1. Molecular characterization, detection & quantitation of biological products Purin Charoensuksai, PhD Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University

2. Example of critical checkpoints influencing biological activity and safety of biological products Vector construction Coding sequence Expression Modification of proteins PTMs Cleavages/truncation Aggregation Host cell proteins/ DNA Residual growth factors Adventitious agents Virus Mycoplasma Stability of cell lines Location of insert Copy number of insert Purification Ability to concentrate target species/clear up contaminants Leachables from columns Re-usage of columns Storage until usage Stability Storage conditions Picture in cover slide taken from: http://www.imapac.com/business_conference /biologics-world-taiwan-2016/the-concept/

3. Various laboratory techniques are routinely used for the characterization of biological products Nucleic acid – AGE – Hybridization – Sequencing – PCR, qPCR – Microarray Protein – Amino acid analysis – Edman degradation – Peptide mapping: HPLC, MS, MS-MS – SDS-PAGE & protein staining – IEF – 2D-gel Immunological assays – Precipitation/ agglutination – Western blot – ELISA Picture taken from: http://www.imapac.com/business_conference/biologics- world-taiwan-2016/the-concept/

4. Molecular assays for biological products Our main focuses: – Brief review of the principle of each assay – Example of their usage in biological product registration Multiple levels: nucleic acids, protein, immunological assays Common types of assays: – Qualitative: detect the presence of something – Quantitative: determine the exact (?) amount of something – Limit test (semi-quantitative): check if the level of something exceeds certain amount

5. Molecular analysis of nucleic acids DNA agarose gel electrophoresis Nucleic acid hybridization DNA sequencing PCR, qPCR Microarray

6. DNA agarose gel electrophoresis (AGE) Separate fragments of DNA based on size 6

7. Porous structure of molecular sieve used to resolve DNA/proteins Picture taken from: ocw.mit.edu/courses/biological-engineering/20-109-laboratory-fundamentals-in-biological-engineering-spring-2010/labs/module-1-day-2-purify-aptamer-encoding-dna/

8. DNA in agarose gels can be visualized by various staining methods Ethidium bromide Fluorescent DNA dye Ethidium bromide

9. Picture taken from: http://www.discoveryandinnovation.com/BIOL202/notes/lecture22.html Picture taken from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/BasePairing.html

10. Hybridization of nucleic acids Southern blot & Northern blot Picture taken from: http://www.gene-quantification.de/mrna.html Southern blot > DNA Northern blot > RNA 10 1 2 34 1.AGE 2.Transfer to solid support (blotting) 3.Hybridization with probe specific for sequence of interest 4.Detection

11. Applications of nucleic acid hybridization UncutTaqISmaI TaqI & SmaI Ladder Uncut TaqISmaI TaqI & SmaI Ladder DNA gel electrophoresisSouthern Blotting Picture taken from: http://www.discoveringdna.com/ Example: roughly map the location of DNA insert (infer genetic stability of cell line)

12. https://www.youtube.com/watch?v=nudG0r9zL2M 12 DNA sequencing: Sanger method

13. 13 Sanger sequencing is also known as a chain-termination method

14. Chain-termination method 5 main components: – DNA template – Primer – dNTPs – fluorescent labelled ddNTPs – DNA polymerase Sequence 1 fragment (read 500-1000 bps) at a time Example: check DNA sequence of insert (DNA or mRNA) 14 DNA sequencing: Sanger method

15. Polymerase chain reaction (PCR) Amplification of target DNA 15

16. Picture taken from: http://www.biosyn.com/tew/taqman-vs-sybr-green-chemistries.aspx Quantitative Polymerase Chain Reaction (qPCR)

17. Picture taken from: http://www.mypols.de/qpcr-probe-2x-master-mix/ A B

18. Some examples of PCR/qPCR applications for the characterization of biological products PCR Amplify desired gene from suitable host for expression Detection of adventitious agents e.g. virus or mycoplasma Detection of host cell DNA qPCR Determine the copy number of insert in master cell, working cell or cell at the end of production

19. DNA microarray Based on hybridization between probes & DNA of interest A Large number of probes are fixed on a solid support (CHIP) enabling the interrogation of multiple targets simultaneously By Squidonius https://commons.wikimedia.org/w/index.php?curid=39422948

20. DNA microarray Picture taken from: http://bitesizebio.com/7206/introduction-to-dna-microarrays/

21. Molecular analysis of proteins Polyacrylamide gel electrophoresis Edman degradation: N-terminal sequencing Peptide mapping: analysis of proteolytic cleavage pattern Mass-spectrometry: MS or MS-MS

22. Polyacrylamide gel electrophoresis (PAGE) Separate proteins based on size 22 Usually performed in a denaturing condition (SDS-PAGE) Can be adapted to resolve proteins in their native conformation (native gel)

23. Proteins in polyacrylamide gels can be visualized by various staining methods Coomassie brilliant blue Silver stain Fluorescent stain e.g. Sypro Ruby http://www.komabiotech.com/product/product_detail.php?item=K14010

24. Edman degradation Phenyl isothiocyanate Phenylthiohydantoin (PTH)- amino acid derivatives are identified through chromatography N-terminal sequencing, up to 30 amino acids Will not work if N-terminal amino group is modified or buried within protein

25. Peptide mapping 1.Fragmentation of protein e.g. proteolytic cleavage by enzymes which cleave specific bond 2.Resolve peptides with appropriate methods e.g. SDS-PAGE, HPLC, MS, etc. www.promega.com

26. Peptide mapping Lys-C = K / X Arg-C = R /X

27. Some examples of peptide mapping applications in the registration of biological products Peptide mapping/finger printing reflects the identity of the parent protein Usage: Identity of drug substance Purity: detect modified forms of drug substance e.g. some PTMs

28. Protein mass-spectrometry (MS) analysis https://pharmchem.ucsf.edu/research/physbio/proteomics

29. 2 types of protein mass-spectrometry (MS) analysis: Top-down and Bottom-up http://circgenetics.ahajournals.org/content/4/6/711/F1.expansion.html

30. Applications of protein MS analysis Mass fingerprint: MS Protein quantitation: quantitative MS Protein sequencing: MS-MS Post-tranlational modification characterization: MS-MS Example of MS application for the registration of biological products: analysis of amino acid variants e.g. deamidation, oxidation, glycation or glycosylation profile of drug substance

31. Immunological assays Exploit antigen-antibody interaction Examples: Precipitation/agglutination reactions Western blot ELISA

32. Precipitation/Agglutination https://www.youtube.com/watch?v=aV8NPSiPh_4

33. Precipitation/Agglutination Antibody/antigen interaction Similarity: antibody crosslink antigen and form precipitate Difference: nature of antigen – Precipitation = soluble antigen – Agglutination = insoluble antigen e.g. RBC, bacteria, antigen fixed on beads (HCG, bacterial toxins, etc.) Picture taken from: https://www.studyblue.com/notes/note/n/final-exam/deck/2914433

34. Example of precipitation/agglutination reaction Hemaglutination assay http://www.cdc.gov/flu/professionals/laboratory/antigenic.htm

35. Applications of hemagglutination assays http://www.vetfolio.com/clinical-pathology/a-primer-on-diagnostic-virology- specimen-selection-and-serology

36. Western blot 36 Resolve proteins by SDS-PAGE

37. Western blot 1.Transfer proteins in gel onto other solid membrane Nitrocellulose PVDF (Polyvinylidene difluoride) 2.Stain with antibody specific to the protein of interest 3.Detect with appropriate methods Colorimetric Chemiluminescent Fluorescent, IR etc. 37

38. Diversity of amino acid

39. Isoelectric focusing

40. 2D-SDS-PAGE

41. ELISA Enzyme-Linked ImmunoSorbent Assay Comparison of test samples with standard antigen with known concentration yield semi-quantitative/quantitative measurement of antigen in test sample

42. Example of ELISA application for the registration of biological products Host cell protein Leachable protein A Antibiotics Insulin Some small molecules: HEPES, resin components, etc.

43. Various laboratory techniques are routinely used for the characterization of biological products Nucleic acid – AGE – Hybridization – Sequencing – PCR, qPCR – Microarray Protein – Amino acid analysis – Edman degradation – Peptide mapping: HPLC, MS, MS-MS – SDS-PAGE & protein staining – IEF – 2D-gel Immunological assays – Precipitation/ agglutination – Western blot – ELISA Picture taken from: http://www.imapac.com/business_conference/biologics- world-taiwan-2016/the-concept/

44. Thank you!