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  5. Improvement of Biological Product Quality by Application of New Technologies to Characterize of Vaccines and Blood Products: NMR Spectroscopy and Light Scattering
  1. Biologics Research Projects

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 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 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 the 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.

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.


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

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

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

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

J Bacteriol 2014 Sep 15;196(18):3271-8
Chemical Structure of the Capsular Polysaccharides (CPS) of Streptococcus pneumoniae Types 39, 47F and 34 by NMR Spectroscopy and their Relation to CPS10A.
Bush CA, Yang J, Yu B, Cisar JO

Prog Nucl Magn Reson Spectrosc 2014 May;79:48-68
NMR of glycans: shedding new light on old problems.
Battistel MD, Azurmendi HF, Yu B, Freedberg DI

Annu Rev Biophys 2014 May 6;43:171-192
Live cell NMR.
Freedberg DI, Selenko P

Carbohydr Res 2014 May 7;389:165-73
Sialo-CEST: chemical exchange saturation transfer NMR of oligo- and poly-sialic acids and the assignment of their hydroxyl groups using selective- and HSQC-TOCSY.
Shinar H, Battistel MD, Mandler M, Lichaa F, Freedberg DI, Navon G

J Phys Chem B 2013 May 2;117(17):4860-9
Direct evidence for hydrogen bonding in glycans: a combined NMR and molecular dynamics study.
Battistel MD, Pendrill R, Widmalm G, Freedberg DI

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 alpha2-8 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