Resolving Biclonal Gammopathy from monoclonal patterns: Diagnostic utility of triple quadrupole mass spectrometry in the era of therapeutic monoclonal antibodies.

Biclonal gammopathy, observed in approximately 1% of myeloma cases, is typically observed using serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Interpretation becomes challenging when dual bands share identical heavy-light-isotypes (e.g., IgGκ/IgGκ), as visual assessment may alone misclassify clonality. Such patterns may arise from structural immunoglobulin variants (dimers, post-translational modification), therapeutic monoclonal antibody (tMAb) interference, or genuine biclonality. In this study, liquid chromatography-triple quadrupole mass spectrometry (LC-TQMS) was applied to determine the light chain masses and differentiate these possibilities with high specificity. Residual serum samples (n = 14) exhibiting identical-isotype dual bands on IFE were subjected to isotype-specific immunoenrichment using camelid antibody-coated agarose beads, followed by chemical reduction and LC-TQMS analysis of light chains. κ light chains exhibited a mass range of 22,973-23,574 Da, while higher κ light chain masses, 24284 Da, 25942 Da were observed in IgM isotype. λ light chains showed masses between 22,604 and 22,820 Da. Among IgG samples (n = 7), biclonality was confirmed in two samples, monoclonality in three samples, tMAb interference (Daratumumab) in two samples. All IgA samples (n = 6) were monoclonal, while IgM sample demonstrated biclonality. Light chain glycosylation (hexose Δm 162 ± 10 Da, sialylation Δm 292 ± 10 Da) was observed in IgG and IgM respectively. LC-TQMS method thus effectively resolved dual-band cases, enabling distinction between biclonality, monoclonality, tMAb interference, and structural variants. IMPACT STATEMENT: LC-TQMS: 1) Addresses diagnostic uncertainty in cases showing dual-band patterns on serum protein electrophoresis or immunofixation. 2) Provides precise mass information of light chains for accurate characterization. 3) Enables distinction between biclonality, monoclonality, therapeutic interference, and glycoforms.4) Demonstrates how therapeutic monoclonal antibody or structural immunoglobulin variants, such as glycosylation can complicate interpretation of monoclonal patterns. 5) Reduces the risk of misclassification, improving diagnostic accuracy and patient management. 6) Advances laboratory diagnostics by integrating molecular precision into routine clinical workflows.