ABSTRACT
With the size of the biopharmaceutical market exponentially increasing, there is an aligned growth in the importance of data-rich analyses, not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships. In monoclonal antibodies, many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known. The importance of their function focuses analytical research efforts on the development of robust, accurate and fast methods to support drug development and quality control. Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however, it is not the only method for quantitative analysis of glycoform heterogeneity. In this study, ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies. While observing good comparability between the quantitative results generated, it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow ac-cording to application and the desired depth of data generated.
ABSTRACT
OBJECTIVE: To evaluate the applicability of UPLC/MS method for the identification test of human serum albumin (HSA) products including plasma derived and recombinant HSA samples. METHODS: ACQUITY UPLC with Vion IMS QT of LC/MS system was used combined with on-line HSA sample desalting with ACQUITY UPLC BEH C18 column. The acquired multiplycharged mass spectrum was processed with MaxEnt1 automatic protein deconvolution software in UNIFI, which can transfer the raw mass spectrometry data to zero charge molecular mass or mass distribution of the intact protein. RESULTS: Intact protein mass analysis not only provided the accurate mass of HSA, but also provided an overall view of the heterogeneity of HSA and the relative amounts of various forms. From this study, a very specific mass signal [(66 437 ± 1), which is the theoretical average MW of human serum albumin ]was obtained from all the six HSA samples. And the characteristic spectra of different samples were also got. CONCLUSION: UPLC/MS method has very good specificity and high sensitivity and can distinguish HSA products made by different manufacturers and processes. The total analytical time is 10 min, which is ideal for the QC identification test of HSA products.