An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies
Transcripción
After preparing buffers and initializing the FPLC system according to the text protocol, use the method editor to set up the run steps as follows. Equilibrate the system with five column volumes or CV of binding buffer.
Load the sample onto the column and collect the sample flow through, and then use five CV of binding buffer to wash the system. Use five CV of elution buffer to elute the column via isocratic fractionation. and collect the purified antibody samples as fractions using the fraction collector.
Finally, equilibrate the system with five CV of binding buffer and collect the flow through into a waste container. Once the method setup is complete, specify the volume of conditioned medium to be applied to the column and save the method.
Next, submerge the sample pump tubing into the vessel containing the conditioned medium. Then, prepare a separate container to collect sample flow through via the specified outlet tubing. Insert collection tubes in the fraction collector and add neutralization buffer to each collection tube. In the system control module of the software, open the method to be used. Then click Start to initiate the run.
When the purification is complete and the evaluation module, check the resulting chromatogram. Combine all the protein-containing fractions into a tube, and then use PBS and a centrifugal filter device with a 30-kilodalton cutoff to concentrate the sample and carry out buffer exchange. Finally, use the BCA assay to measure the antibody concentration according to the manufacturer's instructions.
