Analytical ultracentrifugation: a powerful ‘new’ technology in drug discovery

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  • rire,

    and should become a mainstay for target identification,

    are more and more moving into focus. The method is additionally usefulto better describe the behavior of low-molecular weight drugs in


    The analytical ultracentrifuge is similar to the more familiar

    not depend on a comparison to standards. Consequently,

    sedimentation can be used to analyze the solution behavior

    of nearly any type of molecule over a wide range of concen-

    trations and in a wide variety of solvents. Furthermore, a

    Drug Discovery Today: Technologies Vol. 1, No. 3 2004

    ethpreparative centrifuge, except that the analytical ultracentri-

    fuge is configured to determine the concentration distribu-

    tions of molecules during sedimentation. Analytical

    ultracentrifugation (AUC) provides first-principle hydrody-

    namic and thermodynamic information about the size,

    broad range of particle sizes might be analyzed by using

    different rotor speeds. Sedimentation has the merits of being

    rapid, non-destructive, and simple to use. Advances in data

    analysis software have created new, significantly improved

    tools for obtaining information about protein association

    and the behavior of viruses. Finally, a new, highly sensitive

    fluorescence detection optical system allows the analysis ofE-mail address: (T. Laue) tom.laue@unh.eduURL validation, lead optimization, formulation in drug

    development and QA/QC. Recent studies have used

    AUC to characterize the binding stoichiometry and

    binding sites of an anti-tumor agent; of a hemoglobin-

    stabilizing protein, and of a fibril growth inhibitor, and

    to assess the causes of protein aggregation. The recent

    addition of fluorescence to the existing absorbance and

    interference detectors dramatically extends the flex-

    ibility of analytical ultracentrifugation.

    aqueous solution.Tom Laue from the Center to Advance Molecular Interaction Science,

    an internationally acknowledged expert in the field, reviews latestadvances in technological as well as numerical methods. He summarizes

    useful applications and compares it to other methods.

    shape, molar mass, association energy, association stoichio-

    metry and thermodynamic nonideality of molecules in solu-

    tion. Because sedimentation relies on the principal property

    of mass and the fundamental laws of gravitation, it is a

    primary method for which the results are absolute and doAnalytical ultracenta powerful new tecdrug discoveryTom LaueCenter to Advance Molecular Interaction Science, University of New Hampsh

    Analytical ultracentrifugation (AUC) is a powerful

    means of characterizing the solution behavior of mole-

    cules. Sedimentation velocity analysis, the preferred

    AUC technique for characterizing complex systems,

    has higher resolution, broader applicable range and

    fewer solute/solvent limitations than gel-permeation

    chromatography. The technique is simple to perform1740-6749/$ 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2004.11.012Rudman Hall 379, 46 College Road, Durham, NH 03824, USA

    Section Editor:Oliver Zerbe Institute of Organic Chemistry, University ofZurich, Switzerland

    Many drugs interfere with protein oligomerization or disruption of

    proteinprotein interfaces. Analytical ultracentrifugation is a sensitivemethod to reveal the oligomeric state of proteins and thereby is able to

    detect changes in such. This capability might become of even moreinterest in the context of diseases related to protein misfolding, whichTECHNOLOGIES




    Kelvin Lam Pfizer, Inc., USA

    Henk Timmerman Vrije Universiteit, The N

    Lead optimizationifugation:hnology in 309

  • ability to detect trace quantities of components of interest in

    the presence of high concentrations of background (e.g.

    excipient) molecules.

    These two advances make AUC a first-choice technology

    for the characterization of solutions.

    The first advance: Lamm equation analysis

    Sedimentation is described exactly by the second order,

    differential Lamm equation [3]. In the past, extraction of s,

    D, f and Mb from sedimentation velocity data was difficult,

    particularly from complicated mixtures, because simple solu-

    tions to the Lamm equation analysis did not exist. Seminal

    work by Haschemeyer [10], along with clever computer pro-

    gramming by Schuck [3] and Stafford [4], use computer-

    generated solutions of the Lamm equation to fit sedimenta-

    Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004small samples in complex mixtures, enabling the study of

    valuable molecules under near in vivo conditions.


    Extensive reviews are available that cover the mathematics of

    the sedimentation process [1,2] and data analysis [36].

    Although knowledge of the theory occasionally is useful

    for interpreting some phenomena, it is not needed to appreci-

    ate the quantity and quality of data available from AUC. This

    review will focus on what AUC can do, and will specifically

    address the information available from sedimentation velo-

    city (as opposed to sedimentation equilibrium) analysis.

    The primary quantity of interest in SEDIMENTATION VELOCITY

    (see Glossary) is the sedimentation coefficient, s, which has

    both experimental and molecular definitions (Fig. 1). The

    experimental definition is s v=a, where v is the rate (typi-cally a few microns per second) at which a molecule moves in

    the gravitational field, a. Both v and a are readily measured

    quantities. The molecular definition is s Mb/f, where Mb isthe buoyant mass (the molecules mass less the mass of

    solvent it displaces), and f is the frictional coefficient, which

    depends on the molecules size and shape. Both Mb and f are

    useful parameters for characterizing a molecule.

    It is also possible to determine the diffusion coefficient, D,

    of a molecule from the shape of the sedimenting boundary

    (Fig. 1). Because D = RT/f, where R is the gas constant, T the

    temperature and f the same frictional coefficient as that in the

    definition of s, it is possible to determine the buoyant mass,

    Mb, from sedimentation velocity analysis as s/D = Mb/RT.

    Conversion from the buoyant mass to the anhydrous mass

    is straightforward [7], and has been automated for proteins

    (Sednterp, download available at


    Lamm equation: a second order differential equation which has no

    single analytical solution, but which can be solved using a mathematical

    method called finite element analysis. Used in direct fitting of sedimenta-

    tion velocity data.

    Sedimentation velocity: an analytical ultracentrifuge method in which

    the rate at which concentration boundaries move in a gravitational field is

    monitored.RASMB/), so AUC provides a rigorous means of determining

    solution mass.

    Two recent advances in analytical ultracentrifugation

    Analytical ultracentrifugation is an old method. However,

    two recent advances have increased its utility profoundly.

    The first advance is the use of direct fitting to the LAMM EQUA-

    TION (see Glossary) for the analysis of sedimentation velocity

    data [3]. Programs which use this analysis method are able to

    detect, quantify and characterize tiny quantities of solution

    contaminants. The second advance is the addition of fluor-

    310 www.drugdiscoverytoday.comescence detection [8,9], which extends the useable concen-

    trations into the picomolar range, as well as providing the

    Figure 1. Scans of the absorbance (A) at 230 nm as a function of radial

    position (r) taken at 8 min intervals during a sedimentation velocity

    experiment. Initially, there was a uniform concentration from the air

    liquid meniscus (spike at 6.1 cm) to the bottom of the cell (7.1 cm). The

    left-to-right progress of the boundary is marked by the arrows, and the

    spreading of the boundary as it moves down the cell is highlighted by the

    dots. The shape of the curves is described exactly by the Lamm equation,

    so that analysis of the curves eliminates nearly all of the noise [3].tion velocity data quickly and rigorously. Because neither

    stochastic nor systematic noise are solutions to the Lamm

    equation, they are filtered out, thus allowing very tiny sedi-

    menting boundaries to be detected (Fig. 2) and analyzed

    accurately (Fig. 3). The key point is that very weak signals

    can be detected and provide important molecular informa-

    tion. The sedimentation coefficient of trace quantities (>1%)

    of aggregates or cleavage products might be determined, and

    even complex mixtures of aggregates might be resolved and

    quantified. Although trace quantities less than 1% might be

    detected, reliable estimates of the amount and size of the

  • the analytical ultracentrifuge is useable from 1000 to

    60,000 rpm, a very wide range of gravitational fields might

    be used in AUC. Earlier analysis methods, however, had

    difficulty working with very small molecules (M < 3000)

    and very large molecules (M > 10,000,000). The advent of

    Lamm equation analysis removes both of these limitations.

    Consequently, sedimentation velocity analysis recently has

    provided unique and useful information on the association of

    small peptides [11], as well as on the heterogeneity and

    aggregation of viruses [12].

    In addition to providing s, D, f and Mb for molecules, Lamm

    equation analysis might be used to determine the association

    constants and stoichiometries, even for complex assembly

    schemes (Fig. 4) [3,4]. Recently, these analysis programs have

    been extended to analyze the combined data from several

    experiments, including data acquired using different first-

    principle methods [6]. In addition to Lamm equation analy-

    sis, programs based on thermodynamic first principles are

    available for the analysis of sedimentation equilibrium data

    [13]. Discussion of these programs is beyond the scope of this

    review. It is recommended that interested readers sign up for

    the RASMB e-mail forum (,

    explore web sites (http://www.analyticalultracentrifugation.

    com/, and participate in workshops

    devoted to the sedimentation analysis of interacting systems

    Vol. 1, No. 3 2004 Drug Discovery Today: Technologies | Lead optimization

    Figure 2. The incredible ability of Lamm equation analysis to detect

    trace quantities of sedimenting material is demonstrated here. Shown

    are fluorescence intensity data for a 1.4 pM monoclonal antibody (goat

    anti-mouse IgG) that had been labeled with Alexa-488 dye (5 dye/protein), with the intensities acquired at a 50% gain setting. The totalmaterial can require replicate measurements (see Outstand-

    ing issues).

    signal is12 intensity units (on a scale from 0 to 4095). Data (dots) foronly five scans are shown. The fitted data also are shown (solid lines),

    slightly offset from the raw data for clarity.Lamm equation analysis also broadens the applications for

    sedimentation velocity. For example, because the gravita-

    tional field depends on the square of the rotor speed, and

    Figure 3. The continuous size-distribution (c(s)) analysis for the data in

    Fig. 2 reveals a peak at 6.8 s, which is the correct value for IgG. Theanalysis included all 200 of the scans acquired during the experiment

    (105,000 data points), which were analyzed using Sedfit version 8.9

    ( and fit with an rms of

    0.29 intensity units.Figure 4. Titration of Alexa-488 labeled, goat anti-mouse IgG into a

    fixed concentration of mouse IgG, in 100 mM KCl, 20 mM Tris pH 8,

    0.1 mg/ml ovalbumin. The data have been normalized so that the

    distributions might be presented on a single graph. For each concentra-

    tion of labeled IgG, distributions are shown for samples with and without

    a fixed concentration of mouse IgG. In the absence of mouse IgG, the

    labeled IgG has a peak at 6.8 s, consistent with it being a monomer. The

    shift of the labeled IgG to higher s when unlabeled mouse IgG is added is

    the result of complex formation. Interestingly, at the highest concentra-

    tion of label, the distribution shifts back to lower values of s, consistent

    with the solution being in antibody excess. More detailed analysis of the

    curves would be necessary to determine the size of the complexes. All ofthese data were acquired in a single experiment. 311

  • Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004



    er th

    erea(e.g. that held at the University of Connecticut, http://

    The second advance: fluorescence detection

    The fundamental measurement needed for the analysis of

    sedimentation velocity is the radial concentration distribu-

    tion (Fig. 1). The three optical detectors available for the

    analytical ultracentrifuge are compared in Table 1. The three

    Table 1. Available optical detection systems for AUC

    Property Absorbance

    Sensitivitya 0.1 ODd (190800 nm)

    Radial resolutionb 20 mm

    Data acquisition timec 120 s/sample

    Sensitivity ++

    Selectivity ++

    Precision of analysis ++

    Resolution of analysis ++

    Notes Easiest to use

    When to use Need selectivity

    Added sensitivity


    buffer components

    a Typical minimum useful concentration for analysis. Because the optical systems rely onb The radial distance between independent concentration readings. The smaller this numbc How long it takes to acquire a concentration profile at the radial resolution listed. The short

    The absorbance and interference optical systems acquire data from one sample at a time, wh

    speeds below 6000 rpm, the time to acquire data is determined by the rotor period.d Absorbance unit.detectors have complementary applications. Absorbance and

    interference detectors have been available for some time, and

    an analysis of their strengths and weaknesses is available

    (Technical Bulletin 1821a, available from http://www.beck- The recent addi-

    tion of the fluorescence detector (Aviv Biomedical, Inc, for the Beckman Coulter

    XLA/I analytical ultracentrifuge (

    ) significantly broadens AUC analysis two ways. First, fluor-

    escence detection allows AUC analysis of very dilute solutions

    (Fig. 2), which is required for the analysis of very tight

    interactions or characterizing trace quantities of a solute.

    Second, the selectivity of fluorescence detection means that

    it is possible to perform rigorous AUC analysis of a particular

    molecule of interest in a very complex mixture (e.g. cytosol,

    serum). This means that the rigor of AUC analysis can be

    brought to bear on questions that heretofore were out-of-

    bounds to physical analysis. In particular, it is possible to

    determine the size of complexes under conditions that are far

    closer to in vivo than is possible with most other first-principle

    physical methods. Simple pull-down experiments can be

    performed using fluorescently labeled and unlabeled antibo-

    dies to probe for protein-protein complex formation (Fig. 4).

    312 www.drugdiscoverytoday.comFluorescently labeled ligands, such as a phospholipid, might

    be used as a probe for binding and interactions. Because of

    fluorescence detection, entirely new applications will be

    opened up to AUC analysis.

    Another useful characteristic of fluorescence detection is

    the broad concentration range accessible to fluorescence

    detection (Fig. 4). It is possible in a single experiment to

    monitor the sedimentation behavior of a fluorescent mole-

    Fluorescence Interference

    500 pM (fluorescein) 50 ug/mL

    20 mm 10 mm

    3040 s (all samples) 35 s/sample

    ++++ ++

    ++++ + ++++

    ++ +++

    Requires label No chromophore needed

    Requires sample dialysis

    Need selectivity Buffer absorbs

    Added sensitivity Sample does not absorb

    Non-dialyzable Absorbance variable

    Small quantities Short columns

    rent physical properties for detection, different concentration scales must be used.

    the more data are available for analysis, and the more detailed the analysis will be.

    e time, the more data will be available for analysis, and the more detailed the analysis will be.

    s the fluorescence system can acquire data from all of the samples simultaneously. At rotorcule over a concentration range of 1 pM10 mM. Hence, it

    becomes simple to determine the concentration ranges where

    molecular associations become significant. As is clear in

    Fig. 4, the full complexity of an interacting system is made

    plain. It would be impossible to mistake the data in Fig. 4 with

    a simple monomer-dimer equilibrium. Furthermore, the fact

    that the distributions exhibit an increase in s, followed by a

    decrease in s as the labeled IgG concentration is increased, is

    diagnostic for lattice formation (i.e. the concentration ranges

    of antigen excess and antibody excess are revealed).

    Relationship of AUC to drug discovery

    There are different applications for AUC, depending upon

    whether one is interested in small molecule drugs, or if one is

    developing biopharmaceuticals [14]. For these different areas,

    AUC often is an essential component of protein characteriza-

    tion, target protein validation, drug screening, drug devel-

    opment, drug formulation, and, for biopharmaceuticals,

    product QA/QC.

    If, for the moment, we restrict the discussion to small-

    molecule drugs, AUC can play a critical role in determining

    the strength and stoichiometry of a target molecules inter-

    actions. For example, if a drug target is a protein involved in a

  • signaling cascade, then AUC might be used to address the

    following questions:

    1. What is the native state of oligomerization of the target


    2. How strong are the interactions stabilizing the oligomers?

    3. What other proteins (or other macromolecules) bind to

    the target protein?

    4. What is the stoichiometry and strength of their associa-

    tion with the target protein?

    5. How does the drug affect the oligomerization of the target


    6. How does the drug affect the strength and stoichiometry

    of the interactions of other proteins with the target


    Some of these questions bear on the issue of target valida-

    tion, others bear on the mechanism drug of action, whereas

    others might be important in screening drugs.

    Although we are focused on small molecule drugs, it is

    often assumed that these drugs are monomeric in solution.

    characterization of the stoichiometry and affinity of the drug

    for the target molecule is possible.

    For biopharmaceuticals, AUC is an essential tool in char-

    acterizing the solution behavior of proteins and nucleic

    acids. The fact that AUC might be used with a wide range

    of solvents and over a broad solute concentration range

    makes it a versatile tool as well. Sedimentation velocity

    analysis provides a very sensitive, first-principle method

    for characterizing the size and amount of aggregates (and

    fragments) of biopharmaceuticals, a fundamental charac-

    terization in drug development, and an essential part

    of the QA/QC of protein drugs. It is no wonder, then, that

    the FDA has shown increased interest in having sedimenta-

    tion velocity analysis be a routine part of protein drug


    Because fluorescence is the basis of so many high-through-

    put screening assays, fluorescence-detected AUC will play an

    increasingly important role in understanding the signal

    changes observed in drug screening. All too often, fluores-

    cence-based high throughput screening results find hits that

    Vol. 1, No. 3 2004 Drug Discovery Today: Technologies | Lead optimization






    s co



    ostHigher purchase cConReLower disposable

    Multiple detectors

    s Larger sample volBroadest range ofHigh resolution

    No column matrixPros First-principle meHowever, it is clear that drugs that are marginally soluble in

    aqueous solution (e.g. those requiring DMSO for solubiliza-

    tion) might not remain monomeric. Using the XLI interfer-

    ence optics and sedimentation velocity analysis, it is possible

    to monitor the sedimentation of simple salts (sodium, phos-

    phate, chloride), so AUC analysis certainly should be con-

    sidered to determine the association state of small (Mr < 500)

    molecules in solution.

    If the small molecule drug is a chromophore, its sedimen-

    tation behavior will be influenced strongly by binding to a

    macromolecule. Under these circumstances, an accurate

    Table 2. Comparison summary table

    Technology 1

    Name of specific type of technology Analytical ultracen

    Name of specific technologies with associated

    companies and company websites

    ProteomeLab XL-

    Coulter Inc, http:ferences [3,4]consist of signals that are difficult to interpret in terms of a

    drug-binding event. Fluorescence-detected AUC provides a

    simple means of assessing the solution state of the fluoro-

    phore (e.g. bound versus free) in these systems, thus answer-

    ing the primary question.


    In the 1970s and 1980s, AUC received the reputation of being

    difficult to perform, useful only for highly purified systems

    and useful only for a deep physical chemical understanding

    of a solution. Over that time, textbooks dropped AUC, and

    Technology 2

    ugation Light scattering

    L-I, Beckman

    Dawn, MiniDawn; Wyatt Technology Corp:

    Expert System; Precision Detectors:

    Complete GPC/SEC; Viscotek, Inc:

    BI-MwA; Brookhaven Instruments,


    First-principle method

    Excellent sensitivity for aggregates

    Smaller sample volume

    lecular sizes Lower purchase cost



    Requires separate sample fractionation step by GPC

    Lower sensitivity to smaller molecules

    Higher disposable costs (GPC columns)[15,16] 313

  • 4 Stafford, W.F. (2000) Analysis of reversibly interacting macromolecular

    systems by time derivative sedimentation velocity. In Methods in

    Enzymology, (Vol. 323) Methods in Enzymology (Vol. 323) (Johnson,

    M.L., Ackers, G.K., eds) pp. 302325, Academic Press

    5 Schuck, P. (2003) On the analysis of protein self-association by sedimenta-

    tion velocity analytical ultracentrifugation. Anal. Biochem. 320, 104124

    6 Vistica, J. et al. (2004) Sedimentation equilibrium analysis of protein

    interactions with global implicit mass conservation constraints and sys-

    tematic noise decomposition. Anal. Biochem. 326, 234256

    Drug Discovery Today: Technologies | Lead optimization Vol. 1, No. 3 2004


    Reversible Associations in Molecular Biology:

    Sedfit analysis program and information:

    Alliance Protein Laboratories: Center to Advance Molecular Interaction Sciences:

    Biomolecular Interaction Technologies Center:

    Center for Analytical Ultracentrifugation of Macromolecular Assem-blies:

    Association of Biomolecular Resource Facilities: biologists confined their physical characteriza-

    tions largely to gels and chromatography.

    The fact is, although, AUC is far easier to perform than

    many of the routine methods of molecular biology. Further-

    more, AUC provides a rigorous, direct and easily understood

    glimpse into the solution behavior of molecules, often with-

    out any detailed quantitative analysis the appearance of an

    unexpectedly fast, or unanticipated slow boundary provides

    the insight needed to interpret confusing results from other

    experimental methods. The resolution is higher and the size

    range amenable to analysis is much larger for AUC than the

    nearest competing technique, gel filtration chromatography

    with light scattering detection (GPC-LS) (Table 2) [15]. Both

    AUC and GPC-LS provide first-principle molecular weight

    and size information, with GPC-LS being particularly sensi-

    tive to small quantities of aggregates. With AUC, sample

    fractionation is inherent to the sedimentation process,

    whereas sample fractionation for GPC-LS suffers from the

    limitations of chromatography (e.g. possible interference by

    the gel matrix, sample dilution, possible loss of aggregates in


    National Analytical Ultracentrifuge Facility:, limited useful size range, etc.). Even so, GPC-LS

    is less expensive to implement and can provide excellent

    results. Sample fractionation by field-flow fractionation

    (FFF) also has been coupled with light scattering [16]. This

    7 Laue, T.M. et al. (1992) Computer-aided interpretation of sedimentation

    data for proteins. In Analytical Ultracentrifugation in Biochemistry and

    Polymer Science (Harding, S.E., Horton, J.C., Rowe, A.J., eds), pp. 90

    125, Royal Society of Chemistry

    8 Schmidt, B. and Riesner, D. (1992) A fluorescence detection system for the

    analytical ultracentrifuge and its application to proteins, nucleic acids,

    Related articles

    Laue, T.M. and Stafford, W.F. III (1999) Modern applications of analytical

    ultracentrifugation in annual review of biophysics and biomolecular

    structure. Ann. Rev. 28, 75100

    Arakawa, T. and Philo, J.S. (1999) Applications of analytical ultracentri-

    fuge to molecular biology and pharmaceutical science. Yakugaku Zasshi

    119, 597611

    Hood, W.F. et al. (2001) Modulation of the binding affinity of myelo-

    poietins for the interleukin-3 receptor by the granulocyte colony-sti-

    mulating factor receptor agonist. Biochemistry, 40, 1395813606

    Sukumar, M. et al. (2004) Opalescent appearance of an IgG1 antibody at

    high concentrations and its relationship to noncovalent association.

    Pharm. Res. 21, 10871093

    314 www.drugdiscoverytoday.comviroids and viruses. In Analytical Ultracentrifugation in Biochemistry and

    Polymer Science (Harding, S.E., Horton, J.C., Rowe, A.J., eds), pp. 176

    207, Royal Society of Chemistry

    9 MacGregor, I.K. et al. (2004) Fluorescence detection for the XLI ultra-

    centrifuge. Biophys. Chem. 108, 165185

    10 Todd, G.P. and Haschemeyer, R.H. (1981) General solution to the inverse

    problem of the differential equation of the ultracentrifuge. Proc. Natl.

    Acad. Sci. 78, 67396743

    11 Schuck, P. et al. (1998) Determination of sedimentation coefficients for

    small peptides. Biophys. J. 74, 466474

    12 Lechner, M.D. and Machtle, W. (1999) Determination of the particle size

    distribution of 5-100 nm nanoparticles with the analytical ultracentrifuge.

    Consideration and correction of diffusion effects. Prog. Colloid Polym. Sci.

    113, 3743

    13 Johnson, M.L. et al. (1981) Analysis of data from the analytical ultra-method has the same virtues as GPC-LS, and does not suffer

    from the limitations of the gel matrix. However, FFF works

    best with particles that are larger than proteins, and does not

    have the resolution of GPC.

    Increasingly, AUC is being used to characterize the binding

    behavior, strength and affinity of molecules under considera-

    tion as anticancer pharmaceuticals [17], as fibroid growth

    inhibitors [18], antigens [19] and aggregation blockers [20].

    Given the tremendous amount of information available,

    those who neglect AUC do so at their own risk.

    References1 Fujita, H. (1975) Foundations of Ultracentrifugal Analysis. Wiley-

    Interscience, New York, NY

    2 Wills, P.R. and Winzor, D.J. (2002) Exact theory of sedimentation equili-

    brium made useful. Prog. Colloid Polym. Sci. 119, 113120

    3 Schuck, P. (1998) Sedimentation analysis of noninteracting and self-

    associating solutes using numerical solutions to the Lamm equation.

    Biophys. J. 75, 15031512

    Outstanding issues

    What is the lowest fractional amount of material that can be reliablydetected and characterized?

    How automated can the Lamm equation analysis be made? Is there a good general means for relating fluorescence intensity to

    absolute concentration?

    Material sticking to the cell walls and windows can prevent theanalysis of very low concentrations. What is the best way to prevent

    loss of samples to surfaces?centrifuge by nonlinear least-squares techniques. Biophys. J. 36, 575588

  • 14 Liu, J. and Shire, S.J. (1999) Analytical ultracentrifugation in the phar-

    maceutical industry. J. Pharm. Sci. 88, 12371241

    15 Tarazona, M.P. and Saiz, E. (2003) Combination of SEC/MALS

    experimental procedures and theoretical analysis for studying the

    solution properties of macromolecules. J. Biochem. Biophys. Methods

    56, 95116

    16 White, R.J. (1997) FFF-MALS a new tool for the characterisation of

    polymers and particles. Polym. Int. 43, 373379

    17 Lo, M-C. et al. (2004) Probing the interaction of HTI-286 with tubulin

    using a stilbene analogue. J. Am. Chem. Soc. 126, 98989899

    18 Hatters, D.M. et al. (2002) Suppression of apolipoprotein C-II amyloid

    formation by the extracellular chaperone Clusterine. Eur. J. Biochem. 269,


    19 Rosovitz, M.J. et al. (2003) Alanine-scanning mutations in domain 4 of

    anthrax toxin protective antigen reveal residues important for binding to the

    cellular receptor and to a neutralizing monoclonal antibody. J. Biol. Chem.

    278, 3093630944

    20 Gell, D. et al. (2002) Biophysical characterization of the alpha-globin

    binding protein alpha-hemoglobin stabilizing protein. J. Biol. Chem. 277,


    Vol. 1, No. 3 2004 Drug Discovery Today: Technologies | Lead 315

    Analytical ultracentrifugation: a powerful ?new? technology in drug discoveryIntroductionBackgroundTwo recent advances in analytical ultracentrifugationThe first advance: Lamm equation analysisThe second advance: fluorescence detection

    Relationship of AUC to drug discoveryConclusionReferences


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