Principal Investigator: Daron I. Freedberg, PhD
Office / Division / Lab: OVRR / DBPAP / LBP

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General Overview

The difficulty of characterizing complex biological products makes it especially challenging to ensure that they can be manufactured in a consistent and predictable way. The risk of manufacturing inconsistencies is especially high for novel products, since traditional testing technology might not be able to identify subtle and unanticipated variabilities.

To overcome these difficulties our laboratory uses powerful techniques for characterizing biological products at the molecular level, such as nuclear magnetic resonance (NMR) spectroscopy, laser light scattering, and circular dichroism spectropolarimetry to complement the biological assays traditionally used to characterize these products. NMR spectroscopy is a version of the medical imaging technique called MRI scanning; laser light scattering is a technique for studying molecular weight and size by monitoring the directions of light rays bouncing off the molecule; circular dichroism uses the different ways that chiral molecules absorb light to investigate their structure.

Our laboratory characterizes vaccines that are made from the polysaccharide capsules that surround certain types of pathogenic bacteria. The polysaccharides are long chains of sugar-like molecules. A central idea in Biology is the structure-function relationship, which states that the function of a molecule can be inferred by studying the structure of the molecule. We study the three-dimensional structure of the capsular polysaccharides in vaccines to better understand how they function. This helps us understand how vaccines made from those polysaccharides trigger an effective immune response. This knowledge will contribute to the development of safe and potent vaccines. We are inventing new NMR experiments and improving the technology we use to characterize polysaccharides to help us more accurately and precisely determine polysaccharide structure-function relationships.

Using NMR as a microscope to study polysaccharides at the molecular and atomic levels, we probe the individual atoms and their locations in relationship to each other. This information helps us to determine the shapes of these molecules, which in turn gives us valuable insights into how polysaccharides interact with antibodies and proteins. We also use laser light scattering and circular dichroism to characterize the overall size and shape of polysaccharides, which is crucial for vaccine efficacy and therefore for manufacturing consistency of polysaccharide conjugate vaccines. Together NMR and light scattering, enable the FDA and the pharmaceutical industry to ensure that polysaccharide and polysaccharide conjugate vaccines meet regulatory requirements for safety and effectiveness.

NMR spectroscopy can also be used to gain insight into protein structure. Because our instrumentation is unique within CBER, we also collaborate with groups who seek to understand therapeutic protein and antibody structure and function.

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Scientific Overview

Our laboratory focuses on the relationship between the three-dimensional structures of bacterial capsular polysaccharides and the immune responses these polysaccharides elicit. In many cases, polysaccharides differing subtly in composition show significant immunological differences. Since structure is linked to function, we seek to understand how these subtle compositional differences impact three-dimensional structure.

Our main tool is nuclear magnetic resonance (NMR) spectroscopy, which allows us to probe molecular structure at the atomic level. NMR can be used to report on the structural consequences of even subtle molecular differences.

We divide our projects into two areas: Top-down and bottom-up. In the top-down approach, we study polysaccharide structure either in vitro or in vivo. We used NMR to probe the polysaccharide structure on living E. coli K1 and discounted the idea that polysaccharides on cells have a different structure from those in the free E. coli K1 polysaccharide. This method can potentially be used to delineate polysaccharide-antibody interactions and lead to a better understanding of what structures are critical to effective immunogenicity by correlating this information with readily available immunological data. We also study polysaccharide structure in vitro to understand the overall behavior of these molecules in the absence of cells. A comparison of on-cell structure with that off cell structure will provide insight into polysaccharide structure-function relations.

In the bottom-up approach, we dissect polysaccharides into the repeating units that comprise them and study in detail their three-dimensional structure by NMR. To do this, we use cutting-edge NMR techniques, such as RDCs (residual dipolar coupling), nuclear relaxation and relaxation-dispersion, quantitative J correlation, NOEs (nuclear Overhauser effects) and EXSIDE experiments. We have also developed new experiments to obtain a deeper understanding of molecular structure through direct detection of the hydroxyl groups in poly- and oligosaccharides. Altogether, these experiments provide us with data on the relative orientations of bonds, and consequently the rings that make up the polysaccharide. Detailed studies using the techniques above these help us determine the shape of the repeating units comprising the polysaccharide. These details can then be extended as model units in the polysaccharide. We also utilize our expertise in computational modeling smaller fragments of polysaccharides which help in interpreting our experimental data. Thus, we deduce polysaccharide structures from their constituent, smaller units.

To further our insight into polysaccharide structure, we have prepared isotopically enriched polysaccharides. This technical advance simultaneously increases the sensitivity of our NMR experiments and increases the repertoire of NMR experiments we can use so that we can more precisely quantify polysaccharide structures. We recently used isotopic enrichment to detect hydrogen bonding in a bacterial polysaccharide. This result will help us quantify how hydrogen bonding influences carbohydrate structure.

More recently, we began to detect hidden conformations of carbohydrates indirectly by detecting motion. This will improve our understanding of the observed flexibility in carbohydrates.

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Publications

1. J Biomol NMR 2019 Sep;73(8-9):399
   Editorial. [Special Issue: Torchia]
   Freedberg DI, Kay LE
2. J Magn Reson 2020 Apr;313:106704
   Data processing in NMR relaxometry using the matrix pencil.
   Fricke SN, Seymour JD, Battistel MD, Freedberg DI, Eads CD, Augustine MP
3. ACS Catal 2020 Feb 21;10(4):2791-8
   Size-controlled chemoenzymatic synthesis of homogeneous oligosaccharides of Neisseria meningitidis W capsular polysaccharide.
   Li RY, Yu H, Muthana SM, Freedberg DI, Chen X
4. Isr J Chem 2019 Nov;59(11-12):1039-58
   Solution NMR structural studies of glycans.
   Freedberg DI, Kwon J
5. Sci Rep 2019 Oct 29;9(1):15449
   Effects of codon optimization on coagulation factor IX translation and structure: implications for protein and gene therapies.
   Alexaki A, Hettiarachchi GK, Athey JC, Katneni UK, Simhadri V, Hamasaki-Katagiri N, Nanavaty P, Lin B, Takeda K, Freedberg D, Monroe D, McGill JR, Peters R, Kames JM, Holcomb DD, Hunt RC, Sauna ZE, Gelinas A, Janjic N, DiCuccio M, Bar H, Komar AA, Kimchi-Sarfaty C
6. J Biol Chem 2019 May 10;294(19):7797-809
   Glycosylation of the viral attachment protein of avian coronavirus is essential for host cell and receptor binding.
   Parsons L, Bouwman KM, Azurmendi HF, de Vries RP, Cipollo JF, Verheije MH
7. Anal Chem 2018 Apr 17;90(8):5040-7
   Improving analytical characterization of glycoconjugate vaccines through combined high-resolution MS and NMR: application to Neisseria meningitidis serogroup B oligosaccharide-peptide glycoconjugates.
   Yu H, An Y, Battistel MD, Cipollo JF, Freedberg DI
8. J Bacteriol 2018 Apr 24;200(10):e00051-18
   Iron and zinc regulate expression of a putative ABC metal transporter in Corynebacterium diphtheriae.
   Peng ED, Oram DM, Battistel MD, Lyman LR, Freedberg DI, Schmitt MP
9. Glycobiology 2017 Sep 1;27(9):900-11
   The beta-reducing end in alpha(2-8)-polysialic acid constitutes a unique structural motif.
   Azurmendi HF, Battistel MD, Zarb J, Lichaa F, Negrete Virgen A, Shiloach J, Freedberg DI
10. J Pharm Biomed Anal 2017 Jul 15;141:229-33
    Application of 2D-NMR with room temperature NMR probes for the assessment of the higher order structure of filgrastim.
    Brinson RG, Ghasriani H, Hodgson DJ, Adams KM, McEwen I, Freedberg DI, Chen K, Keire DA, Aubin Y, Marino JP
11. J Med Genet 2017 May;54(5):338-45
    Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.
    Simhadri VL, Hamasaki-Katagiri N, Lin BC, Hunt R, Jha S, Tseng SC, Wu A, Bentley AA, Zichel R, Lu Q, Zhu L, Freedberg DI, Monroe DM, Sauna ZE, Peters R, Komar AA, Kimchi-Sarfaty C
12. J Phys Chem B 2017 Feb 2;121(4):683-95
    Glycan OH exchange rate determination in aqueous solution: seeking evidence for transient hydrogen bonds.
    Battistel MD, Azurmendi HF, Freedberg DI
13. J Am Chem Soc 2015 Oct 28;137(42):13444-7
    Uncovering nonconventional and conventional hydrogen bonds in oligosaccharides through NMR experiments and molecular modeling: application to sialyl Lewis-X.
    Battistel MD, Azurmendi HF, Frank M, Freedberg DI
14. J Magn Reson 2015 Feb;251:65-70
    NMR profiling of biomolecules at natural abundance using 2D 1H-15N and 1H-13C multiplicity-separated (MS) HSQC spectra.
    Chen K, Freedberg DI, Keire DA