The pharmaceutical scientist s Guideto Calorimetry Pharmaceutical Sciences
Overview: This guide is intended for the pharmaceutical scientist wondering whether calorimetry could directly benefit their research. The adsorption or evolution of heat is a universal feature of chemical reactions pertinent to the pharmaceutical industry. The binding of a drug to a macromolecular target, the interaction of an excipient with a drug, the degradation of a protein therapeutic upon storage, or the slow transformation of a drug from a metastable to a stable polymorphic form all give rise to minute changes in heat, the analysis of which provides fundamental insights into the thermodynamics and kinetics controlling the reactions. These insights in turn provide critical input for optimizing research strategies and processing procedures. Calorimeters designed for the pharmaceutical industry rapidly provide accurate and reliable information about potential drug candidates and formulations, sometimes using just nanomoles of material. Calorimetric techniques are direct: there is no requirement for spectroscopic probes, optical transparency, chemical modification or immobilization, and if the reaction is reversible (such as the unfolding of some proteins), the technique is non-destructive. In addition, since the measurement of heat is independent of the complexity of the sample, impurities are tolerated. Three major types of calorimetry are used in the pharmaceutical industry: isothermal, isothermal titration, and differential scanning. These approaches allow the quantitative characterization of a large variety of small molecule and macromolecular systems present either as solutions, membrane soluble suspensions, insoluble suspensions or solids, making calorimetry an ideal technique for studying: Drug stability and shelf life Excipient compatibility Amorphicity and crystallinity Polymorphism Solvent adsorption and water permeation Surface oxidation Binding interactions 2006 All rights reserved
Table of Contents: Theory and Experimental Design.................... 5 What is a calorimeter?......................... 7 Isothermal Calorimetry......................... 8 Isothermal Titration Calorimetry.................. 10 Differential Scanning Calorimetry.................. 13 Applications of Calorimetry to the Pharmaceutical Sciences.... 19 The drug design process....................... 21 Pharmaceutical development..................... 23 Excipient compatibility....................... 23 Amorphous content........................ 24 Shelf life predictions........................ 24 Antibiotic assessment........................ 25 Drug/membrane interactions.................... 25 Drug purity and polymorphism................... 26 Dissolution rates........................... 27 Energetics of solid drug surfaces.................. 27 Drug adsorption........................... 27 Practical Issues to Consider when Choosing a Calorimeter..... 29 Calorimeters for Pharmaceutical Research and Development... 39
theory Theory A calorimeter measures heat generated or absorbed by reactions
Theory
Theory and Experimental Design What is a calorimeter and why is calorimetry such a powerful pharmaceutical research and development technique? A calorimeter designed for pharmaceutical research and development accurately measures extremely small amounts of heat (nanocalories or microcalories) generated or absorbed by reactions occurring in very dilute solution, or in a small amount of suspension or solid sample. Calorimetry can be used either as a screening technique to rapidly determine whether or not a reaction occurs (for example, the undesirable interaction of a drug with an excipient), or it can be used in comprehensive studies to obtain a complete thermodynamic description of a reaction at the molecular level, such as determining the number of protons exchanged with the solvent when a protein and small molecule drug bind. The power of calorimetry arises from the universal association of heat with chemical reactions. Using one of a small number of well-established experimental approaches, the thermodynamics associated with practically any pharmaceutical process can be quickly assessed in a qualitative manner. This qualitative study can be followed directly by an in-depth investigation, often using the same calorimeter, to provide a full quantitative thermodynamic description of the reaction. The power and versatility of calorimetry permits rapid, direct qualitative or quantitative thermodynamic information (depending on the researcher s immediate needs) to be obtained, without the need to establish new experimental protocols for each system studied. Importantly, calorimetry can require as little material as a spectrophotometric investigation. Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory Three major types of calorimetry are used in the pharmaceutical sciences: isothermal, isothermal titration, and differential scanning All three types of calorimetry can provide a thermodynamic description of the reaction. Isothermal calorimetry is primarily used to study the stability of small molecule drugs in the presence of excipients or moisture, isothermal titration calorimetry is principally used to study binding and catalytic reactions, and differential scanning calorimetry is generally employed to characterize the response of drugs (including biomacromolecules) and drug formulations to thermal stress. As explained below, each type of calorimetry answers rather different questions and subjects the sample to significantly different experimental conditions. Depending on the questions being asked and the nature of the sample, a comprehensive thermodynamic and functional study may require employing two or even all three techniques. Isothermal Calorimetry Isothermal calorimetry measures heat flow as a function of time. Isothermal calorimeters designed for pharmaceutical applications provide the most reliable and sensitive test available for determining whether or not a reaction is occurring. All physical and chemical processes are accompanied by an exchange of heat with the environment. If a small sample undergoing dissolution or degradation is maintained under isothermal conditions in a sensitive calorimeter, a temperature gradient will be formed between the sample and its surroundings, causing a minute change in heat flow (typically nanocalories/ second) which can be accurately measured by a sensitive isothermal calorimeter. For example, reactions occurring with rates as low as 0.5% conversion/year (e.g., slow hydrolysis or morphology changes in a small molecule drug) can be characterized in a period of hours to several days, rather than the weeks or months required by less sensitive methodologies. Since getting
a drug to market is a lengthy, multi-step, expensive process, any technique that can expedite the process without compromising the quality of the analyses is an improvement over more traditional approaches such as HPLC. Both kinetic and thermodynamic information about slow processes can be derived from isothermal calorimetry without the need to make assumptions about the reaction mechanism. Sealing the samples in ampoules allows solids, solutions, suspensions, gases or multi-phase systems to be studied under controlled atmospheric conditions. Importantly, the effect of temperature on the kinetics of a process can be studied by simply re-equilibrating the calorimeter at a new temperature and collecting data, without opening the ampoules or adding fresh sample. In addition, because isothermal calorimeters are so sensitive, stability measurements can be conducted at realistic storage temperatures rather than the elevated temperatures commonly used to enhance degradation when other analysis techniques are used. Isothermal calorimetry is often the most rapid and sensitive approach for characterizing the stability, degradation and compatibility of pharmaceutical samples, and is ideal for investigating: Since getting a drug to market is a lengthy, multi-step, expensive process, any technique that can expedite the process without compromising the quality of the analyses is an improvement over more traditional approaches. Excipient compatibility Drug stability Oxidation of drugs Vapor sorption Drug dissolution Drug amorphous/crystalline content Drug polymorphism Glass transition temperature ph stability Metals and metal chelator effects Drug-lipid interactions Drug-biopolymer interactions Cell metabolism Stability of saturated and supersaturated drug formulations Theory
Calorimeters Practical Issues Applications Theory An isothermal experiment can be set up in minutes and can require just milligrams of material. In a typical isothermal experiment, the calorimeter is equilibrated at the desired temperature, then the sample in a sealed ampoule is loaded in the sample chamber. The reference chamber is loaded with an ampoule containing an inert material of similar mass and heat capacity. The sample is maintained under isothermal conditions; as the sample undergoes reaction, a temperature gradient is formed between the sample and its surroundings and the resulting heat flow is measured as a function of time. A typical isothermal calorimeter for pharmaceutical use (such as the Calorimetry Sciences 4500 Isothermal Nanocalorimeter) is capable of maintaining a baseline of ±1 nanocalorie/sec, with a temperature stability of ± 0.0005 C (at 25 C). Because of their sensitivity and stability, such isothermal calorimeters typically require only milligrams of material and, even for samples undergoing very slow reaction, allow a full thermodynamic investigation of the process to be completed in a matter of hours. Isothermal Titration Calorimetry (ITC) Isothermal titration calorimetry is a universally applicable technique for determining the thermodynamic processes controlling dynamic interactions between molecules. Isothermal titration calorimetry (ITC) allows the thermodynamics controlling the interactions between two solute molecules, such as a drug and its protein target, to be directly determined. When two molecules interact noncovalently, they do so primarily through surface hydrophobic patches, with binding specificity provided by the precise pairing of hydrogen bond donors and acceptors, electrostatic interactions, dipole interactions, etc. If two molecules interact, they must display greater affinity for each other than they do for the solvent, as otherwise no binding would occur. 10
Typical incremental ITC data for the titration of 2 -CMP into RNase A. Integration of the areas under each peak with respect to time, followed by normalization per mole of added ligand, allows the enthalpy, the binding constant and the stoichiometry to be calculated. Isothermal titration calorimetry measures the heat changes associated with these interactions. Depending on the calorimeter used, samples can be present either as a dilute solution, a suspension, or as a solid. In ITC, one component of a complex (such as DNA or an enzyme) is present in the calorimeter s sample cell, and the second component (for example, a drug or substrate) is slowly added in an incremental, stepwise fashion. Analysis of the extremely small thermal effects arising from the binding or catalysis reactions allows a full thermodynamic characterization of the reaction and provides fundamental information about the molecular interactions driving the process. Although there are many techniques for studying binding and catalytic reactions, only calorimetry directly provides the enthalpy of the reaction. In the case of a binding reaction, a single ITC experiment also provides the stoichiometry of the reactants and the association constant of the two interacting components. In the case of a catalytic reaction, ITC allows the Michaelis constant, the turnover number and the rate of the reaction to be rapidly and accurately determined. ITC is a highly versatile technique and is the method of choice for characterizing a variety of dynamic interactions including: 11 Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory Biopolymer/drug binding Protein/protein interactions Drug/vesicle/liposome interactions Drug/cell interactions An ITC experiment is fully automated, takes between 20-90 minutes, and requires no more material than a surface plasmon resonance or spectrophotometric experiment. Critical micelle concentration Non-aqueous adsorption ph effects Cell metabolism/drug interactions An isothermal titration calorimeter can quickly and sensitively characterize dynamic reactions such as binding and catalysis. To determine the affinity with which two molecules bind (for example, a protein, polysaccharide or nucleic acid receptor to a drug candidate), approximately 1 ml of a high nanomolar to low micromolar solution of the receptor is loaded into the calorimetric sample cell, and the drug solution is loaded into an injector syringe connected to the sample cell. In a typical experiment, the drug is titrated incrementally into the sample, and heat is generated and registered as a deflection peak by the instrument. Integration of the area under each deflection peak allows the amount of heat produced by each incremental addition of drug to be calculated, until all the binding sites on the receptor are saturated and no further binding is detected. A fully automated incremental ITC experiment takes approximately an hour to run. Alternatively, the drug can be slowly and continuously infused into the sample cell; a continuous titration experiment typically requires less than 20 minutes. Using either approach and the Bindworks software supplied by Calorimetry Sciences, a global fit of the data from a single experiment is often sufficient to allow the association constant (K a ), the binding enthalpy ( H) and the stoichiometry (n) of the reaction to be calculated. Macromolecule/macromolecule interactions (with one macromolecule in the sample cell and the other in the syringe) can be studied in an analogous manner. Importantly, for any titration experiment, conduct- 12
ing the experiment twice, each time using a different buffer having the same ph but different enthalpy of protonation, allows the number of protons exchanged between the ligand and the binding site to be determined, thus providing information on the functional groups involved in the binding reaction. ITC is also ideal for rapidly analyzing essentially any enzymatic reaction, using a wide range of physiological or synthetic substrates and inhibitors, without the need for chromogenic derivatives or coupling enzymes. To determine the rate of catalysis, the enzyme is typically loaded into the calorimeter s sample cell, and the substrate, dissolved in the same buffer as the enzyme, is loaded into the injector syringe. Appropriate concentrations of substrate and enzyme will vary depending on the exact reaction being studied, but generally picomolar to nanomolar enzyme and micromolar substrate concentrations are appropriate. Approximately 20 µl of substrate is injected, and the rate of heat production is monitored continuously until the substrate is depleted. The data are plotted (rate vs. substrate concentration) and fit to yield the catalytic rate constant (k cat ) and the Michaelis constant (K M ). The experiment can be repeated by injecting a second aliquot of substrate: if there is no product inhibition, the same curve will be obtained, whereas if there is product inhibition, its severity can be determined over the course of several injections. The efficiency of various exogenouslyadded inhibitors can be established by repeating the reaction in the presence of a known concentration of the inhibitor. Pseudo-first-order kinetics can also be studied using multiple small injections of substrate rather then one large injection. Differential Scanning Calorimetry (DSC) Differential scanning calorimetry provides fundamental thermodynamic insights into the physical and energetic properties of drugs, formulations and biopharmaceuticals. 13 Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory Partial Molar Heat Capacity (kj K -1 mol -1 ) 110 100 90 80 70 60 50 40 30 20 10 50 60 70 80 90 Temperature ( C) DSC measures the change in energy in a sample as the temperature is raised or lowered, and thus can determine absolute thermodynamic data for thermally-induced transitions. Many small molecules undergo phase transitions or degradation when heated, and many biopharmaceuticals denature and unfold at elevated temperatures. Ultra-sensitive calorimeters can quickly quantify the thermodynamic parameters (and hence stability) of small molecule drugs and macromolecules in their native state, in contact with excipients or stabilizers, following chemical alteration or mutation, or undergoing hydration, dissolution or dehydration. Under a given set of conditions, whether a molecule is folded or unfolded (in the case of a biopharmaceutical), or is crystalline, amorphous, melted or decomposed (in the case of a small molecule drug) depends on the relative contributions of the enthalpic ( H) and entropic ( S) components of the system (i.e., G = H T S). Unfolding of a macromolecule occurs when T S increases sufficiently (for example, by the absorption of heat) to overcome stabilizing enthalpic interactions such as hydrogen bonds, hydropho- T m C p Area = H cal A DSC experiment provides a complete thermodynamic description of a system (in this example, a protein), including the midpoint of the thermal transition, the enthalpy and the change in heat capacity. 14
Heat Flow (mw) 2 0-2 -4-6 -8-10 -12-14 -16 H o I Recrystallization Form I T m I T c Form II -18 130 140 150 160 170 180 190 Temperature ( C) bic interactions and electrostatic interactions. Temperature-induced phase changes of small molecules similarly reflect the strength of intermolecular interactions: depending on the physical state of the sample, DSC can provide the glass transition temperature, recrystallization temperature and enthalpy, and the melting temperature and enthalpy of fusion, as well as detect polymorphic states and quantify degradation and decomposition rates. In a single experiment, DSC can directly measure and allow the calculation of all the thermodynamic parameters characterizing a small molecule drug or biological macromolecule, including: H, the calorimetric enthalpy due to thermal denaturation caused by the breaking of hydrogen bonds, ionic interactions, and the disruption of hydrophobic interactions. H C T m II H o II A DSC scan of a small molecule drug can reveal the enantiotropic conversion of a drug between two forms. In this example, Form I melts at temperature T m I with a characteristic enthalpy of fusion ( H₀ I ), recrystallizes into Form II at temperature T c with a heat of crystallization H c. Form II melts at T m II with an enthalpy corresponding to H₀ II. Theory Applications Practical Issues Calorimeters 15
Calorimeters Practical Issues Applications Theory T m, the transition midpoint, where, in the case of small molecules, half the molecules are in one physical state (e.g., crystalline) and half are in another state (e.g., amorphous), or in the case of macromolecules, where half of the molecules are folded and half unfolded. C p, the change in heat capacity, due to the disruption of crystalline structure upon melting of a small molecule, or to the thermal unfolding of a macromolecule. S, the entropy, a measure of the molecular disorder. G, the Gibbs free energy, a measure of the overall stability of the system. DSC is therefore an equally powerful technique for determining the phase behavior and stability of small molecule drugs and for understanding the thermodynamic parameters controlling the thermal and functional characteristics of a macromolecular therapeutic. In addition, DSC can also provide a global analysis of complex biological systems such as whole cells (for example, determining thermally induced transitions in organelles and in whole cells exposed to various drugs or antibiotics). DSC is thus an extremely versatile and powerful approach to understanding: Small molecule phase changes Drug purity Drug polymorph content Polymorph stability Drug degradation and decomposition Drug-excipient compatibility Drug-hydrate formation Biopolymer conformation and stability Pharmacokinetics Stabilizing effects of ligand binding Organelle and whole cell stability A DSC experiment is fully automated and characterizes reactions faster than most other physical techniques. A DSC instrument contains one or several sample cells and a matched reference cell. If a reaction occurs in a sample cell, the heat produced or 16
absorbed relative to the reference cell provides a very sensitive test for determining whether or not a process is occurring, often allowing characterization of a reaction in a period of an hour or two. The design of a DSC experiment is dependent on whether a small molecule or biological macromolecule is being studied, as described below. Theory Small molecules: DSC studies of small molecules can be conducted on neat solids (to study melting, crystallization, dehydration or hydration, glass transitions and polymorphic transitions), degassed aqueous solutions (to study water-enhanced instability and degradation), and solid mixtures, slurries and suspensions (to study excipient compatibility or dissolution rates). A baseline is first collected, often using empty sealed pans or ampoules in the sample and reference cells. The sealed samples (micrograms to a few milligrams) are placed in the calorimeter sample cells; depending on the instrument, the cells can be maintained in a fixed humidity or oxygen environment during the thermal cycle. User-defined variables (starting and ending temperatures, heating and cooling scan rate, number of scans, etc.) are input, then the instrument software takes control and automatically performs the entire experiment. The samples are heated at a constant rate (typically between 0.5 to 2 C/min) and the absorption or production of heat is monitored. Differences in heat uptake or production by the sample and reference cells are measured and compensated for by the instrument, thus maintaining both cells at the same temperature. The instrument provides a constant readout of the difference in heat absorption or release by the two cells, which corresponds to differences in the apparent heat capacities of the sample and reference. An accurate sample mass measurement, and a steady baseline before and after transitions, is necessary in order to allow quantitative estimates of energetic parameters such as heat capacity and enthalpy of fusion. 17
Theory Biological macromolecules: A few micrograms of sample can provide a complete thermodynamic analysis. When DSC is used to determine the thermal stability of a biological macromolecular sample such as a protein therapeutic, a buffer baseline is first obtained to establish the linearity of the baseline. The sample is then scanned: if the sample is a solution, it should be thoroughly degassed, dialyzed, buffered and of known concentration (high nanomolar to low micromolar). If the sample is a suspension or a solid, dialysis is not required. The sample is loaded into the sample cell, and the reference cell is filled with an equal amount of matching degassed dialysis buffer or solvent. The cells are pressurized at constant pressure (to prevent bubble formation during heating and allow determination of C p ), thermally equilibrated at the chosen starting temperature, then heated and cooled. After cooling both cells to the starting temperature, the experiment can be repeated to determine if the sample exhibits reversible thermal transitions. Because of the extreme stability of buffer baselines obtained on Calorimetry Sciences DSCs designed for biological macromolecules, subtraction of the buffer baseline corrects the data for the partial molar heat capacity of the solvent, allowing the partial molar heat capacity of the biological sample to be directly determined from the thermogram. The software package supplied with the DSC performs this subtraction, and also calculates the calorimetric enthalpy and entropy changes of the sample. In addition, for chemically defined (purified) samples, the data can be fit to twostate or multiple processes to determine the concentrations of folded and unfolded macromolecule in the sample. Thus, a single automated DSC run requiring as little as a few micrograms of sample and limited operator attendance can provide a complete thermodynamic analysis of the thermal stabilities of the sample components. 18
applications How calorimetry can be used to solve research and development problems Applications 19
Applications 20
Applications of calorimetry to the pharmaceutical sciences Calorimetry Sciences has produced a number of detailed Pharmaceutical Sciences application notes which examine specific problems both in the research and in the development phases of drug production. Additional application notes of interest (especially for the drug design process) are available in the Life Sciences series. Each application note outlines the basis of the problem being addressed, discusses how calorimetry can be used to study the problem, points out both the strengths and limitations of the technique, provides practical examples (data, interpretation, discussion), and concludes with a summary of how calorimetry can be used to provide a comprehensive analysis of the problem. Please contact Calorimetry Sciences for a current list and copies of these application notes, or visit our Web site at: www.calorimetrysciences.com. The Drug Design Process: The optimization of a lead compound into a viable drug candidate involves maximizing its binding affinity towards the selected (usually conformationally flexible) target. In the absence of a thermodynamic understanding of the specific interactions between a drug and its target, the design of a drug is often guided only by predictions of the entropic component of the binding interaction. When only entropic contributions are considered and enthalpic contributions are ignored during the design process, highly hydrophobic and conformationally constrained drugs are often designed Theory Applications Practical Issues Calorimeters 21
Calorimeters Practical Issues Applications Theory which exhibit poor binding and specificity. In contrast, drugs with good binding characteristics to their flexible targets pay a significant conformational entropy penalty upon binding, so optimization of their affinity requires substantial favorable binding enthalpy. Enthalpic optimization is less straightforward than entropic (generally hydrophobic) optimization, since specific favorable interactions (hydrogen bonds, van der Waals interactions, electrostatic interactions, etc.) must be designed into the binding interface. Both ITC and DSC measure the enthalpy of a binding reaction and provide a thermodynamic description of the binding of a drug candidate to a target receptor. ITC is the most direct technique for dissecting the free energy of binding into its entropic and enthalpic components. Coupling this information with atomic-level structural data, the relationship between thermodynamics, structure and function can be ascertained. Using this comprehensive approach, new compounds with improved molecular interactions, increased specificity, heightened binding and enhanced efficacy can be designed more rapidly. In addition, ITC is a completely general approach for characterizing enzyme kinetics and catalytic inhibition. Since drugs are increasingly designed to act as specific enzyme inhibitors, ITC can play a major role in both the thermodynamic and functional characterization of enzyme-specific inhibitor drugs (e.g., HIV protease inhibitors). When a drug binds to a receptor, the temperature at which the drug/ receptor complex denatures will be higher compared to the temperature at which the free receptor denatures. DSC thus provides a direct measure of whether a receptor has bound a drug and how tightly, and so can complement studies of binding equilibria obtained by ITC. Indeed, DSC is the preferable technique for assessing drug binding in several circumstances. The first is if binding is very tight (above 10 9 M -1 ). Since DSC compares the 22
extent to which the drug/receptor complex is stabilized towards thermal denaturation compared to the free receptor, DSC allows estimation of binding energies at all binding constants, including extremely tightly associated complexes. The second circumstance is if the bound complex equilibrates very slowly (hours to days) and so is not compatible with the ITC timeframe (seconds to minutes). Finally, the solubilization of very hydrophobic drugs may require the use of organic solvents which can give rise to such large heats of dilution on ITC that the heat of binding is obscured, or the amount of organic required for drug solubilization denatures the receptor. Since DSC studies can be conducted in aqueous, organic or mixed solvent systems, the presence of organics has no effect on the sensitivity or accuracy of the measurement. For detailed descriptions of ITC and DSC applications pertinent to drug design, please see the Life Sciences application notes entitled: Characterizing binding interactions by ITC, Characterizing enzyme kinetics by ITC and Characterizing protein/ligand binding by DSC. Pharmaceutical Development: Excipient compatibility: The formulation of an active pharmaceutical ingredient into a form that can be easily administered (for example, a tablet) requires the addition of stabilizers and binders (excipients). Traditionally, any degradation of the drug in the presence of an excipient is followed by HPLC, however, isothermal calorimetry provides a significantly more rapid and direct approach. To determine the compatibility of an excipient with a drug, the thermal properties of a binary mixture of the drug and excipient are compared to the properties of the excipient and the drug alone. All samples are maintained at the same relative humidity. If the mixture produces heat at a rate different from the two individual components, the compounds are reacting and that excipient is therefore incompatible with that drug. Excipient compatibility studies can also be conducted using DSC, especially if the effects of dynamic temperature changes on excipient/drug mixtures need to be rapidly assessed. Please see the CSC Pharmaceutical Sciences application note entitled: Characterization of drug-excipient compatibility. 23 Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory Amorphous content: Isothermal calorimetry can rapidly detect and quantify the amount of amorphous material in a crystalline drug. Formulation and mechanical processing can increase the amorphous content of a drug, leading to altered physical properties and decreased stability of the product. Even low levels (<1%) of amorphous content generated during processing can be quantified by comparing the heat produced by samples collected at various stages of formulation. The samples, maintained at the same relative humidity by means of a water adsorption cell, typically adsorb water over a period of several hours in the calorimeter. Since adsorbed water acts as a plasticizer, the glass transition temperature (the temperature at which an amorphous compound crystallizes) is lowered, leading to an exothermic recrystallization process that is proportional to the amount of amorphous compound present in the sample. The glass transition temperature can be most reliably determined using DSC. Knowledge of this transition temperature is critical since the amorphous and crystalline forms of a compound can have significantly different stabilities and bioavailabilities. Therefore, choosing storage temperatures and formulation compositions which minimize amorphous/crystalline interconversions is important for maximizing the efficacy and extending the shelf life of a drug. Please see the CSC Pharmaceutical Sciences application note entitled: Characterization of solid state drugs by calorimetry. Shelf life predictions: Isothermal calorimetry is ideal for qualitatively and rapidly assessing the oxidation or degradation of solid or solubilized drugs under various conditions (ph, water content, oxygen concentration, and the presence of metal ions or antioxidants). For example, the thermal output of a drug solubilized at different ph values provides chemical insights into the mechanisms driving degradation: if a drug degrades endothermically at high ph and exothermically at low ph, degradation is driven by entropic considerations in basic conditions and by enthalpic events in acidic conditions. As a second example, oxidation can be determined by first passing an inert gas over a 24
sample and measuring anerobic degradation, then switching the gas flow to oxygen and measuring heat flow after steady state conditions are established; by measuring heat flow at different temperatures, the activation energy of the compound can be determined. It is generally advisable to conduct stability studies at the anticipated storage temperature since multiple temperature-dependant reaction mechanisms are often involved in the aging process, so the overall reaction rate will be dependent on the rate-determining step. However, isothermal calorimetry (and DSC) can be used to accelerate aging processes, so that the stability of the drug at ambient temperature is estimated by measuring the degradative reaction rate at different elevated temperatures, then extrapolating to the desired temperature. Please see the CSC Pharmaceutical Sciences application note entitled: Rapid and practical stability screening by microcalorimetry. Antibiotic assessment: ITC is gaining prominence in characterizing the effect of compounds on cell metabolism. If the titrant inhibits or kills the cells (for example, when an antibiotic is added to a bacterial sample), the resulting decrease in heat output will be quantitatively registered by the calorimeter, providing an accurate measure of the potency of the drug against its microbial target. Calorimetry is also an effective approach for initial toxicity screening of drug candidates against tissues. Regardless of the target, calorimetric measurements are done directly on the sample and do not require time consuming steps to separate the cells from the medium. Drug/membrane interactions: Critical micelle concentration (cmc) can be determined using ITC by injecting a buffered concentrated solution of a detergent (above its cmc) into 25 Theory Applications Practical Issues Calorimeters
Applications either a dilute solution of the same detergent or into buffer. The micelles initially disassociate upon dilution, but as the titration proceeds, the detergent concentration in the sample cell reaches the cmc and no further dissociation occurs. A first-derivative plot of the heat recorded for each injection versus detergent concentration shows a break point corresponding to the cmc. Repeating the experiment in the presence of drugs designed to interact with membranes alters the cmc and provides an assessment of the drug s affinity for membranes. Drug purity and polymorphism: DSC is the ideal technique for obtaining rapid, accurate, unambiguous melting temperatures, for providing a measure of the purity of the sample (impurities and crystal defects lower the melting temperature and broaden the melting endotherm), and for allowing different polymorphs in a sample to be distinguished. Polymorphs have the same chemical composition but different crystalline structures, resulting in different melting temperatures, solubilities, stabilities and bioavailabilities. Polymorph conversion is often characterized by a nucleation temperature at which the kinetics of conversion is significantly increased; the nucleation temperature can be decreased and polymorph conversion accelerated by the presence of a small amount of impurities. Characterization of polymorph conversion and knowledge of the polymorphic composition of a drug formulation is increasingly required by regulatory agencies. Please see the CSC Pharmaceutical Sciences application note entitled: Characterization of solid state drugs by calorimetry. 26
Dissolution rates: Isothermal calorimetry provides a quick method for determining if a tablet or other solid formulation dissolves rapidly, since dissolution is accompanied by significant evolution of heat. Since this simple method does not require any sample filtration or development of a spectroscopic or HPLC protocol, it is a particularly useful tool in quality control, especially in the preformulation stages of product development. Please see the CSC Pharmaceutical Sciences application note entitled: Characterization of solid pharmaceutical systems from heats and rates of solution. Energetics of solid drug surfaces: Direct heats of sorption and wetting can be made using water vapor, solvent vapors, or in the case of wetting, in non-solvent systems. Unlike other methods, calorimetry does not rely upon a model to calculate approximate surface energies. Drug absorption: Scanning calorimetry can be used to monitor changes in the phase composition of liposomes as small or large molecules permeate the outer surface, the bilayer, and finally the interior of the liposome. If drug dissolution or penetration of the bilayer is slow, multiple temperature scans on the same sample will show a gradual change in phase composition over time, providing an indication of how effectively a drug formulation will be taken up by cells prior to the initiation of in vivo testing. Slow temperature scanning results in improved temperature resolution of overlapping peaks. If multi-modal behavior is observed, this may indicate the formation of complexes in the lipid bilayer which could be undesirable if they formed in cell membranes. 27 Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory Notes 28
practical issues What to consider when choosing a calorimeter Practical Issues 29
Practical Issues 30
Practical issues to consider when choosing a calorimeter Several instruments of particular relevance to pharmaceutical scientists are referred to below. Descriptions of these instruments are given on p. 39 of this guide. What technical expertise and support are available? Calorimetry Sciences has been a leader in thermal instrumentation since 1978. The entire process of developing, designing, building, testing and upgrading our hardware and software is done in-house, so our scientists and engineers can competently and promptly answer any questions you have regarding our instruments. In addition, our chemists and biophysicists are available to help you optimize your experimental protocols, assist in analyzing data, inform you when new articles or techniques applicable to your research are published or developed, and in general share our knowledge of pharmaceutical calorimetry. Although the intent of this guide is to Potential customers are encouraged to send a sample for thermodynamic analysis. help researchers decide if calorimetry could benefit their research, sometimes the utility of an approach can only be established by trial. Potential customers are encouraged to send a sample for thermodynamic analysis. We will discuss our findings with you and make recommendations as to which instrument is best suited to your requirements. Our goal is to ensure that you buy the best calorimeter for your specific applications. Calorimetry Sciences instruments are acknowledged to be exceptionally durable, with many units continuing to produce research-quality data 15 to 20 years after purchase. Calorimetry Sciences believes in constantly upgrading and improving our instrumentation: we do not believe in programmed obsolescence! Theory Applications Practical Issues Calorimeters 31
Calorimeters Practical Issues Applications Theory How much sample is required? The amount of sample required is determined by the type of experiment being performed, the enthalpy of the reaction being studied, and the sensitivity of the instrument used. For isothermal measurements of low enthalpy reactions (for example, typical excipient compatibility studies), fractions of a gram to grams of material would be required by the 4400 Isothermal Microcalorimeter (IMC), which can measure three samples simultaneously; similar reactions would require 20 times less material using the 4500 Isothermal Nanocalorimeter (INC). Reactions with larger enthalpies would require substantially less sample for either calorimeter. For ITC binding experiments, the amount of material is very dependent on the binding constant of the reaction, with weak binding systems requiring substantially higher concentrations of material than tight binding systems, as explained in the CSC Life Science application note entitled: Characterizing binding interactions by ITC. The 5300 Nano-ITC would require picomoles to nanomoles of macromolecule in the 1.0 ml sample cell. In the case of enzymatic reactions, nanomolar to picomolar concentrations of enzyme are often sufficient, depending on the enthalpy and rate of the reaction. The 4200 ITC is less sensitive than the 5300 ITC and has a larger sample cell (1.3 ml), but has a larger dynamic range, making it an ideal instrument for studying weak binding reactions. Ultra-sensitive DSC experiments on biomacromolecules are ideally performed using either the 6100 N-DSC II or, if variable pressure control is important, the 6300 N-DSC III. Generally, micrograms of protein or other macromolecule are sufficient to yield a DSC thermogram of sufficient quality to allow a complete and accurate assessment of the enthalpy of unfolding, as well as the T m. If sample size is less of a constraint, the 4100 MC-DSC can simultaneously analyze three macromolecule or small molecule samples, although its sensitivity is less than the 6100/6300 DSCs. 32
How can protein aggregation and precipitation be minimized during DSC scans? The thermal denaturation of proteins exposes previously buried hydrophobic residues, often resulting in protein aggregation and precipitation. If this process causes significant enthalpy changes that cannot be separated from the unfolding process, the process cannot be thermodynamically analyzed. Capillary cells minimize this problem by delaying the thermal effects of aggregation and precipitation, Capillary cells minimize biomacromolecule aggregation and precipitation. often until unfolding of the protein is complete. For example, 100 µm bovine serum albumin (in 0.1 M phosphate buffer, ph 7.2, scanned from 20 to 90 C at 1 C/min) aggregated after a single scan in a Calorimetry Sciences 6300 Nano- DSC equipped with cylindrical cells, whereas under identical conditions aggregation was not observed below 300 µm when capillary cells were used. Calorimetry Sciences supplies its Nano-DSC instruments with capillary cells unless cylindrical cells are specifically requested by the customer. Are pressure perturbation DSC experiments possible? The Calorimetry Sciences model 6300 Nano-DSC III employs a high-precision piston driven by a computer-controlled linear actuator to control the pressure in the cell. Constant pressure is applied during the DSC experiment to obtain constant pressure heat capacity data and to prevent bubble formation or boiling. Importantly, pressure can be varied according to user selectable functions allowing the pressure to be scanned, stepped, or altered in a sinusoidal fashion. This allows exceptional control and flexibility in designing experiments for studying the compressibility and thermal expansivity of samples. Are calorimeter cells difficult to fill? Solid samples can be easily accommodated in instruments with removable cells (4100 MC-DSC, 4200 ITC, 4400 IMC and 4500 INC). These instru- 33 Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory ments can also be used for solution samples: because of the relatively large cell volumes, filling the cells without trapping air bubbles is not a problem provided the solutions are degassed first. Calorimetry Sciences has designed cells for the Nano series that greatly diminish the possibility of a trapped air bubble in small solution volumes. After degassing the sample, a scientist using a Calorimetry Sciences calorimeter for the first time can fill a cell in seconds and start a run in minutes, confident that air bubbles will not affect the experiment. A degassing station and vacuum pump is supplied with every 6300 N-DSC III calorimeter, and is optional with the purchase of the 5300 Nano-ITC. How easy is it to clean the cells? The removable cells found in the 4100, 4200, 4300, 4400 and 4500 can be aggressively cleaned and sonicated, then reassembled and reinserted into the instrument in seconds. The 5300 N-ITC III fixed-in-place conical-shaped cylindrical cell can be very easily cleaned in minutes with acids, bases, organic solvents and water as required (for example, after the analysis of liposomes or membrane proteins) by passing several liters of solvent through the cells using the supplied vacuum pump and special cleaning tool. 34
The 6100/6300 N-DSC instruments come with either fixed-in-place capillary or cylindrical cells which can be cleaned with a variety of solvents. Both the continuous capillary cells and the cylindrical cells can be flushed with liters of cleaning solution in a matter of minutes using the vacuum pump supplied with the instrument. Is it easy to clean and refill the ITC syringe? Removing the syringe from a Calorimetry Sciences ITC instrument is extremely fast and easy. The syringe can be completely removed from the burette assembly in seconds by loosening a thumb screw, allowing quick, thorough cleaning of the syringe and easy refilling. Both a 100 µl and 250 µl syringe are provided with each 4200 ITC and 5300 N-ITC instrument, and are completely interchangeable in the burette assembly. What maintenance do the instruments require? Fixed-in-place cells should be flushed with water between samples, and cleaned with detergent or organics weekly or monthly (depending on the hydrophobicity of the samples). Removable cells should be scrubbed with detergent and water, or with more aggressive solvents if necessary. Water baths should be drained and cleaned every six months to a year. Otherwise, the instruments require no maintenance other than removal, washing and reinstallation of the air filter a few times per year. Is it difficult to design a successful experimental protocol? Calorimetry Sciences is committed to helping researchers maximize useful data from their experiments. To that end, CSC has produced (and continues to expand) a series of application notes aimed at taking the guesswork out of experiment design. The basic experimental approaches for ITC and DSC experiments were presented at the beginning of this guide. Almost all experimental protocols are variations of these approaches, and are described and illustrated in detail in the application notes. In addition, Calorimetry Sciences applications specialists are pleased to work with scientists to troubleshoot procedural problems or develop novel experimental approaches. 35 Theory Applications Practical Issues Calorimeters
Calorimeters Practical Issues Applications Theory Is the data acquisition and analysis software straightforward and flexible? The software included with Calorimetry Sciences instruments was written in-house by our software engineers in consultation with our applications scientists and with input from several leading calorimetry experts. The goal was to develop software that is simple to use, yet sufficiently sophisticated and versatile to be able to handle essentially any data set. Importantly, data analysis is completely transparent: there are no fudge factors built into the analysis regimes, and the user has complete control over the manipulation of the data. The 4400 IMC and 4500 INC both collect heat rate data; the frequency of data collection is set by the operator and depends on the rate of the reaction. The 4300 RSC uses software specific for this instrument which also allows full user control of acquisition parameters. The 5300 Nano-ITC instrument is supplied with three software modules. ITC- Run allows operating temperature, stirring speed, injection volume, the number of injections and other control parameters to be easily specified. User selectable response time, automatic calibration functions and real-time monitoring of data are also provided. The data analysis module, Data analysis is completely transparent: there are no fudge factors built into the analysis regimes, and the user has complete control over the manipulation of the data. 36
BindWorks, is designed to easily fit data to the appropriate binding model. This module produces a graphical representation of the fit in addition to the parameters K a, H, S and the stoichiometry (n). Preprogrammed fitting models are included for three types of binding: independent binding sites, two sets of multiple sites, and cooperative binding. In addition, customer-specific binding models can be easily input. Files are in ASCII format and can be exported to other data analysis, graphical and spreadsheet programs (including Origin and Excel). Data obtained with other manufacturer s instruments can be analyzed with BindWorks. Experiment Design Wizard prevents precious sample material from being wasted. Experiment Design Wizard is an important time-saving feature of Bind- Works. This module allows experimental parameters (such as concentration) and anticipated thermodynamic results to be input, and outputs a simulation of the expected experimental thermogram. In addition to saving numerous hours re-running improperly designed experiments, this tool prevents precious sample material from being wasted on runs that cannot produce useable data due to improper concentration choices. The 4200 ITC also uses BindWorks for data analysis, but data acquisition is controlled by a custom data acquisition package. The 6100/6300 N-DSC acquisition software, DSCRun, allows experimental parameters such as scan rate, temperature range, number of scans, etc. to be specified. All parameters are set and viewed from a single dialog screen, and notes about the experiment can be attached to the data file. Automated calibration functions and real-time monitoring of the collected data are also provided. The DSC data analysis software package, CpCalc, is designed to make the analysis of raw DSC thermograms simple and straightforward. Blank scans are easily selected and subtracted before converting data to molar heat capacity, and calorimetric enthalpy change, entropy change, and partial molar heat capacity of the sample are automatically calculated. Molar heat Theory Applications Practical Issues Calorimeters 37
Calorimeters Practical Issues Applications Theory capacity data can be fit to two-state models to determine van t Hoff enthalpy change, melting temperature and change in heat capacity, and fitting may also be done with two-state processes to help determine the accurate concentration of macromolecule in the sample. Thermograms can be deconvoluted and integrated to provide enthalpic information on the domains and subunits of biological macromolecules. Data are in ASCII format and may be exported into other software, including Origin, PeakFit, SigmaPlot or Excel, at any stage of the analysis, and imported back into CpCalc if desired. CpCalc is utilized by the 4100, 6100 and 6300 DSCs; the 4100, which can operate in either the isothermal or temperature scan mode, utilizes separate data acquisition software. Are training courses available? Following purchase of a Calorimetry Sciences instrument, on-site installation and in-depth training in both instrument hardware and software are available for a nominal fee. In addition, Calorimetry Sciences is initiating a series of seminars and workshops dealing with experiment design, data interpretation and novel applications of calorimetry. Please contact Calorimetry Sciences for details. 38
Calorimetry Sciences instruments for pharmaceutical research and development calorimeters 39 Calorimeters
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Calorimetry Sciences Instruments for Pharmaceutical Research and Development CSC 6300 Nano DSC III The 6300 Nano DSC III (N-DSC III) represents the latest development in high precision biopolymer calorimetry. Lower noise, greater baseline reproducibility, the choice of capillary or cylindrical cells and unparalleled sensitivity in both the heating and cooling scans establish this calorimeter as the ultimate instrument for making partial molar heat capacity measurements on very dilute solutions. Built-in computer controlled cell pressurization allows volumetric properties of biopolymers to be determined. Since many biopolymers are expensive or difficult to obtain, and others behave differently depending on their concentration, there is a need for a scanning instrument that can make exquisite measurements with very little sample. Using the N-DSC III, a full thermodynamic analysis of a biopolymer can be obtained with as little as 2 µgrams of material. CSC 6100 Nano DSC II With the same sensitivity as the 6300, the 6100 Nano DSC II (N-DSC II) is ideal for measuring partial molar heat capacities of biopolymers in dilute solution when volumetric properties are not a consideration. The N-DSC II is a power compensation design using a completely solid-state thermostat and is equipped with the user s choice of Theory Applications Practical Issues Calorimeters 41
Calorimeters Practical Issues Applications Theory either capillary or cylindrical cells. With an operating temperature of -10 to +130 C (up to +160 C in the HT version), scan rates up to 2 C/min in both the heating and cooling directions, and sensitivity at the 1µcalorie/ C level, the N-DSC II can be used to study the thermal denaturation of most biopolymers using just µgrams of sample. CSC 5300 Nano ITC III The 5300 Nano ITC III (N-ITC III) is designed specifically for studying biopolymer-ligand interactions. The N-ITC III is the instrument of choice for those who require the ultimate in sensitivity. State of the art improvements in cell geometry, temperature control, and ease-of-use set the N-ITC III apart as superior for almost all applications. The N-ITC III is a power compensation design employing a self-contained, solid-state thermostat, and fixed-in-place 24K gold cells. Heat effects as small as 30 nanocalories are detectable with 1 nanomole or less of biopolymer, with equilibrium constants in the range of 10 2 to 10 8 M -1 easily determined in a single titration experiment. One experiment can yield the complete set of thermodynamic parameters (K eq, H, S, and n) for almost any reaction. CSC 4500 Isothermal Nanocalorimeter The 4500 Isothermal Nanocalorimeter (INC) is designed to measure very slow or very small heat changes. Since all chemical and physical processes are accompanied by changes in heat, the INC is a universal detector that can be used to follow the progress of slow reactions at modest temperatures. The INC can provide both kinetic and thermo- 42
dynamic information about the stability/reactivity of liquid or solid samples up to 3 ml in volume. The INC can detect changes in heat flow as small as 1 nanocalorie/sec (4 nanojoules/sec) and heat effects as small as 0.1 calorie (0.4 Joules). Typical applications for the INC include shelf life predictions, excipient compatibility, drug polymorphism and cell metabolism. CSC 4400 Isothermal Microcalorimeter The 4400 Isothermal Microcalorimeter (IMC) can detect changes in heat flow as small as 25 nanocalories/sec (0.1 Watt) and heat effects as small as 10 cal (40 Joules). The IMC is available in three temperature ranges: 0 to100 C, -40 to 80 C, and 20 to 200 C. The IMC, which comes with three matched sample cells and one reference cell, can provide both kinetic and thermodynamic information for almost any process occurring in a solid, liquid or gaseous sample. When equipped with accessory flow adsorption, flow mixing, titration and vapor adsorption cells, the IMC can be used to study solute dissolution, solvent and/or solute adsorption at surfaces, binding equilibria, and heats of mixing and/or dilution. CSC 4300 Rapid Solution Calorimeter The 4300 Rapid Solution Calorimeter (RSC) is designed to be used as either an analytical tool for determining sample purity or concentration, or as an instrument for studying the thermodynamics of binding reactions in dilute solutions. With a temperature resolution of 2 µ C, the RSC can measure heat changes as small as 1 millicalorie (4 millijoules). Theory Applications Practical Issues Calorimeters 43
The RSC operates over the temperature range 0 to 100 C, and is designed to perform both batch addition and titration experiments. In addition, ph data may be collected concurrently with the heat data. The RSC is used to measure heats of reaction, equilibrium constants, solution heat capacity and kinetics. CSC 4200 Isothermal Titration Calorimeter The 4200 Isothermal Titration Calorimeter (ITC) can detect heat effects as small as 0.1millicalorie (0.4 millijoule), allowing titrations to be done with nanomoles of biopolymer. Equilibrium constants in the range 10 2 to 10 8 M -1 are easily determined for almost any interaction over the temperature range 0 to 100 C. The ITC, which can also be used as a model 4500 INC, is excellent for studying ligand binding, ph effects and drug/metabolism interactions in cells. Calorimeters CSC 4100 Multi-Cell Differential Scanning Calorimeter The 4100 Multi-Cell Differential Scanning Calorimeter (MC-DSC) is equipped with three sample cells matched to one reference cell, and can analyze liquids, solids, slurries and suspensions. The MC- DSC is designed to study interactions as a function of temperature change, collecting T m, H, and C p data on the three samples in a single experiment. The standard MC-DSC operates over the temperature range -40 to 110 C, while the high temperature version scans up to 200 C. Scan rates are useable selectable from 0 to 2 C/minute. Excellent for studying drug purity, polymorph stability, excipient compatibility and drug degradation. 44
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