Liquid chromatography-mass spectrometry (LC-MS) is fast becoming the gold standard for biopharmaceutical analysis. However, identifying which LC-MS-based technique is best suited to a particular production process can be a challenge, say researchers.
LC-MS, as the name suggests, combines two techniques: a chromatography stage in which components of the process stream are separated; and a spectrographic step that analyzes ionized parts of the components.
The approach provides detailed biochemical and functional information about the compound under analysis such as structure, molecular weight, and quantity, which—in the case of a biopharmaceutical—are vital to ensuring product purity and quality.
But no LC-MS technique is perfect, according to David Paul, PhD, from the National Institute of Pharmaceutical Education and Research (NIPER) in India, who says the growing complexity of modern biopharmaceuticals is proving to be a challenge.
“Large macromolecules, such as intact proteins, can be difficult to analyze using LC-MS because of their complexity and the low ionization efficiency of their constituents.
“Furthermore, it might be challenging to fully fragment big biopharmaceuticals, such as monoclonal antibodies, for in-depth study. This can result in insufficient sequencing coverage and the possible loss of important changes or impurities,” he tells GEN.
Paul also points to issues with the detection of post-translational modifications (PTMs) or low-abundance analytes as shortcomings of LC-MS-based approaches.
There are also challenges from a regulatory compliance standpoint, according to Paul, who says, “At the moment, regulatory bodies mandate the stringent validation of LC-MS techniques used in biopharmaceutical analysis. It can be difficult to meet these requirements, especially for novel or complicated compounds.”
Mix and match
Paul and colleagues discussed such challenges in a recent review and found that industry efforts to address them are focused on using the right LC-MS technique for the right process and in combination with other analytical methods.
They cite the analysis of post-translational modifications—changes that influence the therapeutic or biochemical properties of a protein—as an example of where a combination approach makes sense.
Paul adds, “The structural complexity and variety of some PTMs such as glycosylation and phosphorylation might make it difficult for LC-MS to identify or precisely measure them.
“Even though HPLC with a PDA detector can perform glycan profiling, reduced sensitivity is shown. For this reason, using an HPLC-FLD detector for fluorescence tagging and analysis in combination with MS often yields satisfactory results.”
