BioProof AG The Proof of Concept Company

(1)

Anhang 7

Prüfbericht Stabilität der Prüfsubstanzen im Vehikel

(2)

BioProof AG The Proof of Concept Company

Copy 1 / 2 Copy 1: Sponsor Copy 2: BioProof AG

Short ANALYTICAL REPORT

Stability assays of different dyes in acetone/olive oil vehicle BioProof Study-No.: BS-06-01

F. Anzenberger M. Schulz I. Lamprecht A. v. Nieciecki

Munich, May-2006

Confidential

Test facility: BioProof AG

Weihenstephaner Straße 28 81673 Munich, Germany

Sponsor: BSL BIOSERVICE Scientific Laboratories GmbH

Dr. I. Haist

Behringstrasse 6

82152 Planegg, Germany

(3)

BioProof AG The Proof of Concept Company

1. Synopsis

Study: Stability test of different dyes in acetone/olive oil

Test item: Dyes A - F

Test procedure: High performance liquid chromatography with UV detection

Objective: Stability test of different colours in acetone/olive oil.

Test facility: BioProof AG

Weihenstephaner Straße 28 81673 Munich, Germany

Sponsor: BSL BIOSERVICE Scientific Laboratories GmbH Dr. I. Haist

Behringstrasse 6

82152 Planegg, Germany Head test facility: Dr. Alexander von Nieciecki Study director: Michael Schulz

Study performance: Franziska Anzenberger

Study report: Ingrid Lamprecht

Notebook references: Laboratory notebook BS-06-01

Archiving: BioProof archive

Software: Millenium ³² (vers. 3.2), Excel 2003

(4)

BioProof AG The Proof of Concept Company

2. List of abbreviations and terms

amu atomic mass units

approx. approximately

FA formic acid

Fig. Figure h hours

HPLC high pressure liquid chromatography M molar

min minute mL millilitre

N number of observations

na not applicable

nc not calculable

No. number

ns no sample

nsr no sample for re-analysis sec seconds Tab. Table

(5)

BioProof AG The Proof of Concept Company

3. Table of contents

1. Synopsis ... 2

2. List of abbreviations and terms... 3

3. Table of contents... 4

4. Catalogue of tables and figures ... 5

5. Signatures... 6

6. Introduction ... 7

7. Summary ... 8

8. Principle... 8

8.1. Method description ... 8

9. Experimental part ... 9

Instrumentation and reagents ... 9

Chemicals ... 9

Reference items ... 9

Materials ... 9

Laboratory equipment ... 10

HPLC Systems ... 10

10. Results... 13

11. Archiving ... 15

12. Figures not included in the Text... 16

12.1. Figures ... 16

12.1.1. Chromatograms ... 16

(6)

BioProof AG The Proof of Concept Company

4. Catalogue of tables and figures

Tab. 1: Peak values (main peak) of different dyes in acetone: olive oil (1+3) at different time

points... 13

Fig. 1: Example chromatogram of dye A ... 16

Fig. 2: Example chromatogram of dye B... 17

Fig. 3: Example chromatogram of dye C... 17

Fig. 4: Example chromatogram of dye D ... 18

Fig. 5: Example chromatogram of dye E... 18

Fig. 6: Example chromatogram of dye F ... 19

(7)

BioProof AG The Proof of Concept Company

5. Signatures

Munich, date Franziska Anzenberger

study performance

Munich, date Dr. Michael Schulz

study director

Munich, date Ingrid Lamprecht

study report

Munich, date Dr. Alexander von Nieciecki

head test facility

(8)

BioProof AG The Proof of Concept Company

6. Introduction

The objective of this study was testing the stability of 6 different dyes in a mixture of acetone and olive oil (1+3) as vehicle.

The dyes were suspended in the vehicle in concentrations of 15 % /dye A – E) and 9 % (dye F). The dyes were not soluble in these concentrations in this vehicle.

The following dyes were used:

BSL Nummer Test item 053226A YELLOW E-JD 3442 053226B C.I. Reactive Red 231 053226C Produkt P-4G

053226D C.I. Reactive Yellow 174 053226E NAVY 14 08 723

053226F Dispersionsrot 2754

To simplify matters the dyes were furthermore named A – F.

(9)

BioProof AG The Proof of Concept Company

7. Summary

The dyes were suspended in an acetone/olive oil mixture, so it was difficult to take

homogeneous samples, due to the distribution of different sized dye particles. Therefore some differences in peak size did not result from stability but of difficult sample handling.

Dye B showed no degradation in between 5 hours, also dye F which gave two peaks. Only one peak was selected for calculation. Different peak areas were found for Dyes A and C which are resulting of the difficult handling of the suspension, but also no degradation was observed. The peak areas of Dye D and E were stable if the areas of the two main peaks were added.

8. Principle

8.1. Method description

The pigments were weighed into a PP-tube in a concentration according to the following table:

Conc. (%) weight (mg/mL)

A 15 150

B 15 150

C 15 150

D 15 150

E 15 150

F 9 90

1 ml of the vehicle was pipetted to the dyes and mixed on a vortex mixer. 10 µL were withdrawn at following time points: 0 h, 0.5 h, 1 h, 2 h and 5 h and then diluted with methanol.

Due to the insolubility of the dyes in the vehicle the dilution was very problematic. The different peak values could be explained thereby.

Dye D and E the chromatograms were not reproducible the first time, so they were weighed five- fold (for each time point separately) the half amount and mixed with 0.5 mL of vehicle.

Subsequently 0.5 mL water were added and mixed. After a holding time of 15 minutes the upper phase was suspended and an aliquot of the residue was diluted with water and injected.

(10)

BioProof AG The Proof of Concept Company

9. Experimental part

Instrumentation and reagents Chemicals

Water (prepared by Milli-Q plus) Millipore, 65760 Eschborn, Germany

Methanol Merck KGaA, 64271 Darmstadt,

Germany

Aceton No. 34850 Sigma-Aldrich, 91625 Schnelldorf,

Germany

Reference items

Dyes A -F Sponsor

Materials

0.3 mL PP-short thread micro-vial 32x11.6 mm

AV059321 Alltech Associates, Inc; 82008 Unterhaching, Germany 0.3 mL PP-short thread micro-vial

32x11.6 mm

AV059321 Alltech Associates, Inc; 82008 Unterhaching, Germany

5 mL tubes with caps, 57x15.3 mm Sarstedt, 51588 Nümbrecht, Germany General laboratory equipment and glassware

(11)

BioProof AG The Proof of Concept Company

Laboratory equipment

Half micro balance AT261, DeltaRange, Mettler, 35396 Gießen, Germany Autom. Pipettes, various volumina Eppendorf Reference, 22331 Hamburg, Germany

Multipette pro, Eppendorf, 22331 Hamburg, Germany

Shaker Jahnke und Kunkel KG,

IKA-Vibrax-VXR, 79219 Staufen i. Breisgau, Germany

HPLC Systems

• HPLC equipment Pump

Degasser

Auto sampler (cooled to 5 ºC)

Alliance, Waters 2695 Separations Module, Waters, 65760 Eschborn, Germany

Analytical column ReproSil Pur ODS 3 5µ 150x4.6mm, Maisch , 72119 Ammerbuch 3, Germany

Data system (quantitative evaluation)

Millennium32 (Version 3.20), Waters, 65760 Eschborn, Germany

• HPLC parameters

Solvent Solvent A: 100 % Milli-Q water, Solvent B: 100% Methanol

Flow 0.8 mL/min

Column temperature 35 °C

Injection volume 10 µL

(12)

BioProof AG The Proof of Concept Company

Gradient dye A:

Gradient Time %A %B Flow mL/min 0.0 80.0 20.0 0.8

2.0 80.0 20.0 0.8 16.0 0.0 100.0 0.8 20.0 0.0 100.0 0.8 21.0 80.0 20.0 0.8

Run time 21 min

Gradient dye B:

5.0 98.0 2.0 0.8 10.0 5.0 95.0 0.8 15.0 5.0 95.0 0.8 16.0 98.0 2.0 0.8

Run time 16 min

Gradient dye C:

Gradient

Time %A %B Flow mL/min 0.0 95.0 5.0 0.8

3.0 95.0 5.0 0.8 10.0 5.0 95.0 0.8 15.0 5.0 95.0 0.8 16.0 95.0 5.0 0.8

Run time 16 min

Gradient dye D:

Gradient Time %A %B Flow mL/min 0.0 99.0 1.0 0.8 5.0 99.0 1.0 0.8 10.0 5.0 95.0 0.8 15.0 5.0 95.0 0.8

(13)

BioProof AG The Proof of Concept Company

Gradient dye E:

5.0 98.0 2.0 0.8 10.0 5.0 95.0 0.8 15.0 5.0 95.0 0.8 16.0 98.0 2.0 0.8

Run time 16 min

Gradient dye F:

15.0 0.0 100.0 0.8 20.0 0.0 100.0 0.8 21.0 80.0 20.0 0.8 25.0 80.0 20.0 0.8

Run time 25 min

• Detector

Modell Photodiode Array Detector, Waters 996

Detection wavelength 234 nm

Start wave length 210 nm

End wave length 600 nm

Sampling Rate 1.0

Resolution 2.4

Auto Exposure Yes

Interpolate Yes

Filter Response 1

Channel 1 Off

Channel 2 Off

(14)

BioProof AG The Proof of Concept Company

10. Results

The peaks of each chromatogram were integrated and the peak areas of the main peak at different time points were compared. For none of the dyes a constant loss of peak area was indicating degradation. Especially in the case of dye A large variations were observed, but with no clear tendency. These variations were due to problems in the sampling from

suspension, possibly caused by large variations in the size clots aggregates) formed by the dye particles in this oily suspension.

In addition each chromatogram was checked for peaks of potential degradation products. In none of the chromatograms any additional peaks were raising during incubation time. This confirms the conclusion that no degradation of any of the dyes under investigation is occurring.

Tab. 1: Peak values (main peak) of different dyes in acetone: olive oil (1+3) at different time points

Dye A (repeat measurement) Dye B

Time points Peak area

% Time points Peak area

%

start 69.9 start 95.4

0.5 h 65.9 0.5 h 96.2

1 h 83.7 1 h 95.3

2 h 87.3 2 h 95.0

5 h 55.0 5 h 96.2

Dye C Dye F

Time points Peak area

% Time points Peak area

%

0.5 h 53.8 0.5 h 64.6

1 h 53.0 1 h 65.3

2 h 50.9 2 h 66.2

5 h 52.4 5 h 66.1

(15)

BioProof AG The Proof of Concept Company

Dye D Dye E

Time points

Peak area D1+D2

%

Time points

Peak area E1 + E2

%

0.5 h 97.8 0.5 h 100.0

1 h 98.1 1 h 100.0

2 h 98.2 2 h 100.0

5 h 91.7 5 h 100.0

(16)

BioProof AG The Proof of Concept Company

11. Archiving

After completion of the study all chromatographic raw data (chromatograms, methods, summary tables), MS Excel tables and MS Word documents (study protocol, report) were saved on the BioProof network. The following documents are archived in the BioProof archive:

• laboratory notebooks and sample tables

• raw data and interim results as computer print-outs (calibration lines, chromatograms, reports, excel tables)

• report

• CD-ROM comprising all relevant data

(17)

BioProof AG The Proof of Concept Company

12. Figures not included in the Text

12.1. Figures

12.1.1. Chromatograms

Fig. 1: Example chromatogram of dye A

(18)

BioProof AG The Proof of Concept Company

Fig. 2: Example chromatogram of dye B

(19)

BioProof AG The Proof of Concept Company

Fig. 4: Example chromatogram of dye D

Fig. 5: Example chromatogram of dye E

(20)

BioProof AG The Proof of Concept Company

Fig. 6: Example chromatogram of dye F