June 1, 2005 (Vol. 25, No. 11)
Regulatory-Complaint and Cost-Effective Production Methods for Oligonucleotide Synthesis and Purification
Oligonucleotides are short strands of DNA or RNA that play a major role in drug discovery and molecular diagnostic chip technology. They are used from initial research and screening through to target validation and drug production.
Developers of oligonucleotide-based drugs and molecular diagnostic kits have a clear need for regulatory-compliant and cost-effective production methods. The FDA has formed a working group to develop guidelines for encouraging or requiring the adoption of process analytical technology (PAT) to the biotechnology and pharmaceutical production industries.
PAT has been used extensively in the petroleum and chemical industries for more than ten years. Simply stated, PAT calls for continuous, rather than end-stage, testing and monitoring of a processpotentially from a remote location.
Ideally, that monitoring is cybernetic. That is, the monitoring process is tied to a self-correcting mechanism to solve problems as they are encountered (or to discontinue the process if it is flawed or contaminated). A cybernetic, continuous remote monitoring system produces significant cost savings by minimizing quality-related product discards and lost production time.
PAT will be an important tool to meet the requirements of regulatory bodies such as the FDA. Common monitors like UV and pressure monitors, together with a flexible control software, can be used for PAT in oligonucleotide synthesis.
Oligonucleotide Solid-Phase Synthesis
Oligonucleotide Solid-Phase Synthesis
Solid-phase oligonucleotide synthesis in a flow-through column reactor, using a pump driven system, is carried out according to the following general procedures.
Prior to the synthesis start, the column reactor is filled with a solid support with the first nucleoside attached. This is the anchor for the growing oligonucleotide.
The first reaction, detritylation, removes a 5′-dimethoxytrityl protecting group from the support-bound nucleoside. The release of the protecting group generates a bright red-orange color, and the relative quantities can be measured on-line by UV-VIS. After detritylation, the support is washed with acetonitrile to remove the detritylation reagent.
The second reaction involves coupling of the appropriate phosphoramidite monomer (A, C, G, or T) mixed with an activator. In this reaction, a phosphite triester internucleotide bond is formed in high yields, usually over 99%. For DNA, the coupling is most often done in a single pass of reagents through the column.
RNA, on the other hand, having slower coupling kinetics, requires a longer reaction time. This can be achieved by re-circulating coupling reagents over the column. After coupling, the support is washed with acetonitrile.
The newly formed phosphite triester internucleotide bond is then converted to the corresponding phosphorothioate or phosphodiester with a thiolating or oxidation reagent, respectively. The support is washed with acetonitrile.
The final step in the synthesis cycle is capping of unreacted 5′-hydroxyl groups. After capping, the support is thoroughly washed with acetonitrile before the beginning of the next cycle. The described synthesis is repeated until the full-length oligonucleotide has been synthesized.
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.
The examples described in this article of how PAT can be applied to the process of oligonucleotide synthesis have been implemented in Unicorn software used for control of oligonucleotide synthesis systems from GE Healthcare (www. gehealthcare.com), such as KTA oligopilot, OligoPilot 400, & OligoProcess.