In the poster presented in this blog post, scientists from our Sunnyvale (USA) research lab describe a straightforward method for creating designer assays for QC analysis of monoclonal antibodies (mAb) using our HPLC-compatible, streptavidin-coated monolith column. This affinity column produces narrow concentrated peaks with minimal diffusion, allows for titer analysis over a wide concentration range, and is applicable for a range of applications from small-scale protein to antibody titer analysis.
Titled, How to Create Your Own Affinity Titer Column, (downloadable PDF), the poster was presented at this year’s HPLC conference and states, “Leveraging the benefits of reactive polymeric monoliths, affinity media can be created with low non-specific binding and a pore structure that provides excellent elution kinetics. Efficient convective mass transfer and minimal diffusion allows for much narrower elution bands, resulting in more concentrated fractions and a higher level of sensitivity required for titer analysis at low sample concentrations. In addition, high binding capacity extends the range of quantification limiting the need for sample dilution.”
The image is of a schematic of monolith bound streptavidin, conjugated with biotinylated Protein A, used for antibody analysis.
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The poster includes detailed information on ligand preparation and addition and also loading the column with the biotinylated ligand; a schematic on how the column is loaded is also included. One of our HPLC systems (Thermo Scientific Dionex UltiMate 3000 HPLC system) equipped with a UV detector and a prototype, protein-identification HPLC column (Thermo Scientific ProSwift Streptavidin HPLC column) was used in the method development.
As per the results, the researchers conclude that the column can bind over 8 mg/mL bed biotin-BSA, the efficient binding kinetics result in minimal loss of precious ligand, and excellent elution characteristics allow concentrated peaks to be collected for further analysis.
- Do check out a growing library of monoclonal antibody applications on this blog.
- Our recently released Chromatography Solution Online Center features many useful and complimentary chromatography tools which can help speed up your analysis. The site is updated on a monthly basis; therefore, do check out the Archives section to see what was previously featured.
If you have questions on the method or the techniques, do enter them in the Comments box below; our experts look forward to hearing from you