Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comment

The manuscript entitled “Optimization of a Simple Protocol for IgG Purification from Baboon (Papio anubis) Serum Using Caprylic Acid” is generally well written and clearly structured. The experimental protocol is simple and easy to follow, and the results obtained are overall good and convincing.

However, the study would have been strengthened by the use of a design of experiments (DoE) or a more systematic optimization strategy, which could have allowed a better evaluation of the influence of key parameters and resulted in a more robust optimization process. Despite this limitation, the reported results remain acceptable and relevant.

Originality and relevance of the topic
The topic addressed in this manuscript is relevant to the field, particularly in the context of simplifying IgG purification protocols. While the use of caprylic acid for immunoglobulin purification is already well established, applying a simplified protocol to baboon (Papio anubis) serum is of practical interest, especially for laboratories working with non-human primate models. That said, the manuscript would benefit from a clearer explanation of the specific gap it aims to address within the existing literature.

Contribution compared with existing studies
The main contribution of this work lies in its practical and methodological aspect, rather than in conceptual novelty. The protocol is simple and accessible, which is a positive point. However, the authors should better highlight how their results compare with previously published studies in terms of yield, purity, efficiency, or practical advantages, in order to clearly position their work within the field.

Methodological aspects and possible improvements
The experimental procedure is clearly described and appears technically sound. Nevertheless, the study would have been strengthened by a more systematic optimization approach, such as a design of experiments (DoE), to better assess the influence of key parameters (e.g., caprylic acid concentration, pH, incubation time). Providing clearer justification for the selected experimental conditions would also improve the methodological rigor of the study.

Conclusions and consistency with the data
Overall, the conclusions are supported by the data presented. However, they could be slightly toned down to reflect the exploratory nature of the optimization and the absence of a comprehensive experimental design. Emphasizing the practical applicability of the protocol rather than its optimization would make the conclusions more balanced.

In summary, this manuscript addresses a relevant topic and presents generally acceptable results. With a clearer positioning within the literature, some methodological clarifications, and more balanced conclusions, its scientific quality and impact would be improved.

Specific comments / Minor revisions

1. Units and SI notation
• Lines 26, 31, 104, 116, 153, 163, 386, 481, 487:
  “minutes” should be written as “min”, in accordance with internationally accepted SI notation.
• Lines 126, 144, 154, 164, 172, 189, 199, 225:
  “1 hour” should be written as “1 h”.
• Line 127:
  “16 hour” should be written as “16 h”.
• Line 224:
  “volts” should be written as “V”.
2. Numerical data
• All numerical values should be consistently rounded to two or three decimal places throughout the manuscript.
3. Figures and graphical presentation
• The figures are ambiguous in terms of color, making it difficult to clearly distinguish between experimental conditions.
• In several figures, the figure legends are disproportionately large compared to the graphs themselves, which affects readability. The authors are encouraged to improve color contrast and adjust the size of legends for clearer data visualization.

 

Overall recommendation

With these minor revisions and clarifications, the manuscript would be significantly improved in terms of clarity, readability, and compliance with international publication standards.

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Introduction

The Introduction is well written, comprehensive, and scientifically robust, providing a clear and coherent background that effectively supports the objectives of the study. The authors present a logical progression from the general biological role of immunoglobulins to the specific relevance of Papio anubis as a biomedical model, highlighting its genetic and immunological similarity to humans. This contextualization is well supported by appropriate and up-to-date references.

A major strength of the Introduction is the clear justification for the use of baboon-derived immunoglobulins, supported by evidence of their applicability in immunological studies, vaccine development, and diagnostic and therapeutic applications. The discussion of traditional and non-chromatographic purification methods is balanced and technically accurate, clearly outlining their advantages and limitations, particularly in terms of cost, scalability, selectivity, and safety.

The rationale for selecting caprylic acid (CA) precipitation as the purification strategy is particularly strong. The authors convincingly describe its mechanism of action, advantages regarding viral safety, preservation of IgG functionality, and suitability for low-resource laboratory settings. The review of previous studies applying CA precipitation to other species further strengthens the scientific foundation of the work.

Importantly, the authors clearly identify a knowledge gap, noting the absence of studies addressing the optimization of caprylic acid–based IgG purification from baboon serum. This gap is well articulated and provides a strong justification for the present study.

Minor Suggestions for Improvement

1. Clarification of the study objective:
   The final sentence of the Introduction could be slightly expanded to explicitly state the main objective(s) of the study. Clearly indicating which parameters are being optimized (e.g., pH, caprylic acid concentration, reaction time) and which outcomes are evaluated (e.g., yield, purity, IgG integrity) would further strengthen the closing of the Introduction.
2. Textual flow and formatting:
   Minor formatting issues related to line numbering and paragraph breaks should be corrected in the final version to improve readability. These do not affect the scientific quality of the text.

Overall, the Introduction is strong, well-structured, and appropriate for publication, requiring only minor refinements to further enhance clarity and impact.

Materials and Methods

1. Ethical approval
   Please include an explicit ethics statement indicating the approving committee, protocol/approval number, and confirmation that all procedures involving Papio anubis complied with relevant animal welfare regulations.
2. Blood volume collected per animal
   The manuscript reports collection of 6 × 100 mL of blood per baboon. Please clarify the total volume collected per animal, whether collections were performed in a single or multiple sessions, and whether this volume is within recommended safe limits for non-human primates.
3. Sedation and animal handling
   Please specify whether animals were sedated and/or anesthetized during femoral venipuncture, and briefly describe the measures taken to minimize stress and pain.
4. Pooling of serum samples
   Serum samples were pooled prior to purification. Please justify the rationale for pooling and indicate the number of independent pools used in the analyses.
5. Definition of optimal purification conditions
   Several parameters were evaluated during optimization. Please clarify which outcome(s) were primarily used to define the “optimal” purification conditions.
6. Centrifugation parameters
   For reproducibility, please report centrifugation conditions in relative centrifugal force (× g), in addition to rpm.

Reviewer’s Overall Assessment

The manuscript is well written, methodologically sound, and scientifically solid. The Results and Discussion sections are clear, coherent, and appropriately interpreted, with conclusions well supported by the data presented. The Conclusion effectively summarizes the main findings and highlights the relevance of the optimized caprylic acid fractionation protocol, and does not require further revision.

The comments raised above are limited to minor clarifications and ethical transparency within the Introduction and Materials and Methods sections. Addressing these points will improve clarity, reproducibility, and compliance with editorial standards but does not require additional experiments or changes to the results, discussion, or conclusions.

Overall, the manuscript is suitable for publication after minor revisions, focused exclusively on the points outlined above.

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors The manuscript describes a methodological gap by optimising a caprylic acid-based protocol for IgG purification from baboon serum, a relevant non-human primate model with high translational value. The study is well motivated, the experimental rationale is sound, and the work has practical relevance for laboratories in low-resource settings. The manuscript is generally well written, logically structured, and supported by appropriate literature. However, several critical issues must be addressed before publication, particularly regarding experimental details, validation of IgG identity and functionality, and clarity of conclusions.   Major points 1. Title: The phrase “Optimisation of a Simple Protocol” is appropriate, but the title could be more precise. I suggest “Optimisation of a Caprylic Acid–Based Protocol for IgG Purification from Baboon (Papio anubis) Serum.” 2.The title indicates purification from two baboons, but the implications of this small sample size are not sufficiently discussed. It is unclear whether experiments were performed independently on both sera or pooled. Clarify whether sera were processed independently or pooled. How reproducibility was assessed. Explicitly acknowledge this limitation in the Discussion and Conclusions. 3. The Introduction is comprehensive but somewhat repetitive regarding the advantages of CA purification. The section on non-chromatographic methods could be streamlined. Condense background on alternative methods and focus more explicitly on: Why baboon IgG presents a unique case. Why is it required despite success in other species? 4.Purity is assessed primarily by SDS-PAGE and albumin:globulin ratios, which confirm enrichment but not functional integrity. Claims that the IgG is “comparable to commercial-grade products” are not fully substantiated without functional assays. I recommend including at least one functional or immunochemical assay. If not feasible, explicitly temper claims regarding therapeutic or diagnostic suitability. 5. The manuscript frequently refers to “highest yield”, “increased protein”, or “minimised albumin”, but numerical values, replicates, and variability are not consistently described in the provided sections. Also, it is unclear how protein concentration was measured and how many replicates were performed. Ensure that data are presented with mean ± SD (or SEM) and number of replicates. Statistical comparisons (if any) are described. 6. The manuscript repeatedly frames the method as suitable for therapeutic antibody production, including virus safety and comparability to antivenoms. However, no direct testing of endotoxin levels, viral clearance, sterility, or in vivo safety is presented. Reframe claims more conservatively: Emphasise research-grade and preclinical utility. Clearly state that additional downstream processing and validation would be required for therapeutic applications. 7. The comparison with commercial IgG products is interesting but insufficiently detailed. It is unclear whether comparisons are based on side-by-side SDS-PAGE, concentration only, or additional metrics. Clarify which parameters were compared and avoid implying regulatory equivalence unless supported by appropriate data. 8. The Conclusions restate results clearly but could better distinguish what was demonstrated versus what is proposed. Explicitly separate (yield, purity under defined conditions) and the potential applications (future therapeutic or large-scale use)] 9. Minor language issues should be corrected.  

Author Response

Please see the attachment. Thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made appropriate revisions and provided clear and satisfactory explanations in response to my previous comments. These changes have improved the manuscript. I now recommend the publication in its present form.