Monoclonal Antibodies

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Chapter: Pharmaceutical Microbiology : The Manufacture And Quality Control Of Immunological Products

Monoclonal antibodies are immunoglobulins or a fragment of an immunoglobulin, with defined specificity, produced by a single clone of cells. They can be obtained from immortalized B-lymphocytes that are cloned and expanded as continuous cell lines (murine and human monoclonal antibodies) or from rDNA-engineered mammalian or bacterial cell lines (engineered monoclonal antibodies).


MONOCLONAL ANTIBODIES

 

A)                   Preparation

 

Monoclonal antibodies are immunoglobulins or a fragment of an immunoglobulin, with defined specificity, produced by a single clone of cells. They can be obtained from immortalized B-lymphocytes that are cloned and expanded as continuous cell lines (murine and human monoclonal antibodies) or from rDNA-engineered mammalian or bacterial cell lines (engineered monoclonal antibodies). Production of monoclonal antibodies is based on a seed lot system using a master cell bank and a working cell bank derived from the cloned cells. Two approaches are currently in use: single harvest (produc tion at finite passage level) and multiple harvest (continuous-culture production). In the first method, the cells are cultivated up to a defined maximum number of passages or population doublings (in accordance with the stability of the cell line). In the second method, cells are continuously cultivated for a defined period (in accordance with the stability of the system and production consistency). In this case, monitoring is necessary throughout the life of the culture; the required frequency and type of monitoring will depend on the nature of the production system. Bulk harvests can be made by pooling individual harvests before purification. The purification process to remove unwanted host cell derived proteins, nucleic acids and carbohydrates is also designed to remove and/or inactivate non-enveloped and enveloped viruses and other impurities.

 
B)                Quality Control

 

The quality of therapeutic monoclonal antibodies is controlled by rigorous characterization of the products by chemical and biological methods. The final product should be tested for bioburden and bacterial endotoxins, purity, integrity and potency by suitable analytical methods, comparing with a reference preparation. The identity test can be done by suitable methods comparing the product with the reference preparation. Molecular size distribution can be determined by size-exclusion chromatography. Depending on the nature of the monoclonal antibody, its microheterogeneity and isoforms, a number of different tests can be used to demonstrate molecular identity and structural integrity. These tests may include peptide mapping, isoelectric focusing, ion-exchange chromatography, hydrophobic interaction chromatography, oligosaccharide mapping, monosaccharide content and mass spectrometry. The purity can be examined by SDS–polyacrylamide gel electrophoresis under reducing and non-reducing conditions or capillary electrophoresis. For monoclonal antibodies with specific antimicrobial activity, an appropriate assay is performed to determine the level of this. Monoclonal antibodies have been developed for postexposure prophylaxis of anthrax (anti-Protective Antigen), botulism (anti-toxins A–G) and plague (anti-V antigen). Monoclonal antibodies directed against the core region of Gram-negative bacterial endotoxins and intended for treatment of sepsis, have met with little success.


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