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

DRUG DISCOVERY

TODAY

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

Editors-in-Chief

Kelvin Lam – Pfizer, Inc., USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Lead optimization
TECHNOLOGIES
Analytical ultracentrifugation:a powerful ‘new’ technology indrug discoveryTom LaueCenter to Advance Molecular Interaction Science, University of New Hampshire, Rudman Hall 379, 46 College Road, Durham, NH 03824, USA

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 perform

and should become a mainstay for target identification,

target 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.

E-mail address: (T. Laue) [email protected] http://www.camis.unh.edu/

1740-6749/$ � 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2004.11.012

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

Many drugs interfere with protein oligomerization or disruption of

protein–protein 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, which

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

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.

Introductionsedimentation can be used to analyze the solution behavior

of nearly any type of molecule over a wide range of concen-
The analytical ultracentrifuge is similar to the more familiar
preparative 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,

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 do

not depend on a comparison to standards. Consequently,

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

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 of

www.drugdiscoverytoday.com 309

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

Glossary

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.

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].

small samples in complex mixtures, enabling the study of

valuable molecules under near in vivo conditions.

Background

Extensive reviews are available that cover the mathematics of

the sedimentation process [1,2] and data analysis [3–6].

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 is

the buoyant mass (the molecule’s mass less the mass of

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

depends on the molecule’s 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 http://www.bbri.org/

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.com

escence detection [8,9], which extends the useable concen-

trations into the picomolar range, as well as providing the

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-

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

http://www.bbri.org/rasmb/


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 total

signal is �1–2 intensity units (on a scale from 0 to 4095). Data (dots) for

only five scans are shown. The fitted data also are shown (solid lines),

slightly offset from the raw data for clarity.

material can require replicate measurements (see Outstand-

ing issues).

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. The

analysis included all 200 of the scans acquired during the experiment

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

(http://www.analyticalultracentrifugation.com/) and fit with an rms of

0.29 intensity units.

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 (http://www.bbri.org/rasmb/),

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

com/, http://www.ap-lab.com/) and participate in workshops

devoted to the sedimentation analysis of interacting systems

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 of

these data were acquired in a single experiment.


http://www.analyticalultracentrifugation.com/

http://www.ap-lab.com/


http://www.camis.unh.edu/



Table 1. Available optical detection systems for AUC

Property Absorbance Fluorescence Interference

Sensitivitya 0.1 ODd (190–800 nm) 500 pM (fluorescein) 50 ug/mL

Radial resolutionb 20 mm 20 mm 10 mm

Data acquisition timec 120 s/sample 30–40 s (all samples) 3–5 s/sample

Sensitivity ++ ++++ ++

Selectivity ++ ++++ �

Precision of analysis ++ + ++++

Resolution of analysis ++ ++ +++

Notes Easiest to use Requires label No chromophore needed

Requires sample dialysis

When to use Need selectivity Need selectivity Buffer absorbs

Added sensitivity Added sensitivity Sample does not absorb

Non-dialyzable Non-dialyzable Absorbance variable

buffer components Small quantities Short columns

a Typical minimum useful concentration for analysis. Because the optical systems rely on different physical properties for detection, different concentration scales must be used.b The radial distance between independent concentration readings. The smaller this number, the more data are available for analysis, and the more detailed the analysis will be.c How long it takes to acquire a concentration profile at the radial resolution listed. The shorter the time, the more data will be available for analysis, and the more detailed the analysis will be.

The absorbance and interference optical systems acquire data from one sample at a time, whereas the fluorescence system can acquire data from all of the samples simultaneously. At rotor

speeds below �6000 rpm, the time to acquire data is determined by the rotor period.d Absorbance unit.

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

www.ucc.uconn.edu/�wwwbiotc/UAF.html).

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

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-

man.com/resourcecenter/literature/BioLit). The recent addi-

tion of the fluorescence detector (Aviv Biomedical, Inc,

http://www.avivbiomedical.com/) for the Beckman Coulter

XLA/I analytical ultracentrifuge (http://www.beckman.com/

) 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).


Fluorescently 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-

cule over a concentration range of 1 pM–10 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 molecule’s inter-

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

http://www.bitc.unh.edu/



http://www.cauma.uthscsa.edu/


http://www.abrf.org/JBT/1999/December99/dec99cole.html

http://www.ucc.uconn.edu/~wwwbiotc/uaf.html


signaling cascade, then AUC might be used to address the

following questions:

1. W

Ta

Na

Na

c

Pro

Co

Re

hat is the native state of oligomerization of the target

protein?

2. H
ow strong are the interactions stabilizing the oligomers?
3. W
hat other proteins (or other macromolecules) bind to
the target protein?

4. W
hat is the stoichiometry and strength of their associa-
tion with the target protein?

5. H
ow does the drug affect the oligomerization of the target
protein?

6. H
ow does the drug affect the strength and stoichiometry
of the interactions of other proteins with the target

protein?

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.

However, 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

ble 2. Comparison summary table

Technology 1

me of specific type of technology Analytical ultracentrif

me of specific technologies with associated

ompanies and company websites

ProteomeLab XL-A/X

Coulter Inc, http://ww

s First-principle method

High resolution

No column matrix

Broadest range of mo

Lower disposables co

Multiple detectors av

ns Larger sample volume

Higher purchase cost

ferences [3,4]

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

validation.

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

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.

Conclusion

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

w.beckman.com/

Dawn, MiniDawn; Wyatt Technology Corp:

http://www.wyatt.com/

Expert System; Precision Detectors:

http://www.precisiondetectors.com/

Complete GPC/SEC; Viscotek, Inc:

http://www.viscotek.com/

BI-MwA; Brookhaven Instruments,

Inc: http://www.bic.com/

First-principle method

Excellent sensitivity for aggregates

Smaller sample volume

lecular sizes Lower purchase cost

st

ailable

Requires separate sample fractionation step by GPC

Lower sensitivity to smaller molecules

Higher disposable costs (GPC columns)

[15,16]








Links

� Reversible Associations in Molecular Biology: http://www.bbri.org/

rasmb/

� Sedfit analysis program and information: http://www.analyticalultra-

centrifugation.com/

� Alliance Protein Laboratories: http://www.ap-lab.com/

� Center to Advance Molecular Interaction Sciences: http://www.ca-

mis.unh.edu/

� Biomolecular Interaction Technologies Center: http://www.bitc.un-

h.edu/

� Center for Analytical Ultracentrifugation of Macromolecular Assem-

blies: http://www.cauma.uthscsa.edu/

� Association of Biomolecular Resource Facilities: http://www.abrf.org/

JBT/1999/December99/dec99cole.html

� National Analytical Ultracentrifuge Facility: http://www.ucc.ucon-

n.edu/�wwwbiotc/uaf.html

Outstanding issues

� What is the lowest fractional amount of material that can be reliably

detected 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 the

analysis of very low concentrations. What is the best way to prevent

loss of samples to surfaces?

molecular 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

pre-filtration, 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

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, 75–100

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

fuge to molecular biology and pharmaceutical science. Yakugaku Zasshi

119, 597–611

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, 13958–13606

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

high concentrations and its relationship to noncovalent association.

Pharm. Res. 21, 1087–1093


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.

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