Mass Spectrometry Characterization of HIV Broadly Neutralizing Antibody PGT 121

Broadly neutralizing antibodies (bNAbs) such as PGT 121.414.LS (PGT 121) represent a promising therapeutic approach for preventing and treating HIV-1 infection. Traditional ligand-binding assays (LBAs) used in pharmacokinetic/pharmacodynamic (PK/PD) studies often struggle with the structural complexity and proteoform diversity of these antibodies. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) provides a powerful alternative, enabling both quantitative analysis and detailed structural characterization.

PGT 121 is a V3 glycan-dependent bNAb produced in mammalian cells, and its structure is influenced by various post-translational modifications (PTMs) such as glycosylation, which affect stability, receptor binding, and therapeutic efficacy. To comprehensively characterize PGT 121, researchers at the University of Buffalo combined middle-up and bottom-up LC–MS/MS workflows. The middle-up analysis examined intact subunits (Fc/2, Lc, and Fd), while bottom-up peptide mapping (using trypsin and chymotrypsin digests with PNGase F treatment) provided high-resolution information on PTMs and glycoforms. Their study identified 4 Fc glycoforms (mainly G0F and G1F), 13 unique glycoforms at Asn-124 on the heavy chain, and an unexpected N-terminal serine on the light chain. Together, these findings illustrate the heterogeneity of PGT 121 proteoforms and underscore the importance of integrating middle-up and bottom-up mass spectrometry for full structural elucidation. Such detailed characterization is critical for ensuring quality control, supporting PK/PD interpretation, and guiding the clinical development of therapeutic bNAbs.

LCGC International spoke to Troy Wood of the University of Buffalo, corresponding author of the paper (1) that resulted from this study, about his group’s work and their findings.

What advantages does LC–MS/MS offer over traditional LBAs for pharmacokinetic and pharmacodynamic studies of therapeutic antibodies like PGT 121.414.LS?
LC–MS/MS provides enhanced specificity compared to traditional LBAs in the quantification of therapeutic antibodies. LBAs rely on the selective binding of the target antibody to an immobilized capture antibody. Method development can be time-consuming due to the requirements and synthesis of specialized reagents (2). Additionally, their accuracy and reproducibility can be impacted by non-specific interactions, causing reduced specificity at lower concentrations. In contrast, LC–MS/MS enables direct quantitation of the antibody by measuring enzymatic peptides derived from the antibody’s unique complementarity-determining regions. This approach not only improves specificity but can also be used to measure several analytes simultaneously. The additional ability of LC-MS/MS to measure several antibodies is becoming increasingly desired with co-administered antibody therapies.

How does liquid chromatography contribute to the separation and identification of proteoforms in complex biotherapeutics such as monoclonal antibodies?
Liquid chromatography allows for separation of peptides resulting from enzymatic digestion. Liquid chromatography is used to separate the modified peptides allowing for sensitive detection of lower abundance modifications. A traditional limitation of electrospray ionization is the suppression of low abundance species by higher abundance species. Enzymatic digestions of antibodies result in a complex mixture with many peptides with varying signals. The ability to separate this mixture is critical to identifying peptides containing modifications in low stoichiometric abundance.

Can you explain the main differences between bottom-up and middle-up LC–MS workflows when characterizing monoclonal antibodies?
Bottom-up and middle-up LC–MS workflows differ primarily in the size of the antibody fragments analyzed, the enzymes used, and mass spectrometer parameters used. In a bottom-up workflow, antibodies are enzymatically digested, commonly with trypsin, into small peptides. These peptides are then separated by LC and analyzed by mass spectrometry, allowing for the primary sequence confirmation and detection of subtle PTMs, such as deamidation and oxidation, due to the sensitivity of tryptic peptides. Bottom-up workflows are used both in characterization workflows as well as quantitation methods.

Middle-up workflow implements more specific enzymes, often combined with reduction of disulfide bonds, to generate larger fragments such as intact light chains, half crystallizable fragments (Fc/2) and heavy chain fragments of the Fab (Fd). This approach enables rapid characterization of major structural features and larger PTMs like glycosylation, with less extensive sample preparation and shorter LC run times. Middle-up workflows offer rapid insight into the major structural features of an antibody of rapid characterization, while bottom-up provides detailed mapping of small modifications.

Why is combining bottom-up and middle-up chromatographic workflows advantageous for the comprehensive characterization of PTMs?
Combining middle-up and bottom-up workflows offers detailed insight into the structure of the antibody. Bottom-up provides detailed mapping of small modifications, while middle-up facilitates efficient profiling of larger structural attributes. In our study, we were able to view glycosylations present in both the Fc and Fd locations of the antibody. Fc glycosylations were identified in a conserved site of the Fc/2 fragment as expected using the high-resolution masses for the Fc/2 fragments. The annotations were further confirmed using the high resolution and tandem mass spectra of the tryptic peptides containing the glycosylation site. Additionally, the combination of workflows was advantageous as we identified a complex glycosylation site located in the Fd fragments. In the middle-up workflow, deconvolution was difficult due to the large number of glycoforms simultaneously present, causing signal suppression in the mass spectrum. Following up with our bottom-up workflow, we were able to separate the glycosylated peptides from the region and with better sensitivity, were able to annotate the glycosylations present using the high-resolution masses of the tryptic peptides. This application demonstrated how multiple workflows provide complementary information and can improve the annotations of PTMs.

Describe how enzymatic digestion is integrated into LC–MS/MS workflows and how it affects chromatographic peptide mapping.
Enzymatic digestion is typically performed post sample extraction or purification and is typically one of the final steps prior to sample analysis. Predigestion preparation steps, such as denaturation of the antibody and reduction of disulfide bonds, are commonly performed prior to digestion to improve digestion efficiency. Intact antibodies are large (~150 kDa), complex biomolecules. Direct analysis of intact, non-denatured antibodies is difficult and requires high concentration antibody samples due to low sensitivity. The large size of the antibodies also produces mass spectra with broad isotopic patterns, making the identification of small mass shifts due to oxidation or deamidation difficult to distinguish. Enzymatic digestion is implemented to overcome this limitation by reducing the intact antibody into peptides that can be separated by LC and provide a higher sensitivity for mass spectrometry. For characterization, small modifications can be observed using enzymatic digestion that cannot be observed when analyzing the intact antibody.

What types of post-translational modifications can be detected and quantified using LC–MS/MS, and why are these important for understanding bNAb quality attributes?
There are several post-translational modifications that are examined using LC-MS/MS. A major PTM of interest for broadly neutralizing antibodies is glycosylation. Glycosylation is a common modification among immunoglobulin Gs with a conserved glycosylation site located in the crystallizable fragment (Fc) of the antibody. Glycosylation is of interest in bNAbs for their role in Fc-mediated functions that are associated with optimal activity in vivo. Interest in the role of glycosylation has led to investigations into glycoengineering of bNAbs to enhance the elimination of HIV-1 in cells (3). The interest in glycosylations on bNAb activity demonstrates the need for characterization of these sites. Additionally, LC-MS/MS can investigate other amnio acid specific modifications located in the complementary determining region of the antibody. PTMs located in this region can have direct impacts on antigen binding. The bNAb CAP256-VRC26 for example, contains an O-sulfated tyrosine located in the heavy chain complementary determining region H3 loop that is important for the antibody’s function (4). The implementation of LC-MS/MS to characterize these sites is important for the continued development of engineered bNAbs.

How can LC–MS/MS–based peptide mapping be used to confirm the primary sequence and identify glycosylation sites in antibodies like PGT 121.414.LS?
LC-MS/MS peptide mapping can be used to confirm the primary antibody sequence by comparing the MS/MS data with the expected peptides based on the expected amino acid sequence of the antibody. The fragmentation spectra of the peptides are used to identify the amino acids present in the peptide. In collisional induced dissociation, the peptide backbone is fragmented and generate fragments that differ by the masses of amino acids. The fragmentation spectra are then used along with the high resolution MS¹ mass to identify the peptide based on the theoretical peptides from the amino acid sequence. High sequence coverage is commonly achieved and can confirm agreement between the predicted sequence and the observed sequence. Discrepancies between observed peptides and theoretical peptides can also be annotated in the case of non-agreement. The glycosylation sites can be localized using the high-resolution masses of the enzymatic peptides which have masses that differ from their expected masses by the masses of expected glycans. The fragmentation of the antibody by enzymatic digestion allows us to localize the location of glycosylations.

In the context of PGT 121.414.LS, what does the identification of Fc glycoforms such as G0F and G1F through chromatographic analysis reveal about product consistency and therapeutic function?
The identification of the various glycoforms in our study shows that our PGT 121 product contains a mixture of glycoforms. The glycoforms of PGT 121 are being investigated for their effect on therapeutic function. The Fc-mediated functions of bNAb activity are currently of interest for their effect on activity and possible role in protection against infection. Glycoengineering studies are ongoing for plant derived PGT 121 for investigations into the impacts of glycosylation on activity (3).

What key parameters must be optimized and validated in LC–MS/MS methods to ensure accurate quantitation and structural characterization of monoclonal antibodies in clinical development?
Ensuring accurate quantitation and structural characterization of monoclonal antibodies is critical for obtaining accurate and reproducible data. In our study, we optimized the key parameters in sample preparation with the most important preparation step being enzymatic digestion. The parameters in enzymatic digestion include digestion time, enzyme-to-protein ratio, and temperature. Reproducible digestion is essential for reliable peptide generation. Additionally, liquid chromatography conditions were optimized for both the middle-up and bottom-up workflows to provide efficient separation, particularly for detecting PTMs. Optimal separation increases sensitivity and detection for PTM characterization. Tuning and optimization of the mass spectrometer source parameters is especially important to obtain high-quality mass spectra of larger antibody fragments. Proper optimization across these parameters ensures reproducibility, and confidence in both quantitative and qualitative assessments.

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References

1. Gould, C. E,: Ma, Q.; Cha, R. et al. Proteoform Characterization of HIV-1 Broadly Neutralizing Antibody PGT 121.414.LS Product Through Middle-Up and Bottom-Up Proteomics for Clinical Support. J. Am. Soc. Mass Spectrom. 2025. DOI: 10.1021/jasms.5c00198
2. Hagman, C.; Ricke, D.; Ewert, S. et al. Absolute Quantification of Monoclonal Antibodies in Biofluids by Liquid Chromatography−Tandem Mass Spectrometry. Anal. Chem. 2008, 80 (4), 1290-1296. DOI: 10.1021/ac702115b
3. Anand, S. P.; Ding, S.; Tolbert, W. D. et al. Enhanced Ability of Plant-Derived PGT121 Glycovariants To Eliminate HIV-1-Infected Cells. J. Virol. 2021, 95, 10.1128/jvi.00796-21. DOI: 10.1128/JVI.00796-21
4. Singh, A. A., Pooe, O., Kwezi, L. et al. Plant-Based Production of Highly Potent Anti-HIV Antibodies with Engineered Posttranslational Modifications. Sci. Rep. 2020, 10, 6201. DOI: 10.1038/s41598-020-63052-1