Cell culture processes for monoclonal antibody production.docx - Cell culture processes for monoclonal antibody production Animal cell culture

Cell culture processes for monoclonal antibody production.docx

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Cell culture processes for monoclonal antibody production Animal cell culture technology requires optimization of a number of variables 1. Cell lines capable of synthesizing the required molecules at high productivities that ensure low operating cost 2. Culture media and bioreactor culture conditions that achieve both the requisite productivity and meet product quality specifications 3. Appropriate online and off line sensors capable of prodiving information that enhances process control 4. Good understanding of culture performance at different scales to ensure smooth scale- up Successful implementation also requires appropriate strategies for process development, scale up and process characterization and validation that enable robust operation that ensure compliance with current regulations Introduction - Antibody therapies may require large doses over a long period of time, manufacturing capacity becomes an issue because the drug substance must be produced in large quantities with cost and time efficiency - Mammalian cells have been considered difficult to work with due to low yield, medium complexity, serum requirement and shear sensitivity - Cell culture process development starts with cell line generation and selection, followed by process and media optimization in small scale systems Mammalian expression systems - Therapeutic antibodies are mainly produced in mammalian host cell lines including NS0 myeloma cells, PER6 human cells, and Chinese hamster ovary cells o This expression system is determine by its ability to deliver high productivity - NS0 cells lack endogenous glutamine synthetase enzyme activity making them suitable for use with GS as a selectable marker for recombinant antibody expression - Mouse derived cell lines, including NS0 produce N-glycolylneuraminic acid ( this cannot be synthesized by humans) - Murine cells, including NS0 produce alpha-gal-alpha (1,3) gal linkages which humans have expressed anti-bodies against it o NS0 cells are limitly used due to potential immunogenicity aspects - CHO cells are predominant host used to produce therapeutic proteins Cell line development - Mammalian expression vectors contain one cassette for antibody genes and selectable marker gene(s) for expression in mammalian cells, and a second cassette for the genes enabling plasmid replication in bacteria - CMV promoter and EF1a promoter are used to drive antibody heavy and light chain expression
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o Often an intron sequence in the 5’ untranslated region is included after the promoter/enhancer to increase export of transcribed mRNA to the cytoplasm from the nucleus, and one or more 3’ polyadenylation signal sequences are included to maximize mRNA levels - Transcription, translation and secretion are also required for antibody production - Electroporation and lipofection are the most common choices of transfection - Transfection cells are then selected, relying on different selectable markers that can be categorized into two groups; metabolic selectable markers and antibiotic selectable markers -
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