Current Pharmaceutical Biotechnology - Volume 6, Issue 3, 2005

Calorimetric experiments on complex biological systems have been conducted since the 18th century. Lavoisier and Laplace developed an ice calorimeter to study the metabolism of a guinea pig through weighing the ice melted over the observation period and knowledge of the latent enthalpy of fusion of water. This early experiment set the scene for a number of other researchers but limitations in instrument design restricted their studies to relatively rapid reaction systems. A more advanced instrument was developed by Calvet in the first half of the 20th Century and marked an increase in the application of calorimetry to a wide variety of biological systems. As more advanced instruments were developed quantitative kinetic and thermodynamic analysis became possible. The emphasis then began to shift from more complex biological systems to relatively simple chemical systems. With the advent of more sophisticated analysis techniques for calorimetric data and with modern pharmaceutical therapies becoming increasingly designed for specific bio-therapeutic targets, for example protein-substrate interactions, we are now witnessing a shift back to investigations of complex systems of biological origin. In recognition of this revolution manufacturers are beginning again to modify and improve their products in order to facilitate this renewed interest with high throughput instruments being developed specifically for systems of a biological nature. The articles in this issue reflect the resurgence of calorimetry as a useful tool in the study of biologically important systems. A wide range of topics and calorimetric methods are covered they include: stability assessment of biopharmaceuticals, investigation of biopharmaceuticals by solution calorimetry, characterisation of freeze dried systems, assessment of polymorphic content, studies of microbiological systems and advances in calorimetric data analysis. In a manner analogous to the renaissance of calorimetry in the biopharmaceutical arena, the authors for this special issue were selected because they are all at the start of their research careers and thus represent the next generation of calorimetrists entering this field.

The assessment of stability (of actives, excipients and/or formulated products) is an important, and often timeconsuming, part of pharmaceutical product development. Conventionally, HPLC is used to quantify the concentrations of a parent compound and any degradation products as a function of storage time. HPLC, however, is relatively insensitive to small changes in concentration and it is often the case that stability assays are conducted under stress conditions, in order to accelerate any degradation processes. The Arrhenius relationship is then employed to give an initial prediction of stability under storage conditions while long-term studies, under storage conditions, are conducted to confirm these predictions. The properties of isothermal calorimetry, such as its intrinsic sensitivity to small changes in heat and invariance to the physical form of a sample, make it ideally suited for stability assessment because it obviates the need for an Arrhenius analysis. In addition, the ability to conduct titration or gas perfusion experiments vastly increases its range of applications. Recent advances in instrumental design and data analysis have made it easier to analyse data quantitatively for complex systems. It is the purpose of this review to highlight some of these developments, discuss them in the context of pharmaceutical and biopharmaceutical examples and explore some of the future challenges and applications of the technique.

A great number of pharmaceutical substances exist in crystalline solid-state. Because of the complexity of their chemical structure many different polymorphs of a given substance can exist. Polymorphic forms of solid pharmaceuticals influence not only their dissolution behavior, i.e. bioavailability but also their solid-state stability. It is well known that only one polymorphic form is thermodynamically stable and all other metastable forms will convert, eventually, to the more stable form. Hence it is essential to choose the most suitable polymorphic form in the early stage of pharmaceutical development. The following article reviews the recent applications of solution calorimetry that allows characterization of pharmaceutical polymorphs through accurate determination of enthalpy of solution. Each crystalline form possesses a defined enthalpy of solution, therefore solution calorimetry is used for the quantitative analysis of the desired polymorphic form and determination of enthalpy of transition corresponding to the difference in enthalpies of solution for a polymorphic pair. More recently this technique has been applied to the estimation of thermodynamic transition temperature, which is useful for the evaluation of thermodynamic stability relationships between polymorphs. This article will also describe the kinetics and thermodynamics of polymorphic transitions, from a metastable form to the thermodynamically stable form, through studies using ampoule-based isothermal microcalorimetry. Such studies are particularly useful when metastable forms are to be selected in order to enhance bioavailability. If the metastable form, or the pharmaceutical product containing it, can be shown to be sufficiently stable, it could then be used in a formulation where its therapeutic effects could be exploited.

Isothermal calorimetry is rapidly becoming an indispensable tool for the quantitative determination of a variety of kinetic and thermodynamic parameters for a wide range of systems. In particular calorimetry is finding increased application to the investigation of stability and incompatibility of pharmaceutical materials. In order to draw meaningful conclusions and to predict behaviour in related systems it is necessary to have the means to calculate accurately parameters such as the rate constant and enthalpy. To this end several groups have been developing equations which describe calorimetric output in these terms. This paper will briefly outline some of these equations and discuss some of the limitations that currently exist in their application. A particular emphasis is placed on the recent developments relating to the application of these equations to flow calorimetric data. The main application of these equations is usually found in the pharmaceutical industry. Pharmaceutical formulations are usually extremely complex mixtures consisting of many different excipients as well as the active drug. Because of these large numbers of ingredients it is often observed that multiple chemical and physical process occur over the lifetime of the study. This complexity is then reflected in the calorimetric data rendering the application of the simple equations useless. Dealing with this complexity is a major issue amongst the calorimetric community and some of the recent advances in this field are also discussed.

In solution calorimetry the heat of solution (ΔsolH) is recorded as a solute (usually a solid) dissolves in an excess of solvent. Such measurements are valuable during all the phases of pharmaceutical formulation and the number of applications of the technique is growing. For instance, solution calorimetry is extremely useful during preformulation for the detection and quantification of polymorphs, degrees of crystallinity and percent amorphous content; knowledge of all of these parameters is essential in order to exert control over the manufacture and subsequent performance of a solid pharmaceutical. Careful experimental design and data interpretation also allows the measurement of the enthalpy of transfer (ΔtransH) of a solute between two phases. Because solution calorimetry does not require optically transparent solutions, and can be used to study cloudy or turbid solutions or suspensions directly, measurement of ΔtransH affords the opportunity to study the partitioning of drugs into, and across, biological membranes. It also allows the in-situ study of cellular systems. Furthermore, novel experimental methodologies have led to the increasing use of solution calorimetry to study a wider range of phenomena, such as the precipitation of drugs from supersaturated solutions or the formation of liposomes from phospholipid films. It is the purpose of this review to discuss some of these applications, in the context of pharmaceutical formulation and preformulation, and highlight some of the potential future areas where solution calorimetry might find applications.

Many features of microorganisms make them pre-eminently suitable for study by microcalorimetry. They have thus, in the past, been the basis of fundamental studies in metabolism and cellular physiology. In this review we look at the application of calorimetry to the impact of bacteria and fungi on the pharmaceutical industry both in the exploitation of useful microorganisms and the fight against harmful ones. Obviously they are of great relevance to the pharmaceutical industry as agents of human disease, with more antimicrobial products registered for production than for any other type of human affliction. Microcalorimetry offers the opportunity to study microorganisms in real time and in heterogeneous systems, allowing for more descriptive and representative analysis. Other advantages that microcalorimetry confers over traditional microbiological techniques are reductions in time, better reproducibility and simplicity. Also the manufacture of all pharmaceutical products requires the exclusion of microorganisms to a greater or lesser degree. The enumeration and identification of such contaminants is of great importance for the well-being of patients and to maintain the integrity of the product. New techniques are required to increase the reliability and sensitivity over conventional methods. Finally, as our understanding of biology develops, the sophistication of therapeutic agents available, such as vaccines, cytokines and engineered antibodies, is increasing. Necessarily, prokaryotic and eukaryotic cells, possibly transformed with the appropriate genes, are the producers of such proteins. Microcalorimetry offers a sensitive means of developing the conditions for optimum production of such products in active form since it gives instantaneous information on the physiology of the producer cell.

A key challenge facing the pharmaceutical industry is the production of biotechnological drug products such as proteins in a stable form. Freeze-drying is preferred for manufacturing such products because of the low temperatures used. However, the protein may still degrade during the process necessitating the inclusion of a protectant. This review describes the range of thermal analysis techniques that have been used to investigate the properties of formulations to be freeze dried and the resultant products. This approach has allowed insight into the key parameters required for design of formulations and processes that will generate the best possible products.