Wanted: Native Protein LC-MS

The biologics field is booming and showing no signs of slowing down. As the biotech industry has developed, it has adopted advanced engineering techniques to produce antibody-drug conjugates (ADCs), bispecific monoclonal antibodies, new types of fusion protein scaffolds – and the products are becoming increasingly complex. Yet complexity is no excuse for compromise when it comes to patient safety or lot-to-lot reproducibility, so analytical assays must be up to the challenge of converting complicated biomolecular puzzles into tractable, well-characterized molecules.

There is no one-size-fits-all method for addressing every characterization challenge presented by these new modalities, but it is reasonable to suggest that LC and MS will play a central role. Advances in LC-MS have made it easier than ever to confirm recombinant protein sequences and to investigate product-related impurities. However, LC separations coupled to MS detection have relied largely on denaturing conditions that afford sensitive detection but also restrict our ability to investigate protein conformations and interactions. In short, techniques that couple native separations to MS analysis are needed.

The biologics field is booming and showing no signs of slowing down. As the biotech industry has developed, it has adopted advanced engineering techniques to produce antibody-drug conjugates (ADCs), bispecific monoclonal antibodies, new types of fusion protein scaffolds – and the products are becoming increasingly complex. Yet complexity is no excuse for compromise when it comes to patient safety or lot-to-lot reproducibility, so analytical assays must be up to the challenge of converting complicated biomolecular puzzles into tractable, well-characterized molecules.

There is no one-size-fits-all method for addressing every characterization challenge presented by these new modalities, but it is reasonable to suggest that LC and MS will play a central role. Advances in LC-MS have made it easier than ever to confirm recombinant protein sequences and to investigate product-related impurities. However, LC separations coupled to MS detection have relied largely on denaturing conditions that afford sensitive detection but also restrict our ability to investigate protein conformations and interactions. In short, techniques that couple native separations to MS analysis are needed.

Genuine limitations in reagent and column technologies have long stifled progress. But that’s not to say we’re not moving forward: for example, Bifen Chen, Ying Ge, and colleagues successfully used volatile salts to directly connect hydrophobic interaction chromatography with a mass spectrometer in 2016 (1). They then applied this method to the interrogation of monoclonal antibody samples (2), highlighting the potential for us to selectively separate intact drug isoforms and immediately access MS information for peak identification. This is particularly beneficial to those looking to characterize cysteine-linked ADCs and bispecific antibodies that would otherwise dissociate under denaturing conditions. 

In parallel, Yann LeBlanc and Guillaume Chevreux have started to establish robust, MS-compatible ion exchange separations, including the demonstration that ammonium formate and acetate can be used to carry out salt-mediated pH gradient separations of monoclonal antibodies (3). This seminal work has ushered in a new wave of publications, each describing equally interesting separations – everything from traditional isoelectric focusing to pH gradients supplemented with significant increases in ionic strength (4, 5). But when will these novel methods find their way into routine labs?

Adoption of novel technologies is never easy, but the potential rewards make the venture more than worthwhile. Of course, if we are to develop robust methods that can reproducibly yield easy-to-interpret mass spectra, it will be important to remain scrupulous about reagent quality. And that will require that we learn from those working on high sensitivity trace metal quantitation; here, the most important factor to consider is purity. Certified reagents and high-quality plastics should be used for mobile-phase preparation to avoid the formation of salt adducts. Likewise, the use of different types of volatile salts will require diligent investigation to better understand their effects on electrospray ionization. Nevertheless, by wielding high resolution, MS-compatible native separations, the field will be able to explore new depths of sample characterization; these new separations may be coupled to the detection of native-like gas-phase conformations using a cyclic ion mobility separator (6), or applied to the detection of large megadalton complexes using charge detection MS (7).

The future of native protein LC-MS is bright, and the ever-evolving complexity of biopharmaceuticals gives just cause for investigators to make fast-paced improvements to emerging approaches. Only time will reveal the upper capabilities of these approaches, but I for one am confident and excited to see where such research takes us.