PHARMACEUTICAL TECHNOLOGY-II

(PRACTICAL)

MONOCLONAL ANTIBODIES

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

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.

ANTIBODIESPOLYCLONAL 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.

Types of Monoclonal Antibodies

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.

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.

METHODS OF PRODUCTION

PURIFICATION TECHNIQUES:

1) FILTRATION METHOD:

Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 um filter.

2) CHROMATOGRAPHY:

a) Most of the charged impurities are usually anions such as nucleic acids and endotoxins. These are often separated by ion exchange chromatography.

b) column chromatography can also use which is much quicker method.

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.

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.

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.

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.

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.

APPLICATIONS OF MONOCLONAL ANTIBODY

1) IN CANCER:-

Radio immunotherapy (RIT)

Antibody-directed enzyme prodrug therapy (ADEPT)

Immunoliposomes

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.

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.

LIST OF FDA’s APPROVED MONOCLONAL ANTIBODIES

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 degenerationBrentuximab

VedotinAdcetris Chimeric Hodgkin lymphoma

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

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