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Intact Protein Characterization by HPLC-MS
Intact protein characterization by mass spectrometry plays a vital role in the biopharmaceutical industry by enabling direct determination of molecular weight, confirmation of molecular integrity, and detection of select post-translational modifications (PTMs) without the need for enzymatic digestion. This approach allows for rapid assessment of mass heterogeneity and conjugation levels—such as drug-to-antibody ratio (DAR) in antibody-drug conjugates (ADCs)—and supports both early-phase development and late-stage comparability studies. Thanks to its speed, specificity, and minimal sample preparation, intact mass analysis has become a routine first-line tool in the analytical workflows for monoclonal antibodies, fusion proteins, and other complex biologics.
As biologics become increasingly diverse and structurally complex, selecting the appropriate LC-MS platform is critical to ensure accurate intact mass analysis. The table below compares key HPLC-MS techniques—including RPLC-MS, SEC-MS (native and denaturing), CEX-MS, AEX-MS, and HIC-MS—highlighting their separation mechanisms, operational modes, advantages, and limitations. This overview is intended to guide analytical scientists and biopharma teams in choosing the optimal method.
Comparison of Different HPLC-MS for Intact Protein Characterization
| Name | LC Separation Mechanism | Condition | Comments | Advantages | Disadvantages |
| RPLC-MS | Hydrophobicity | Denaturing | Most commonly used type | Offers high sensitivity due to the use of ESI-compatible, low pH mobile phases (e.g., 0.1% formic acid in water and acetonitrile) and elevated column temperatures. Capable of characterizing mispaired bispecific antibodies and degradation fragments, such as light chain, which is among the most common fragment species formed during storage. | Carryover Artificial fragments due to high column temperature or in-source fragmentation. Denaturing only. Cannot detect any species with non-covalent binding, such as aggregates. |
| SEC-MS | Size | Native | To ensure MS compatibility and maintain native protein structure, a volatile aqueous buffer such as 50–100 mM ammonium acetate is often used. An extended m/z range instrument, such as a Q-ToF or specialized Orbitrap, is necessary for detecting higher-mass species such as dimers. | Capable of detecting non-covalent complexes, such as protein aggregates, AOC with siRNA, and protein–protein assemblies. Exhibits lower carryover compared to RPLC-MS. | Provides lower sensitivity compared to RPLC-MS. SEC columns are more fragile and require gentler handling than RP columns. |
| Denaturing SEC-MS | Size | Denaturing | 0.1% FA in 15-20% acetonitrile is often used | Fragments should be able to separated from intact protein. Exhibits lower carryover compared to RPLC-MS. | SEC columns must be compatible with organic solvents and low pH environments. SEC columns are more fragile and require gentler handling than RP columns. |
| CEX-MS | Charge | Native | Separates proteins by differences in net positive charge. Good for IgG1-based mAb, which possess a high isoelectric point (pI) of usually ≥ 8. Requires the use of volatile buffers to ensure MS compatibility. | Enables characterization of each charge variant peak by intact mass, allowing correlation to specific modifications such as deamidation, isomerization, or succinimide formation—modifications that are typically undetectable by RPLC-MS. | Provides lower sensitivity compared to RPLC-MS. |
| AEX-MS | Charge | Native | Separates proteins by net negative charge Good for IgG4-based mAb, which possess a low isoelectric point (pI) of usually < 8. Requires the use of volatile buffers to ensure MS compatibility. | Enables characterization of each charge variant peak by intact mass, allowing correlation to specific modifications such as deamidation, isomerization, or succinimide formation—modifications that are typically undetectable by RPLC-MS. | Provides lower sensitivity compared to RPLC-MS. |
| HIC-MS | Hydrophobicity | Native | Requires the use of volatile buffers to ensure MS compatibility. | Often used for ADC characterization | Provides lower sensitivity compared to RPLC-MS. |