The proposed mass spectrometry method to monitor glycosylation was considered to be cumbersome and unsuitable, as it lacked sensitivity and accuracy to monitor batch-to-batch consistency of glycosylation. The company was asked to provide convincing ([semi]-quantitative) data with regard to the batch-to-batch consistency of the glycosylation pattern (e.g., by fluorophore-assisted carbohydrate electrophoresis [FACE]).

FACE, using the enzymic release of oligosaccharides followed by electrophoresis, was investigated. An early semi-quantitative version of this showed good correlation between the relative quantities of the oligosaccharide species released from the samples and so provided evidence for a consistent glycosylation pattern between batches, both in terms of relative amounts and species present. Although this gel-based method could not differentiate between oligosaccharide species with the accuracy of MS, it was considered by the regulators to be less of a risk than adopting the then-experimental MS technique.

Eventually, however, state of the art succeeds. Even if a state-of-the-art technique such as MS is rejected in favor of an old tried-and-trusted method in some situations, it can still prevail when a change is forced by driving factors, such as the power of MS to separate similar protein isoforms, which cannot easily be done with accuracy by any other method. ¹³

The ability to separate molecules based on size and charge was first described in 1912 by JJ Thompson. Despite years of intense development of MS methodology and equipment, the goal of analyzing large macromolecules remained elusive for over 70 years.¹⁴

Before 1982, several tools were available to characterize natural proteins physico-chemically. These were:

• Edman degradation
• UV and visible spectroscopic methods
• Protein content tools (such as the Bradford method)
• Constituent chemical content (e.g., nitrogen)
• Electrophoretic analysis (e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis [SDS-PAGE])

The emphasis on the "process defines product" attitude during the early development of biological products was due to the relative inability to fully characterize protein products to ensure safety and efficacy. The big breakthrough in protein characterization of the 1980s was the advent of high-performance liquid chromatography (HPLC). HPLC provides high peak resolution and can be demonstrated by the separation of species-specific insulins, many of which differ by a single amino acid.¹⁵ HPLC was employed for the characterization of insulin as the first recombinant protein therapeutic to be approved. HPLC is now a mainstay of the protein characterization toolbox.

Similarly with the advent of better detector technology, MS was able to resolve higher molecular weights. This allowed increased assurance of product quality and a better understanding of the relationship between structure and function. For instance, the total molecular weight of proteins up to approximately 500 kDa can be determined by MS with a far greater mass accuracy than by using other methods such as SDS-PAGE and size-exclusion chromatography. Accuracy and resolution are further increased as the mass of the analyte decreases, such as when proteins are fragmented to peptides during peptide mass fingerprint. The exceptional mass accuracy of MS permits the characterization of major post-translational modifications, for example disulphide bonding, glycosylation, and lipidation.

Until recently MS was rarely used as an in-process or release test, probably because MS has been considered a highly specialized procedure that is assumed to be unsuitable for routine manufacturing. However, the rise of proteomics has led to mass spectrometers that are high-throughput and robust, and therefore, ultimately more cost-effective and easier to use. So it may yet transpire that validated MS will become more commonly used as an in-process test. This may actually be a necessity in certain cases where purification involves the separation of isoforms that may only be differentiated by a technique with high mass accuracy.