MONOCLONAL ANTIBODIES

1. PHARMACEUTICAL TECHNOLOGY-II ( PRACTICAL) MONOCLONAL ANTIBODIES

2. Monoclonal Antibody:- “An  antibody  produced by a single clone of cells. Monoclonal antibody  is therefore a single pure type of   antibody ”

3. CONT’D It is a protein used by the immune system to identify and neutralize foreign objects like bacteria and viruses. Each monoclonal antibody recognizes a specific antigen.  

4. ANTIBODIES POLYCLONAL MONOCLONAL Derived from different B Lymphocytes cell lines. Batch to Batch variation affecting Ab reactivity & treatment. NOT Powerful tools for clinical diagnostic tests . Derived from a single B cell clone. No Batch to Batch variations. Effectiveness of Ab is much more predictable. Enable the development of secure immunoassay systems.

5. Types of Monoclonal Antibodies

6. ADVANTAGES : 1) Homogeneity: Monoclonal antibody represents a single antibody molecule that binds to antigens with the same affinity and promote the same effectors functions. 2) Specificity : The product of a single hybridoma reacts with the same epitope on antigens. 3) Immunizing Antigen: Need not be pure or characterized and is ultimately not needed to produce large quantities of antibody. 4) Selection: It is possible to select for specific epitope specificities and generate antibodies against a wider range of antigenic determinants. 5) Antibody Production: Unlimited quantities of a single well-defined monospecific reagent.

7. Disadvantages: 1) Affinity: Average affinity of monoclonal antibodies are generally lower than polyclonal antibodies. 2) Effector Functions : Because antibody is monoclonal, it may not produce the desired biologic response. 3) Specificity: Monoclonals against conformational epitopes on native proteins may lose reactivity with antigens. 4) Cross reactions : Antibodies sometimes display unexpected crossreactions with unrelated antigens. 5) Time and effort commitment : VERY LARGE .

8. METHODS OF PRODUCTION

9. PURIFICATION TECHNIQUES: 1) FILTRATION METHOD: Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 um filter.

10. 2) CHROMATOGRAPHY : Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography . column chromatography can also use which is much quicker method.

11. 2 ) ANTIBODY HETEROGENEITY: Product heterogeneity is common to monoclonal antibody and other recombinant biological production and is typically introduced either upstream during expression or downstream during manufacturing. These variants are typically aggregates,  deamidation  products,  glycosylation  variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions. These seemingly minute changes in a monoclonal antibody’s structure can have a profound effect on preclinical stability and process optimization as well as therapeutic product potency,  bioavailability , and immunogenicity . The generally accepted method of purification of process streams for monoclonal antibodies includes capture of the product target with Protein A, elution, acidification to inactivate potential Mammalian viruses, followed by cation exchange chromatography, and finally anion exchange chromatography.

12. 3) RECOMBINANT: Recombinant antibody engineering involves the use of  viruses or  yeast  to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different  amino acid  sequences from which antibodies with desired specificities can be selected.  These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detect ability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.

13. 4 ) CHIMERIC ANTIBODIES: In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells, and the expression of this chimeric DNA through cell culture yielded partially mouse, partially human monoclonal antibody. For this product, the descriptive terms "chimeric" and " humanised " monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in the recombinant process.

14. Cont’d Chimeric antibodies take their name from the chimera, a mythical beast with the head of a lion, the body of a goat and the tail of a dragon. The Rituxan chimera is a hybrid of antibodies from both human and murine (mouse) sources. The CD20 antigen is injected into a mouse, encouraging the production of antibodies. Antibody producing cells are then isolated from the spleen of the animal. These are then combined with immortal cells called myeloma cells. This results in a cell line that will go on producing the antibody.

15. 5) FULLY HUMAN MONOCLONAL ANTIBODIES: A method to generate fully human monoclonal antibodies using blood from normal donors without previous exposure to the antigen. The technology uses to generate an antigen presentation system, which emulates the  natural presentation of antigens  by the immune system .

16. APPLICATIONS OF MONOCLONAL ANTIBODY

17. 1) IN CANCER :- Radio immunotherapy  (RIT ) Antibody-directed enzyme prodrug therapy (ADEPT) Immunoliposomes

18. 2) IN AUTOIMMUNE DISEASES: Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in  rheumatoid arthritis , Crohn's disease and ulcerative Colitis . Basiliximab and   daclizumab   activated  T cells and thereby help preventing acute rejection of kidney transplants. Omalizumab inhibits human immunoglobulin E ( IgE ) and is useful in moderate-to-severe allergic asthma.

19. 3) IN DIAGNOSTIC TEST: Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones . e.g . monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits. Another diagnostic uses of antibodies is the diagnosis of AIDS by the ELISA test.

20. LIST OF FDA’s APPROVED MONOCLONAL ANTIBODIES

21. Antibody Brand name Type Indication Abciximab ReoPro chimeric Cardiovascular disease Adalimumab Humira human Several auto-immune disorders Alemtuzumab Campath humanized Chronic lymphocytic leukemia Basiliximab Simulect chimeric Transplant rejection Belimumab Benlysta human Systemic lupus erythematosus Bevacizumab Avastin humanized Colorectal cancer, Age related macular degeneration Brentuximab Vedotin Adcetris Chimeric Hodgkin lymphoma

22. Antibody Brand name Type Indication Canakinumab Ilaris Human Cetuximab Erbitux chimeric Colorectal cancer, Head and neck cancer Certolizumab pegol Cimzia humanized Crohn's disease Daclizumab Zenapax humanized Transplant rejection Denosumab Prolia , Xgeva Human Postmenopausal osteoporosis Eculizumab Soliris humanized Paroxysmal nocturnal hemoglobinuria Efalizumab Raptiva humanized Psoriasis

23. Antibody Brand name Type Indication Palivizumab Synagis humanized Respiratory Syncytial Virus Panitumumab Vectibix human Colorectal cancer Ranibizumab Lucentis humanized Macular degeneration Rituximab Rituxan, Mabthera chimeric Non-Hodgkin lymphoma Tocilizumab Actemra Humanised Rheumatoid arthritis Tositumomab Bexxar murine Non-Hodgkin lymphoma Trastuzumab Herceptin humanized Breast cancer