1. Structures and functions of biomolecules & applications Purin Charoensuksai, PhD Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University

2. Definitions of Biological products http://www.fda.gov/AboutFDA/Transparency/Basics/ucm194516.htm Picture in cover taken from: http://chemistry.mtu.edu/~thompson/ch4710/quizes/final/final2005/final2005.html

3. Types of Biological products http://www.slideshare.net/robpuopolo/061713-biological-product-fda-basics-webinar-2

4. New biological products approved by US FDA in 2015 Trade NameActive ingredientCompanyIndicationType CosentyxsecukinumabNOVARTISplaque psoriasismAb Natparaparathyroid hormoneNPS PHARMShypocalcemia in hypoparathyroidismpolypeptide UnituxindinutuximabUNITED THERAPneuroblastomamAb PraluentalirocumabSANOFI AVENTIShypercholesterolemia (PCSK9 inhibitor)mAb RepathaevolocumabAMGENhypercholesterolemia (PCSK9 inhibitor)mAb Tresibainsulin degludec injectionNOVO NORDISKdiabetes mellituspolypeptide PraxbindidarucizumabBOEHRINGER INGELHEIMreverse Pradaxa’s blood-thinning effectsmAb Strensiqasfotase alfaALEXION PHARMhypophosphatasia (HPP)polypeptide NucalamepolizumabGLAXOSMITHKLINEasthmamAb DarzalexdaratumumabJANSSEN BIOTECHmultiple myelomamAb PortrazzanecitumumabELI LILLYadvanced (metastatic) squamous NSCLCmAb EmplicitielotuzumabBRISTOL MYERS SQUIBBmultiple myelomamAb Kanumasebelipase alfaALEXION PHARMlysosomal acid lipase (LAL) deficiencypolypeptide http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugInnovation/ucm430302.htm Of the 45 newly approved drugs in 2015, 13 are biological products The majority of new biological products are proteins

5. Complexity of biological products compared to conventional small molecules http://www.slideshare.net/robpuopolo/061713-biological-product-fda-basics-webinar-2

6. Key concerns of biological products Biological activity and safety Activity: proteins function through their structures Safety: modified forms, contaminants from production process give rise to safety concerns Any factors affecting these attributes must be addressed, characterized and reported for evaluation upon registration

7. Scope of today’s talk Molecular foundation of proteins From basic building blocks to three dimensional structure to mature and active proteins Concept of the production of biological products: Natural VS. Engineered Review how proteins are naturally produced from their blueprints within cells and how this process can be engineered for biopharmaceutical industries

8. The building blocks of proteins: Amino acids Picture modified from: http://www.nutrientsreview.com/proteins/amino-acids

9. Diversity of amino acid 20 different types of aa The difference is in the side chain (R) Nonpolar Polar Charged negative (acidic) positive (basic) Each has designated abbreviation 3 letters system 1 letters system Valine - Histidine - Serine Val - His - Ser VHS

10. Proteins Natural polymers made of amino acids Each amino acid is linked through peptide bond Two ends: N-terminus (amino) C-terminus (carboxyl) https://en.wikibooks.org/wiki/Structural_Biochemistry/Protei ns/Structures#/media/File:Aasequencedirection**.png https://www.mun.ca/biology/scarr/MGA2_03-21.html

11. Three dimensional structure of proteins "Main protein structure levels en" by LadyofHats

12. Three dimensional structure of proteins Immunoglobulins http://myplace.frontier.com/~dffix/medmicro/igs.htm

13. Forces stabilizing three dimensional structure of proteins Any factors disturbing these forces may then affect 3D structure of proteins and their activity

14. The diversity of protein post-translational modifications http://www.esciencecentral.org/ebooks/applications-of- molecular-genetics/posttranslatonal-modfcatons-of- proteins.php

15. Post-translational modifications regulate functions of proteins Jensen Nature Reviews Molecular Cell Biology 7, 391-403 (June 2006) | doi:10.1038/nrm1939

16. Protein glycosylation https://www.thermofisher.com/th/en/home/life-science/protein-biology/protein-biology-learning- center/protein-biology-resource-library/pierce-protein-methods/protein-glycosylation.html https://www.neb.com/applications/glycobiology

17. Effect of glycosylation on biological functions of antibodies Affect conformation of antibodies and their functions Efficacy, immunogenicity, stability, pharmacokinetic properties Factors affecting glycosylation include: species, tissue, clone, culture conditions Glycoengineering: KO/KI glycosyltransferases to obtain antibodies with desired glycosylation profile Jefferis R, Biotechnol. Prog. 2005, 21, 11-16 Liu H, et.al, mAbs. 6:5, 1145-1154

18. How proteins are naturally made within cells https://www.youtube.com/watch?v=gG7uCskUOrA

19. How proteins are naturally made within cells

20. Nucleic acids: DNA and RNA

21. Codons: Alteration in nucleotide sequence may alter amino acid sequence of protein, which may affect the biological activity or lead to undesirable outcomes

22. Recombinant DNA technology for the production of recombinant proteins Expression (Mammalian cells) Protein purification 22 http://www.bio.davidson.edu/Courses/Molbio/MolStudent s/spring2003/Keogh/plasmids.html

23. Key components of a plasmid Binding of transcription machineries Desired gene to be expressed For the propagation of plasmid Selectable marker: For the selection of desired clones “ATG” 23

24. From basic knowledge to applications: Identification of critical checkpoints influencing biological activity and safety of biological products 24 Vector construction Coding sequence Expression Modification of proteins PTMs Cleavages/truncation Aggregation Host cell proteins/ DNA Residual growth factors Adventitious agents Virus Micoplasma Stability of cell lines Purification Ability to concentrate target species or clear up contaminants Leachables from columns Re-usage of columns Storage until usage Stability Storage conditions

25. Summary Most of the new biological products submitted for registration are proteins Proteins exert their functions through their 3D structures. Any factors disturbing the forces stabilizing native conformation of proteins will affect their activities. DNA sequence Certain modification e.g. oxidation and reduction Storage conditions Multiple PTMs decorate proteins and may regulate their functions

26. Thank you!