Introduction to Enteris BioPharma, Inc.
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
Actual Bioavailability Data Peptides and Small Molecules 25
Absolute Bioavailability (%)
proprietary peptide
20
15
zanamivir tigecycline CR-845 kanamycin tobramycin
10 proprietary peptides proprietary peptide
5
octreotide *GLP-1 analog
0
0 Studies in beagle dogs Except *Rat Study
1000
2000
calcitonin
PTH 1-34
3000
4000
insulin
5000
6000
Molecular Weight (Da) 5
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
Chemical Stability • x-S-S-x Resistant to Proteolysis?
Modified? Physical Stability • Aggregation rate Projected Dose (mg)
Injectable Dose (mg)
Net Charge • Anionic charges
Bioavailability (%) if known
• Cationic charges
• Rat
• pI
• Dog 6
Criteria for Selection of Small Molecules
ENTERIS SMALL MOLECULE ASSESSMENT MATRIX Molecular Weight (Da)
Moisture sensitive/hygroscopic
BCS Class, if known
Is the API plastic, elastic or brittle?
Describe the solid (salt form, hydrate, solvate, etc.
Any known excipient incompatibilities?
Solubility
Primary mechanism(s) of chemical degradation
•
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
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
•
Safety • Excipient Safety Profile
• Peptides
• LLC Toxicology Studies
• Small molecules
• LLC Regulatory and Clinical
• BCS Class III
•
Patents
• BCS Class II
•
Business Development
•
Manufacturing
8
Mechanism of Drug Delivery
9
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
Components of Enteris’ Solid Dosage Formulation
14
Peptide Experience
15
Dog Model Predicts Bioavailability in Humans 4500 4000
Linear Regression for Cmax: Y=A+B*X 2 R =0.9801
1000
Human Cmax (pg/ml)
Plasma Cmax (pg/ml)
5000
3500 3000 2500
2000 1500 1000 500 0 0
1
2
3
Dose (mg)
4
5
y=.995x - 0.7548 R=0.958 R2=0.918
100
10
10
100
1000
10000
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
Sponsored
No. of Amino Acids
Study Type
Ranges of Unformulated Bioavailability (%) for 12 Peptides
Ranges of Formulated Bioavailability* (%) for 12 Peptides
Rat
0.35 – 6.0
10.7 – 26.0
Dog
0.39 – 22.8
4 – 39
* 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 All D Amino Acid Peptide
Enteris
Enteris
Enteris
18
Absorption of LHRH Analog in Dogs as a Function of Enteric-Coating
A
B
D
L30D-55 (10%)
Tmax (min)
Capsule Formulation in Dogs Bioavailability (% F)
12000
Formulation A 10000
111
L30D-55 (15%)
116
L30D-55/ FS30D (12%)
152
3.0 4.6 7.2
LHRH (pg/mL)
Formulation
Enteric coat (weight gain)
Formulation B Formulation D Formulation B
8000
Unformulated Unformulated C 6000 Formulation D 4000 2000 0
Unformulated
L30D-55 (10%)
0
130
0.1
100
200
300
400
500
Time Relative to T max (minutes)
19
Bioavailability of Cara’s CR845 in Preclinical & Phase I
16%
20% 13%
13%
Rat
Dog
15% 10% 5%
0%
Man
20
Phase I Oral CR845 Study
CR845 Demonstrated 16% Oral Bioavailability N = 8/group
100
CR845 (ng/mL)
0.5 mg 1 mg 3 mg 10 mg
10
1
0.1 0
4
8
12
16
Time (hours)
20
24 Mean + SEM
21
PTH Phase II Study Mean PTH Cmax Values for Subjects Receiving Oral PTH(1-31)NH2 and Forsteo®
22
Phase III Oral Calcitonin Study Phase III Study for Oral sCT: Primary Endpoint (Change in LS BMD) Achieved Mean % Change LS-BMD
3.00 2.50
2.00 1.50
1.00
1.5
0.50 0.00
0.8 p<0.001*
rsCT Tablet
p=0.014*
0.5 p=ns*
Nasal Spray
Placebo
23
Small Molecule Experience
24
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
Tigecycline Case Study KANAMYCIN PROJECT REVIEW AMY STURMER 1.0 / 31JULY2013 26
BCS Class III Molecule Tigecycline Case Study •
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
27
BCS Class III Molecule Tigecycline Rat Study
(mcg/mL) Concentration Plasma Tigecycline (mcg/mL) Plasma
Animals dosed via intraduodenal administration to simulate oral dosing 2.25
(0.64 mg/kg IV or 4.8 mg/kg ID)
2.00
IV ID PBS ID 100mM CA, 26mM LLC ID 400mM CA, 26mM LLC
1.75 1.50 1.25 1.00 0.75 0.50 0.25 0.00 0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Time (hrs)
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
Plasma Tigecycline Concentration Plasma Concentration (ng/mL) (ng/mL)
180
150
15 mg 30 mg 45 mg
120
90
60
30
0 0.0
• 0.5
1.0
1.5
2.0
2.5
3.0
3.5
API was not detected in any “unformulated” arm
4.0
Time (hr)
31
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
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 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
Tigecycline Human PK Data
34
Aminoglycoside (Kanamycin, Tobramycin) Case Studies KANAMYCIN PROJECT REVIEW AMY STURMER 1.0 / 31JULY2013 35
Project Overview •
Background • • •
•
Indication: • •
•
Interacts with the 30S subunit of prokaryotic ribosomes. Induces substantial amounts of mistranslation and indirectly inhibits translocation during protein synthesis
Contraindications: • • •
•
Used to treat wide variety of infections Effective against gram negative bacteria
Mechanism of action: •
•
Aminoglycoside bactericidal antibiotics Consist of two amino sugars glycosidically linked to deoxystreptamine Available in oral, IV, IM, and inhaled
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
6
67 (9)
118 (18)
9167 (983)
2.8 (0.3)
0 mg CA, 1
0 mg LLC, uncoated
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)
31
147 (147)
195 (NA)
8573 (8573)
2.6 (2.6)
50 mg CA, 5
100 mg LLC, coated
37
IV Tobramycin Study
38
Plasma Levels in 8 Dogs Given Formulated Oral Tobramycin Capsules
39
Mean Plasma Levels in Dogs Given Oral Tobramycin
(Adjusted for Tmax and Dose)
40
PK of Tobramycin
No of Dogs/No of Responders Cmax (ng/mL) Tmax (min) AUC(0-t) (ng*min/mL) AUC(0-t) (ng*min/mL/mg) %F
Unformulated Capsules (SEM)
Formulated Capsules (SEM)
3/8 3 (1) 145 (35) 270 (140) 27 (14) 0.15 (0.08)
8/8 314 (46) 144 (20) 25829 (4738) 2583 (474) 14.5 (2.7) 41
Pharmacokinetic Parameters for Kanamycin and Tobramycin Mean Pharmacokinetic Parameters for Kanamycin and Tobramycin Following Oral Administration to Beagle Dogs (±SEM)
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
BCS Class III Molecule Zanamivir Case Study
43
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
Zanamivir plasma concentration (ng/mL)
700
IV [0.083 mg/kg] unformulated [1.25 mg/kg]
600
formulated: 500 mg CA, 100 mg LLC [1.25 mg/kg] 500
400
300
F0-4h = 19.8% (56% CV) 200
100
0 0
30
60
90
120
150
180
210
240
Time post-dose (minutes) 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
Small Molecule Experience BCS Class II
47
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
Fenofibrate Equilibrium Solubility with Increasing Concentrations of LLC 400 O
Fenofibrate Concentration (mcg/mL)
350
CH3
O CH3
300
H3C
O H3C
O
Cl
250 200
Solubility in 1:3 DMF:PBS pH 7.2 0.25mg/mL
150 100 50 0 0.0
2.5
5.0
7.5
10.0
LLC Concentration (% w/v)
49
Safety
50
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
Control Tissue
2.0 mM LLC
LeCluyse E.L., et al. (1993). J. Pharm. Exp. Therap. 265(2):955-962.
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
Modeling of perturbed membrane in Caco-2 cells indicates an effective pore radius ca. 20Å
Modified from: Brayden, D. Permeation Enhancers and Oral Peptide Delivery. Presented at the Roche Colorado Peptide Symposium, Sept. 12, 2011. 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
LLC Toxicology Studies
57
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
LLC Regulatory and Clinical
60
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 ninemonth repeat dose studies in rats and dogs respectively, would serve to support the use of LLC as an excipient in drug products.
61
Registration Requirements
• 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)
62
LLC Type V DMF Submitted to FDA
“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
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
65
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
Business Development
67
Working with Enteris
Formulation Development
In-Vitro • Solubility • Permeability In-Vivo • Intra-duodenal: Rat Study In-Vivo • Capsule: Dog Study
Tablet Formulation Optimization
3 months
In-Vitro & In-Vivo • Stability; Design Space • Tablet: Dog Study
3 months
68
Manufacturing
69
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
DirectExpression ExpressionofofPeptides Peptides Direct • • •
Patented E. coli-based recombinant peptide expression technology Peptide secreted directly into culture medium Enzymatic amidation of recombinant precursor
Enteris’ Peptide Manufacturing Advantages • • • •
Robust yield and purity Scalable Efficient Cost-effective
76
Enteris’ Recombinant Manufacturing Process
77
High Yield and High Product Purity at Low Cost
PTH analog-gly
Glucose regulatory peptide analog-gly
Glucose regulatory peptide-gly
kDa
sCTgly
Recombinant Peptide Manufacturing
66.2 45.0 31.0 21.5 14.4 6.5
SDS PAGE and HPLC Trace of Crude Medium • Enriched Starting Material • Extracellular yields of 400 to 1300 mg/L • Reduced purification steps 78
Amidated Peptide Manufacturing Example rhPTH(1-34)NH2 Step 1. Recombinant production of rhPTH(1-34)Gly35OH precursor The peptide: human PTH(1 -34)-Gly
rhPTH(1-34)GLY Production and Fermentation Feed Rate
SVSEIQLMHNLGKHLNSMERVENLRKKLQDVHNFG MW = 4,116.8 Da P1 P2
KAN-R
ompA
800
0.300
PTH(1-34)gly 700
Term
0.250
P1
PTH(1-34)gly
pPTH(1-34)G-03
Term
7799 bp
P3
Ori
PRLA-4
0.200 PTH(1-34) mg/L
ompA
500 400
0.150
300
Feed Rate mL/min
600
P2
0.100 200 0.050 100
Term SecE P/O LAC-IQ
SEC-E
0
0.000 1
5
9
13
17
21
25
29
Hours Post Induction
Term
The host : E. coli B strain, BLM-6
PTH(1-34) mg/L
Feed Rate ml/min
79
Thank You
