle Dog PK Study
• API was not detected in
any “unformulated” arm 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0
30
60
90
120
150
180
15 mg
30 mg
45 mg
Time (hr)
Pla
sm
a T
igecycli
ne C
on
cen
trati
on
(ng
/mL
)
Pla
sm
a C
on
ce
ntr
ati
on
(n
g/m
L)
31
• Summary
• %F of formulated ID dosing in rat model increased by 10 to 20 fold
• Enabled the development of a solid oral dosage form with desirable level of
bioavailability and commercial viability in dog model
• Achieved high oral bioavailability where other formulation technologies have
failed
BCS Class III Molecule (Tigecycline)
Study Summary
PK Data: Enteric Coated
Capsules in Dogs
Mean
5 mg IV
Mean
15 mg PO
Mean
30 mg PO
Mean
45 mg PO
Cmax (ng/mL) 335.0 75.51 121.13 177.01
Tmax (h) 0.08 1.84 1.58 1.67
AUC(0-t) (ng*hr/mL) 410.86 132.65 366.65 574.32
%Fest -- 12.2 14.87 15.53
F%CV -- 72.0 43.3 67.7
32
Synergy of CA and LLC
Demonstrated on Tigecycline
Mean %F
400mM CA/26mM LLC 21.64
0 CA/26mM LLC 8.86
(m)
33
KANAMYCIN
PROJECT REVIEW
AMY STURMER
1.0 / 31JULY2013
Aminoglycoside (Kanamycin, Tobramycin)
Case Studies
35
Project Overview
• Background • Aminoglycoside bactericidal antibiotics
• Consist of two amino sugars glycosidically linked to deoxystreptamine
• Available in oral, IV, IM, and inhaled
• Indication: • Used to treat wide variety of infections
• Effective against gram negative bacteria
• Mechanism of action: • Interacts with the 30S subunit of prokaryotic ribosomes. Induces substantial
amounts of mistranslation and indirectly inhibits translocation during protein
synthesis
• Contraindications: • Side effects include tinnitus, toxicity to kidneys, and allergic reactions to the drug
• Presence of intestinal obstruction
• Generally not indicated for long-term therapy due to nephrotoxocity and ototoxicity
• Marketed by BMS, Alcon, Novartis etc. • Wide veterinary use for certain indications
36
Mean PK for Kanamycin Following
Oral Administration to Beagle Dogs (±SEM)
Formulation Key Excipients N Cmax
(ng/mL)
Tmax
(min)
AUC(0-t)
(ng*min/mL) %F
1
0 mg CA,
0 mg LLC,
uncoated
6 67 (9) 118 (18) 9167 (983) 2.8 (0.3)
2 500 mg CA, 100
mg LLC, coated 4 428 (88) 101 (13) 46216 (6593) 14.2 (2.0)
3 250 mg CA, 100
mg LLC, coated 3 408 (72) 125 (22) 36970 (10465) 11.4 (3.2)
4 100 mg CA, 100
mg LLC, coated 3 489 (196) 160 (10) 40336 (18038) 12.4 (5.5)
5
50 mg CA,
100 mg LLC,
coated
31 147 (147) 195 (NA) 8573 (8573) 2.6 (2.6)
37
PK of Tobramycin
Unformulated
Capsules
(SEM)
Formulated
Capsules
(SEM)
No of Dogs/No of
Responders 3/8 8/8
Cmax (ng/mL)
3
(1)
314
(46)
Tmax (min)
145
(35)
144
(20)
AUC(0-t)
(ng*min/mL)
270
(140)
25829
(4738)
AUC(0-t)
(ng*min/mL/mg)
27
(14)
2583
(474)
%F
0.15
(0.08)
14.5
(2.7)
41
Pharmacokinetic Parameters
for Kanamycin and Tobramycin
Formulation Key Excipients N Cmax
(ng/mL)
Tmax
(min)
AUC(0-t)
(ng*min/mL) %F
JSV-003-005 10 mg Kanamycin, 500
mg CA, 100 mg LLC 4 428 (88) 101 (13) 46216 (6593) 14.2 (2.0)
JSV-003-051
10 mg Tobramycin,
500 mg CA, 100 mg
LLC
8 314 (46) 144 (20) 25829 (4738) 14.5 (2.7)
42
Mean Pharmacokinetic Parameters for Kanamycin and Tobramycin
Following Oral Administration to Beagle Dogs (±SEM)
BCS Class III Molecule
Zanamivir Case Study
• PK studies of zanamivir in beagle dogs included the following arms: • I.V. (n=3 dogs)
• 0.083 mg/kg
• dosed as 1 mL of 1 mg/mL zanamivir in PBS
• Formulated enteric-coated capsule (n=5 dogs)
• 1.25 mg/kg
• 15 mg zanamivir, 500 mg citric acid, 100 mg LLC, Prosolv
• Un-formulated enteric-coated capsule (n=3 dogs)
• 1.25 mg/kg
• 15 mg zanamivir, Prosolv
• Plasma samples collected to 4 hours
44
Zanamivir Dog Study Results
0
100
200
300
400
500
600
700
0 30 60 90 120 150 180 210 240
Zan
am
ivir
pla
sm
a c
on
cen
trati
on
(n
g/m
L)
Time post-dose (minutes)
IV [0.083 mg/kg]
unformulated [1.25 mg/kg]
formulated: 500 mg CA, 100 mg LLC [1.25 mg/kg]
F0-4h = 19.8% (56% CV)
45
Conclusions from
Zanamivir PK Study
• Bioavailability
• Unformulated: 0.95% (88% CV)
• Formulated: 19.8% (56% CV)
• %F is somewhat underestimated, as the elimination phase of the
oral dose is not complete at 4 hours
• Compared to Relenza® Diskhaler (marketed inhalation product, GSK)
• Relenza %F∞ ranges from 4% to 17% post-inhalation
• Dosage form requires inhaler device and specially-packaged blisters
of Relenza®
• High variability due to differences in inhalation performance
• Enteris technology shows ~20% bioavailability over just 4 hours
46
In-vitro Fenofibrate Solubility Study
• BCS class II compound
• Insoluble in water
• Slightly soluble in ethanol (1 mg/mL)
• Soluble in DMF (30 mg/mL) and DMSO (15 mg/mL)
• Solubility in 1:3 DMF:PBS pH 7.2 reported at 250 mcg/mL
• Equilibrium solubility in water and increasing levels of LLC
• Excess fenofibrate weighed into individual PP vials
• Solutions mixed at 125rpm at 25°C for 4 days
• Fenofibrate concentration measured by HPLC against a standard curve
prepared in neat CH3CN
• This experiment measured water solubility at 4.3 ng/mL
48
0.0 2.5 5.0 7.5 10.00
50
100
150
200
250
300
350
400
Solubility in 1:3 DMF:PBS pH 7.20.25mg/mL
O
ClO
CH3
CH3
O
OCH3
CH3
LLC Concentration (% w/v)
Fen
ofi
bra
te C
on
cen
trati
on
(mcg
/mL
)
Fenofibrate Equilibrium Solubility with
Increasing Concentrations of LLC
49
Excipient Safety Profile
• Protease Inhibitor: Organic Acid • Pharmaceutically accepted ingredient
• Transport Enhancer: Acyl Carnitine • Scope of non-clinical safety package has been confirmed by FDA
• Genotoxicity and respiratory toxicity completed
• 6 month rat toxicology study completed
• 9 month dog toxicology study completed
• Part of tablet formulation in 12 clinical studies (328 subjects )
• All other Excipients • Pharmaceutically accepted ingredients
• Drug Master File (Type V Safety Data)
• Available for cross-reference
51
Acylcarnitines as Permeation Enhancers
• Acylcarnitines are fatty acid esters of L-carnitine that have a single
aliphatic hydrocarbon chain of variable length.
• 3-O-lauroyl-L-carnitine, LLC, is the carnitine ester of lauric acid, a 12
carbon aliphatic fatty acid.
• Plasma acylcarnitine concentrations in healthy human subjects
ranges from 6 μM to 15 μM, with the majority of studies reporting
values ≥10 μM.
• Function to transfer long chain fatty acids across the
mitochondrial membrane for β-oxidation and subsequent
adenosine triphosphate (ATP) production
52
The Permeation
Enhancement by LLC is Transient
LeCluyse E.L., et al. (1993). J. Pharm. Exp. Therap. 265(2):955-962. 53
LLC’s Effects are Reversible
Within 15 to 30 Minutes of Removal
LeCluyse E.L., et al. (1993).
J. Pharm. Exp. Therap.
265(2):955-962.
Control Tissue 2.0 mM LLC
54
Little Potential for Opportunistic
“Piggyback” Permeation
Molecule Type MW (kDa) 3-Dimensional
Radius (Å)
Macromolecules 1 – 10 10 – 30
LPS > 100 100 – 1000
Enteric Toxins 70 – 900
Viruses 600 – 1000
Bacteria > 1000
Modified from: Brayden, D. Permeation Enhancers and Oral Peptide Delivery.
Presented at the Roche Colorado Peptide Symposium, Sept. 12, 2011.
Modeling of
perturbed
membrane in
Caco-2 cells
indicates an
effective pore
radius ca. 20Å
55
Tight Junction Modifiers are Common
• Drug Compounds:
• Aspirin
• NSAIDS
• Phenothiazines
• Food and Drug
Additives/Excipients:
• EDTA
• C8-C18 fatty acids
• Various polymers
• Poly-L-lysine
• Natural/Food Products:
• ZOT
• ATP
• Chitosan and chitosan
derivatives
• Wheat gluten
• Oat saponins
• Capsaicin
• Alcohol
56
3-O-Lauroyl-L-Carnitine
Preclinical Safety Studies
• Completed Safety Pharmacology Studies
• Acute neurotoxicity in rat
• Acute respiratory in rat
• Acute cardiovascular in dog
• hERG
• CYP450 inhibition/ induction
• Metabolic profiling in hepatocytes (multiple species)
• Completed Toxicology Studies
• Oral MTD studies in rat and dog
• 4 day oral repeat dose finding in rat and dog
• 1 month oral toxicology with toxicokinetics in rat and dog
• Standard genotoxicity
• 6 month oral repeat dose in rat
• 9 month oral repeat dose in dog
58
All Safety Pharmacology Studies
Generated Desirable Outcomes
• Lauroyl-L-Carnitine did not:
• Inhibit hERG tail current in vitro, at doses up to ca. 90 μg/mL
• Affect ECG parameters in dogs dosed up to 100 mg/kg/day PO
for 1 month
• Produce cardiovascular effects in dogs at up to 100 mg/kg
• Induce any respiratory effects in rats at up to 100 mg/kg by
gavage
• Produce any adverse effects on neurobehavioral function in rats
at up to 100 mg/kg
59
FDA Feedback on LLC program
• FDA Advice Letter received August, 2009
...the studies that have been completed to date in your
development program and the proposed six- and nine-
month repeat dose studies in rats and dogs respectively,
would serve to support the use of LLC as an excipient in
drug products.
61
• Reproductive toxicology • Studies required for populations of premenopausal females, women
of child bearing potential, or males
• Embryo-fetal studies – for registration • Recommended embryo-fetal studies in two species, with an
assessment of teratogenicity as a minimum to complete drug approval applications in postmenopausal women
• Carcinogenicity – likely not required • The nonclinical findings to date could support the proposal that
carcinogenicity studies are not necessary
• If there are no preneoplastic lesions or serious adverse toxicologic effects in these studies, FDA would concur that carcinogenicity studies will not be necessary (None observed – reports submitted Sept, 2012)
Registration Requirements
62
“3-O-Lauroyl-L-Carnitine Hydrochloride
(LLC) Preclinical and Clinical Data.”
• Contains full study reports of all non-clinical and
clinical studies
• Non-clinical and clinical overview documents
• Available to partners for cross-referencing
LLC Type V DMF Submitted to FDA
63
Summary
• Extensive preclinical toxicology package
• Observed no preneoplastic lesions or serious adverse
toxicologic effects
• Completed toxicology study of 9 months duration
• Extensive Clinical Experience
• < 8 single dose Phase 1 studies
• 8 week Phase 2a study for oral sCT program
• 24 week Phase 2 study for oral PTH program
• Type V DMF containing safety data of LLC
64
Patents Summary
Oral Delivery Patents
• 7 issued U.S. patents
• 2 allowed U.S. patent applications
• 3 pending U.S. provisional patent applications
• 29 issued Foreign patents
• 5 pending Foreign patent applications
• Key issued patents extend through 2030
66
Working with Enteris
In-Vitro
• Solubility
• Permeability
In-Vivo
• Intra-duodenal:
Rat Study
In-Vivo
• Capsule: Dog
Study
In-Vitro & In-Vivo
• Stability; Design Space
• Tablet: Dog Study
Formulation
Development
Tablet Formulation Optimization
3 months 3 months
68
Manufacturing
• Enteris cGMP Manufacturing
• 32,000 ft2 cGMP facility located in Boonton, NJ
• Separate tableting and nasal spray filling suites
• Full QA/QC and regulatory support
• Commercial product in US distribution
70
Recent Technical Achievements
• Identified coated organic acid as compatible excipient with peptides
and small molecules
• Simple and scaleable manufacturing process
• Optimized release characteristics and bioavailability
• Demonstrated room temperature stability of peptide tablets for 24
months
• Supplied CTM for Phase 1 and Phase 2 studies
71
Tableting and Capsuling Line
• Comil conical mill
• V-Blender
• Korsh XL-100 10 station tablet press - up to
10,000 tablets/hr
• Natoli NP-RD10a single station press for 1 to
300 tablets
• Vector LDCS coating pan for enteric coating
• Capsugel Profill capsule filler
• Phase 1 and 2 clinical supplies
• Clinical packaging
• Open label, double blind
72
Quality Control
• Raw Material Release Testing
• Final Product Release
• Assay Development
• Assay Transfer, Optimization & Validation
• Special Projects and Investigations
• Stability Studies
• In process, intermediate and facility testing
capabilities
73
Quality Assurance
• Systems compliant with FDA, EMEA, MHRA and ICH
• Customer focused, providing real time feedback on all quality related
issues.
• Full project participation:
• process development → batch record design → GMP manufacture.
• Manufacturing oversight through concurrent batch record review.
• Vendor auditing and qualification program.
74
Inspection History
Regulatory Inspection History
• Most recent FDA inspection – October, 2011 - No FDA-483 issued
• Successful Pre-Approval Inspection – June, 2003
• Most recent EMA inspection – October, 2006 - GMP Certificate
issued
• Successful Pre-Approval Inspection – February, 1998
• GMP inspections by QPs from UK & Germany for Phase 3 - Clinical
Trial – 2008
• GMP Certificates issued
75
E. coli Recombinant Peptide Manufacturing
Enteris’ Peptide Manufacturing Advantages
• Robust yield and purity
• Scalable
• Efficient
• Cost-effective
• Patented E. coli-based recombinant peptide
expression technology
• Peptide secreted directly into culture medium
• Enzymatic amidation of recombinant precursor
Direct Expression of Peptides Direct Expression of Peptides
76
kDa
66.2
45.0
31.0
21.5
14.4
6.5
sC
Tg
ly
Glu
co
se r
eg
ula
tory
pep
tid
e-g
ly
Glu
co
se r
eg
ula
tory
pep
tid
e a
nalo
g-g
ly
PT
H a
nalo
g-g
ly
SDS PAGE and HPLC Trace of Crude Medium
• Enriched Starting Material
• Extracellular yields of 400 to 1300 mg/L
• Reduced purification steps
High Yield and High Product Purity
at Low Cost
Recombinant Peptide Manufacturing
78
Amidated Peptide Manufacturing Example
rhPTH(1-34)NH2
0
100
200
300
400
500
600
700
800
1 5 9 13 17 21 25 29
Hours Post Induction
PT
H(1
- 34)
mg
/L
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Feed
Rate
mL
/min
PTH(1-34)
mg/L
Feed Rate ml/min
0
100
200
300
400
500
600
700
800
1 5 9 13 17 21 25 29
Hours Post Induction
PT
H(1
- 34)
mg
/L
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Feed
Rate
mL
/min
0
100
200
300
400
500
600
700
800
1 5 9 13 17 21 25 29
Hours Post Induction
PT
H(1
- 34)
mg
/L
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Feed
Rate
mL
/min
0
100
200
300
400
500
600
700
800
1 5 9 13 17 21 25 29
Hours Post Induction
PT
H(1
- 34)
mg
/L
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Feed
Rate
mL
/min
0
100
200
300
400
500
600
700
800
1 5 9 13 17 21 25 29
Hours Post Induction
PT
H(1
- 34)
mg
/L
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Feed
Rate
mL
/min
0
100
200
300
400
500
600
700
800
1 5 9 13 17 21 25 29
Hours Post Induction
PT
H(1
- 34)
mg
/L
0.000
0.050
0.100
0.150
0.200
0.250
0.300
Feed
Rate
mL
/min
rhPTH(1-34)GLY Production and
Fermentation Feed Rate
The host : E. coli B strain, BLM-6
The peptide: human PTH(1 - 34) - Gly
SVSEIQLMHNLGKHLNSMERVENLRKKLQDVHNFG MW = 4,116.8 Da
Step 1. Recombinant production of rhPTH(1-34)Gly35OH precursor
pPTH(1-34)G-03
7799 bp
PTH(1-34)gly
ompA
SEC-E LAC-IQ
KAN-R
PRLA-4
ompA
PTH(1-34)gly
P1
P2
P3
SecE P/O
P2
P1
Ori
Term
Term
Term
Term
79
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