jm cc sirius 2015
TRANSCRIPT
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New In Vitro Methods for Bio-Relevant Analysis of Both Small Molecules and Proteins
Jon Mole
Executive Vice President, Sirius Analytical Inc.
Beverly, [email protected]
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An introduction to Sirius
Sirius was founded in 1989. We are a manufacturer and vendor of instrumentation for measurement of physicochemical parameters.
We also provide an Analytical Service, and measure thousands of samples for hundreds of customers, worldwide, each year.
In the US, we are based in Beverly, MA.
www.sirius-analytical.com
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My presentation today:
- The need for improved in-vitro testing models
- Sirius inForm – dynamic dissolution testing instrument
- Introducing Sirius Scissor – a new instrument for biotherapeutics
- Example studies from both systems
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GI Conditions & traditional dissolution
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Historically:
• Develop pharmaceutical products to be manufactured in bulk
• Test criteria based on reproducibility of manufacture
The Rise of Formulation
Currently:
• Enabling techniques to improve bioavailability of BCS class 2 and 4 API’s.
• More recently formulation has become more focussed towards pharmacokinetics
• Requires a deeper understanding of the physicochemical properties of API’s in the presence
of the formulation ingredients for achieving required exposure levels
• Understanding the solubility and dissolution behaviour of API’s is an important part of
formulation design
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Traditional dissolution testing is a quality performance test (drug release and QC)
Traditional Dissolution is not biorelevant…
?
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European IMI-OrBiTo* Program
* OrBiTo = Oral Biopharmaceutics Tools
8http://www.orbitoproject.eu/
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Platform for automated biorelevant dissolution and solubility testing with support for final dosage forms
http://www.sirius-analytical.com/products/inform
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Sirius inForm- new instrument for use in Formulation studies
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Automated dispensing of acid, base, solvents, buffers and SIFs during an assay
Stirrer, pH and UV probes. Sample vial in Peltier block. Automated sample addition.
Dispensers for Automated addition of media, titrants, solvents
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Automated dispensing of acid, base, solvents, buffers and SIFs during an assay
Stirrer, pH and UV probes. Sample vial in Peltier block. Automated sample addition.
Robotic arms for vial handling and assay probe handling
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Automated dispensing of acid, base, solvents, buffers and SIFs during an assay
Stirrer, pH and UV probes. Sample vial in Peltier block. Automated sample addition.
20 Vial autoloader, HPLC vial tray, sonicator, automated cleaning, vacuum manifold, tablet holder
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The inForm measurement cell
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pH electrode
UV probe
Temperature probe
Reagent capillaries
Stirrer
Inert gas
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Sample tablet holder can be lowered from
above.
Basket for powders and
dosage forms.
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Three ways to measure concentration
• pH-metric
– Good for solutions whose pH is < 3 units from pKa
• In-situ UV
– Good for drugs that absorb UV
• Automated Off-line sampling
– Best method if samples are very turbid
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Dru
g co
nce
ntr
atio
n
Time
“Spring”
Equilibrium solubility
Diagram adapted fromBrouwers, J. et al. J. Pharm. Sci. 2009, 98 (8), 2549-2572.
Crystalline sample
“Spring” with “Parachute”
TRADITIONAL DISSOLUTION
“Spring and Parachute” model
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Supersaturated, sample is precipitating
Subsaturated, sample is dissolving
Supersaturated, sample is fully dissolved
Precipitate appears
Equilibrium solubility
Time
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0
2
4
6
8
10
0 30 60 90 120
Co
nce
ntr
atio
n (
μM
)
Time (minutes)
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Dissolution of Warfarin* powder at pH 3.1
* Warfarin is an acid, pKa 4.94
Equilibrium solubility
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0 30 60 90 120
Time (minutes)
0
100
200
300
400C
on
ce
ntr
ation
(µ
M)
Same graph, different scaling
Equilibrium solubility
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0
100
200
300
400C
on
ce
ntr
ation
of n
eu
tra
l sp
ecie
s (
µM
)
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Warfarin aqueous CheqSol: a “Spring”
Equilibrium solubility
Precipitate appears
Kinetic solubility
0 30 60 90 120
Time (minutes)
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0
100
200
300
400C
on
ce
ntr
ation
of n
eu
tra
l sp
ecie
s (
µM
)
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0 30 60 90 120
Time (minutes)
Warfarin and PVP*: a “Parachute”
* Polyvinylpyrrolidone. With thanks to Ashland Specialty Chemicals
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How we measure concentrations by in-situ UV
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In-situ UV
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pH Versus Time
20:00 30:00
Time(minutes:seconds)
3
5
7pH
N
O
O
CH3
OH
Cl
OCH3
IndomethacinAcid, pKa 4.1
IndomethacinTitration in linear buffer solutionMeasure spectra at each pH point
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2 4 6 8
pH (Concentration scale)
0.0
0.5
1.0
1.5
2.0
Absorb
ance
0
50
100
150
200
Specie
s C
oncentra
tion (µ
M)
5.11
250 300 350 400
Wavelength (nm)
0.0
0.5
1.0
1.5
2.0A
bsorb
ance
0
5000
10000
15000
20000
25000 Mola
r absorp
tion (/c
m/M
)
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Molar Absorption Coefficients (MECs)
N
O
O
CH3
OH
Cl
OCH3
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Comparing MEC with dissolution data
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250 300 350 400
Wavelength (nm)
0.0
0.5
1.0
1.5
Ab
so
rba
nce
N
O
O
CH3
OH
Cl
OCH3
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inForm case studies
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Client Evaluation 1 – A major US pharma company
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• API is a weak acid with pKa = 9 and log P = 3
• Four forms supplied
– Crystalline API, powder
– Formulated crystalline API, extracted from a capsule
– Amorphous solid dispersion, powder
– Formulated amorphous solid dispersion, part of
tablet
• Equal weight of API used in each experiment
• All experiments at 37°C
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Dilution, FaSSIF introduced
Gastric period: Acetate-phosphate
buffer + HCl
Gastric emptying. pH raised with NaOH
Biorelevant dissolution. FaSSIF added
Intestinal period
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Biorelevant dissolution
1
2
3
4
5
6
7
0
5
10
15
0 2 4 6
pH
Mas
s o
f dis
solv
ed
AP
I (m
g)
Time (hours)
Formulated crystal API
Crystal API
Solid dispersion
Formulated solid dispersion
pH
Crystalline samples
1
2
3
4
5
6
7
0
200
400
600
800
0 1 2
pH
Mas
s o
f dis
solv
ed
AP
I (μ
g)
Time (hours)
Formulated crystal API
Crystal API
pH
Crystalline
Amorphous
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Biphasic dissolution. Nonanol added
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Gastric period: Acetate-phosphate
buffer + HCl
Dilution, lipid layer introduced
Gastric emptying. pH raised with NaOH
Drug dissolving in aqueous solution
Drug partitioned into lipid
Total amount dissolved
Intestinal period
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1
2
3
4
5
6
7
0
5
10
15
0 1 2
pH
Mas
s o
f dis
solv
ed
AP
I (m
g)
Time (hours)
Crystal API, lipid
Formulated crystal API, lipid
Formulated crystal API, aq.
Crystal API, aqueous
pH
1
2
3
4
5
6
7
0
5
10
15
0 1 2
pH
Mas
s d
isso
lve
d o
f AP
I (m
g)
Time (hours)
Formulated solid dispersion, lipid
Solid disperion, lipid
Formulated solid dispersion, aq.
Solid dispersion, aqueous
pH
Biphasic dissolution
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Crystalline API-may be overstimated
Formulated solid dispersion -may be overestimated
Crystalline
Amorphous
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Client Evaluation 2 – Small US Pharma
• Controlled supersaturation
• Solvent quench method: concentrations by UV
• Two examples
– Bifonazole, Droperidol
• FaSSIF and various additives used to test the effect on supersaturation/precipitation rate
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Tyndall-Rayleigh scattering correction
BEFORE
AFTER
OK for data analysis
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Aqueous
Pluronics
PVP
Soluplus
FaSSIFHPMC
DroperidolBase, pKa 8.2Controlled supersaturation at pH 6.8
BifonazoleBase, pKa 6.6Controlled supersaturation at pH 5
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Summary of inForm PlatformThe Sirius inForm instrument is a useful tool for early development and
formulation scientists providing more predictive tools for drug performance
• Sirius inForm can set up a wide range of experimental conditions
• Automated biorelevant solubility & dissolution
• Automated biphasic dissolution
• Measuring supersaturation & precipitation behavior
• Dealing with turbidity
• Innovative tests for investigating IVIVC
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What about Protein formulations?
Introducing Sirius “Scissor”: Sub Cutaneous Injection Site SimulatOR
A New In Vitro Test for Injection Site Events for Biopharmaceuticals
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Importance of Subcutaneous Injections
1989 to 2012 Biotechnology products (mostly proteins and peptides)
grew in number from 13 to 210
Sales increased to US$163 billion.
2001 to 2012 Biotech products accounted for 71% revenues for the
ten top-selling pharmaceuticals in 2012, up from 7% in 2001.
Move to subcutaneous (SC) injections Currently, ~ 40 protein and peptide drugs are given SC
Therapies shifting care to home treatment will increase this number of SC drugs
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Potential Issues for SC and IO injections
Current formulations are designed to• Keep API stable in a vial for several years• Minimize injection volume (high concentration)• Minimize pain upon injection
APIs are stressed upon injection by• Transition from formulation to homeostatic conditions immediately
after injection- Physical stress due to change in pH- Transition through isoelectric point?- Change in concentration of stabilizing agents- Altered interactions with stabilizing agents
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Possible Events After Injection
There are currently no in vitro methods available to examine potential events that
might be experienced by an API during its transition from an injected formulation to the
steady-state conditions of the injection site environment.
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What We Know and What is Needed
What we know
• Site to site and patient to patient variability is seen for bioavailability (%BA) of many biopharmaceuticals.
• No animal model correlates to (%BA) observed in man.
• Conditions/characteristics of physical and chemical environments of the injection site are species specific.
• Insolubility/precipitation upon injection can lead to cellular responses and macrophage clearance.
What we need
• A versatile in vitro model to examine the potential impact of specific, individual post-injection events.
• A dynamic system that emulates approximate time and conditions for post-injection transitions.
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Design of the Scissor instrument
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Schematic of the Scissor system chamber featuring: injection cartridge acting as asimulated injection site; pH probe for monitoring the pH within the cartridge; light sourceand detector for monitoring aggregation events; chamber of physiological buffer;thermocouple and heater/stirrer
Sirius Scissor - Schematic
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In vitro study of subcutaneous injection of two insulin formulations
Two insulin formulations; Insuman Rapid (fast acting) and Insuman Basal (slow acting)
Light %T of Insuman Rapid and Basal
PK of Insuman Rapid and Basal
Fraction appearing in ISF buffer (data by HPLC)
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Pharma Study - Outcome Potentially Important for mAbs@
6h
rs
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Our Approach
• No pre-clinical model has been identified that will correlate with human in vivo outcomes
• This model is not intended to examine cell-mediated or immune responses
• Our in vitro model simulates dynamic events at the site of injection site of a drug
• The model monitors ECM interactions, pH changes, protein turbidity, excipients fate and spectroscopic properties.
• The system models events for several hours at physiological conditions
• Future studies will focus on correlations pre-clinical human in vivo outcomes to establish a predictive tool.
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Scissor - Acknowledgements
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Genentech
Vikas Sharma
Stefan Ficsher
Sreedhara Alavattam
Tom Patapoff
Ann Daugherty
Sirius Analytical
John Comer
Karl Box
George Butcher
Brett Hughes
University of BathRandy MrsnyHanne KinnunenJenni SobleAlison EvansMatt Young
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• Thanks for listening!
• Any questions?
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www.sirius-analytical.com
Make formulation decisions earlier with detailed bio-relevant data