New In Vitro Methods for Bio-Relevant Analysis of Both Small Molecules and Proteins

Jon Mole

Executive Vice President, Sirius Analytical Inc.

Beverly, MAJon.Mole@sirius-analytical.com

1

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

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

GI Conditions & traditional dissolution

4

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/

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.

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

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

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)

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

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

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

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

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

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

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

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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35

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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

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

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.

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

• Thanks for listening!

• Any questions?

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Jon.Mole@sirius-analytical.com

www.sirius-analytical.com

Make formulation decisions earlier with detailed bio-relevant data