PAT Applied to the Process
By applying multiwavelength/ channel UV monitoring during the synthesis cycle, the complete process can be visualized. Visualization of the complete process based on UV-VIS monitoring is depicted in Figure 1.
A cybernetic control solution ensures that maximum yield and purity, as well as cost-efficient production, is obtained. This solution takes advantage of the continuous monitoring enabled by the multiwavelength UV monitor and conditional programming of the control software.
Measurement by UV
The releases of the 5'-dimethoxytrityl protecting group can be measured by UV, and the signal is integrated on-line by the control software. The amount of dimethoxytrityl eluted is a direct measure of oligonucleotide sequences still active in the column reactor for further coupling of bases, and thus can be used as a quantitative measurement of the ongoing synthesis.
The information can be used to terminate the ongoing synthesis should the integrated value drop below a pre-set limit, an important function that helps avoid costly prolongation of an oligonucleotide not meeting final QC criteria (Figure 1).
In order to ensure that complete detritylation has occurred, conditional programming can be used in such a way that the detritylation reagent will continue to be pumped through the column until the absorbance at 436 nm is below a pre-set level.
Incomplete wash-out of reagents between the synthesis steps could generate unwanted side reactions and modifications to the oligonucleotide. Required wash volumes are both dependent on known physical parameters like column reactor diameter and/or bed height (column volume), but wash volumes are also dependent on changed diffusion characteristics during the synthesis and hard to predict.
By using the UV signal and control software with conditional method programming it is possible to monitor the wash performance on-line and control the execution of next step conditionally, based on a fully complete wash. See wash steps depicted in Figure 1.
3. Base Identification
and Reagent Detection
UV measurement can also be used to identify and detect reagents as they enter and/or leave the column reactor. Different synthesis reagents require different wavelengths (Figure 1).
In addition to variations in column volume due to diameter and bed height, different solid support nucleoside loadings are required for synthesis of different lengths of oligonucleotides. For this reason, one and the same column volume may represent different synthesis scales.
At the same time the system hold-up volume is static, thus the ratio of system volume/column volume differs with reagent volume due to the difference in synthesis scale and flow rate required to achieve the correct linear flow rate for the column diameter.
To save process time, the reagent can be charged by the system at a high flow rate. As the reagent front is detected by a UV monitor pre-column, the flow rate can be reduced to the linear flow rate needed for the reagent to pass the column.
Detection of the coupling reagents is especially important for RNA synthesis where the monomers are re-circulated. UV can be used to ensure that the monomers are in the re-circulation loop, i.e., that they reach the loop but are not pushed to waste, before the loop is closed.
By making a quotient from two different UV wavelengths it is also possible to distinguish between A, C, G, and T monomers (Figure 2). Using the control software, conditional programming can be applied to the obtained signal, and thus rules can be set up for on-line identification of the monomer added.
4. Column Back-Pressure
As a result of the growing oligonucleotide, the diffusion properties of the solid support change and the column pressure drop increases during synthesis. Because of this, wash flow rates would normally be set low to allow for the pressure increase. However, to reduce the overall process time it is desirable to carry out wash steps as quickly as possible.
This problem can be addressed by utilizing a pressure flow control where a pressure limit is set rather than a flow set point. The pumps, controlled by the software, will thus generate the set pressure. This results in maximum wash flow rate being obtained throughout the process, without exceeding system pressure limits.
In practice this means that the actual flow rate during the wash steps is lower at the end of the process than at the beginning. This function, in conjunction with UV to detect complete wash, gives a fast and efficient wash, which would not be possible without monitoring and conditional programming.
If the process is sensitive to high flow rates, the actual pressure and/or flow can be kept within an acceptable window by employing conditional programming.