Mass spec is poised to fulfill its potential in the QC lab, say researchers who claim that a new intact protein analysis method overcomes challenges that have up to now limited adoption. Growing biopharma industry interest in critical quality attributes—characteristics that determine product quality—has increased demand for analytical techniques that are able to provide more detailed information about protein drug products.
Mass spectrometry, which measures the mass-to-charge ratio of molecules, has emerged as a potential solution with the development by Amgen in 2015 of an LC-MS technique called the multi-attribute method (MAM).
“There is a strong movement towards the implementation of MS in QC that is being driven by initiatives like the multi-attribute method,” says Jonathan Bones, PhD, from the National Institute for Bioprocessing Research and Training in Ireland.
“The driving force,” he explains, “is the power of the data that is generated. And MAM, as the name suggests, enables the replacement of a number of individually focused methods with a single analytical method. This is an attractive feature.”
But despite this potential, use of MAM in the QC lab has been limited. One possible reason for this, suggest Bones and colleagues in a new study, could be the complicated sample preparation steps required to produce the digests analyzed by the technique.
“Traditional MAM used proteolytic digestion of the sample, usually with trypsin, to generate peptides, which are then analyzed using the LC-MS MAM workflow for attribute analysis and new peak detection,” notes Bones.
Aiming to reduce these onerous sample prep requirements, Bones and colleagues came up iMAM, a variation of the technique that can analyze intact proteins.
“Analysis on the intact level offers a number of advantages, including minimal or simplified sample preparation, which reduces the potential for induction of a sample prep-related modification; capture of the natural dynamic range of modifications that exist on the protein; and the ability to analyze size variants such as dimers or other multimeric species, information that would be lost during proteolytic digestion,” says Bones.
“The other motivation is that methods that involve the analysis of protein therapeutics on the intact level—such as size exclusion, reversed phase, or cation exchange-based charge variant analysis methods—are often routine analysis methods. A focus of my research group recently was to adapt these methods for MS coupling, and then to develop and implement the MAM ethos as an add-on.”
To test the iMAM, Bones and colleagues used it to analyze three drugs: Infliximab with the focus being on identification; Adalimumab to detect post-translational modifications; and Trastuzumab to search for new components.
And, according to their study, “The method generated reproducible data using either native or denatured intact mass analysis and was used with three different applications, delivering both qualitative and quantitative results.”
The next step is to tweak the iMAM approach for other therapeutic modalities.
“We’re continuing to develop iMAM,” Bones says. “We are tailoring the method for use with mAb subunits, and we are also implementing iMAM in our process analytical technology (PAT) initiatives.
“We’ve successfully integrated LC-MS for online monitoring during mAb upstream processing. iMAM enables us to bring our PAT platform another step closer to deployment in the regulatory environment.”