Proteoform Characterization of HIV-1 Broadly Neutralizing Antibody PGT 121.414.LS Product Through Middle-Up and Bottom-Up Proteomics for Clinical Support

Broadly neutralizing antibody (bNAb) therapies are under development for the prevention and treatment of HIV-1 infection as an alternative to conventional antiretroviral therapy because of their potential for sustained passive immunotherapy. Currently, bNAbs targeting the HIV-1 viral envelope proteins are included in research protocols to evaluate their role in preventing transmission and in combination antiviral regimens to achieve viral suppression. While quantification of bNAbs is of clinical importance, validation of the structural fidelity of therapeutic antibodies is also an important consideration to assess the clinical efficacy. Here, we applied middle-up and bottom-up proteomics workflows for the quality assurance of the primary structure of the HIV-1 bNAb PGT 121.414.LS product. Middle-up and bottom-up proteomics workflows were performed using liquid chromatography coupled to an Orbitrap mass spectrometer. Middle-up analysis of the crystallizable fragment (Fc/2), light chain (Lc), and fragment denaturation (Fd, N-terminal fragment of the heavy chain) regions of PGT 121.414.LS was performed by using IdeS digestion, which revealed proteoform heterogeneity. To complement the middle-up approach, a bottom-up workflow combining trypsin and chymotrypsin digests was performed for detailed glycoform mapping and annotation. The bottom-up results indicated that Lc contained an additional N-terminal Ser residue. For the Fc, two abundant and two lower-abundance glycoforms of the heavy chain were detected that correspond to Asn-312 (Asn-297 in the consensus heavy chain sequence of IgG). For Fd, bottom-up analysis revealed eight sialylated glycoforms and five nonsialylated glycoforms at Asn-124 of the heavy chain. The results emphasize bNAb heterogeneity, which should be considered in affinity binding studies.