1. New In Vitro Methods for Bio-Relevant Analysis of
Both Small Molecules and Proteins
George Butcher
Technical Product Manager, Sirius Analytical
Forest Row, UK
george.butcher@sirius-analytical.com
1

2. 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

3. 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

4. GI Conditions & traditional
dissolution
4

5. 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
5

6. Traditional dissolution testing is a quality
performance test (drug release and QC)
Traditional Dissolution is not biorelevant…
?
6

7. 7

8. European IMI-OrBiTo* Program
* OrBiTo = Oral Biopharmaceutics Tools
8http://www.orbitoproject.eu/

9. Platform for automated biorelevant dissolution and
solubility testing with support for final dosage forms
http://www.sirius-analytical.com/products/inform

10. 10
Sirius inForm
- new instrument for use in Formulation studies

11. 11
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

12. 12
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

13. 13
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

14. 14

15. The inForm measurement cell
15
pH
electrode
UV probe
Temperature
probe
Reagent
capillaries
Stirrer
Inert gas

16. 16
Sample tablet
holder can be
lowered from
above.
Basket for
powders and
dosage forms.

17. 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
17

18. 18
Drugconcentration
Time
“Spring”
Equilibrium
solubility
Diagram adapted from
Brouwers, J. et al. J. Pharm. Sci. 2009, 98 (8), 2549-2572.
Crystalline sample
“Spring” with “Parachute”
TRADITIONAL DISSOLUTION
“Spring and Parachute” model

19. 19
Supersaturated,
sample is precipitating
Subsaturated,
sample is dissolving
Supersaturated,
sample is fully
dissolved
Precipitate appears
Equilibrium
solubility
Time

20. 0
2
4
6
8
10
0 30 60 90 120
Concentration(μM)
Time (minutes)
20
Dissolution of Warfarin* powder at pH 3.1
* Warfarin is an acid, pKa 4.94
Equilibrium
solubility

21. 21
0 30 60 90 120
Time (minutes)
0
100
200
300
400Concentration(µM)
Same graph, different scaling
Equilibrium
solubility

22. 0
100
200
300
400Concentrationofneutralspecies(µM)
22
Warfarin aqueous CheqSol: a “Spring”
Equilibrium
solubility
Precipitate appears
Kinetic
solubility
0 30 60 90 120
Time (minutes)

23. 0
100
200
300
400Concentrationofneutralspecies(µM)
23
0 30 60 90 120
Time (minutes)
Warfarin and PVP*: a “Parachute”
* Polyvinylpyrrolidone.
With thanks to Ashland Specialty Chemicals

24. 24
How we measure concentrations by in-situ UV

25. In-situ UV
25
pH Versus Time
20:00 30:00
Time(minutes:seconds)
3
5
7
pH
N
O
O
CH3
OH
Cl
O
CH3
Indomethacin
Acid, pKa 4.1
Indomethacin
Titration in linear buffer solution
Measure spectra at each pH point

26. 2 4 6 8
pH (Concentration scale)
0.0
0.5
1.0
1.5
2.0
Absorbance
0
50
100
150
200
SpeciesConcentration(µM)
5.11
250 300 350 400
Wavelength (nm)
0.0
0.5
1.0
1.5
2.0Absorbance
0
5000
10000
15000
20000
25000
Molarabsorption(/cm/M)
26
Molar Absorption Coefficients (MECs)
N
O
O
CH3
OH
Cl
O
CH3

27. Comparing MEC with dissolution data
27
250 300 350 400
Wavelength (nm)
0.0
0.5
1.0
1.5
Absorbance
N
O
O
CH3
OH
Cl
O
CH3

28. 28
inForm case studies

29. Client Evaluation 1 – A major US pharma company
29
• 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

30. 30
Dilution,
FaSSIF
introduced
Gastric period:
Acetate-phosphate
buffer + HCl
Gastric emptying.
pH raised with NaOH
Biorelevant dissolution. FaSSIF added
Intestinal period

31. 31
Biorelevant dissolution
1
2
3
4
5
6
7
0
5
10
15
0 2 4 6
pH
MassofdissolvedAPI(mg)
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
MassofdissolvedAPI(μg)
Time (hours)
Formulated crystal API
Crystal API
pH
Crystalline
Amorphous

32. Biphasic dissolution. Nonanol added
32
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

33. 1
2
3
4
5
6
7
0
5
10
15
0 1 2
pH
MassofdissolvedAPI(mg)
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
MassdissolvedofAPI(mg)
Time (hours)
Formulated solid dispersion, lipid
Solid disperion, lipid
Formulated solid dispersion, aq.
Solid dispersion, aqueous
pH
Biphasic dissolution
33
Crystalline API-
may be overstimated
Formulated solid dispersion -
may be overestimated
Crystalline
Amorphous

34. 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
34

35. 35

36. 36
Tyndall-Rayleigh scattering correction
BEFORE
AFTER
OK for
data
analysis

37. 37
Aqueous
Pluronics
PVP
Soluplus
FaSSIF
HPMC
Droperidol
Base, pKa 8.2
Controlled supersaturation at pH 6.8
Bifonazole
Base, pKa 6.6
Controlled supersaturation at pH 5

38. Summary of inForm Platform
The 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
38

39. What about Protein formulations?
Introducing Sirius “Scissor”:
Sub Cutaneous Injection Site SimulatOR
A New In Vitro Test for Injection Site Events for
Biopharmaceuticals
39

40. 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

41. 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
41

42. 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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43. 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.
43

44. 44
Design of the Scissor instrument

45. 45
Schematic of the Scissor system chamber featuring: injection cartridge acting as a
simulated injection site; pH probe for monitoring the pH within the cartridge; light source
and detector for monitoring aggregation events; chamber of physiological buffer;
thermocouple and heater/stirrer
Sirius Scissor - Schematic

46. 46
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)

47. 47
Pharma Study - Outcome Potentially Important for mAbs
@6hrs

48. 48
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.

49. Scissor - Acknowledgements
49
Genentech
Vikas Sharma
Stefan Ficsher
Sreedhara Alavattam
Tom Patapoff
Ann Daugherty
Sirius Analytical
John Comer
Karl Box
George Butcher
Brett Hughes
University of Bath
Randy Mrsny
Hanne Kinnunen
Jenni Soble
Alison Evans
Matt Young

50. • Thanks for listening!
• Any questions?
50
george.butcher@sirius-analytical.com
www.sirius-analytical.com
Make formulation decisions earlier with detailed bio-relevant data