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All concerns of both reviewers were addressed and revised manuscript is acceptable now.
[# PeerJ Staff Note - this decision was reviewed and approved by Gwyn Gould, a PeerJ Section Editor covering this Section #]
The manuscript is well-written with clear structure, appropriate language, and professional presentation. The authors have carefully addressed issues regarding figure/table descriptions and have improved literature relevance by incorporating key references, thus enhancing the clarity and completeness of the revised manuscript.
The experimental design is methodologically sound and adheres to ICH and Chinese Pharmacopoeia guidelines. The authors have clarified issues regarding specificity, robustness testing, and statistical methodology. Their responses provide sufficient detail to support reproducibility and reliability of the assay.
The findings are robust, supported by extensive validation across multiple parameters. The authors clearly acknowledge limitations such as small sample size and have proposed reasonable plans for future expansion. Additional justification for method selection and standardization further strengthens the scientific integrity.
The manuscript contributes practically to drug quality control, especially for low molecular weight heparins. The authors’ revisions have significantly improved clarity, methodological transparency, and overall quality. Minor formatting and grammar issues were corrected in the latest version.
The reviewer was satisfied with the author's response to my concerns regarding the manuscript. I agree with accepting the manuscript in its current form.
The experimental method is well-designed, demonstrating a clear and systematic approach. Additionally, the analytical merits of the study have been thoroughly evaluated.
The method presented in the study is suitable for rapid drug quality testing and has the potential to be effectively applied in routine pharmaceutical analysis.
Please address concerns of both reviewers and amend the manuscript accordingly.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
1. BASIC REPORTING
The manuscript adheres well to the PeerJ standards for basic reporting, with a clear structure, professional English, and a logical flow. However, some areas could be improved for clarity and completeness.
Minors:
1) Incomplete Figure and Table Descriptions
While tables (e.g., Tables 1-8) are included and relevant, they lack detailed captions or footnotes in some cases. For example, Table 1 mentions "The sample weight of the test substance may vary slightly during sample preparation," but this variability is not quantified or explained further. I suggest adding descriptive captions and clarifying abbreviations (e.g., "Water R") directly in the table or a legend to improve accessibility for readers unfamiliar with the terminology.
2) Literature Relevance
The references are relevant and up-to-date (e.g., ICH 2023a, Europe 2024c), but the introduction could benefit from briefly citing additional studies that highlight the challenges of traditional anti-Xa assays (beyond the authors’ assertions). This would strengthen the justification for the study.
2. EXPERIMENTAL DESIGN
The experimental design is rigorous, well-defined, and aligns with the study’s aim to establish a reproducible anti-Xa potency assay. However, some methodological details require clarification or expansion.
Minors:
1) Specificity Testing
The specificity results (Table 3) show that the blank control absorbance (0.9457) is higher than test samples (e.g., T4 = 0.5732), but no negative controls (e.g., samples lacking enoxaparin sodium or AT-III) are described to confirm the absence of interference from other components. I suggest adding a brief description of such controls in the methods and results to strengthen this claim.
2) Robustness Testing Scope
Robustness testing (Page 13, Lines 198-217) evaluates temperature, incubation time, and standing time, which is commendable. However, it does not address variability in reagent batches (e.g., factor Xa or chromogenic substrate), which the discussion acknowledges as a potential limitation (Page 17, Lines 319-320). I recommend either including preliminary data on batch variability or justifying its exclusion.
3) Statistical Analysis Detail
The use of the 4×4 parallel line assay method (Vølund, 1978) for potency calculation is appropriate, but the statistical approach (e.g., software used, assumptions of linearity) is not detailed. Please specify the tools or formulas used for regression and T-tests (e.g., P > 0.7 in Table 5) to enhance transparency.
3. VALIDITY OF THE FINDINGS
The findings are robust, statistically sound, and well-supported by the data, with clear links to the research question. However, some limitations could be better addressed.
Minors:
1) Small Sample Size for Reproducibility
The authors acknowledge that only three batches were tested across two labs (Page 17, Lines 316-318), limiting statistical power. While the results are promising, I suggest either expanding this dataset or clarifying why three batches are sufficient for initial validation.
2) Control Method Justification
The choice of the European Pharmacopoeia method as a control is justified by the lack of a Chinese Pharmacopoeia method (Page 16, Lines 277-284), but the discussion could briefly address why the USP method (Convention, 2024) was not adapted instead, given its broader range of acceptable conditions.
3) Address Limitations More Proactively
The discussion notes limitations (e.g., batch variability of factor Xa, small sample size), but solutions are vague (e.g., “preliminary experiments” for Xa adjustment, Page 18, Lines 321-326). I recommend specifying actionable steps (e.g., testing multiple Xa batches) to mitigate these issues in future studies.
4. General Comments
This manuscript presents a well-executed study with significant practical implications for enoxaparin sodium quality control. The authors deserve commendation for their detailed validation, clear presentation of results, and focus on operational efficiency (e.g., reduced reagent volumes, single-operator design). The English is professional, though minor grammatical adjustments could enhance readability (e.g., “s pecificity” in Table 3 title should be “specificity”). The primary weaknesses—small inter-laboratory sample size—is addressable with minor revisions. The study’s strengths outweigh these issues, making it a valuable contribution to the field.
In this manuscript, the authors evaluate the analytical merits of the anti-Xa factor potency of enoxaparin sodium using an ELISA-based UV-Vis spectrophotometer. The study focuses on an important aspect of drug quality control. The manuscript is well-written and provides the scientific community with a better understanding of method evaluation for the LMWH anticoagulant drug enoxaparin sodium.
The authors use appropriate methods to assess the analytical merits of the drug quality test, such as accuracy, precision, and reproducibility. This evaluation ensures that the method is suitable for its intended purpose.
The reviewer believes this is a routine method evaluation protocol and does not present any novelty or significant scientific advancement.
1. The equation for estimating the activity of test sample T (IU/mL) appears to be overlapped. A standardized format for equation writing is required for better readability.
2. In the Anti-Xa factor potency assay, how do you evaluate the completeness of the factor Xa and antithrombin III complex reaction?
3. Besides inter-laboratory reproducibility, have the authors evaluated inter-day and intra-day reproducibility?
4. In the linearity experiment, the reviewer requests that the authors clearly show the limit of detection and limit of quantitation.
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