monoclonal antibody : production & application.pptx

monoclonal antibody : defination, type, importance, nomenclature, production, application, approved marketed products

1. Monoclonal Antibodies ; Preparation and Application Guided by : Mr. Arya. S. Vyas Presented by : Harshid Kukadiya M.Pharm (Sem II) Department of Pharmaceutics L. M. College of Pharmacy, Ahmedabad

2. TABLE OF CONTENTS Antigen, Antibody, Monoclonal Antibody; structure & type Introduction Production major steps of mAb production Application D ifferent application of in research & industry A pproved mAb Approved mAb for treatment by authority 01 02 03 04

3. Introduction A ntigen-Antibody S tructure of antibody M onoclonal antibody 01

4. A ntigen-Antibody An antigen is a foreign substance that enters your body. This can include bacteria, viruses, fungi, allergens, venom and other various toxins. An antibody is a protein produced by our immune system to attack and fight off these antigens. A ntigen Antibody

5. Antibody structure Also known as an  immunoglobulin  ( Ig) They are glycoproteins comprising of 82–96% polypeptides and 4–18% carbohydrates. They are large, Y-shaped protein having four peptide chains, produced by plasma cells Two of these fragments are identical and retain the antigen binding capacity associated with an intact antibody. The two identical long chains are called heavy chains and the two identical short chains are called light chains .

6. They are the antibodies that are identical because they were produced by one type of immune cell, all clones of a single parent cell. Monoclonal antibody

7. Monoclonal antibodies (mAb) are a single type of antibody that are identical and directed against a specific epitope (antigen, antigenic determinant). They are produced by B-cell clones of a single parent or a single hybridoma cell line. A hybridoma cell line is formed by the fusion of a one B-cell lymphocyte with a myeloma cell. Monoclonal antibodies can have monovalent affinity , in that they bind to the same epitope. The idea of " magic bullets " was first proposed by Paul Ehrlich , who, at the beginning of the 20th century, postulated that, if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity.

8. Nomenclature of Monoclonal Antibody

9. TABLE OF CONTENTS Antigen, Antibody, Monoclonal Antibody; structure & type Introduction Production major steps of mAb production Application D ifferent application of in research & industry A pproved mAb Approved mAb for treatment by authority 01 02 03 04

10. P roduction of monoclonal antibodies 02

11. Production steps Immunization of mouse Cell fusion Isolation of hybridomas of single specificity Characterization and storage Screening of products Selection of Hybridomas Cloning and propagation

12. Immunization of mouse Mouse is immunized by injecting with an appropriate antigen along with adjuvant. Injection of antigens at multiple sites are repeated several times for increased stimulation of antibodies. 3 days prior to killing of an animal a final dose is given intravenously. Spleen is aseptically removed and disrupted to release the cells. By centrifugation lymphocytes are separated from rest of the cells.

13. Cell fusion Lymphocytes are mixed with myeloma cells and is exposed to PEG for a short period. The mixture is then washed and kept in fresh medium. The mixture contains hybridomas, free myeloma cells, and free lymphocytes.

14. Selection of Hybridomas HAT selection H = Hypoxanthine A = Aminopterin Blocks De nova pathway T = Thymidine Only cells capable of nucleotide synthesis by the salvage pathway survive in HAT medium. HAT are metabolites for the salvage pathway. ( Key enzyme : HGPRT )

15. Isolation of hybridomas of single specificity This is done by method known as limited dilution. The cells are distributed in multi well culture plates at very low density

16. Screening, cloning and propogation of products The two techniques namely ELISA and RIA are commonly used for the screening purpose. In next step desired antibody are identified and they are isolated and cloned. Separate clones of activated B cells, each producing antibodies of a single specificity known as monoclonal antibody.

17. Characterization and storage In the final step, these monoclonal antibodies are characterized and stored. Mostly they are stored in liquid nitrogen. Now this monoclonal antibodies are ready to use in treating and diagnosis diseases.

18. TABLE OF CONTENTS Antigen, Antibody, Monoclonal Antibody; structure & type Introduction Production major steps of mAb production Application D ifferent application of in research & industry A pproved mAb Approved mAb for treatment by authority 01 02 03 04

19. Application of mAb 03

20. Therapeutic application : Immune‐mediated diseases Antibody Type Medical uses Alemtuzumab Humanized, mAb, IgG1 Multiple sclerosis Brodalumab IgG2Human, mAb, IgG2 Plaque psoriasis Canakinumab Human, mAb, IgG1 Cryopyrin‐associated periodic syndrome Monoclonal antibodies have revolutionized the treatment of autoimmune diseases. It can target different components of the immune system .

21. Oncology Antibody Type Medical uses Trastuzumab Humanized, mAb, IgG1 Metastatic breast cancer Rituximab Chimeric, mAb, IgG1 non‐Hodgkin lymphoma anfd eukemia Avelumab Human, mAb, IgG1 Merkel‐cell carcinoma treatment of various neoplasias , including both hematologic ma‐ lignancies and solid tumors To target tumor antigens and kill cancer cells

22. Infectious diseases Antibody Type Medical uses Bezlotoxumab Human, mAb, IgG1 Clostridium difficile colitis Raxibacumab Human, mAb, IgG1 Anthrax (prophylaxis and treatment) There are advantages of mAbs for the treatment of infections, over immune sera-derived preparations, such as low lot-to-lot variability, low risk of pathogen transmission, and no immunological complications associated with the use of heterologous sera.

23. Diagnostic application M onoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents.  Anti β HCG antibody can be used to detect pregnancy only 14 days after conception. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal infections.

24. Diagnostic application M onoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents.  Anti β HCG antibody can be used to detect pregnancy only 14 days after conception. Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes, streptococcal infections.

25. As therapeutic agent In destroying disease causing organisms : mAb promote efficient opsonization of pathogenic organisms (by coating with antibody) and enhance phagocytosis. In the immunosuppression of organ transplantation : in the normal medical practice, immunosuppressive drugs such as cyclosporin and prednisone are administered to overcome the rejection of organ transplantation. In recent years, mAbs specific to T-lymphocyte surface antigens are being used for this purpose.

26. Protein purification Monoclonal antibodies can be produced for any protein and can be used conventionally for the purification of the protein against which it was raised. These include the specificity of the mAb to bind to the desired protein, very efficient elution from the chromatographic column and high degree of purification.

27. Blood group Typing Before blood transfusion, blood group typing is essential. Blood group typing is carried out to find out compatibility between donor and recipient blood groups. in all cross-match tests, a specific chemical reaction of antibodies with erythrocyte antigens is carried out to monitor agglutination Recently at the National Institute of Immunology monoclonal antibodies against blood group antigens A and B have also been developed.

28. As a vehicle for drug delivery An antibody can be used as an attached drug (magic bullets) or toxin (immunotoxin) at cancer cells. By this approach, higher concentration of the drug can be built up locally, minimizing systemic toxicity. Indeed a far better survival rate is observed in experimental mice given Daunamycin loaded on mAb raised against mouse lymphomas as compared to those receiving the drug alone. mAb covalently bound to a toxin or toxin-subunit knowm as immuno-toxin have been observed to be effective in therapy of some cancers

29. TABLE OF CONTENTS Antigen, Antibody, Monoclonal Antibody; structure & type Introduction Production major steps of mAb production Application D ifferent application of in research & industry A pproved mAb Approved mAb for treatment by authority 01 02 03 04

30. Approved mAb 04

31. Product name Dosage form Concentration (mg / ml) indication Approved year Sponsor Susvimo (Ranibizumab) Liquid   100 Neovascular (wet) Age-related macular degeneration 2021 Roche/Genentech Aduhelm (Aducanumab- avwa ) Liquid   100 Alzheimer’s disease 2021 Biogen and Eisai Vyepti ( Eptinezumab-jjmr ) Liquid   100 Migraine   2020 Lundbeck Beovu (Brolucizumab- dbll ) Liquid 120 Neovascular (wet) AMD 2019 Novartis Aimovig ( Erenumab-aooe ) PFS 70,140 Migraine 2018 TEVA PHARMS USA

32. GTU Questions : Explain in detail the method of producing monoclonal antibodies by hybridoma technology. ( winter 2018 ) Describe in brief the applications of monoclonal antibodies. ( winter 2018 ) 3. Discuss method of preparing monoclonal antibodies. ( winter 2019 ) 4. Discuss preparation and applications of Monoclonal Antibodies. ( winter 2021 ) 5. Write a short note on monoclonal antibodies. ( winter 2022 ) 6. Describe the pharmaceutical applications of monoclonal antibodies.’ ( summer 2022 ) 7. Describe the method of producing monoclonal antibodies by hybridoma technology. ( winter 2018 ) 8. State the applications of monoclonal antibodies in drug targeting giving Examples. ( summer 2021 )

33. Reference : [1] Bayer, V. An Overview of Monoclonal Antibodies. In Seminars in oncology nursing ; Elsevier, 2019; Vol. 35, p 150927. [2] Fesseha , H. Monoclonal Antibody and Its Diagnostic Application- Review. BJSTR , 2020 , 30 (4). https://doi.org/10.26717/BJSTR.2020.30.004997 [3] Berger, M.; Shankar, V.; Vafai , A. Therapeutic Applications of Monoclonal Antibodies. The American Journal of the Medical Sciences , 2002 , 324 (1), 14–30. https://doi.org/10.1097/00000441-200207000-00004 [4] Payne, W. J.; Marshall, D. L.; Shockley, R. K.; Martin, W. J. Clinical Laboratory Applications of Monoclonal Antibodies. Clinical Microbiology Reviews , 1988 , 1 (3), 313–329. https://doi.org/10.1128/cmr .1.3.313  

34. [5] Production of Monoclonal Antibodies - Yokoyama - 2013 - Current Protocols in Immunology- WileyOnlineLibrary https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471142735.im0205s102 (accessed Mar 16, 2024) [ 6 ] Wang, S. S.; Yan, Y. (Susie); Ho, K. US FDA-Approved Therapeutic Antibodies with High-Concentration Formulation: Summaries and Perspectives. Antib Ther , 2021 , 4 (4), 262–272. https://doi.org/10.1093/abt/tbab027

35. Thank you..