Download ProteomeLab™ XL-A/XL-I Protein

PROTEOMELAB XL-A/XL-I PROTEIN CHARACTERIZATION SYSTEM
TM
NATIVE CONDITION PROTEIN CHARACTERIZATION
• Stoichiometry
• Molecular Conformation
The ProteomeLab XL-A or XL-I provides the ideal
solution to questions of protein characterization by
uniquely measuring therapeutic targets as interacting
elements instead of in isolation or bound to a substrate.
In addition, it provides a complementary approach to
traditional instrument methods and technologies like
size exclusion chromatography, surface plasmon
resonance, and mass spectrometry.
P-10119B
B2006-7120-MG-3
MW ~ 144,000 Da
Two mechanisms of inhibition
Compound B
0.45
0.4
Absorbance 360 nm
0.4
0.3
0.2
0.1
0
6.9
6.95
7
Mechanism A
Mechanism B
Inhibition
timedependent
not timedependent
[Enzyme]
sensitive
not sensitive
SPR kinetics
slow
fast
SPR stoichiometry
variable
low
Sedimentation eq.
aggregated
monomeric
MW ~ 35,000 Da
7.05
7.1
radius, cm
0.35
0.3
0.25
7.15
2
6.4
6.45
6.5
radius, cm
6.55
6.6
Sedimentation analysis
provides a complementary
approach to analyze and
confirm stoichiometry and
mechanism of assembly.
Sedimentation for Success, Characterizing small molecule / protein interactions by
sedimentation analysis helps validate lead series early in drug-discovery processes,
Michelle Arkin, Modern Drug Discovery, November 2004.
Two compound series shown to result in a relative change in the sedimentation
profile of the molecular weight complex. The aggregate species is clearly shown
to produce an exponential-like profile.
Better Lead Optimization
Aggregation
Sedimentation analysis is ideally suited for quantifying protein
aggregation with measurements made in free solution under
physiological conditions and temperatures, typically in the same
formulation buffer and concentrations as the stored product.
Bioactivity
Immunogenicity
Sedimentation
coefficient distribution for a highly
stressed monoclonal
antibody sample
showing aggregates
at levels as low as
0.1%, by weight.
heptamer, 0.1%
hexamer, 0.4%
0.2
tetramer 5.3%
0.4
trimer 14.6%
? free light chain, 1.4%
0.6
pentamer 1.4%
dimer 30.6%
0.8
c(s) vs S plot
0.14
0.12
% Monomer – 68.2%
% Aggregate – 23.1%
% Empty capsid – 7.9%
Monomer
0.10
0.08
c(s)
main peak (monomer), 45.5%
1.0
? HL half molecule, 0.8%
Characterizing biopharmaceuticals prior to pre-clinical and clinical trials
can save millions of dollars in development costs by eliminating expensive
reformulations. Aggregation, which affects bioactivity, pharmacokinetics,
and immunogenicity is of particular interest in the area of protein therapeutics.
High dose protein drugs, such as monoclonal antibodies administered
subcutaneously, require formulations at high concentration (>50 mg/mL)
which can exacerbate the potential for aggregation. In addition to stability
issues, concerns from small amounts of aggregates generating an immune
response is causing a growing regulatory interest for including complementary methods of analysis beyond the industry standard of size
exclusion chromatography.
Aggregates
0.06
Empty capsid
0.04
0.02
0.0
0
2
4
6
8
10
12
14
16
18
20
22
24
0.00
10
sedimentation coefficient (Svedbergs)
0.5
Analysis of
two different
formulations
of an antibody
reveals differences
in aggregation
that could
not be seen by
size exclusion
chromatography.
shift of main peak is due to
differences in buffer viscosity
0.4
0.3
a low ionic strength
formulation produces
significantly less dimer
0.2
0.1
0.0
2
4
6
8
10
12
sedimentation coefficient (Svedbergs)
• Heterogeneity / Aggregation
• Interacting / Self-Associating systems
Compound A
0.5
Absorbance 360 nm
Understanding the binding of small molecule antagonists helps validate
lead series early in the drug-discovery process. In high-throughput drug
discovery projects, initial “hits” are often identified by the inhibition
of protein function. “False positives” arising from functional screening
can include compounds that act through high stoichiometry binding
or protein aggregation. By contrast, compounds that have well-defined
stoichiometry and binding site are more likely to generate successful
drug leads. Sedimentation analysis offers a straightforward approach
for characterizing binding stochiometry and affinity early in the drug
discovery process and at the same time identifies binding artifacts or
anomalies.
Characterizing the Aggregation and Conformation of Protein Therapeutics.
John S. Philo, American Biotechnology Laboratory, October 2003.
110
210
310
410
510
610
710
810
910
S
Sample concentration = 1.720 fringes, can be converted to vp/mL
Viral Vectors & Vaccines Conference, Nov. 2004, Steven Berkowitz, Ph.D., Biogen Idec.
Assessing adenovirus homogeneity employed
in a gene therapy delivery system allows for
determination of the relative concentrations
of both monomer and aggregate.
Demonstrated Comparability Testing
Conformation
2
0.02
Characterizing the homogeneity of biopharmaceuticals provides an efficient means of
demonstrating that samples from different manufacturing lots, or material from different
purification processes, all contain the same molecular conformation. Sedimentation analysis
uses a unique approach for demonstrating comparability by precisely measuring sedimentation
coefficients to reveal detailed information on molecular conformation and aggregate level
and types of protein therapeutics. Indeed, one of the benefits of this approach is that
sedimentation coefficients are absolutely calibrated (not relying on molecular standards)
and can be measured to a precision of +/- 0.2% or better for comparisons within the same
run and +/- 0.5% run-to-run.
normalized c(s)
An operating system that does not require calibration
or use of standards enables measurements based on
a redistribution of a sample’s molecular mass. Simple
yes/no answers provide the flexibility to get quick,
top level answers to questions regarding:
Interacting Systems
c(s), normalized (total area = 1)
Beckman Coulter’s ProteomeLab ™ XL-A/XL-I
Protein Characterization System with native
condition capability provides the ideal solution to
satisfying the demands of the drug discovery process
by characterizing a protein’s behavior in free solution
under physiological concentrations, temperatures
and buffer conditions. The XL-A with absorbance
optics provides the selectivity and sensitivity to
measure protein behavior between 190 – 800 nm.
The XL-I adds an additional optical system based
on Rayleigh Interference Optics for use in UV
absorbing buffers that provides higher concentration
capability and greater precision.
Early Lead Validation
g(s*) (AU/Svedberg)
A growing demand for protein, antibody, and
small molecule therapeutics is challenging the biopharmaceutical industry to improve drug discovery
processes and accelerate time to market, reduce drug
development costs, and ensure optimum dose delivery.
To satisfy these demands, high-throughput screening
assays are including steps to validate “hits” and
reduce the number of “false positives” in lead series.
More robust methods for protein characterization
are being employed to avoid the expense of adjusting
a drug formulation during pre-clinical / clinical
trials and minimize the risk of side effects by
quantifying lot-to-lot variations in product
comparability testing.
X100
lot 1
lot 2
0.01
1
0.95%
0.42%
0.05%
0.30%
0.03%
0.07%
Comparability of conformation and aggregation for two different
manufacturing lots of a monoclonal antibody with results from both
lots showing good homogeneity (98.6% main peak or better) and the
same molecular conformation based on reproducible sedimentation
coefficients (6.339 S for lot 1 and 6.335 S for lot 2).
0.10%
0.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
sedimentation coefficient (svedbergs)
www.beckmancoulter.com/xla
Characterizing the Aggregation and Conformation of Protein Therapeutics.
John S. Philo, American Biotechnology Laboratory, October 2003.
© 2006 Beckman Coulter Inc.
Printed in U.S.A. on recycled paper.