Determining the Stoichiometry of a Protein–Polymer Conjugate Using Multisignal Sedimentation Velocity Analytical Ultracentrifugation



Susan M. Clardy1, David H. Lee1, Peter Schuck2


  1. Mersana Therapeutics Inc., Cambridge, Massachusetts 02139, United States
  2. National Institutes of Health, Bethesda, Maryland 20892, United States


Advances in polymer science have broadened the applications of protein–polymer conjugates as biopharmaceuticals and biotechnology reagents. The complex nature of these conjugates makes characterization challenging. Here, we describe the use of multisignal sedimentation velocity analytical ultracentrifugation to measure the polymer-to-protein ratio. To demonstrate the principle, we applied this approach to a series of antibody–drug conjugates with different polymer-to-protein ratios and various degrees of heterogeneity, and validated results with orthogonal analytical techniques. We found that multisignal sedimentation velocity can provide accurate information on key attributes including polymer-to-protein ratio, which is important for maximizing the therapeutic potential of future protein–polymer conjugates.

CovalX Technology Used



Amid the growing development of protein-polymer conjugate therapeutics, the protein-to-polymer ratios (PPR) of these large and complex compounds can be difficult to analyze. However, the use of a multisignal sedimentation velocity analytical ultracentrifugation (MSSV) has been shown to accurately provide spectral determinations of protein-polymer conjugate PPRs alongside other quality attributes. This is crucial for broadening the therapeutic applications of protein-polymer conjugates. The MSSV utilized an Autoflex III MALDI-ToF Mass Spectrometry with an HM4 interaction module from CovalX, which can accommodate large molecules of up to 2MDa with nanomolar sensitivity. By employing CovalX systems, the MSSV was able to ascertain the various PPRs in a series of antibody-drug conjugates, demonstrating that multisignal sedimentation velocity can provide accurate characterization of protein-polymer conjugates.


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Categories : Publications, High-Mass MS