2. Advanced Library Generation Tobias Knaute / Johannes Zerweck Peptide Arrays JPT Peptide Technologies, Berlin

3. 2 What´s the Problem? The peptide library generation for determining antibody / antigen interactions gets complex if many proteins are involved Post-translational modifications increase complexity even more Number of peptides is limited due to experimental setup (max. 7000 peptides per experiment) Problem: How to generate the most efficient library concerning coverage and content

4. 3 Conventional Method Fixed Overlap Overlap = Length - Offset CON: Insufficient for many homologous sequences Could lead to bloated peptide lists Coverage not adjustable PRO: Easy to understand & implement Sufficient for simple screenings and epitope mappings MGARASVLSGGELDR RASVLSGGELDRWEK VLSGGELDRWEKIRL GGELDRWEKIRLRPG LDRWEKIRLRPGGKK... N C Generation of overlapping peptides based on primary structure MGARASVLSGGELDR RASVLSGGELDRWEK VLSGGELDRWEKIRL GGELDRWEKIRLRPG LDRWEKIRLRPGGKK...

5. 4 Advanced Method Dynamical Overlap Algorithm enabling optimal coverage of protein sequences by peptide libraries Task 1: find optimum between number of peptides and coverage Task 2: generate peptide-score according to frequency of motifs (e.g. in other homologous sequences) Task 3: ability to inject fixed sequences (e.g. from previous projects) Task 4: ability to adjust coverage and/or number of peptides to specific needs

6. 5 Example Task: - 2 homologous sequences - make peptide scan - peptide length 15 - maximum overlap 11 - make sure every residue is covered by at least 3 peptides Protein 1 Protein 2

7. 6 Example - Convential Scan Convential scan Fixed overlap 15/11 Peptides of protein 1 Protein 1 Peptides Protein 2

8. 7 Example - Convential Scan Convential scan Fixed overlap 15/11 Peptides of protein 2 Peptides Protein 1 Protein 2

9. 8 Example - Convential Scan Convential scan Fixed overlap 15/11 All individual peptides, mapped to protein 1 Redundancy introduced by homology.  Several peptides from protein 2 fit into sequence of protein 1 Peptides 4567778754 Number of peptides covering specific amino acid in protein sequence  amino acid coverage Protein 1 Protein 2

10. 9 Example - Dynamical Overlap All peptides, mapped to protein 1  Redundant peptides are rejected by algorithm Overlap adjustable by coverage constraint. Peptides overlap 8 334444443 Amino acid coverage is optimized Protein 1 Protein 2

11. 10 Case Study: HIV1 ENV gp160 24 Sequences of Group M Subtype B highly conserved region highly variable region Task: Minimum coverage: 3 peptides/residue Length: 15 Max. overlap: 11 3283 peptides needed for traditional method 2303 peptides needed for advanced method

12. 11 Case Study: HIV1 ENV gp160 Residue Coverage Map Advanced 3283 peptides mean density: 6.7 peptides/residue 2303 peptides mean density: 3.7 peptides/residue Conventional

13. 12 Summary Algorithm to build optimal libraries developed Coverage / number of peptides can be adjusted Elimination of redundant motifs Addresses diversity, best suited for proteome-wide sequence alignments in complex libraries Applicable to PepStar TM microarrays, PepTrack TM Libraries, PepMix TM peptide pools... Based on the input of protein sequences, JPT will develop an optimized library for seroprofiling or immune monitoring experiments

14. 13  JPT Peptide Technologies

15. 14 History 1994Jerini Spin-Off from Charité Clinics Berlin 2002 Jerini Peptide Technologies - Business Unit 2004 JPT Peptide Technologies GmbH 2005 JPT Inc. in Boston, MA, USA 2006 New JPT Facilities in Berlin 2009Acquisition by BioNTech AG 2011 50 Employees, 13 PhD‘s Offices & Reps Berlin, Boston, Denver, Brussels 1500 sqm wet lab and 1200 sqm office space Capabilities for organic & biological laboratory work Approved S2 lab for infectious patient samples Cleanroom microarray capabilities Business develoment & marketing History, Quality Assurance & Business Model Quality Assurance 2002 Quality Management System 2004 DIN ISO 9001:2000 Certification Since 2008 GCLP Compliance Audits (NIH, IAVI, BCM, Cellmedica, Genticel...) 2010 DIN ISO 9001:2008 Certification Business Model http://jpt.com http://rnd.jpt.com http://shop.jpt.com

16. 15 Major Application Fields of Peptides Immunology Vaccine development B- and T-cell epitope discovery Diagnostic tools for infectious, autoimmune diseases, allergies & cancer Immune monitoring & immune therapies Proteomics & Enzyme Profiling Enzyme profiling and protein/protein interaction studies Quantification of protein expression levels Biomarker discovery Drug Discovery Biomedical screening Peptide therapeutics SAR, structure activity relationship studies

17. Feel free to discuss your specific needs with our experts ! JPT Peptide Technologies GmbH Volmerstrasse 5 12489 Berlin, Germany T +49-30-6392-5500 X +49-30-6392-5501 info@jpt.com www.jpt.com -> http://jpt.com for Custom Products & Services - > http://rnd.jpt.com for Peptide Related Collaboration Projects - > http://shop.jpt.com for Catalog Products