In the last issue of Elab, the principles behind ultrasonic spectroscopy were introduced. Here, Cormac Smyth,Evgeny Kudriashov, Breda O'Driscoll and Vitaly Buckintake a closer look at its practical applications.
Pharmaceutical production normally involves the manufacture of the finished product, followed by laboratory analysis to verify quality. The disadvantages of this approach are associated with the requirements for continual process optimisation, recurring manufacturing difficulties, and the expense of failed batches. A novel technique,high-resolution ultrasonic spectroscopy, can address many of these concerns.
Capable of dealing with a wide range of samples and dynamic processes,high-resolution ultrasonic spectroscopy is a non-destructive tool that generates product quality information in real process time. It allows in-line, or fast off-line, testing as a primary means of process monitoring, yielding rich data on the variations stages of the process.
This article takes describes two applications the monitoring of denaturation and aggregation of proteins in antibody solution, and crystallisation.
A good example of the practical use ofhigh-resolution ultrasonic spectroscopy is the monitoring of thermal transitions in biopolymers such as protein denaturation, aggregation and gelation.
Fig. 1 shows the ultrasonic monitoring of heat-induced transitions in a solution of antibody during consequent heating and cooling ramps between 20 and 80oC.
Antibodies of the immunoglobulin E (IgE) play a key role in the immune response to host defence against parasitic infection and in the development of allergic and inflammatory responses. These include inflammation, itching, coughing, lacrimation, bronchoconstriction, mucus secretion, vomiting and diarrhoea; all common symptoms in allergic disorders. The effective functions of all antibodies depend on their ability to sensitise cells for antigen-induced activation by binding to cell surface receptors.
The ability to regulate these interactions offers the potential to control the harmful effects of IgE. Heat-induced transformations in the protein structure have a great impact on their functional properties and assessing these changes is one of most important parameters required for manufacturing of drugs and vaccines based on the antibody.
To examine the thermal properties of antibody, 1ml of 5mg/ml solution of IgE in PBS buffer (pH 7.4) was loaded into one ultrasonic cell, and a reference cell was filled with the buffer. The ultrasonic velocity and attenuation in IgE solution were continuously monitored upon consecutive heating(at 0.5oC/min) and cooling (at 0.25oC/min) ramps.
The inserts in Fig. 1 (a and b) show the ultrasonic temperature profiles in the IgE solution. As seen in the figure, relative ultrasonic velocity (solution buffer) and attenuation decrease monotonically within the temperature range between 2050oC. The decrease in ultrasonic velocity (baseline) is attributed to the different temperature properties of ultrasonic velocity (such as density and compressibility) in the hydration shell of protein in comparison with bulk water.
Temperature dependence of ultrasonic parameters when heating between 20 and 55oC were used as a baseline. The ultrasonic data after subtraction of the baseline are shown in main graphs in Fig. 1 (a and b).
Sharp drop in ultrasonic velocity and increase in attenuation at T~54oC demonstrate the heat-induced structural transition (unfolding) in the protein solution. Changes in the ultrasonic velocity can be used to make quantitative analysis of this transition.
An aideal' unfolding of a globular protein (increase accessibility of atomic groups to water) will increase the value of ultrasonic velocity, because of the lower compressibility of water in the hydration shells of atomic groups in the proteins.
The observed decrease of ultrasonic velocity shows that the real unfolded IgE structure has a higher compressibility compared with the native structure. This is explained by an immediate aggregation of unfolded hydrophobic parts of the molecule and the formation of hydrophobic acores', which are highly compressible.
Simple quantitative comparison of observed changes in ultrasonic velocity with those measured for coil-globule transition in polymers and micelle formation, shows poor packing of antibody aggregates and significant accessibility of atomic groups of protein to water. The aggregation caused by the heat transition IgE is clearly seen on the attenuation curves (Fig. 1b).
The sharp rise of ultrasonic attenuation observed around 54oC at all frequencies can be attributed to the formation protein particle aggregates.
The scattering of ultrasonic wave on these particles results in the growth of overall ultrasonic attenuation. The scattering contribution is dependent on the ratio of particle size and frequency and the packing of the particle - the attenuation at higher frequencies is more sensitive to the formation of small particles.
Therefore, multi-frequency ultrasonic attenuation measurements allow analysis of the particle structure and size. As also seen in Fig.1 a, ultrasonic attenuation starts decrease above after 70oC. This could be explained by a second thermal transition in protein solution at temperatures around and above 70oC. This second transition can also bee seen on ultrasonic velocity data as an extra decrease on the heating curve and a breakpoint on the cooling curves.
Overall ultrasonic attenuation does not essentially decrease after the cooling ramp, showing that the protein aggregates do not dissociate after cooling. Ultrasonic velocity does not return to the initial value for the native protein. This suggests the irreversibility of the heat-induced protein aggregation.
A clear break-point in ultrasonic velocity at about 70oC shows some rearrangement of the protein structure at this temperature. This is not accompanied by any change in ultrasonic attenuation, showing that this rearrangement does not affect the overall the aggregated state of the protein.
At about 40 to 45oC a small deviation on the velocity curve Fig. 1 a shows a possible presence of transition reversed to the main transition on the heating curves observed at 54oC. However the small change in ultrasonic velocity and absence of any drop in attenuation show the aggregated structure of protein at low temperatures.
As illustrated in this example, high-resolution ultrasonic spectroscopy can be used to analyseheat-induced denaturation of the protein, detect aggregation and secondary temperature transitions, characterise of the state of protein in thepost-transition phase and, after cooling, verify the irreversibility of the transition.
Measurement of the amount of crystallised compounds in raw materials, intermediates and batches of final products, as well as assessment of the size of crystals and interaction between crystals is an important part routine analytical in the pharmaceutical industry.
The HR-US series of spectrometers allow you to measure the crystal grows with time, temperature or just assess the amount of the crystals is the sample.
Figs 2a and 2b show the ability of ultrasonic spectroscopy to monitor crystallisation. The monitoring of crystal formation, in particular the kinetics of this reaction is essential for the optimisation of process control in the batch crystallisation of pharmaceutical compounds. In this example high-resolution ultrasonic spectroscopy is used to assess the crystallisation in palm oil subjected to different thermal treatments.
Palm oil is a complex natural oil consisting of two main triglyceride fractions, palamatic and oleic acid as well as small amount of other triglycerides and diglycerides. In this example, an ultrasonic cell filled with palm oil at 40°C. The temperature was cooled down from 40°C to 10°C at 1°C/min and at 0.2°C/min, and the crystallisation under cooling was monitored by the measurements of both ultrasonic velocity and attenuation.
Fig. 2 shows two distinctive transitions, one at 35°C and the other below 20°C. Both transitions are seen clearly in the slow cooling regime. High temperature transition is attributed to crystallisation of palmitic fatty acid, while low temperature point associated with the crystallisation of oleic fatty acid. Rise in ultrasonic velocity is proportional to the growth of solid phase (micro-crystals), and thus it can be used to make quantitative analysis of kinetics of the crystallisation.
At 17°C, the solid fat content in the oils of the two different cooling rates is the same, as indicated by similar values of ultrasonic velocity. The crystal network formation is also shown by a clear transition resulting in an increase in ultrasonic attenuation for both cooling rates (Fig. 2b). The sharp peak at 35°C shows a high cooperativity of crystallisation at low cooling rates.
This example demonstrates high potential of new ultrasonic technique for the analysis of crystallisation processes. Temperature transition, kinetics of the transition and solid phase content growth can be monitored easily using the HR-US spectrometers.
Recognition of the potential of high-resolution ultrasonic spectroscopy lead to the Silver Award for best new product in the show at the PittCon given to the first HR-US series of ultrasonic spectrometers (HR-US 101) and the R&D 100 Award in 2002.
Cormac Smyth, Evgeny Kudriashov, Breda O'Driscoll are with Ultrasonic Scientific, Dublin, Ireland. Vitaly Buckin is with the Department of Chemistry, University College Dublin, Dublin 4, Ireland.
More information, plus a free introduction to the principles of operation of the ultrasonic spectrometer, is available directly from Ultrasonic Scientific,tel: 00 353 1 218 0600, fax: 00 353 1 2180601,email: firstname.lastname@example.org