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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Improvement of Biological Product Quality by Application of New Technologies to Characterize of Vaccines and Blood Products: NMR Spectroscopy and Light Scattering

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


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 this difficulty our laboratory uses powerful techniques for characterizing biological products, such as NMR spectroscopy, light scattering, and circular dichroism to complement the biological assays traditionally used to characterize these products. (NMR spectroscopy is the laboratory version of the more elaborate medical imaging technique called MRI scanning; 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 individual types of molecules absorb light to investigate their structure.)

Our laboratory characterizes vaccines that are made from polysaccharides--long chains of sugar-like molecules. These vaccines are made from the polysaccharide capsules that surround certain types of bacteria. We study the connection between the structure of the capsule polysaccharides in vaccines and the ability of vaccines made from those polysaccharides to trigger an effective immune response. This knowledge will contribute to the development of safe and potent vaccines.

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 molecular 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, together with NMR, enable the FDA and the pharmaceutical industry to ensure that polysaccharide vaccines meet regulatory requirements for safety and effectiveness.


Scientific Overview

Our laboratory focuses on the relationship between the three-dimensional structures of bacterial capsular polysaccharides and the immune responses these polysaccharides elicit.

Our main tool is nuclear magnetic resonance, which allows us to probe molecular structure at the atomic level. 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.

In the bottom-up approach, we dissect polysaccharides into the repeating units that make them up 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 and EXSIDE experiments. These experiments provide us with data on the relative orientations of bonds, and consequently the rings that make up the polysaccharide. Detailed studies such as these help us determine the shape of the repeating units. In collaboration with colleagues with expertise in computation, we can deduce polysaccharide structures from their constituent, smaller units.

To facilitate these studies we have prepared isotopically-enriched polysaccharides. This technical advance increases the sensitivity of NMR so that we can more precisely quantify polysaccharide structures. We recently used this method 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.


Publications

J Magn Reson 2013 Mar;228:130-5
Accurate determinations of one-bond 13C-13C couplings in 13C-labeled carbohydrates.
Azurmendi HF, Freedberg DI

J Magn Reson 2013 Mar;228:159-65
Constant time INEPT CT-HSQC (CTi-CT-HSQC) - A new NMR method to measure accurate one-bond J and RDCs with strong 1H-1H couplings in natural abundance.
Yu B, van Ingen H, Freedberg DI

J Am Chem Soc 2012 Jul 4;134(26):10717-20
Evidence for helical structure in a tetramer of α2-8 sialic acid: unveiling a structural antigen.
Battistel MD, Shangold M, Trinh L, Shiloach J, Freedberg DI

Biopolymers 2012 Mar;97(3):145-54
Transient hydrogen bonding in uniformly 13C, 15N labeled carbohydrates in water.
Norris SE, Landström J, Weintraub A, Bull TE, Widmalm G, Freedberg DI

J Magn Reson 2012 Feb;215:10-22
More accurate 1J(CH) coupling measurement in the presence of 3J(HH) strong coupling in natural abundance.
Yu B, van Ingen H, Vivekanandan S, Rademacher C, Norris SE, Freedberg DI

J Biomol NMR 2011 Sep;51(1-2):163-71
NMR detection and characterization of sialylated glycoproteins and cell surface polysaccharides.
Barb AW, Freedberg DI, Battistel MD, Prestegard JH

Carbohydr Res 2011 May 1;346(6):759-68
Utility of coupled-HSQC experiments in the intact structural elucidation of three complex saponins from Blighia sapida.
Mazzola EP, Parkinson A, Kennelly EJ, Coxon B, Einbond LS, Freedberg DI

Curr Protoc Nucleic Acid Chem 2008 Dec;Chapter 3:Unit 3.17
Release of DNA oligonucleotides and their conjugates from controlled-pore glass under thermolytic conditions.
Grajkowski A, Cieslak J, Norris S, Freedberg DI, Kauffman JS, Duff RJ, Beaucage SL

Nat Struct Mol Biol 2008 Aug;15(8):868-9
NMR structure of chaperone Chz1 complexed with histones H2A.Z-H2B.
Zhou Z, Feng H, Hansen DF, Kato H, Luk E, Freedberg DI, Kay LE, Wu C, Bai Y

Bioconjug Chem 2008 Aug;19(8):1696-706
Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH.
Grajkowski A, Cieslak J, Kauffman JS, Duff RJ, Norris S, Freedberg DI, Beaucage SL

Proc Natl Acad Sci U S A 2007 Jul 10;104(28):11557-61
Extracellular structure of polysialic acid explored by on cell solution NMR.
Azurmendi HF, Vionnet J, Wrightson L, Trinh LB, Shiloach J, Freedberg DI

Anal Chem 2006 Jul 1;78(13):4634-4641
Chemical Characterization of Diaspirin Cross-Linked Hemoglobin Polymerized with Poly(ethylene glycol).
Buehler PW, Boykins RA, Norris S, Alayash AI

Dev Biol 2005;122:77-83
Using nuclear magnetic resonance spectroscopy to characterize biologicals.
Freedberg DI

Anal Chem 2005 Jun 1;77(11):3466-78
Structural and Functional Characterization of Glutaraldehyde-Polymerized Bovine Hemoglobin and Its Isolated Fractions.
Buehler PW, Boykins RA, Jia Y, Norris S, Freedberg DI, Alayash AI

Carbohydr Res 2005 Apr 11;340(5):863-74
The utility of residual dipolar couplings in detecting motion in carbohydrates: application to sucrose.
Venable RM, Delaglio F, Norris SE, Freedberg DI

Proc Natl Acad Sci U S A 2005 Apr 12;102(15):5564-9
Escherichia coli K1 polysialic acid O-acetyltransferase gene, neuO, and the mechanism of capsule form variation involving a mobile contingency locus.
Deszo EL, Steenbergen SM, Freedberg DI, Vimr ER

J Am Chem Soc 2004 Aug 25;126(33):10478-84
Discriminating the helical forms of peptides by NMR and molecular dynamics simulation.
Freedberg DI, Venable RM, Rossi A, Bull TE, Pastor RW

 

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