By the time Pittcon 2022 was canceled due to the COVID-19 pandemic, key elements of the technical program were already in place, leading the organizers reformat portions of the program for electronic distribution. This included the Keynote speech, called the Wallace. H. Coulter Award Lecture, presented by professor Albert J. R. Heck of Utrecht University (Netherlands) on March 9.
Heck, a professor of chemistry and pharmaceutical Sciences at Utrecht University and scientific director of the Netherlands Proteomics Center, is a leading expert in protein mass spectrometry. His group develops and implements innovative mass spectrometric methods for the more efficient and detailed characterization of proteins in relation to their biological function. In short, Heck applies protein mass spectrometry to problems in proteomics, glycoproteomics and structural biology.
Heck’s Pittcon lecture addressed two topics: characterization of single molecules or particles of large biopolymers by mass spectrometry; and characterization of antibodies in blood of individual donors to study variability in the human immune response to pathogens.
First, Heck discussed the mass spectrometry-based analysis of Adeno-Associated Virus (AAV), nowadays often used as a vector in gene therapy. Here, the idea is to encapsulate a human transgene inside the cavity of the viral particle and deliver it to target cells.1
For QC of these gene therapy products, one needs to measure the detailed structure of the AAV and its cargo. Using high-resolution native mass spectrometry and charge detection mass spectrometry (CD-MS), Heck’s lab developed technologies to precisely determine the viral content. By using single particle techniques, resembling the Coulter principle, the team can successfully characterize AAV and other virus-like particles with very high sensitivity and precision, notwithstanding the fact that these have molecular weights of above a few million Da. By conventional standards for mass spectrometry, these masses are gigantic. The sweet spot of MS is less—usually much less—than 50,000 Da. Citations of MS in the mega Da range are notable exceptions.
As a technique, CD-MS involves measuring the charge on the species in the Orbitrap as well as its mass-to-charge ratio, m/z. This allows studying ions within a single charge state.2 For single molecule CD-MS, the key is to dilute the sample so only a few 10s to 100s of ions are trapped in the Orbitrap at the same time. Conventional wisdom was that Orbitraps needed an ion load of about 100,000 in the trap for quality spectra, but dilution is simple and fast. So, if only a few ions are present, the charge can be determined along with the conventional m/z.
In his lecture, Heck showed examples of CD-MS of engineered antibodies being studied as potential therapeutic agents for various diseases, including tumors. Particularly impressive was the development of complex structures of antibody-based formats made up of multiple (3 to 16) Fab units. These ranged in mass into the mega Dalton range. Heck went on to show that CD-MS is useful in analysis of single cells, tissues, and proteomics. For most topics in biology, the examples were analyzed by native MS, i.e. with ions with low charge. High charge states need to be avoided, since charge-charge repulsion could perturb the native structure. CD-MS as practiced with the Orbitrap seemed to have no trouble with this.
In a sign of the times, Heck also discussed his study of antibodies directed at the spike trimer-protein on SARS-CoV-2. Details of the antibody binding to the spike trimer protein of SARS-CoV-2 are important in the target of vaccines and rational design of biotherapeutics. A priori, the stoichiometry of the binding was predicted to be three antibody ligands. With CD-MS, it was easy to show masses corresponding to naked plus one and two antibodies—but not three. This limitation seems to be steric hinderance precluding trimeric binding of the spike. This may explain the unanticipated loss of binding avidity of some affinity constructs.3
The study of affinity response led Heck to the question of how unique is the immune response from individual to individual? After all, with COVID-19, many are infected, some die and some are asymptomatic. Antibodies are produced by B cells in blood, with each cell producing one unique antibody. Upon infection with a pathogen, the body produces a large number of B cells. Only a few of these will produce the antibodies that will interact with the pathogen to mark it for destruction.
One idea proposed was to treat people with antibodies of COVID-19 survivors; however, the approach did not work Why? Heck compared the antibody profiles of two, then eight, infected individuals. Were they the same? For the case of two people, the vast majority of antibodies were completely different. Increasing the cohort size to eight individuals repeated the story leading to the conclusion that individuals have a unique antibody profile, even when sick with a common pathogen.4 This uniqueness is due to structures in the Fab region producing different structures in the Complimentary-Determining Region (CDR).
Heck also touched on the significance of this observation in mothers’ milk. The chemistry professor reports that lactating mothers produce IgA1 with unique clonal profiles. So, now that we can measure them, what is the difference? What does it mean? This could be a very interesting and important future area of research.
“Each person makes its own repertoire of antibodies against each pathogen. In this vast resource, there should be ideal candidates for biotherapeutics. Our approaches may provide a way to find them,” Heck said during his Keynote presentation.
Pittcon’s Wallace. H. Coulter Award Lecture is named after the inventor of the Coulter principle, which was developed in the 1940s to quickly count blood cells by measuring the changes in electrical conductance as cells suspended in a conductive fluid passed through a small orifice. This measured the number and size of cells. For 70 years, Coulter Counters have been used in hematology research and diagnostics. Recently, the market has expanded with the increasing interest in nanoparticles and batteries.
References
- Snijder J, et al. JACS 136 (2014) 7295-9
- Worner, T. et al. Nature Comm (2021)
- Yin, v. et al. ACS Cent. Sci. 2021,7,11, 1863-1873.
- A. Brandt, et al, DOI:10.1016/jcels2021108.008