AUC 2022

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Sedimentation Velocity Studies of Pooled Human Serum
Submitter: John Correia
Authors: John J Correia, Reid Bishop, Patrick Kyle, P. J. Sherwood
Corresponding Author: John J Correia
Title: Sedimentation Velocity Studies of Pooled Human Serum
Contribution Type: Full Talk
Selected for Presentation Yes
Abstract: There is great interest in the biotech industry in investigating therapeutic proteins in high concentration environments like formulation conditions and human serum. The fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity (SV) AUC experiments in tracer or what has been called BOLTS protocols. Here we compare pooled (six) human serum samples by AUC SV techniques and demonstrate the potential and advantage of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin-HSA complex slowed by solution nonideality and exhibiting a JO effect due to presence of IgG. Absorbance SV experiments on diluted serum samples verifies the HSA-IgG composition as well as a significant IgM boundary at 19s. Alexa-488 labeled Simponi is utilized as a tracer to investigate the behavior of a therapeutic Mab in serum. Dilution series allow extrapolation to extract so, while direct boundary SEDANAL fitting verifies the utility of a matrix of phenomenological nonideality ks and BM1 parameters and the source of the JO effect. As a valadation of future applications, secondary Mabs to human IgG and IgM verify the formation of ~10 s 1:1 complexes with human IgG and 19 s complexes with human IgM pentamers. These data demonstrate that FDS AUC allows a wide range of potential approaches to investigating therapeutic antibodies in human serum environments.