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In pre-clinical in vivo studies of therapeutic antibody candidates, it is essential that the antibody products used for animal studies be free of endotoxin and antibody aggregates. Therefore, it is always required to remove antibody fragments and aggregates generated in cell culture and downstream purification processes as well to remove endotoxin. Some antibodies are prone to aggregation, especially during elution from protein A/G column at low pH. Chromatographic purification steps are needed to remove aggregates and increase monomer antibody purity.
Endotoxin is another big challenge in pre-clinical production using transient transfection. This is because a large amount of expression plasmid is usually added to the cell culture and the plasmid has to be produced in E coli. fermentation. Endotoxin's property is quite similar to the DNA plasmid used for transient transfection. Therefore, one needs to remove as much as possible endotoxin from the DNA plasmid production and purification process, and control endotoxin level throughout the transient transfection and purification process to avoid introducing endotoxin into the process and removal any residual endotoxin from the antibody product. Experience and expertise is critical to achieve effective endotoxin control. In many cases, process development is needed to fine-tune process parameters for effective endotoxin removal.
Sino Biological has extensive experience in producing high-quality monoclonal antibodies meeting requirements for GLP studies. For monomer purity, the minimum requirement is typically >95%. In some cases where customers may need to use very high doses in animal studies, we have produced many antibodies with a monomer purity >99%. For endotoxin, most customers require us to achieve a level below 1 EU/mg. Again, in cases where very doses are employed in animal studies, we have produced antibodies with an endotoxin level <0.1 EU/mg. The higher the monomer purity and the lower the endotoxin level, the higher the production cost. In you service request, please specify a suitable monomer purity and endotoxin level so that we can provide the most economic quote for producing your antibodies that can meet your requirement.
Antibody monomer purity is controlled through removing antibody aggregates and fragments with polishing chromatographic steps in downstream purification. Aggregates and fragments can be separated from the monomer form of an antibody by surface charge differences, hydrophobic interaction, and by hydrodynamic size differences. Chromatographic methods include anion exchange, cation exchange, hydrophobic chromatography, and size exclusion column. One would need to combine experience and experiments to determine the best polishing steps and process parameters to remove aggregates and fragments effectively. Achieving a monomer purity of great 99% can be a real challenge, especially for antibodies that are prone to aggregate.
Endotoxin, derived from bacteria and typically introduced into cell culture through raw materials and containers, can be hard to remove from the antibody products. In a transient transfection, it is even more difficult because one would need to use DNA plasmids for cell transfection and the DNA plasmid is produced and purified from E. coli. fermentation, and hence it usually contains very high levels of endotoxin unless special cares and measures are taken to remove endotoxin from the plasmid. For some antibodies that bind to endotoxin, it is even a bigger challenge as the endotoxin molecules can co-purify with the antibody product.
Sino Biological has developed advanced platform technologies for rapid and effective antibody production and purification. Our successful production of >10,000 antibodies in the past seven years (2007-2014) demonstrates our capability and capability. We are proud to have served many customers worldwide in supporting their antibody discovery and development programs. We expect to fully leverage our platform technology and experience to serve more customers and make a bigger impact on supporting antibody product development.
How to Submit a Request for Antibody Production and Purification Service?
If you are interested in using our antibody purification services to support your antibody discovery and development program, please contact us by leaving an on-line message or send us your detailed request to CROfirstname.lastname@example.org. Based on the quantity and quality requirement of your request, a formal quote will be send back to you for your review, comment, and/or acceptance.