1. Purity profiling and the associated functional effects of a new peptide
Mathieu Verbeken1, Romain Lefebvre2, Els Goossens3, Walter Luyten4,
Liliane Schoofs5 and Bart De Spiegeleer1,*
1 DruQuaR (Drug Quality and Registration) group, Faculty of Pharmaceutical Sciences,
Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
2 Department of Pharmacology, Faculty of Medicine and Health Sciences, Ghent University,
De Pintelaan 185, B-9000 Ghent, Belgium
3 VAR-CODA-CERVA, Groeselenberg 99, B-1180 Ukkel, Belgium
4 Faculty of Biology, Leuven University, Naamsestraat 59 box 2469, B-3000 Leuven, Belgium
5 Department of Animal Physiology and Neurobiology, Faculty of Biology, Leuven University, Naamsestraat 59 box 2469, B-3000 Leuven, Belgium
*Corresponding author: Tel.: ; Fax: , b

2. 1. Introduction
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
Identification of new peptides or peptide leads with the
potential of serving as a basis towards new peptidic drugs!
[1] Croner B. Peptides as drugs: Discovery and development. Wiley VCH (2002).
BGFW Forum Spa DruQuaR, Fac. Pharm. Sciences, Ghent University

3. 1. Introduction Mouse peptidomics Synthetic peptides
2. Objective
3. Methods
4. Results
5. Conclusion
Mouse peptidomics
Synthetic peptides
Functional screening
Decision: Yes/No
BGFW Forum Spa DruQuaR, Fac. Pharm. Sciences, Ghent University

4. 2. Objective Knowing: small changes in peptide structure
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
Knowing: small changes in peptide structure
big functionality changes
Initially “active” peptide coded SBO215_7:
impurity profiling functionality ?
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5. 3. Methods - Functionality
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
Tissue organ bath (TOB) experiments – screening of a peptide-database:
Principle: In vitro recording of smooth muscle contractions in isolated preparations using a
force transducer
Tissues (isolated):
Mouse vas deferens
Rat aortic ring
Guinea pig ileum and trachea
Screening: administration of peptides and interpretation of smooth muscle recordings
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6. 3. Methods - Analytics
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
HPLC-DAD/UV-ESI/iontrap MSn:
Column: Grace/Vydac C18 monomeric (+ guard), 250 x 4.6 mm, 5 µm
Column temperature: 30°C
Gradient method: in 45 min from 95:5 H2O/ACN % FA (m/V) to 5:95 H2O/ACN % FA (m/V)
Flow rate: 1.0 ml
Detection: from 195 nm to 400 nm [quantification: 210 nm]
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7. Crude vs. high purity peptide material (one supplier)
4. Results
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
TOB results of a peptide coded SBO215_7 – baseline induced contractions:
Crude vs. high purity peptide material (one supplier)
Guinea pig ileum
BGFW Forum Spa DruQuaR, Fac. Pharm. Sciences, Ghent University

8. Three crude suppliers vs. a high purity peptide: 10-4 M
4. Results
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
Three crude suppliers vs. a high purity peptide: 10-4 M
Guinea pig ileum
GLB
> 95 %
GLB
The Sci
Pi Prot
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9. 4. Results GLB GLB > 95 % NOT OR Three HPLC peaks The Sci Pi Prot
Requirements for the synthesis related impurity causing the functional effect:
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
Reporting threshold: 0.5 %
List up impurities using Boolean operators: Yes/No
GLB
GLB
> 95 %
NOT OR
Three HPLC peaks
of peptide synthesis related impurities
(in range of 10-6 M)
The Sci
Pi Prot
BUT
AND
Currently: identification MSn
Synthesis for confirmation
Functionality on GPI
Observation: completely different impurity-quality profile
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10. 5. Conclusion
1. Introduction
2. Objective
3. Methods
4. Results
5. Conclusion
Functionality screening of new peptides in tissue bath experiments: Quality aspects are generally neglected in this phase of investigation, leading to wrong fail/pass decisions:
e.g. crude vs high purity SBO215_7 material: different pharmacological outcome due to low level impurities
QUALITY OF PEPTIDES IS AN IMPORTANT ASPECT IN THEIR FUNCTIONAL SCREENING USING TOB EXPERIMENTS TO AVOID FALSE POSITIVE FUNCTIONALITY CONCLUSIONS.
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11. Thank you for your attention!!!
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