What happens if a protein-based biotherapeutic includes unexpected isoforms? Traditional, analysis might not even reveal very similar isoforms in a biotherapeutic. So, Jonathan Bones, PhD, principal investigator at the National Institute for Bioprocessing Research and Training in Dublin, and his colleagues applied microchip capillary electrophoresis-mass spectrometry (CE-MS) to isoform analysis.

When asked about the main dangers of missing isoforms in the analysis of biopharmaceuticals, Bones says: “Danger might not be the correct word, simply because we don’t know if that is the case.” Instead, Bones thinks of this from a risk-based perspective. “To better understand the risk associated with missing an isoform, we would need to follow up the analysis with a functional study to see whether that particular isoform had any effect on potency or any potential implication with regard to safety,” he says. “The challenge here is from the analytical point of view, if we do not know that such an isoform is present.” In some cases, isoforms can co-elute in separations, which leaves some forms unidentified. As Bones explains, “We need the analytical power to separate the various different protein isoforms present on our molecule so that we can annotate the data accordingly and better understand product microheterogeneity.”

CE-MS might solve that problem through several benefits: excellent separation selectivity, low sample consumption, and coupling the electrophoresis with high-resolution MS. In combination, Bones says, this “enables us to perform confident mass-based annotation of the various charge variants present.”

Microfluidic CE interface

To test this approach, Bones and his colleagues used the ZipChip microfluidic CE interface coupled to a high-resolution Orbitrap mass spectrometer. “The ZipChip is really simple to use,” Bones says. “You simply select the chip, sample buffer, and background electrolyte needed for your particular application of interest and then you are all set.” Plus, the kit-based methods of the ZipChip minimizes the need for optimization.

“For charge-variant profiling, the ZipChip offers an orthogonal approach to our other platform method, which is pH gradient–cation exchange chromatography coupled to mass spectrometry,” Bones explains. “Combining the data enables us to really perform a deep characterization of the sample.”

But it’s not all about the ZipChip. “The mass spectrometer is also playing a key role,” Bones says. “In the present study, an Orbitrap mass spectrometer offered high resolution–accurate mass capabilities and, when combined with the excellent frontend separation, proved to be an excellent method for understanding the complexity of the molecule understudy,” which was panitumumab, an IgG2 type monoclonal antibody.

For now, CE-MS is considered a method for specialists. “For sure, it’s not the easiest coupling of analytical separations and mass spectrometry, but instrument formats such as the 908 Devices ZipChip interface make application of the technology really simple,” Bones says. “The ZipChip interface simply connects to the frontend of the mass spectrometer, and it’s just a matter of taking off the standard electrospray source and replacing it with the ZipChip.”

As a result, commercial bioprocessors could now make use of CE-MS to find hidden isoforms in biotherapeutics. As Bones concludes: “Everything has been streamlined to make the application of the technology as easy as possible, which is a really nice feature and makes what was once considered specialist, mainstream.”

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