Pharmaceutical Applications of microCalorimetry


Excipient Screening

Author: Jeff Rachford



Development of a new pharmaceutical product is a complex and time-consuming process.  The discovery of a new chemical entity with promising ADME efficacy and toxicity is followed by a series of studies commonly know as the “development process”.   These studies include investigation of the stability of the active pharmaceutical ingredient (API) under various conditions of heat, moisture, acidity, light, etc.  Studies to determine crystallinity, morphology, hygroscopicity, solubility, etc. are also a part of the development process.


Since API needs to be formulated into, for example, a solid dosage format, early phase investigation of the compatibility of the API with excipients has gained increasing attention. It has become recognized that early phase characterization of API incompatibilities with common excipients can significantly reduce the time required to arrive at a final product formulation1


HPLC Excipient Compatibility Method


HPLC analysis is generally considered to be the most successful analytical method to study API/excipient blends for incompatibilities2.    Physical mixtures of API and excipient are made and studied at elevated temperatures.  The samples are often subjected to elevated humidly to enhance reactivity.  However, these studies can require weeks of waiting until a sufficient amount of API has reacted to make conclusions about incompatibilities.  They can also be somewhat labor intensive since samples must be pulled during the waiting period and other time consuming steps may be involved, for example when the excipient is not soluble in the mobile phase and/or filtrations are required before injection into the HPLC instrument.


A method has been proposed using HPLC to rapidly screen excipients1.  Mixtures of API and excipients with 20% water are prepared followed by storage at 50C for 1 and 3 weeks when the samples are analyzed by HPLC.  The addition of 20% water tends to accelerate any degradation processes between API and excipient. Samples are also analyzed by other techniques to determine if physical changes such as conversion from crystalline to amorphous form have taken place. 


While this represents a significant improvement over other HPLC methods where simple physical mixtures of API and excipient are studied, the time and effort required are still significant.  Hence, often only limited excipient compatibility studies are carried out before clinical trials.  Once promising efficacy and toxcity is shown from the clinical trials, the late phase formulation scientists are faced with development of a robust formulation with perhaps little, if any knowledge of excipient compatibility.


Microcalorimetry Excipient Compatibility Method


Microcalorimetry offers the formulation scientists a simple and less time consuming alternative for determining if chemical and/or physical interactions can be expected between API and excipients.  These studies can be carried out in early and/or late phase development.  Excipients showing promise after screening by microcalorimetry can then be studied using more traditional methods (HPLC), resulting in potentially significant less time and effort compared to using conventional methods on all the excipients.3


In a typical compatibility experiment a solution, suspension, or solid mixture of API and excipient is placed in the calorimeter and the thermal activity (heat flow) at a constant temperature is monitored.  The basic assumption is that the rate of heat production is proportional to the rate of chemical and/or physical processes taking place in sample.


 A simple example would be conversion of A to B:


A -------> B


Thermal activity = dq/dt = DH * dn/dt


Where q is the heat (e.g., calories), t is time in seconds, DH is the heat of reaction, and n is the number of moles of B formed.  Therefore, the output of the calorimeter is directly proportional to the rate of the reaction at any given time t.


The thermal activity of API and excipient are measured individually, then the output of the blend is compared to the “non-interaction” curve constructed from the individual components:



Figure 1.  Basic principle used for excipient compatibility studies.


If an experimentally significant difference is observed the excipient is considered to be potentially incompatible with the API.  Because the signal may be the sum of numerous chemical and physical processes, one should exercise proper caution before attempting to correlate the signal with rate of degradation.  Instead, the method should be used as an indicator of potential incompatibility.  Applying this simple testing criteria reduces the number of samples that must be screened using time consuming HPLC, X-ray, ect. methods, thus saving valuable time and effort during the formulation process.


Figure 2 shows an example where the heat output of the API/excipient blend matched the sum of the heat output from the API plus excipient:



Figure 2.  API and excipient show no sign of incompatibility.


Notice the difference between the non-interaction curve and the API/excipient blend was less than the range of error (usually estimated from previous historical data on the given excipient).  Figure 3 shows the same API mixed with a different binder where clearly there is evidence of a potential incompatibility:



Figure 3.  The significant interaction suggests incompatibility.


As mentioned earlier, there should be no attempt made to correlate the signal with “how much” or “how fast” any processes is taking place using this method.  This is because a small signal could in fact be a physical change.   For example,  that a particular excipient acts a nucleation site where polymorph conversion is enhanced, or it could be a very exothermic chemical reaction, such as oxidation, taking place very slowly.  Therefore, the “pass” or “fail” screening criteria should be based on the simple observation if a “chemical or physical process was detected” versus “no chemical or physical process was detected”.




Modern microcalorimeters are capable of measuring extremely small heat flows.  Based on assumptions about molecular weight, enthalpy, and reaction mechanism Angberg4 et al stated it is possible to detect degradation rates of only .68% per year.  Of course it is important to remember that the observed thermal activity can be due to a variety of processes, not only a chemical reaction.  Also, in a practical lab setting, measurements of such small heats can require significant effort and care,  negating the advantage of rapid screening by microcalorimetry.  The autor has used most of these instruments first at Eastman Kodak then Eli Lilly and future publications will include more details of the experimental method (including instrument(s) used).


Regadless of the equipment used, one must keep in mind the classical rule of thumb:  "The advantage of microcalorimetry is that it sees everything.  The disadvantage of microcalorimetry is that it sees everything".


To elaborate, processes that can be detected, in addition to chemical degradation include conversion of amorphous to crystalline material, polymorph conversions, dissolution processes, etc.  Also, within a single batch of API or excipient there can be significant differences observed in heat output from one sampling to the next (hence the use of the range of values as part of the criteria for determining compatibility versus incompatibility). 

Microcalorimetry experiments are usually run for a few hours with the idea that non-chemical processes will come to completion (e.g., dissolution) early in the experiment.  Typically if any chemical reaction (s) are taking place these events are followed by an output consistent with pseudo zero order degradation. For practical studies, Schmitt5 et al recommends adding 20% water to enhance reactivity and running isothermally at 50C, similar to the HPLC method mentioned above.


As is the case with HPLC screening methods, the output from a microcalorimeter can be amplified by performing experiments at elevated temperatures.  Scanning microcalorimeters (also know as HSDSC for “high sensitivity differential scanning calorimeter”), while less sensitive than dedicated isothermal systems, can be programmed to step through a number of temperatures in a single run.  This makes it possible to study the compatibility starting at modest temperatures (e.g., 35C) up to very high levels of thermal stressing (e.g., 85C) on the same sample in a single 24 – 48 hour run.  An example is shown below:



Graphing the log (heat flow) versus the reciprocal temperature results in a classical Arrhenius plot, from which one may be able to determine at what temperature apparent non-Arrhenius behavior begins.  This can be very useful information when long-term stability/excipient testing begins in the later phases of the development process. 


The following examples serve to illustrate how microcalorimetry can be used to quickly screen excipients during the formulation process.


Example 1: The Maillard Reaction


A common incompatibility pharmaceutical formulation scientists encounter is between lactose (anhydrous or monohydrate) with APIs containing primary or secondary amines.  Know as the Maillard reaction, the example below shows how this common incompatibility can easily be detected using microcalorimetry. 


Figure 4.  Interaction of API containing a primary amine with lactose.


The red line represents the calculated combined output from the API and lactose which were each run separately (100mg solids).  All samples contained 20% water as suggested by Schmitt.  Notice the presence of a large exothermic process during the first 10-12 hours that appeared to level off to a pseudo zero order reaction after about 14 hours.  The initial exothermal process is likely a combination of the Maillard condensation reaction and other processes, for example conversion of amorphous to crystalline material, formation of hydrates, etc.  As mentioned above, these processes are assumed to come to completion rapidly compared to the degradation of API that would generally be assumed to take weeks if not longer to complete.  Therefore, the data above would be consistent with a potential chemical interaction resulting in chemical incompatibility between the excipient and API.



Example 2: Compatible Alternative for Lactose


Figure 5 shows the results for the same API but with a different excipient.  Notice that in this case mannitol did not show any significant difference between the non-interaction curve and the blend results.   This suggests mannitol would be expected to be a promising excipient in a solid dosage formulation for this API.


Figure 5.  Interaction of API containing a primary amine with mannitol.



Example 3: Solution Compatibility


Many pharmaceutical compounds are formulated as solutions.  Microcalorimetry can be used to screen excipients in these systems just like those used for solid dosage formulations.  Figure 6 illustrates that lactose had no apparent net influence on the degradation of an antibiotic at an elevated temperature.  The high temperature was used to accelerate the degradation of the API so as to obtain a large signal on the microcalorimeter.



Figure 6.  Testing compatibility between an antibiotic and lactose in solution.



Additional examples from the literature can be found in the references.




Microcalorimetry offers a rapid and easy to perform method for screening excipients for use in solid and liquid formulations.  The experimental method is simple, and always the same, eliminating the need to develop a robust HPLC method before compatibility studies can begin on each API, or API salt form in the search for a stable formulation with excipients.  In a single experiment both physical and chemical interactions are screened, eliminating the need to use expensive and time-consuming methods, such as XRPD to monitor processes such as conversion from one solid form to another.


By quickly eliminating excipients with obvious incompatibility with API only those, which have a good prospect for performing well in formulations, can then be tested using more traditional methods such as HPLC.  This results in significant savings in time and effort compared to testing all possible excipients using more labor-intensive methods. 


Late in the development of a formulation, problems may arise (e.g., compressibility, dissolution rate) and require replacement of an excipient.  Microcalorimetry is a very powerful tool in these cases, since numerous potential replacements for binders or diluents, ect. can be quickly screened, and then tested in the formulation. 


The same process can also be used to rapidly screen alternatives to excipients if post formulation chemical or physical development stability issues arise.  Manufacturing changes, and their effect on compatibility can also be screened with minimal effort. Common examples would be when suppliers of an excipient change their manufacturing processes, or when cost or capacity issues require a different vendor be chosen to supply an excipient.



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Quoted References


1.      Serajuddin, A. T. M. et al. (1998) Compatibility Testing.  Journal of Pharmaceutical Sciences vol. 8 no. 7, 696 – 704

2.      Higgins, J. D. et al.  (2003) A Stop Along The Drug Development Highway.  Today’s Chemist at Work.  July issue.  22 – 26.

3.      Phipps, M. A. et al.  (2000) Application of Isothermal Microcalorimetry in Solid State Drug Development.  Pharmaceutical Sciences & Technology Today vol. 3 no. 1, 9 – 17.

4.      Angberg, M. (1995) Lactose and Thermal Analysis with Special Emphasis on Microcalorimetry.  Thermochimica Acta 248, 161 – 176.

5.      Schmitt, E. A. et al.  (2001) Rapid, Practical and Predictive Excipient Compatibility Screening using Isothermal Microcalorimetry.  Thermochimica Acta 380, 175 – 183.



General References


Selzer, T. / Radau, M. / Kreuter, J., (1998) Use of isothermal heat conduction microcalorimetry to evaluate stability and excipient compatibility of a solid drug.  International Journal of Pharmaceutics, vol. 17, Issue 2, 227-241.


Skaria, C.V. / Gaisford, S. / O'Neill, M.A.A. / Buckton, G. / Beezer, A.E., (2005) Stability assessment of pharmaceuticals by isothermal calorimetry: two component systems.  International Journal of Pharmaceutics, vol. 292, Issues 1-2.  127-135.


Thompson, K.C.,  (2000) Pharmaceutical applications of calorimetric measurements in the new millennium. Thermochimica Acta, vol 355, Issues 1-2.  83-87.


Chadha, R. / Kashid, N. / Jain, D.V.S.,  (2004) Evaluation of the in vitro compatibility of amoxicillin/clavulanic acid and ampicillin/sulbactam with ciprofloxacin. Journal of Pharmaceutical and Biomedical Analysis, vol. 36, Issue 2.  295-307.