Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase

Verónica Avello; Mauro Torres; Mauricio Vergara; Julio Berrios; Norma A. Valdez-Cruz; Cristian Acevedo; Maria Molina Sampayo; Alan J. Dickson; Claudia Altamirano

doi:10.1371/journal.pone.0277620

Peer Review History

Original SubmissionApril 12, 2022
19 Aug 2022 Decision Letter - Ioscani Jimenez del Val, Editor PONE-D-22-10823Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase.PLOS ONE Dear Dr. Altamirano, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. To accept your manuscript for publication, we ask that you provide time profiles for the continuous cultures (e.g., VCD, Glc, Lac, and Titre), indicating when during the culture samples for cell cycle phase and transcriptomic analyses were taken. We ask for this to allow readers to ascertain that the cultures indeed reached steady state conditions. Please submit your revised manuscript by Oct 03 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Ioscani Jimenez del Val, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please amend your current ethics statement to address the following concerns: a) Did participants provide their written or verbal informed consent to participate in this study? b) If consent was verbal, please explain i) why written consent was not obtained, ii) how you documented participant consent, and iii) whether the ethics committees/IRB approved this consent procedure. 3. Thank you for stating in your Funding Statement: “This work was financially supported by funding (1200962 and 11190488) Fondo Nacional de Desarrollo Cientifico y Tecnologico, (180146) REDES from the National Research and Development Agency of Chile, and (BB/N022041/1) from the Biotechnology and Biological Sciences Research Council of the UK.” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 4. Thank you for stating the following in the Acknowledgments Section of your manuscript: “This work was financially supported by funding (1200962 and 11190488) Fondo Nacional de Desarrollo Cientifico y Tecnologico, (180146) REDES from the National Research and Development Agency of Chile, and (BB/N022041/1) from the Biotechnology and Biological Sciences Research Council of the UK.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “This work was financially supported by funding (1200962 and 11190488) Fondo Nacional de Desarrollo Cientifico y Tecnologico, (180146) REDES from the National Research and Development Agency of Chile, and (BB/N022041/1) from the Biotechnology and Biological Sciences Research Council of the UK.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments (if provided): Based on reviewer comments, I recommend that the authors consider including time profiles for the continuous cultures performed under different treatments (mild hypothermia, NaBu, and control). This will allow the readers to confirm that steady states were indeed achieved and will strengthen the conclusions presented by the authors. It may be as simple as adding a supplementary file with the data and referencing it in the manuscript. Once data confirming steady state during continuous cultures is made available, I the manuscript will be ready for publication in PLoS One. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Manuscript Number: PONE-D-22-10823 Manuscript Title: Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase. PLOS ONE This paper focused on the gene expression under continuous CHO cultivation. It seems that this paper contains valuable information. I think that the manuscript should be accepted with major revision. Chemostat data should be shown in the manuscript. Authors mentioned that all chemostat cultures were operated at a steady-state. All analytical data were analyzed on the basis of chemostat steady-state. Without this information, it is very difficult to elucidate the obtained data. However, detail time course of cell and limiting-substrate concentrations were not shown. To confirm chemostat steady-state, the detail time course should be shown in the revised manuscript. Reviewer #2: This is a good paper that examines the mechanism of the effect of low temperature and NaBu, which is considered effective in a production system using CHO cells. It is especially excellent that it has been validated in CHEMOSTAT culture. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0277620.r001
Revision 1
12 Sep 2022 Author Response Editor comments: “Based on reviewer comments, I recommend that the authors consider including time profiles for the continuous cultures performed under different treatments (mild hypothermia, NaBu, and control). This will allow the readers to confirm that steady states were indeed achieved and will strengthen the conclusions presented by the authors. It may be as simple as adding a supplementary file with the data and referencing it in the manuscript. Once data confirming steady state during continuous cultures is made available, I the manuscript will be ready for publication in PLoS One.” A: We have included the data regarding our chemostat cultures during the steady state as requested. This data was included in the supplementary file and mentioned in the manuscript to provide the readers an understanding how cells behaved during the continuous cultivation. Reviewer #1: General comments: “This paper focused on the gene expression under continuous CHO cultivation. It seems that this paper contains valuable information. I think that the manuscript should be accepted with major revision. Chemostat data should be shown in the manuscript. Authors mentioned that all chemostat cultures were operated at a steady-state. All analytical data were analyzed on the basis of chemostat steady-state. Without this information, it is very difficult to elucidate the obtained data. However, detail time course of cell and limiting-substrate concentrations were not shown. To confirm chemostat steady-state, the detail time course should be shown in the revised manuscript”. A: Thank you for the time invested in the review of this manuscript and for the positive comments. We have included the data that supported the achievement of a steady state in our chemostat cultures. This data includes profiles of cell growth, hr-tPA production, glucose and lactate throughout the cultures. We included this data in the Supplementary file as S2 Fig, and mentioned in the main manuscript. Reviewer #2: General comments: “This is a good paper that examines the mechanism of the effect of low temperature and NaBu, which is considered effective in a production system using CHO cells. It is especially excellent that it has been validated in CHEMOSTAT culture”. A: We thank the reviewer for the time taken to review this paper and for the positive comments. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0277620.r002
12 Oct 2022 Decision Letter - Ioscani Jimenez del Val, Editor PONE-D-22-10823R1Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase.PLOS ONE Dear Dr. Altamirano, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Minor revisions should be included to briefly discuss deviations from steady state and potential implications on the broader conclusions (i.e., that cell cycle arrest underlies increased productivity) presented in the manuscript. Please submit your revised manuscript by Nov 26 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Ioscani Jimenez del Val, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments (if provided): The authors have successfully addressed all comments made by the reviewers. However, additional questions have been raised regarding the existence of a true steady state during the continuous cultures. As suggested by Reviewer 1, minor revisions should be included in the manuscript to briefly discuss deviations from steady state and potential implications on the broader conclusions (i.e., that cell cycle arrest underlies increased productivity) presented in the manuscript. Whit these minor revisions, the manuscript will be ready for publication in PLoS One and will be of high interest to the journal's readership and the broader CHO community. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: No ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This paper focused on the gene expression under continuous CHO cultivation. It seems that this paper contains valuable information. I think that the manuscript should be accepted with minor revision. Steady-state is not clear According to time course of chemostat culture as shown in S2 Fig., I think that it is not steady-state. For example, VCD, lactate concentrations, other concentrations are not constant. Also, viability data is necessary. Viability (Nt/Nv) should be constant for steady-state. Limiting-substrate is not clear. According to the difference of limiting substrate, the steady-state should be changed (i.e.,doi: 10.1007/s00449-009-0351-8). Authors should clearly mention it and discuss about it in the revised manuscript. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0277620.r003
Revision 2
25 Oct 2022 Author Response Editor comments: The authors have successfully addressed all comments made by the reviewers. However, additional questions have been raised regarding the existence of a true steady state during the continuous cultures. As suggested by Reviewer 1, minor revisions should be included in the manuscript to briefly discuss deviations from steady state and potential implications on the broader conclusions (i.e., that cell cycle arrest underlies increased productivity) presented in the manuscript. With these minor revisions, the manuscript will be ready for publication in PLoS One and will be of high interest to the journal's readership and the broader CHO community. A: Thank you for the time taken for reviewing this paper and the positive comments. We addressed the concerns of Reviewer #1 in the section below. Reviewer #1: General comment: This paper focused on the gene expression under continuous CHO cultivation. It seems that this paper contains valuable information. I think that the manuscript should be accepted with minor revision. Steady-state is not clear. According to time course of chemostat culture as shown in S2 Fig., I think that it is not steady-state. For example, VCD, lactate concentrations, other concentrations are not constant. Also, viability data is necessary. Viability (Nt/Nv) should be constant for steady-state. Limiting-substrate is not clear. According to the difference of limiting substrate, the steady-state should be changed (i.e.,doi: 10.1007/s00449-009-0351-8). Authors should clearly mention it and discuss about it in the revised manuscript. A: We agree with the reviewer that Fig S2 did not show clearly the steady state, although we believe that this issue was associated with the number of conditions plotted in the graphs that made it difficult to identify the steady state from the adaptation process to the feeding regime. To address this point, we have modified Fig S2 (see below new figure) to show the initiation of the feeding regime, the corresponding adaptation process and the steady state. Cell viability was above 95% for all cultures and showed in FigS1 for the steady state. We also agree with the reviewer and the editor that the slight variations during the steady state, particularly for glucose and lactate in low temperature at high dilution rate should be discussed. We have addressed this point in the discussion section. Discussion section (Lines 441-457, Pages 13-14): “This study has provided novel insights into the regulation of the cell cycle and its relationship to the productive phenotypes of CHO cells. Nonetheless, these results must be interpreted with caution, and potential limitations should be borne in mind. An initial limitation concerns the use of chemostat cultures, a cultivation method that does not represent the reality of current industrially-relevant manufacturing settings, where cells can reach high cell density and product yields. However, continuous culture represents an excellent tool for investigating the isolated effects of environmental parameters on cell physiology and performance. Additionally, findings made in continuous cultures have been shown to be relevant in other cultivation systems [25,43,44]. Another important consideration of using continuous culture is the cellular adaptation to the feeding regime. This adaptation often leads to changes in cell density, r-protein production and metabolism during the initial residence times (time when medium is renewed in the vessel) (Fig S2). In our experimental design, we consider that steady state was attained when the concentration of viable cells and metabolites are in a 5% deviation range between consecutive points. Our data showed that four residence times enabled the achievement of steady state conditions. Whilst we found small variation in glucose and lactate at HD-33°C conditions (despite the constant cell density and hr-tPA production), these slight deviations did not disrupt the steady state conditions and the biological effects of both low temperature and sodium butyrate observed in this study.” Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0277620.r004
1 Nov 2022 Decision Letter - Ioscani Jimenez del Val, Editor Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase. PONE-D-22-10823R2 Dear Dr. Altamirano, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ioscani Jimenez del Val, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): I believe the authors have adequately addressed all reviewer comments/suggestions. I believe the manuscript is now ready for publication in PLoS One. Reviewers' comments: https://doi.org/10.1371/journal.pone.0277620.r005
Formally Accepted
4 Nov 2022 Acceptance Letter - Ioscani Jimenez del Val, Editor PONE-D-22-10823R2 Enhanced recombinant protein production in CHO cell continuous cultures under growth-inhibiting conditions is associated with an arrested cell cycle in G1/G0 phase. Dear Dr. Altamirano: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ioscani Jimenez del Val Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277620.r006

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