Evaluation of albumin structural modifications through cobalt-albumin binding (CAB) assay

•A cobalt albumin binding (CAB) assay was developed with a reaction buffer (50mM NaH2PO4, pH 7.4).•Dithiothreitol (DTT) concentration and minimal sample volume were set for optimal reaction.•It was confirmed that structural changes of human serum albumin (HSA) were detectable by CAB assay.•CAB assay could detect conformational changes of HSA induced by various stimulants.•CAB assay has the potential to be used for quality control of albumin injections. Human serum albumin (HSA) is the most abundant protein in the human body. HSA injections prepared by fractionating human blood have mainly covered the demand for albumin to treat hypoalbuminemia, the state of low concentration of albumin in blood. HSA in solution may exist in various forms such as monomers, oligomers, polymers, or as mixtures, and its conformational change and/or aggregation may occur easily. Considering these characteristics, there is a great chance of modification and polymer formation during the preparation processes of albumin products, especially injections. The albumin cobalt binding (ACB) test reported by Bar-Or et al. was originally designed to detect ischemia modified albumin (IMA), which contains the modified HSA N-terminal sequence by cleavage of the last two amino acids. In this study, we developed a cobalt albumin binding (CAB) assay to correct the flaws of the ACB test with improving the sensitivity and precision. The newly developed CAB assay easily detects albumin configuration alterations and may be able to be used in developing a quality control method for albumin and its pharmaceutical formulations including albumin injections.