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Organic peptides - new technologies for a bio-based
Organic peptides new technologies for a bio-based production Dr. Christian Schwarz, Synthesis of Peptides (SynPep) Peptides Chains of ≤ 100 amino acids § Advantages § high potency § high specificity § low toxicity § novel properties § Disadvantages § manufacturing costs § sensitivity to proteolytic digestion 2 SynPep Christian.Schwarz@hhu.de Global peptide market § 14.1 Billion USD in 2011 § Almost exclusively therapeutical peptides § Key segments § cancer § metabolic disorder § central nervous system § anti-infectives 3 Transparency Market Research, 2013 SynPep Christian.Schwarz@hhu.de Market Growth § 2010 § 67 marketed peptides § 150 in clinical phases § > 400 in advanced clinical phases Frost and Sullivan, 2010 4 SynPep Christian.Schwarz@hhu.de Challenges § Manufacturing strategies applicable on all scales § Quality of peptides § Downstreaming processing and isolation è Costs of goods 5 Frost and Sullivan, 2010 Lonza, 2006 SynPep Christian.Schwarz@hhu.de Production strategies Chemistry 6 Biotechnology SynPep Christian.Schwarz@hhu.de Chemical synthesis § Currently applied for ± 90% of peptides § nowadays ‘chemical peptides’ Advantages good for short peptides (< 30 mer peptides) automatable and scalable (short production times) 7 Disadvantages expensive raw materials challenging for larger peptides - insertions, deletions,… chain aggregation yield purification atom economy SynPep Christian.Schwarz@hhu.de Chemical synthesis Raw materials for 20 kg of an average 18 mer peptide Raw Material Requirements Amino acids 0.8 tonnes Fine chemicals 14 tonnes Organic solvents 430 tonnes Water 600 tonnes è Rather expensive approach 8 SynPep Christian.Schwarz@hhu.de Biotechnological production § Currently applied for < 10% of peptides Advantages relatively cheap scalability easy to perform short production time Disadvantages development time and costs isolation from expression host ‘only’ 20 natural building blocks è ‘organic peptides’ è No general production strategy available 9 SynPep Christian.Schwarz@hhu.de Chemistry vs. Biotechnology Proportion of overall costs Raw materials Production Chemistry 30-40% of overall costs 60% including puri#cation and isolation Biotechnology 5% 95% including puri#cation and isolation Leong, S., Singapore, Expression Systems for Peptide Production, 2007 10 SynPep Christian.Schwarz@hhu.de Biotechnological production § Challenges during expression 11 SynPep Christian.Schwarz@hhu.de Biotechnological production § Strategies § (large tandem polymer repeated units) 12 SynPep Christian.Schwarz@hhu.de Biotechnological production § Strategies § (large tandem polymer repeated units) § fusion with carrier protein 13 SynPep Christian.Schwarz@hhu.de Biotechnological production § Procedure § expression of fusion § § § § protein cell disruption purification separation of peptide from carrier protein purification è 5-50 mg of peptide/l Leong, S., Singapore, Expression Systems for Peptide Production, 2007 14 SynPep Christian.Schwarz@hhu.de Biotechnological production § Procedure § expression of fusion needless? § § § § 15 protein cell disruption purification separation of peptide from carrier protein purification SynPep Christian.Schwarz@hhu.de Biotechnological production § Procedure § expression of fusion needless? § § § § 16 protein cell disruption purification separation of peptide from carrier protein purification SynPep Christian.Schwarz@hhu.de Type 1 Secretion System in E. coli outer membrane protein kDa cell ex MW tract m cult arker ure sup erna tant Gram-negative cell 120 membrane fusion protein 50 30 ABC transporter 10 è up to 1 g/l pure protein 17 SynPep Christian.Schwarz@hhu.de Proteins HlyA HlyA1 LipA SprP PlaB MBP PlbF CalB Vif IFNA8 IFNA1 IFNA16 IFNA21 IFNA2 TesA IFABP Pep-des NisA HCRF Mab42 Mab40 Fuzeon® Calcitonin (human) 18 Calcitonin (salmon) Func-on Toxin -‐-‐-‐ Lipase HypotheCcal protease Phospholipase Maltose binding HypotheCcal lipase Lipase Virion infecCvity factor Interferon Interferon Interferon Interferon Interferon Esterase FaRy acid binding Func-on AnCbioCc CorCcoliberin Alzheimer‘s disease Alzheimer‘s disease HIV therapy Osteoporosis Osteoporosis SynPep MW [kDa] 110.5 23.7 64.8 62.7 49.5 41.9 35.0 33.8 22.7 19.5 19.4 19.3 19.3 19.2 18.9 15.1 MW [kDa] 6.0 4.9 4.6 4.5 4.3 3.6 3.6 Yields (mg/liter) > 100 > 900 ± 2 ± 7 ± 6 ± 4 ± 9 ± 2 ± 1 ± 1 ± 1 ± 1 ± 3 ± 3 ± 4 ± 1 Yields (mg/liter) ± 5 ± 6 ± 5 ± 3 ± 2 ± 2 Christian.Schwarz@hhu.de ± 1 Proteins HlyA HlyA1 LipA SprP PlaB MBP PlbF CalB Vif IFNA8 IFNA1 IFNA16 IFNA21 IFNA2 TesA IFABP Pep-des NisA HCRF Mab42 Mab40 Fuzeon® Calcitonin (human) 19 Calcitonin (salmon) Func-on Toxin -‐-‐-‐ Lipase HypotheCcal protease Phospholipase Maltose binding HypotheCcal lipase Lipase Virion infecCvity factor Interferon Interferon Interferon Interferon Interferon Esterase FaRy acid binding Func-on AnCbioCc CorCcoliberin Alzheimer‘s disease Alzheimer‘s disease HIV therapy Osteoporosis Osteoporosis SynPep MW [kDa] 110.5 23.7 64.8 62.7 49.5 41.9 35.0 33.8 22.7 19.5 19.4 19.3 19.3 19.2 18.9 15.1 MW [kDa] 6.0 4.9 4.6 4.5 4.3 3.6 3.6 Yields (mg/liter) > 100 > 900 ± 2 ± 7 ± 6 ± 4 ± 9 ± 2 ± 1 ± 1 ± 1 ± 1 ± 3 ± 3 ± 4 ± 1 Yields (mg/liter) ± 5 ± 6 ± 5 ± 3 ± 2 ± 2 Christian.Schwarz@hhu.de ± 1 ence carri er pr otein secre ted i nterf minu eron s car rier Activity of secreted interferons refer untr eate d § highly active interferons § formation of disul#de bonds 20 SynPep Christian.Schwarz@hhu.de General Secretion System for E. coli § Advantages § good yields for all expressed polypeptides in batch cultures § high initial purity for simplified downstreaming § formation of disulfide bonds § high activity of various proteins shown ✓ ✓ ✓ ✓ è ‘organic peptides’ 21 SynPep Christian.Schwarz@hhu.de General Secretion System for E. coli § Road map § up-scaling to increase yields § cost-efficient separation of proteins/peptides from carrier protein è Alternative route to organic peptides? 22 SynPep Christian.Schwarz@hhu.de Alternative route to organic peptides? § Production of 18 mer peptide ≈ 300 – 400 mg/l cell culture! 23 SynPep Christian.Schwarz@hhu.de Alternative route to organic peptides? è one-step puri#cation for ≥ 95% purity 24 SynPep Christian.Schwarz@hhu.de Alternative route to organic peptides? Raw Material Requirements for 20 kg of: 18 mer 18 mer Chemical Synthesis Alternative route* Amino acids 0.8 tonnes --- Fine chemicals 14 tonnes 0.1 tonnes Bu&er compounds --- 11 tonnes Organic solvents 430 tonnes 34 tonnes Water 600 tonnes 292 tonnes * calculated values based on the production of 50 mg of 18 mer peptide, no scaling e&ects considered. 25 SynPep Christian.Schwarz@hhu.de Alternative route to organic peptides! § Advantages § high yields (hundreds of mgs/l) § one-step purification § scalabel process § cost-efficient separation of carrier protein ✓ ✓ ✓ ✓ è ‘organic peptides’ 26 SynPep Christian.Schwarz@hhu.de Bio-based technologies of SynPep Organic peptides with the: 1. Type 1 Secretion System § production of proteins (and peptides) § high initial purity § high activity 2. Alternative route § several hundred mgs of peptides per liter § cost-effective production 27 SynPep Christian.Schwarz@hhu.de Acknowledgements Prof. Dr. K.-E. Jaeger 28 SynPep Christian.Schwarz@hhu.de Our team Dr. C. Schwarz Dr. T. Schwarz Prof. Dr. Dr. S. Smits L. Schmitt Questions? Christian.Schwarz@hhu.de 29 SynPep Christian.Schwarz@hhu.de
