Monoclonal antibodies (mAbs) are susceptible to chemical modification and degradation during production, formulation, and storage. It is therefore important to observe the purity and molecular weight of mAbs during the manufacturing process.
Monoclonal antibody characterization with reversed-phase LC/MS
Reversed-phase LC/MS is a well-established analytical technique for the analysis of mAbs, at intact or fragment level, to provide accurate molecular mass. Choosing the correct reversed-phase column and LC method is critical to achieve fast analysis times and reproducible, high-resolution separations.
Reverse phase characterization and workflow solutions include:
- Sample preparation: The sample may be analyzed intact or may be reduced and alkylated.
- Separation (RP): Different pore sizes of polymeric reversed phase (PLRP-S) columns provide high efficiency separations over the full range of protein and peptides.
- Detection and data analysis: Providing full analysis of the data produced.
PLRP-S: The versatile choice
The aim of reverse-phase LC/MS characterization of antibodies is to assess any changes to the mAb primary structure. In addition to setting up a new method for a new protein molecule, there are many challenges to consider:
- Insufficient resolution can lead to poor accuracy and precision, limiting confidence in results.
- Long analysis times result in a low sample throughput.
- Short column lifetimes mean that researchers must buy new columns often, which is a poor use of resources.
- Method transfer to multiple columns requires different columns for UV and MS, which is time-consuming.
To address these challenges, PLRP-S columns offer durable, resilient polymer particles that deliver reproducible results and long column lifetimes. These features mean that the column can be changed less often, resulting in a lower cost of analysis and increased confidence in the results generated. The thermal and chemical stability of PLRP-S for separations at extremes of pH and high temperature allows for flexibility in the analysis of difficult samples.
Furthermore, the multipurpose column results in lower costs. There is also choice for a large range of sample types, as PLRP-S columns are available in a range of pore sizes from 100–4000 Å. This range of pore sizes provides high-efficiency separations over the full range of protein and peptides. Excellent peak shapes with various ion pairing agents means that the same columns and mobile phase can be used for both UV and MS, with no redevelopment for different detectors.
The PLRP-S column shows great versatility in the characterization of mAbs. Reversed-phase LC/MS methods using the column are useful for both intact mAbs and mAb fragments. Agilent polymeric PLRP-S columns contain rigid, macroporous, spherical particles of polystyrene divinylbenzene. The particles are inherently hydrophobic, so there is no bonded phase or alkyl ligand required for reversed-phase LC/MS separations.
Preparing samples with AdvanceBio Desalting-RP cartridges
Agilent AdvanceBio Desalting-RP cartridges are designed and manufactured for online removal of salt ions before MS detection in (U)HPLC analysis of biomolecules. The cartridges contain a 10 µm 1000 Å reversed-phase polymeric particle that will effectively remove the salt and improve the quality of the MS data generated. These cartridge-style columns can be used on their own on any LC system to desalt collected fractions. They allow for the use of MS detection with traditionally non-MS friendly chromatography methods like IEX, SEC, and Affinity.
With Agilent AdvanceBio Desalting-RP cartridges, you can save time by allowing desalting of samples online. They reduce instrument maintenance and cut downtime by avoiding salt contaminating MS.
To find out how Agilent can enhance your confidence in analysis with reversed-phase LC/MS, click here.