Bispecific antibodies and fusion proteins are novel biologics that combine the specificities of two different molecules and simultaneously target two epitopes. Such biologics with ‘two target’ functionality can interfere with multiple surface receptors and aid in enhanced bio-therapeutic efficacy. Assembling a bispecific molecule from two different parental molecules expressed in the same producer cell may result in various non-functional forms with respect to bispecificity. Here we look into the active and inactive forms of a bispecific Fc-fusion protein. A mass spectrometric analysis of IdeS subunits revealed the identical amino acid sequence of the two forms, but significant differences in inter-chain hinge region disulfide bonding and some variations in O-linked glycosylation. Majority of the inactive form was found to lack inter-chain hinge region disulfide bonds and therefore present as ‘half-molecules’. SE-HPLC analysis was performed on 40oC heating time-course samples, where a rapid formation of ‘half-molecules’ for the inactive form validates the previous findings. Interestingly, the hinge region cysteines were all in oxidized form as shown by peptide map LC-MS analysis, indicating the occurrence of intrachain disulfide bond shuffling, instead of simple under-disulfide bonding. Disulfide bond mapping is currently underway to resolve the detailed disulfide bonding structures of both active and inactive forms. Intrinsic tryptophan fluorescence study has shown the upper-level structural differences between the two, and further biophysical characterization will be performed to demonstrate the inactive form is a conformer of the active form.

Mass spectrometric characterization of a recombinant ‘two-target’ fusion protein

256th National Meeting (2018)

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