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User reviews and opinions
|Dak||2:15pm on Sunday, October 17th, 2010|
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Data file 28-9785-70 AA MicroCal label-free interaction analysis
MicroCal VP-Capillary DSC system
Differential scanning calorimetry (DSC) is a powerful analytical tool for characterizing the stability of proteins and other biomolecules. DSC directly measures the enthalpy (H) and temperature (TM) of thermally induced structural transitions in solution. This information gives valuable insights into factors that stabilize or destabilize proteins, nucleic acids, micellar complexes, and many other macromolecular systems, alone or as complexes. This information can be used to predict shelf lives, develop purification strategies, and characterize and evaluate protein constructs or other biotherapeutic entities. In addition ligand affinities to a protein target in small molecule drug discovery programs can be ranked rapidly. MicroCal VP-Capillary DSC System (Fig 1) provides high throughput and sensitivity in an automated, integrated platform.
Key features and benefits:
Industry-proven stability-indicating technique Direct measurement of biomolecular stability in solution Minimal assay development required Screen up to 50 samples per day with unattended operation MicroCal VP-Capillary DSC system has an active cell volume of 130 l allowing for thermodynamic measurements of even precious samples (sample concentrations of about 0.2mg/ml are typically needed). A fully integrated autosampler enables running of up to 50 samples per day. All filling, injection, and cell cleaning functions are fully automated for walk-away operation. For applications where higher throughput and unattended operation and sample handling are not required, MicroCal Manual Capillary DSC without autosampler is also available. MicroCal VP-Capillary DSC system is controlled by an intelligent user interface, VPViewer and data analysis is performed with Origin software (OriginLab Corporation). A schematic diagram of the thermal core of MicroCal VP-Capillary DSC system is shown in Figure 2. Matched capillary cells provide fast scan rates and fast temperature equilibration. The cells allow user selectable scan rates up to 250C/h. Precise temperature control is maintained with Peltier elements. The inside surface of the cell is covered with Tantalum 61 for chemical resistance and to ensure inertness when working with proteins and other biomolecules.
Fig 1. MicroCal VP-Capillary DSC system.
The matched sample and reference capillary cells are fixed in place within a silver adiabatic jacket ensuring reproducible and ultrasensitive performance with low maintenance. Three user-selectable response times are available, high gain, low gain, and passive. Passive mode is the most sensitive and is used to scan broad transitions (protein denaturation) while the high gain mode is recommended for sharp transitions (e.g., lipids). The system has a self-contained pressurizing system (0 to 45 psi) for the study of solutions above their boiling point.
Inlet/outlet tubes (To distributor valve) T-1
Fig 3. MicroCal VP-Capillary DSC software user interface.
Fig 4. Microsoft Excel sample list and experiment template.
Automated data analysis
The automated data analysis feature in MicroCal VPCapillary DSC software works seamlessly with Origin software to significantly reduce the data analysis bottleneck. Buffer scans and baselines are automatically subtracted and data is automatically normalized to concentration and immediately ready for the user to analyze. The user can also set baseline detection and subtraction limits to select the desired peaks for a particular experiment. All key data parameters, including TM, TM, H, and TM onset, are automatically generated and tabulated. All data are easily exported to Microsoft Excel.
Fig 2. Schematic diagram of the thermal core of MicroCal VP-Capillary DSC system.
Flexible experiment design and setup
MicroCal VP-Capillary DSC software facilitates experiment design and setup by providing a single, intuitive graphical user interface for selecting and entering all sample and experiment run parameters (Fig 3). For maximum productivity, the software includes a Microsoft Excel sample list and experiment template (Fig 4) that allows offline experiment design and setup. A completed template can be saved and reused reducing the time it takes to design and set up future experiments. Additional sample trays can be appended to a running experiment without having to interrupt the run, providing flexibility for multiuser operation. Efficiency in experiment design is further enhanced with intelligent run parameter defaults and optional data fields for completely describing samples.
The DSC experiment
DSC measures the heat absorbed when a protein or other biological macromolecule undergoes a melting between a native, biologically active conformation and an unstructured, inactive conformation. A protein is placed in the calorimetric cell and is heated at scan rates typically between 10C and 240C/h. Protein unfolding is an endothermic event and is observed in DSC as a positive displacement in the signal (the heat capacity).
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The midpoint of this melting transition is known as the TM, and the area under the melting curve is the enthalpy, H, of the process (Fig 5). In addition to the classical application of understanding the noncovalent forces responsible for macromolecular assembly and stability, these data can be used to understand the factors that contribute to the folding and stability (shelf life) of the macromolecules.
Cp (Kcal mol-1 C-1) 15
be an exceptionally good indicator of the relative stability of liquid formulations*. Figure 6 shows an example where a MicroCal VP-Capillary DSC system has been used to rapidly screen formulation conditions for a monoclonal antibody (MAb). The stability study was done as part of a preliminary biophysical study and was used to narrow down the solution conditions (pH, buffer and excipient concentration) to be used in the subsequent optimization cycle.
Cp (Kcal mol-1 C-1)
Fig 5. A protein is denatured in the DSC. The peak maximum is the TM and the area under the curve is the enthalpy, H, of the process.
In many cases, decisions can be made on which excipients and buffers will lead to the most favorable conditions for the protein or biotherapeutic simply by observing shifts in the TM. Shifts to a higher TM indicate more favorable stabilizing conditions. Strong correlations between TM data and shelf life are frequently observed. In addition to giving insight into the thermodynamics of protein stability, the area-under-the-curve data (H, enthalpy) reflect the extent of folded molecules making DSC ideal for quantitation of the intact protein after storage, purification, or manufacture.
55 None Sodium chloride Sorbitol Sucrose
Fig 6. (A) DSC data for a therapeutic antibody in several different buffer types. (B) Thermal transitions as a function of buffer type and excipients.
Stability screening for formulations development
Liquid biotherapeutics are highly desirable because they are easy to administer and less expensive to produce than lyophilized drug product. The challenge is finding conditions where they remain active with no aggregation and no precipitate formation. TM data has been shown to
The buffer-excipient combinations containing the sample protein were scanned at 60C/h, using a protein concentration of 2mg/ml. The raw data (Fig 6A) was buffer baseline subtracted, the concentrations normalized, and the thermal transitions plotted as a function of buffer types and excipient (Fig 6 B). These data show that acetate buffer is not suitable and a combination with sodium chloride, in particular, should be avoided in further optimization studies.
*Data and images courtesy of Alex Tracy, Novartis (formerly with KBI Biopharma).
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Optimization of purification conditions
Biotherapeutic downstream production processes must achieve high purity and adequate yield on a large scale and within a given economic framework. Using DSC, the most stabilizing loading and elution conditions can be established during process development. In Figure 7, DSC is used to establish the stability of an antibody in a range of buffers.
When a drug candidate binds to the target protein, the TM of the drug-target complex is shifted relative to that of the target molecule alone. The binding constant can be calculated from the TM shift at a single ligand concentration(1). The expressions describing this relationship are in the software provided with MicroCal VPCapillary DSC instrument. Ribonuclease A (RNAseA, 0.03 mM in 50mM potassium acetate, pH 5.5) was studied with MicroCal VPCapillary DSC in the presence of 0, 0.5, and 1mM of 2CMP, and data from a series of 96measurements performed at a scan rate of 240C/h taken over 2 days (approx. 50/day) are shown in Figure 8.
Cp (Kcal mol-1 C-1) 18
Citrate + mannitol, pH 3.5 Glycine + Arg, pH 3.0
Glycine + Ile, pH 3.mM glycine, pH 3.0
50 mM glycine, pH 3.0
Glycine + Leu, pH 3.0 Citrate, pH 3.90 Temperature (C)
0 -Temperature (C)
Fig 7. DSC unfolding profiles of an antibody in seven elution buffers. The black lines are the concentration-normalized data and the blue lines are the deconvoluted peaks using the integrated software provided with the instrument.
The established purification protocol required elution at low pH, a condition in which the antibody was unstable and had a high tendency to aggregate. To minimize the effect, the proteins were loaded and eluted at low concentrations. The DSC study showed that the stability of the antibody was significantly enhanced in the presence of mannitol (see the top DSC profile in Fig 7) and, when included in the elution buffer, resulted in a 7.5-fold increase in the functional capacity of the purification medium and a comparable reduction in the cost to purify the antibody as a result of the better utilization of this purification medium.
Fig 8. DSC scans of RNAse A with no 2CMP (8 scans overlaid at the lowestTM), 0.5 mM 2CMP (7 scans, overlaid at the middle TM) and 1 mM 2CMP (7scans overlaid at the highest TM).
Data and image courtesy of Prathima Archarya, Biosonata Consulting (formerly with Diosynth Biotechnologies).
The scans were automatically analyzed to generate TM and H values. TM was 61.62 0.06C in the absence of 2CMP, 66.7 0.03C in the presence of 0.5 mM 2CMP, and 67.77 0.03C in the presence of 1 mM 2CMP. The binding constant determined at both concentrations was approx. 1 M, at 25 C, under both conditions and compares favorably with direct isothermal titration calorimetry measurements.
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The relationship between affinity and TM shift means that DSC can be used to rank order the affinities of related compounds. TM data for RNase (0.09 mM in 50 mM acetate buffer, pH 5.5) obtained with MicroCal VP-Capillary DSC at 200C/h in the presence of a number of ligands at a concentration of 10 mM are shown in Table1 together with KD as determined by isothermal titration calorimetry (ITC) at 25C under the same buffer conditions.
Table 1. TM and KD values for a series of RNase ligands
MicroCal VP-Capillary DSC and MicroCal Manual Capillary DSC system Short-term noise (RMS average) Baseline repeatability Experimental temperature range Minimum response time (high gain) 0.6 cal/C1 1.5 cal/C2 -10C to 130C 5s Tantalum 130 l, capillary type, fixed-in-place, nonremovable 8.2 kg 10 kg 43 16.cm 82.8 38.5 64.8 cm
TM (C) No ligand Phosphate 3UMP Pyrophosphate 2CMP 61.08 62.95 67.47 68.24 70.71
KD (M) at 25C 12 1.7
Weight, calorimeter Weight, autosampler Dimensions , calorimeter (W H D) Dimensions, autosampler (W H D)
The rank order of TM data generated by DSC is the same as the affinity data determined directly by ITC, demonstrating the utility of DSC for comparing the binding of related ligands. It should be noted that certain assumptions may need to be made for the quantitative determination of KD using this method, and that these values should be considered as approximate values in all but the most favorable instances. The magnitude of the TM shift is related to both the concentration and affinity of the added ligand. A weak binder will typically induce a smaller TM shift than a tight binder at the same concentration. This has practical consequences for those involved in fragment-based or more traditional high-throughput screening campaigns in small molecule drug discovery. To screen for the weak (approx. 1mM) binders typical for fragment-based programs, the DSC measurements would require ligand concentrations close to 1 mM to cause a 1C shift. In more traditional hit-validation campaigns, ligand concentrations close to 50 M could be used. Compounds with affinities of 5 M or lower would induce TM shifts of 2C or more during these circumstances.
Using upscan mode at 200C/h with 10 s filter and over a temperature range of 10C to 110C, in passive mode. In upscan mode at 60C/h. When using upscan mode at 200C/h, the upper temperature limit is 115C.
Product MicroCal VP-Capillary DSC MicroCal Manual Capillary DSC MicroCal VP-Capillary DSC Software 2.0 Upgrade Code no. 28-4289-48 28-4289-46 28-9784-80
1. Brandts, J.F. and Lin, L.N. Study of strong to ultratight protein interactions using differential scanning calorimetry. Biochemistry 29, 6927-6940 (1990).
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GE, imagination at work, and GE monogram are trademarks of General Electric Company. MicroCal is a trademark of GE Healthcare companies. All third party trademarks are the property of their respective owners.
For local office contact information, visit www.gelifesciences.com/contact www.gelifesciences.com/microcal GE Healthcare Bio-Sciences AB Bjrkgatan 84 Uppsala Sweden
2010 General Electric Company All rights reserved. First published May 2010 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse 5 D-79111 Freiburg Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327 Piscataway, NJ 08855-1327 USA GE Healthcare Japan Corporation Sanken Bldg., 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073 Japan
imagination at work
28-9785-70 AA 05/2010
GE Healthcare Life Sciences
Application note 28-9870-39 AA MicroCal label-free interaction analysis
Shelf-life determination of low molecular weight compounds
Product stability is an important parameter in pharmaceutical development. This application note demonstrates the use of calorimetry to determine the stability of an aqueous sample of cefazolin. The approach is widely applicable to low molecular weight materials in both aqueous and nonaqueous solvents. Primary areas of applications are drug formulation and stability, small molecule characterization, nucleic acid analogs, and pesticide development.
O O N N N N O N H
Fig 1. Structure of cefazolin.
Estimates of product stability are often made using accelerated thermal decomposition studies at high temperatures followed by extrapolation of the data to the temperature of interest. When the temperature range under investigation is much higher than the temperature of interest, the extrapolation of results is less certain since the mechanism of decomposition can be different at different temperatures. Previously, calorimetry has rarely been used for stability studies since limitations in sensitivity prevented meaningful measurements at low temperatures (0C to 25C) where decomposition rates are very low. In this temperature range, heats of decomposition are so small as to be virtually undetectable by most calorimeters. The sensitivity of MicroCal VP-DSC is high enough to overcome this limitation. Here we show how MicroCal VP-DSC can be used to provide stability information at lower temperatures previously unattainable by differential scanning calorimetry.
Materials and methods
DSC scans were performed using a 9.5 mg/ml aqueous solution of the antibacterial drug cefazolin (Sigma Biochemicals, Fig 1). The sample was scanned using MicroCal VPDSC from approximately 20C to 130C at a heating rate of 3.8C per hour. After rapid cooling, the sample was scanned a second time. Results were compared against a water-water baseline determined using the same experimental conditions. For isothermal studies, another solution of cefazolin at the same concentration was observed for 16.3 hours at 60C using the isothermal mode of MicroCal VP-DSC. A waterwater baseline was determined in the same manner over a 25hour period.
DSC scanning study
The results of the two cefazolin scans and water-water baseline are shown in Figure 2. In the first scan, the heat of decomposition is readily apparent at temperatures above 35C, leading to a large exotherm with a minimum at 92C. Above 120C, another exothermic process is apparent. The shape of the repeat scan is almost identical to the baseline below 92C. Above this temperature, the second exothermic process can be seen again.
Cp (mcal min-1) 0
Since more than one decomposition process occurs at high temperature, we have chosen to study the low temperature range (20C to 60C) further.
DSC isothermal study
Isothermal scans at 60C are shown in Figure 4, including a water-water control scan which establishes the zeroheat baseline. At this temperature there are two separate decomposition processes occurring in the cefazolin solution. During the first five hours of the isothermal study, a minor impurity appears to decompose faster than cefazolin itself. After this period, cefazolin decomposition shows a nearlinear decrease in rate of decomposition with time, which is the expected behavior for a first-order decay.
Temp. (C) Cp (mcal min-1) Control 0
-0.8 1st scan -1.120
Fig 2. DSC scans of an aqueous cefazolin solution.
The excess heat associated with the primary mechanism of decomposition at lower temperatures is obtained by subtraction of the second scan data from the first. The result of this operation is shown in Figure 3A. Excess heat is developed already at a temperature slightly above 20C (Fig3B).
Excess Cp (mcal min-1) 0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.120 Temp. (C)
Fig 4. Isothermal study at 60C.
Excess Cp (mcal min-1) 0 -0.02 -0.04 -0.06 -0.08 -0.Temp. (C)
When the total integrated area between the control line and the cefazolin line in Figure 4 is compared with that fraction of area in the early region, the early phase is found to represent only 2.5% of the total heat for the process. Hence it is justified to remove it from the calculations by using an integration based on an extrapolation from data obtained after the early phase, as shown by the dashed line in Figure4. Over a period of 16.3 hours, the total heat obtained from this integration is -55 mcal for the decomposition process. Assuming the process is first-order, the total heat of decomposition of the entire sample (1.0 10-5 moles) is -170mcal which gives an estimated enthalpy change (H) for the process of -17.0kcal/mole.
Fig 3. Excess heat for irreversible chemical decomposition.
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To calculate rates (moles/min) for the decomposition process at all temperatures, the excess heat capacity (Cp) data shown in Figure 3B (mcal/min) is divided by the H for the process (mcal/mole) at each temperature. The rate constant (k) can then be calculated from a knowledge of Cp and H at a given temperature, along with the molar amount of unreacted material at that temperature. If the natural logarithm of the rate constant is plotted as a function of the inverse temperature as shown in Figure5, a linear correlation is obtained, the slope of which is equal to the activation energy (Ea) of the first-order decomposition process. In the case of cefazolin this is equal to 21.1kcal/mole. If this relationship is extrapolated to 20C, the rate of decomposition at this temperature (k20) is calculated to 7.910 -6 min-1, consistent with a half-life of 61days. It is important to note that measurements of this type describe thermal stability which may or may not be related to the presence of contaminants in, or potency of, a compound.
20 In k -Temp. (C)
Ea = 21.1 kcal mole-1 k20 = 7.9 10-6 min-1 Half-life at 20C = 61 days
Fig 5. Arrhenius plot of isothermal decomposition of cefazolin.
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GE, imagination at work, and GE monogram are trademarks of General Electric Company. MicroCal is a trademark of GEHealthcare companies. All third party trademarks are the property of their respective owners. 2010 General Electric Company All rights reserved. First published Nov. 2010
For local office contact information, visit www.gelifesciences.com/contact www.gelifesciences.com/microcal GE Healthcare Bio-Sciences AB Bjrkgatan 84 Uppsala Sweden
All goods and services are sold subject to the terms and conditions of sale of the company within GEHealthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse 5 D-79111 Freiburg Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327 Piscataway, NJ 08855-1327 USA GE Healthcare Japan Corporation Sanken Bldg., 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073 Japan
imagination at work
28-9870-39 AA 11/2010
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