HPLC with Post-Column Derivatization with Alizarin for Determination of OATD-02, an Anticancer Arginase Inhibitor in Clinical Development
Round 1
Reviewer 1 Report
The goal of this work was to create an analytical technique for determining OATD-02 selectively using HPLC with post-column derivatization and fluorescence detection. OATD-02, a novel boronic acid derivative under clinical development, is an anti-cancer arginase inhibitor. In my opinion, several points must be carefully addressed.
In the introduction, the authors should add more detail about the previous analysis method of OATD-02 or its derivatives.
In methods, the purity data of the standard have to be provided in MS and the details of crude mixtures from synthesis. Section 2.3 should be moved to discussion. The chromatographic conditions and validation parameters have to be displayed in more detail. In line 125, "extremely sensitive and very selective" should be omitted. The different properties of each column have to be indicated.
In the results and discussion, the standard chromatogram has to be illustrated. The analysis time is too long. In a chromatogram, there is a peak at around one minute. The authors should discuss these points. Please provide the reason why the authors selected the analysis concentration as 0.8–1.2 mg/mL and provide the concentration of each injection for linearity. The authors should report the details of the robustness results.
The last sentence of the conclusion should be omitted: "It was tested for boronic acid analogues, which were synthesised in Molecure S.A., while optimizing compounds with the most balanced structural and physicochemical properties and the best pharmacokinetic profile The developed method was successfully transferred to contract manufacturing or organization (CMO) and applied in the routine analysis of drug active ingredient OATD-02 in the samples manufactured in GMP standard" because it is not related to the objective of the study.
The authors should update the citation since a lot of old references were used.
no
Author Response
Reviewer #1:
Thank you very much for your time and your very helpful comments.
The goal of this work was to create an analytical technique for determining OATD-02 selectively using HPLC with post-column derivatization and fluorescence detection. OATD-02, a novel boronic acid derivative under clinical development, is an anti-cancer arginase inhibitor. In my opinion, several points must be carefully addressed.
- In the introduction, the authors should add more detail about the previous analysis method of OATD-02 or its derivatives.
Author’s response:
In the Introduction, the details about the analytical method to determine of OATD-02 were added.
- In methods, the purity data of the standard have to be provided in MS and the details of crude mixtures from synthesis. Section 2.3 should be moved to discussion. The chromatographic conditions and validation parameters have to be displayed in more detail. In line 125, "extremely sensitive and very selective" should be omitted. The different properties of each column have to be indicated.
Author’s response:
In section 2.1 Reagents, there were added some information about the standard of OATD-02, which was well characterized by NMR, MS, XRPD, ICP, GC, HPLC, Karl Fischer methods and it is under provisional patent application. Section 2.3 was moved to discussion. The chromatographic conditions were presented in Table 3. The validation results were added in Section 3.2. The different properties of each column were presented in Section 3.1.3.
- In the results and discussion, the standard chromatogram has to be illustrated. The analysis time is too long. In a chromatogram, there is a peak at around one minute. The authors should discuss these points. Please provide the reason why the authors selected the analysis concentration as 0.8–1.2 mg/mL and provide the concentration of each injection for linearity. The authors should report the details of the robustness results.
Author’s response:
In the discussion Figure 7 was added (Comparison of chromatograms of OATD-02 crude mixture with all synthesis impurities and blank). Due to the all impurities in synthesis process, the time of analysis was set to 23 minutes, which was displayed on Figure 7. Additionally the peak around one minute was presented in comparison of blank to prove that it is the injection peak. In section 3.2 Method validation, there were added the concentrations of each injection for linearity. The concentrations as 0.8-1.2 mg/mL were selected because in accordance with ICH Guidelines (Validation of Analytical Procedures: Text and Methodology Q2(R1)) the analytical method should provide an acceptable linearity from 80 to 120 percent of the test concentration, which was set to 1 mg/mL. This depended on the intended application of the analytical procedure for the assay of a drug substance. The details about the robustness results were added in Table 6.
- The last sentence of the conclusion should be omitted: "It was tested for boronic acid analogues, which were synthesised in Molecure S.A., while optimizing compounds with the most balanced structural and physicochemical properties and the best pharmacokinetic profile The developed method was successfully transferred to contract manufacturing or organization (CMO) and applied in the routine analysis of drug active ingredient OATD-02 in the samples manufactured in GMP standard" because it is not related to the objective of the study.
Author’s response:
The last sentence was omitted.
- The authors should update the citation since a lot of old references were used.
Author’s response:
Some citations were updated.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript, applsci-2527472 is well presented based on a systematic study of analysis of OATD-02 by post-column derivatization of the target compound. The manuscript can be published after minor revision.
Comments
1. The selection of the analytical column is important fort the method development. According to the result in Table 1, peak area by XSelect CSH-C18 is not the largest value. However, the recovery data in Table 4, are fine. These data appear to be inconsistent, what is the argument for this? If the sample was injected without the columns, how large peak area would be found? Please add some discussion on it.
2. The derivatization will be sensitive against oxygen, since such a phenol can be readily oxidized under basic conditions. Do the authors require any special care for the issue?
3. The final conditions seem to be relatively basic, and the base tolerance of the column should be discussed (the reviewer already checked that the Waters XSelect CSH C18 can be used under pH 11).
Author Response
Reviewer 2:
Thank you very much for your time and your very helpful comments.
The manuscript, applsci-2527472 is well presented based on a systematic study of analysis of OATD-02 by post-column derivatization of the target compound. The manuscript can be published after minor revision.
Comments
- The selection of the analytical column is important for the method development. According to the result in Table 1, peak area by XSelect CSH-C18 is not the largest value. However, the recovery data in Table 4, are fine. These data appear to be inconsistent, what is the argument for this? If the sample was injected without the columns, how large peak area would be found? Please add some discussion on it.
Author’s response:
The comparison of columns in Table 2 was prepared for OATD-02 crude mixture (with impurities), so there is no data for sample injection without the column. In Section 3.1.3 there were added some information about the comparison of 3 columns with the largest value of peak area for OATD-02. The peak areas of OATD-02 were the largest values on columns: Luna C18, XSelect HSS T3 and XSelect CSH-C18, but only on column XSelect CSH-C18 the tailing factor of peak of OATD-02 was below 1.5. Moreover the column XSelect CSH-C18 can be used in the widest range of mobile phase pH values (under pH 11), which is very important when using a basic mobile phase with sodium bicarbonate.
- The derivatization will be sensitive against oxygen, since such a phenol can be readily oxidized under basic conditions. Do the authors require any special care for the issue?
Author’s response:
There are no phenols in the structure of OATD-02 and its related substances, only cyclohexane derivatives. In the injection sequence, it was verified that there are no peak area decreasing during the analysis and the drug substance and analytical method are stable under basic conditions.
- The final conditions seem to be relatively basic, and the base tolerance of the column should be discussed (the reviewer already checked that the Waters XSelect CSH C18 can be used under pH 11).
Author’s response:
In Section 3.1.3 there were added information about the column XSelect CSH-C18. “Moreover the column XSelect CSH-C18 can be used in the widest range of mobile phase pH values (under pH 11), which is very important when using a basic mobile phase with sodium bicarbonate.”
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
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