microcal dsc systems introduction to technology ... · preformulations most stable construct...
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MicroCal DSC systems
Introduction to technology,
instruments and
information provided
MicroCal DSC systemsInstruments & Software, Expertise and Services
Instrument
PlatformSoftware Expertise
Service
Used in leading academic, pharmaceutical and biotech laboratories
– Life Science research
– Drug development (primary biotherapeutics)
– Process development and formulation
30 40 50 60 70 80 9002468
101214
oC)
Native Unfolded
Protein stability
DSC experimental principle
Protein unfolding followed through heat changes
30 40 50 60 70 80 90
0
2
4
6
8
10
12
14
Cp
(kca
l/m
ole
/o C)
Temperature (C)
Folded Unfolded
T=20°C T=90°C
2
6
10
14
Cp
(kca
l/m
ole
/o C)
30 40 50 60 70 80 90
oTemperature(C)
Tm
DH
Tm (transition midpoint) is an indicator of stability
ΔH (enthalpy) is an indicator of stabilizing forces/energetics
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Protein unfolding
Applications for stability determination using
Tm shift analysis
Tm shift reflects
changesin stability
Optical clarity of
samples is unimportant
Comparative studies:
− Identify the most stable
construct or clone
− Identify the most
stabilizing conditions
• Control (e.g.
native)
• Mutant form
• Post-translational
change
• Alternative buffer
composition
• Long term storage
effects
Example: Tm shift analysis between native and mutant proteins
Effects of removal of an α-helix on thermal stability
Measurements of Tm using MicroCal DSC detects any
changes
Result: Increases in thermal stability observed
by Tm shifting
With differential scanning calorimetry
you can…
Characterize and select the most
stable protein or biotherapeutic
candidate
Optimize expression, purification and
manufacturing conditions in days
Rapidly and easily determine
optimum conditions for liquid
formulations
MicroCal VP-Capillary DSC
MicroCal VP- DSC
MicroCal VP-Capillary DSC
Get it right from the start – Improved productivity,
assess protein stability quickly and easily
Sensitivity and data quality for confident decision-making
Fully automated unattended run
Minimum assay development with direct in solution
technique
Streamlined workflow and
data analysis for faster results
• Key data parameters (Tm, ΔH)
generated automatically
Also available as a manual, single
sample system
• Upgradable to full automation
MicroCal VP-DSC system
When stability counts
Directly measures the heat of binding (enthalpy, ΔH) and transition midpoint (Tm)
Single sample
Manual sample loading
Unattended operation
MicroCal DSC systems
Customers & Key applications
Where we find our MicroCal DSC
customers
Application areas
Manufacturability
Optimization of purification conditions
– Determine optimal bioprocessing conditions
Biocomarability
– Process changes - ensure comparability of the product and the formulation
– Biosimilars
Formulation Development
– Develop correct liquid formulation
– Effects of excipients
– Long-term stability studies
Quality control &
ManufacturingTarget discovery Lead discovery
Process development
& Formulation
Preclinical & Clinical
Research
Biotherapeutics & Vaccines
Target customers
Pharma/Biotech/Contract
Reserach Organization (CRO)
Biochemists and molecular
biologists
Screening departments, antibody
analytics, characterization,
protein/Ab engineering,
biophysical analysis
departments, assay development,
process development and QC
MicroCal VP-Capillary DSC in preformulation
development
› Preformulations:
Identify the most stable
construct or clone
Identify the most
stabilizing conditions
Identify the most
destabilizing conditions
Preformulations
Most stable
construct
Stabilizing
conditions
Destabilizing
conditions
Example 1: The use of DSC to aid in the selection of
antibody mutants
Most stable antibody constructs identified
Stability of each domain
can be assessed
Minor differences in
primary sequence can
have a big impact on
antibody stability
The least stable
expressed poorly and
quickly formed high
MW aggregates
Demarest et al, Application note
Results from a pH primary
screen of a therapeutic
antibody
DSC was the most accurate
and fastest predictor of
suitable formulations
Example 2: The use of DSC for stability screening during formulation developmentFind stabilizing conditions faster
Black - T=0
Grey - 2 weeks at 40°C
Red - 4 weeks at 40°C
F. Ollila, Novartis Pharma AG.
application note
Size Exclusion
Chromatography
Laser Light
Scattering
DSC Tm
MicroCal VP-Capillary DSC system in process
development
Process
development
Optimize yields
Reduce costs
Process development:
Identify stable elution conditions
Optimize yields of
biotherapeutic from purification
Reduce costs
Example 1: The problem 1(3)
Low utilization of resin
capacity (2 mg/ml)
Increased loading caused
precipitation in column eluate
Further Processing
Protein A
Capture
Elution
Hold Step
UF/DF
P. Acharya, application note
Example 1: The solution: identify stabilizing
elution conditions using DSC 2(3)
DSC identified the
most stabilizing
elution conditions
This allowed
increased
functional loading
capacity since
mAb is stabilized
P. Acharya, application note
Example 1: Benefits of increasing antibody
eluate stability 3(3)
Increasing the functional loading capacity
of the Protein A column resulted in time
savings
Eluting the antibody at higher
concentrations eliminated a UF/DF step
resulting in time and cost savings
P. Acharya, application note
Only DSC offers universal thermal stability
assay
Heat as universal readout
No optical artifacts, works
in turbid solutions
Works under controlled
conditions in most of
buffers
Allows to study protein
samples as they are
Minimum assay
development
Summary
Major application areas:
Preformulation & Process
development
• Predict long term stability
• Understand the non-covalent
forces responsible for protein
or lipid
micelles stability
• Compare native, altered and
mutant forms
MicroCal DSC systems measure
the stability of biomolecules
Stability
Mechanism