In order to determine the structure of molecules, researchers have been using the information obtained by using circular dichroism (CD) spectroscopy for decades. Although a CD measurement is known to be easy to perform, the serial measurement of a large number of samples has been proven difficult so far especially due to the time requirement for the thorough cleaning and refilling of the short optical path length cuvettes, also evaluating the results and performing the necessary calculations required a lot of manual work. However, the growing number of biotechnologically produced pharmaceutical products, especially the spread of biosimilar products following original medicines and the analytical tests necessary for their characterization, required the acceleration of the existing methods. Due to to this challenge, the JASCO HTCD Plus (high throughput) system has been completed, which is capable of performing automated and reproducible pharmaceutical and biotechnological measurement tasks, such as screening and biosimilarity testing of proteins, peptides, DNA/RNA, antibodies, and monitoring secondary and tertiary structural changes of molecules.
The average measurement time of the samples placed in the microplates or HPLC vessels is only about 7.5 minutes. Every single step of the operations performed by the system is optimized so that the measurements are performed in a short time, using few samples, with high sensitivity and as little cross-contamination as possible. The samples placed in the 120 position HPLC vial rack or the 2×96 position microplates of the autosampler can be kept at the desired, constant temperature with a circulator thermostat. The 80 – 150 µl sample required for the measurement is transferred by a syringe pump to a 25 µl flow-through cell block with an optical path length of 1 mm and a volume of 25 µl placed in the Peltier thermostated cell holder of the JASCO J-1500 spectrometer. Optionally, JASCO cell blocks with optical path lengths of 0.1, 0.2 and 2 mm or flow-through cells of other brands can also be placed into the sample holder. The system tracks the movement of the sample with a photometric flow monitoring system, which ensures that the measurement is performed only when the sample has completely filled the cuvette. Recording simultaneous transmission and fluorescence spectra are also possible. After the measurement, the sample can be retrieved for further tests.
To avoid cross-contamination, the sample transfer system must be thoroughly washed between the measurement of each sample. For this, 3 different washing liquids are available, and the software allows the detailed setting of the washing steps. To prevent sample dilution caused by the washing liquids remaining in the tubes, the system also dries the tubes with a separate air pump.
The control of the subunits and creation of sample sequences are provided by the JASCO Spectra Manager 2 or Spectra Manager CFR program package. The sample sequences can also be imported from a spreadsheet software. The Spectra Manager CFR ensures the compliance with 21 CFR Part 11 recommendations indispensable for the pharmaceutical industry. For sample measurement the software packages contain the following program modules: spectrum measurement (Spectra Measurement), detector signal measurement as a function of temperature (Variable Temperature Measurement), spectrum measurement as a function of temperature (Temperature Interval Scan Measurement) and detector signal measurement as a function of temperature and spectrum recording at the given temperatures (Temperature/Wavelength Scan Measurement).
For biosimilarity studies and comparative studies of higher order structure proteins and peptides the qHOS (higher order structure similarity evaluation software) module is available. The comparison of the recorded spectra can also be carried out by weighing the spectrum regions and noise level. The evaluation can be done following 3 different statistical methods: Student’s t-test, Welch’s t-test and TOST Equivalence t-test.
Another advantageous feature of the J-1500 HTCD-Plus system is that it has a sample holder system that can be easily assembled and replaced by the user, a feature usual for JASCO spectroscopic instruments. Due to this, if the task requires a more specialized measurement, the HT (high throughput) module can be easily replaced with the sample holders or other accessories of the basic instrument.