Mass spectrometry has been a highly effective tool in planetary exploration to investigate the chemical composition of a wide range of targets. In particular, in situ mass spectrometry has been utilized to evaluate the habitability of different planetary environments by searching for evidence of essential nutrient availability, prebiotic chemistry, and potential biomarkers such as isotopic signatures and chiral preference. Yet the deployment of mass spectrometers across the solar system presents a myriad of challenges, such as the requirements for low mass, low power, sample processing and analysis autonomy, and high reliability against punishing environmental conditions. In this talk, I will chronicle the successful Sample Analysis at Mars (SAM) instrument investigation, which has made measurements on the surface of Mars for nine years as part of the Curiosity rover scientific payload. I will describe the Dragonfly Mass Spectrometer (DraMS), which is in development for an ambitious rotorcraft mission to Saturn’s moon Titan. Dragonfly will characterize Titan’s habitability and determine how far prebiotic chemistry has progressed in this exotic, icy environment known to provide the necessary ingredients for life. I will also introduce upcoming missions that will use mass spectrometers to both further our understanding of Mars and kick off a new era of Venus exploration.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has claimed the lives of over 3.8 million people globally. The fast-paced development of virus vaccine candidates while maintaining safety and efficacy highlighted the need of an advanced analytical toolkit, including mass spectrometry for product and process characterization. Leveraging prior live virus vaccine development and commercialization experience at our company, both vesicular stomatitis virus (VSV) and measles virus (MeV) vaccine candidates were developed. Both vaccine candidates were discontinued at Phase 1 due to inferior clinical response compared to convalescent serum.1
During vaccine development, a number of MS methods were developed to characterize both vaccine virus structural and surface proteins as well as residual host cell proteins from the production process. Additionally, these MS methods were leveraged for the characterization of glycosylation of SARS-CoV-2 spike protein standards. More specifically, charge detection mass spectrometry was utilized to characterize highly heterogeneous intact spike glycoprotein trimers and demonstrated higher levels of glycan heterogeneity than bottom-up glycoproteomics would suggest. This new-to-industry method enables molecular weight characterization of highly heterogeneous reagents used in vaccine development while requiring significantly lower amounts of starting material than traditional methods such as SEC-MALS. Finally, the need to determine the identity and clearance of host cell proteins (HCP) across process intermediates drove the development of a relative quantification proteomics method that enables simultaneous characterization of Vero HCPs and virus structural proteins. This method was then utilized to demonstrate the clearance of potential enzymes that could impact spike glycoprotein stability.

The HCP and virus structural protein characterization by MS provided orthogonality to more traditional ELISA and gel-based assays. Overall, the development of mass spectrometry methods to characterize SARS-CoV-2 vaccine candidates provided orthogonal data sets to enable rapid vaccine development. The established MS methods can be leveraged for future vaccine development.

Marketing application (MA) enabling product characterization (PdC) studies is a critical workflow in identifying product-related substances vs. impurities to define molecule attribute criticality, and ultimately the commercial control strategy. PdC studies are labor-intensive, often requiring multiple rounds of column enrichment/fraction collection and concentration of charged isoforms.

In this work we combined ion exchange chromatography with native mass spectrometry (native IEX-MS) to characterize the charged variants and associated post-translational modifications. Generally, IEX-HPLC is not amenable to direct MS analysis given incompatibilities of the eluent and MS ionization source; therefore, a IEX separation was optimized using MS-friendly volatile buffers. The method was capable of simultaneously resolving and identifying the following modifications: deamidation, isomerization, O-glycosylation, and amino acid substitution (or misincorporation) modifications in the acidic variants, and isomerization, dimerization, in the basic variants. We also developed an orthogonal limited digestion method that cleaves at a major site resulting in formation of two separate domains antigen binding domain-1 and antigen binding domain-2 with the helped of the developed IEX-MS method, we were able to identify modifications specific to each domains. Using this approach, we were able to efficiently and directly identify several post-translational modifications in the native and stressed samples, as well as provide information about modification clearance in the downstream purification process. Peptide mapping was performed to confirm the results.
This native IEX-MS workflow augmented with limited digestion affords method affords faster analysis and provides unbiased information of important PTMs associated with bispecific protein. The developed workflow can be applied to different protein therapeutic modalities at different stages of product development.

Cellular therapies have shown considerable clinical activity in recent years. A goal of CAR-T process development (PD) is to produce consistent drug product. To achieve this goal, PD scientists employ process analytical technologies (PAT) to monitor upstream, downstream, and final product attributes. This talk will focus on how the cell therapy field is leveraging mass spectrometry-based PAT for cell-based therapies.

Oligonucleotides are an emerging type of therapeutic molecules, with hundreds of projects currently in development at all major pharma companies. Most oligonucleotides are synthesized by classic solid phase synthesis using phosphoramidite chemistry. Therefore, they are classified as synthetic molecules by the authorities. However, their chemical and biological properties are between classic small molecules and large molecule biologics. Thus, CMC development is challenging.

Due to their high molecular mass, complex structure and impurity profile, high-resolution mass spectrometry is an invaluable analytical technique during development.

We will show two examples for analyses performed with HRMS resulting in valuable information for process development.

In the first example, different N-1 impurities were analyzed by MS/MS to elucidate the sequence of the oligonucleotide impurity. In this case, N-first and N-last impurities had the same mass as the first and the last residue of the oligonucleotide are the same. We were able to distinguish N-first and N-last structures in purified and crude sample providing valuable information about the synthetic process.

In another example, we analyzed samples taken at different stages of the synthesis and purification process. The impurity profile of the “in-process” samples were analyzed allowing conclusions about the process.

Mass spectrometry (MS) plays a key role throughout all stages of drug development 1, 2 and is now as ubiquitous as other analytical techniques such as surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and supercritical fluid chromatography (SFC), among many others.

Here in I will describe how high throughput solid-phase extraction mass spectrometry (HT-SPE-MS) was used to rapidly screen and characterize the covalent binding of different irreversible inhibitors (acrylamide based) to a protein target containing a non-native cysteine residue. This technology is now used in regular project support.

And from a more research and scientific interest perspective, I will also discuss how ion mobility (IM) measurements, molecular dynamics (MD) and quantum mechanical (QM) calculations (performed within biopharma) have been used infer ionic gas-phase structure, ranging from small molecules and monoclonal antibodies (mAbs).

1. Campuzano, I. D. G.; Sandoval, W., Editorial: Special JASMS Focus on Mass Spectrometry in Industry. J Am Soc Mass Spectrom 2021, 32 (8), 1850-1851.
2. Campuzano, I. D. G.; Sandoval, W., Denaturing and Native Mass Spectrometric Analytics for Biotherapeutic Drug Discovery Research: Historical, Current, and Future Personal Perspectives. J Am Soc Mass Spectrom 2021, 32 (8), 1861-1885.